This article was downloaded by: [Dr Nizar Nasri] On: 10 December 2011, At: 03:11 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Chemical compounds from Phoenician juniper berries (Juniperus phoenicea) Nizar Nasri
a c
a
b
a
, Nizar Tlili , Walid Elfalleh , Emna Cherif , Ali
b
c
Ferchichi , Abdelhamid Khaldi & Saida Triki
a
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 To link to this article: http://dx.doi.org/10.1080/14786419.2010.523827
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
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 (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; antioxidant 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:
[email protected]
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
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
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 (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
Moisture content (%) Total lipids (%) Total proteins Kjeldahl assay (%) Bradford assay (mg g1 DW) Total reduced sugars (mg g1 DW)
EK
OK
KS
Means SD
13.4 10.2 1.4
9.6 18.2 0.07
10.7 07.0 0.4
11.2 1.9 11.8 5.8
6.0 0.9 75.9 1.7
9.4 0.33 34.7 0.36
9.67 0.4 19.6 0.9
8.4 2.0 43.4 10.6 (4.3%)
228.1 18.6
168.8 25.4
180.8 13.8
192.6 31.3
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS, Kesra (Siliana) population.
Table 2. Tunisian J. phoenicia fatty acid compositiona (in percent of total fatty acids) from seeds. Populations Fatty acids 14 : 0 16 : 0 16 : 1 cis 16 : 1 trans 17 : 0 18 : 0 18 : 1D9 18 : 2D9/12 18 : 3D9/12/15 20 : 0 20 : 1D11 20 : 2D5/11 20 : 2D11/14 20 : 3D5/11/14 20 : 3D11/14/17 20 : 4D5/11/14/17 24 : 0 cis-D5 fatty acids saturated fatty acids
Retention time (min)
EK
OK
KS
Mean SD (n ¼ 3)
2.23 3.16 3.46 3.56 3.79 4.18 4.39 4.80 5.46 5.78 6.34 6.65 7.08 7.41 8.24 8.65 14.07
0.5 7.8 0.4 0.7 0.2 4.2 12.5 31.4 29.4 0.1 0.3
0.4 7.2 0.2 0.2 0.1 4.6 12.8 34.8 26.1 0.2 0.8 0.2 1.8 3.8 0.3 6.1 0.8 10.0 13.3
0.5 8.3 0.0 0.5 0.0 4.4 13.2 32.7 29.5 0.1 0.0 0.0 1.0 3.0 0.0 6.3 0.7 9.3 14.0
0.5 0.1 7.8 0.6 0.2 0.2 0.5 0.3 0.1 0.1 4.4 0.2 12.8 0.3 33.0 1.7 28.3 1.9 0.2 0.0 0.3 0.4 0.1 0.1 1.5 0.4 3.3 0.4 0.2 0.2 6.3 0.2 1.0 0.4 9.6 0.4 13.4 0.6
1.8 3.1 0.3 6.4 1.4 9.5 12.9
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
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
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 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 OK KS Mean
60.2 68.1 63.0 63.8
153.6 502.6 465.7 373.9
82.8 97.2 103.6 94.6
37.0 47.9 110.7 65.2
0.9 0.7 1.5 1.1
0.7 0.8 1.8 1.1
1.3 2.2 1.6 1.7
0.2 0.4 0.4 0.3
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
Table 4. Total polyphenol and flavonoid contents of juniper berry extracts. Populations 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 gallic acid per 100 g DW Total flavonoids as mg 919.0 7.0 835.0 25.0 916.0 73.3 rutin per 100 g DW
1764.0 174.3 890.0 47.6
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 Table 5. extracts.
N. Nasri et al. DPPH and ABTSþ antioxidant capacity and reducing power of juniper berry
Populations DPPH and ABTSþ antioxidant capacity
KS
EK
OK
DPPH mM 1430.6 108.2 1387.0 157.7 1193.4 128.2 TEAC per 100 g DW ABTSþ mM 1184.5 127.4 1133.9 215.6 998.9 108.5 TEAC per 100 g DW
Mean SD (n ¼ 3) 1337 126.2 1105.7 95.9
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS, Kesra (Siliana) population.
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-Garcı´ a & 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).
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
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, 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´ 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.
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
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, 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.
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
References Adams, R.P. (2004). The junipers of the world: The genus Juniperus. Victoria, BC: Trafford Publishing. Bellakhdar, J. (1997). La pharmacope´e marocaine traditionnelle. Me´decine arabe ancienne et savoirs populaires. Paris: Ibis Press. Benavente-Garcı´ a, O., & Castillo, J. (2008). Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry, 13, 6185–205. Bowen, J.H., & Watkins, C.B. (1997). Fruit maturity, carbohydrate and mineral content relationships with watercore in ‘Fuji’ apples. Postharvest Biology and Technology, 11, 31–38. Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Brandwilliams, W., Cuvelier, M.E., & Berset, C. (1995). Use of a free-radical method to evaluate antioxidant activity. Food Science and Technology, 28, 25–30. Bravo, L. (1998). Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews, 56, 317–333. Chu, Y.H., Chang, C.L., & Hsu, H.F. (2000). Flavonoid content of several vegetables and their antioxidant activity. Journal of the Science of Food and Agriculture, 80, 561–566. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. Dulau, I., & Thebaudin, J.Y. (1998). Functional properties of leguminous protein: applications in food. Grain Legumes, 20, 15–16. Elfalleh, W., Nasri, N., Marzougui, N., Thabti, I., M’rabet, A., Yahya, Y., . . . , Ferchichi, A. (2009). Physico-chemical properties and DPPH–ABTS scavenging activity of some local pomegranate (Punica granatum) ecotypes. International Journal of Food Sciences and Nutrition, 60, 197–210. Harborne, J.B., & Williams, C.A. (2000). Advances in Favonoid research since 1992. Phytochemistry, 55, 481–504. Hayouni, E.A., Abdrabba, M., Bouix, M., & Hamdi, M. (2007). The effects of solvents and extraction method on the phenolic contents and biological activities in vitro of Tunisian Quercus coccifera and Juniperus phoenicea L. fruit extracts. Food Chemistry, 105, 1126–1134. Heim, K.E., Tagliaferro, A.R., & Bobilya, D.J. (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. The Journal of Nutritional Biochemistry, 13, 572–584.
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011
1742
N. Nasri et al.
Hertog, X.G., Feskens, E.M., & Kromhout, D. (1997). Antