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Chemical compounds from Phoenicianjuniper berries (Juniperus phoenicea)Nizar Nasri a c , Nizar Tlili a , Walid Elfalleh b , Emna Cherif a , AliFerchichi b , Abdelhamid Khaldi c & Saida Triki aa Laboratoire de Biochimie, Département de Biologie, Faculté desSciences de Tunis, Université Tunis El-Manar 2092, Tunisiab IRA, Laboratoire d’Arido-culture & cultures oasiennes, MédenineBP. 4119, Tunisiac INRGREF, Unité de Recherche Gestion et Valorisation desRessources 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, AbdelhamidKhaldi & Saida Triki (2011): Chemical compounds from Phoenician juniper berries (Juniperusphoenicea), Natural Product Research, 25:18, 1733-1742

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Natural Product ResearchVol. 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

aLaboratoire de Biochimie, Departement de Biologie, Faculte des Sciences de Tunis,Universite Tunis El-Manar 2092, Tunisia; bIRA, Laboratoire d’Arido-culture &cultures oasiennes, Medenine BP. 4119, Tunisia; cINRGREF, Unite de Recherche Gestionet Valorisation des Ressources Forestieres, BP: 10 Ariana 2080, Tunisia

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

Natural chemical compounds are a widely researched topic worldwidebecause 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 alsoquantified like Na (63.8mg per 100 gDW) or K (373.9mg per 100 gDW).Total reduced sugars are ca 192.6mg g�1DW. Polyphenols and flavonoidsfrom berries are highly present with an average of 1764� 174.3mg gallicacid per 100 gDW and 890� 47.6mg rutin per 100 gDW, respectively.Mean free radical scavenging activities, determined by DPPH and ABTS,are 1337� 126.2mM TEAC per 100 gDW and 1105.7� 95.9mM TEACper 100 gDW, respectively. All findings improve the possible presence ofbiologically active fractions in phytocomplex that could be used as suchand/or extracted for the formulation of supplements and/or ingredients forthe 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 aprogressive increase in the demand for vegetable sources for domestic purposes.Plants containing natural compounds are of particular interest. The use of naturalcomponents for reducing cardio, cerebrovascular diseases and cancer mortality hasgained considerable attention (Hertog, Feskens, & Kromhout, 1997).

Polyunsaturated fatty acids are minor lipid phytochemicals. They are structurallydistinguished by the presence of repeating methylene units. These units produce anextremely Fexible chain rapidly reorienting through conformational states andconstitute 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

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(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 compoundshave 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 alsorecognised to be an integral part of the human and animal diet metabolites

(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). InTunisia (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 heightof 10m. 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 berrieshas 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 anaverage 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.

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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

Table 2. Tunisian J. phoenicia fatty acid compositiona (in percent of total fatty acids) fromseeds.

Populations

Fatty acids Retention time (min) EK OK KS Mean� SD (n¼ 3)

14 : 0 2.23 0.5 0.4 0.5 0.5� 0.116 : 0 3.16 7.8 7.2 8.3 7.8� 0.616 : 1 cis 3.46 0.4 0.2 0.0 0.2� 0.216 : 1 trans 3.56 0.7 0.2 0.5 0.5� 0.317 : 0 3.79 0.2 0.1 0.0 0.1� 0.118 : 0 4.18 4.2 4.6 4.4 4.4� 0.218 : 1D9 4.39 12.5 12.8 13.2 12.8� 0.318 : 2D9/12 4.80 31.4 34.8 32.7 33.0� 1.718 : 3D9/12/15 5.46 29.4 26.1 29.5 28.3� 1.920 : 0 5.78 0.1 0.2 0.1 0.2� 0.020 : 1D11 6.34 0.3 0.8 0.0 0.3� 0.420 : 2D5/11 6.65 0.2 0.0 0.1� 0.120 : 2D11/14 7.08 1.8 1.8 1.0 1.5� 0.420 : 3D5/11/14 7.41 3.1 3.8 3.0 3.3� 0.420 : 3D11/14/17 8.24 0.3 0.3 0.0 0.2� 0.220 : 4D5/11/14/17 8.65 6.4 6.1 6.3 6.3� 0.224 : 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 p¼ 0.05and tr, traces.

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� 1.9Total lipids (%) 10.2� 1.4 18.2� 0.07 07.0� 0.4 11.8� 5.8Total proteinsKjeldahl assay (%) 6.0� 0.9 9.4� 0.33 9.67� 0.4 8.4� 2.0Bradford assay(mg g�1DW)

75.9� 1.7 34.7� 0.36 19.6� 0.9 43.4� 10.6 (4.3%)

Total reduced sugars(mg g�1DW)

228.1� 18.6 168.8� 25.4 180.8� 13.8 192.6� 31.3

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

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unknown but it has been hypothesised that it might be related to a coldacclimatisation 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 acid20 : 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) fattyacids 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 juniperonicfatty acid can minimise hepatic reperfusion injury by reducing cell death in thepericentral regions of the liver lobule by 75%. The same authors confirm that trypanblue distribution time, an indicator of the hepatic microcirculation, was reduced byapproximately 25% with fish oil and over 50% by juniper berry oil diets, particularlydue to extensive incorporation of the fatty acid 20 : 3D5/11/14 into phospholipids inliver tissue, especially phosphatidylinositol.

2.2. Protein and reduced sugar contents

Juniperus phoenicea seed protein contents (by Kjeldahl, 1883) ranged from6.0� 0.9% (El-Ala population, coded EK) to 9.6� 0.4% (KS population) with anaverage of 8.4� 2.0% and between 75.9� 1.7mg g�1DW (EK population) to19.6� 0.9mg g�1DW (KS population), averaging 43.4mg g�1DW (4.3%) usingBradford (1976) assay (Table 1).

To compare, we see that the seeds of J. phoenicea store a quantity of proteincomparable 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) to228.1� 8.6 (EK population), with an average of 192.6� 31.3 as mg glucose per1 gDW (Table 1).

Storage chemicals from seeds of non-conventional plants should be explored.Proteins, for example, play a significant role in human health. In developingcountries, where the average protein intake is less than required, it is essential to findnew sources of edible protein and other nutrients to overcome the populationproblem.

2.3. Mineral contents of J. phoenicea seeds

Quantified minerals in J. phoenicea berries are shown in Table 3. The mean averageof Na is 63.8mg per 100 gDW; OK population has the highest value (68.1mg per100 gDW). Ca and Mg average 94.6mg per 100 gDW and 65.2mg per 100 gDW,respectively. Cu, Zn, Fe and Mn minerals are present in few amounts

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1.1mg per 100 gDW, 1.1mg per 100 gDW, 1.7mg per 100 gDW and 0.3mg per100 gDW, respectively.

Juniperus phoenicea berries contained high levels of potassium (373.9mg per100 gDW) slightly more than those found in pomegranates (211mg per 100 gDW;Elfalleh et al., 2009); K contents are 153.6mg per 100 gDW, 502.6mg per 100 gDWand 465.7mg per 100 gDW for EK, OK and KS populations, respectively. Contentsof minerals obtained in the studied populations remain lesser than some commonlyedible 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 rangedfrom 1638.0� 52.0 (EK population) to 1963.0� 108.0 (KS population), as mg gallicacid per 100 g dry weight (mg gallic acid per 100 gDW). Total Favonoids varied from835.0� 25.0 (EK population) to 919.0� 7.0 (KS population), as mg rutin per 100 gof dry weight basis (mg rutin per 100 gDW).

2.5. DPPH and ABTSþ antioxidant capacity and reducing power ofjuniper berry extracts

The antioxidant capacity of juniper berry extracts was evaluated with the DPPH andABTS tests (Table 5). The free radical scavenging activity determined by DPPHvaried from 1193.4� 128.2 (OK population) to 1430.6� 108.2 (KS population)mM

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

Populations

Mineral contents

Na K Ca Mg Cu Zn Fe Mn

EK 60.2 153.6 82.8 37.0 0.9 0.7 1.3 0.2OK 68.1 502.6 97.2 47.9 0.7 0.8 2.2 0.4KS 63.0 465.7 103.6 110.7 1.5 1.8 1.6 0.4Mean 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.

Total polyphenol andflavonoid contents

Populations

KS EK OK Mean� SD (n¼ 3)

Total polyphenols as mggallic acid per 100 gDW

1963.0� 108.0 1638.0� 52.0 1691.0� 106.0 1764.0� 174.3

Total flavonoids as mgrutin per 100 gDW

919.0� 7.0 835.0� 25.0 916.0� 73.3 890.0� 47.6

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

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trolox equivalent antioxidant capacity per 100 g dry weight (TEAC per 100 gDW)with an average of 1337.0� 126.2mM TEAC per 100 gDW and the valuesdetermined by ABTS ranged from 998.9� 108.5mM TEAC per 100 gDW (OKpopulation) to 1184.5� 127.4mM TEAC per 100 gDW (KS population), with anaverage of 1105.7� 95.9mM TEAC per 100 gDW.

Because of the multiple reaction characteristics and mechanisms involved in theestimation of the total antioxidants, no single method could accurately reFect all theantioxidants 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 theresults from the DPPH method assay since both methods are based on a similarantioxidant mechanism and the extracts used in both tests were methanol-soluble.All juniper extracts are comparable without significant differences. The richness ofjuniper berries with phenolic compounds confers to this species a pharmaceuticalimportance. Indeed, phenolic compounds exhibit a wide range of physiologicalproperties, such as anti-allergenic, anti-atherogenic, anti-inflammatory,anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatoreffects (Benavente-Garcıa & Castillo, 2008; Hayouni, Abdrabba, Bouix, & Hamdi,2007). Moreover, flavonoids have been shown to act as scavengers of variousoxidising species, that is super oxide anion (O�2 ), hydroxyl radical or peroxy radicals(Harborne & Williams, 2000). Samoylenko et al. (2008) reported that ethanolextracts of J. phoenicia berries showed anti-malarial and anti-leishmanial activities.The same authors were able to demonstrate that anti-bacterial activities of theseberry extracts are due to the presence of terpenoids, especially diterpenes totarol andferruginol. Despite the fact that it is not clear which components of the totalpolyphenols or flavonoids compounds are responsible for the total observedantioxidant capacity, the antioxidant capacity of juniper berry extracts is confirmed.

Total polyphenols and flavonoids from Phoenician juniper berries are highlypresent with an average of 1764.0� 174.3mg gallic acid per 100 gDW and890.0� 47.6mg rutin per 100 gDW, respectively. Free radical scavenging activity,determined by DPPH and ABTS, are 1337.0� 126.2mM TEAC per 100 gDW and1105.7� 95.9mM TEAC per 100 gDW, respectively. Nevertheless, because of theuse of these extracts as pharmaceutical resources, extracts should not be introducedunless their safety has been suitably confirmed. Some examples illustrate the need toexplore adequately the composition of some functional foods: pomegranate fruits

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

DPPH and ABTSþ

antioxidant capacity

Populations

KS EK OK Mean� SD (n¼ 3)

DPPH mMTEAC per 100 gDW

1430.6� 108.2 1387.0� 157.7 1193.4� 128.2 1337� 126.2

ABTSþ mMTEAC per 100 gDW

1184.5� 127.4 1133.9� 215.6 998.9� 108.5 1105.7� 95.9

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

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were familiarly reported to have higher antioxidant activities (Li et al., 1996) andgreat pharmaceutical usefulness. However, Sanchez-Lamar et al. (2008) reportedthat some galenic preparations of pomegranate were toxic because of their alkaloidcontents. There were also some reports of immunological disturbance afterconsumption 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�370N, longitude 09�330E, altitude 467m; Om Jedour(Kasserine): latitude 35�350N, longitude 08�560E, altitude 819m; Kesra (Siliana):latitude 35�480N, longitude 09�210E, altitude 740m. The plant was identified byDr A. Khaldi from I.N.R.G.R.E.F-Tunisia and voucher specimens (VS1-JP2010,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 ‘Faculte desSciences 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 coneswere selected for antioxidant properties and mineral analysis. Seeds selected forstorage chemical analysis (fatty acids and storage proteins) were separated manuallyfrom berries.

3.2. Oil, protein, mineral and reduced sugar extractions from seeds

Seeds were grounded in a mortar and extracted with Soxhlet apparatus as describedpreviously in Nasri, Khaldi, Fady, and Triki (2005).

Fatty acid composition was determined by gas chromatography as fatty acidmethyl esters (FAMEs). FAMEs were prepared by vigorous shaking of a solution ofJ. phoenicea oil sample in n-hexane (0.2 g in 3mL) with 0.4mL methanolic potassiumhydroxide solution (2�N). Chromatographic analysis was performed as describedpreviously in Nasri et al. (2005). Fatty acids were identified by comparing retentiontimes 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 andTriki (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 ina muffle furnace at 550�C. After cooling, the sample was added to 5mL deionisedwater and 1mL of hydrochloric acid and subjected to boiling. The capsule contentwas filtered. The filtrate was adjusted by deionised water to 100mL. The mineralconstituents (Ca, Na, K, Mg, Cu, Zn, Fe and Mn) from J. phoenicea seeds wereanalysed separately, using an atomic absorption photometer (Shimadzu A 6800,Kyoto, Japan). All measurements were performed in triplicate. Each sample wasconverted into mg per 100 g DW.

The procedure of reduced sugar analysis is as follows: 1 g of seeds of J. phoeniceais added in a test tube to a picric acid solution (5 g of picric acid and 27.5 g of

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

& Smith, 1956, modified). The solution is well mixed, and then the test tube is dipped

into the boiling water for 15min, and the colour produced is compared with standard

colour solution using glucose solution (0–1mg) prepared under the same condition

as cited below.

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

Singh, & Sakariah, 2001). Distilled water (7.9mL) was added to 0.1mL juniper berry

extract (1 g of filtered homogenised powder in 10mL of methanol) and 0.5mL

Folin-Ciocalteu reagent (1 : 1 with water). After 1min, 1.5mL of sodium carbonate

(1M) 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 (50–500mgL�1). Results were expressed as mg gallic acid per 100 g of

dry weight basis (mg gallic acid per 100 gDW). 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 0.3mL of each

extract. Then, 0.15mL of NaNO2 (0.5M) was added, followed by 0.15mL of

AlCl3–6H2O (0.3M). Test tubes were incubated at ambient temperature for 5min,

and then 2mL NaOH (1M) was added in the mixture. The volume of reaction

mixture was made to 10mL with distilled water. The mixture was vortexed and the

absorbance was measured at 506 nm. A calibration curve was prepared with rutin

(10–300mgL�1) and the results were expressed as mg rutin per 100 g of dry weight

basis (mg rutin per 100 gDW). 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 7mM

ABTS solution with 2.45mM 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 2mL of diluted ABTSþ solution, absorbance at 734 nm

was measured at 5min. Results were expressed as mM trolox equivalent antioxidant

capacity per 100 g dry weight (mM TEAC per 100 gDW; 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 2mL of

methanolic DPPH solution (100mM), 25 mL of different juniper berry extracts was

added. After the reaction was allowed to take place in the dark for 30min, and the

absorbance at 517 nm was recorded to determine the concentration of remaining

DPPH. Results were expressed as mM TEAC per 100 gDW.

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3.4. Statistical analyses

All chemical compounds contents were carried out in duplicate or triplicate andexpressed as mean� SD. Statistical analyses were performed using STATISTICAsoftwear (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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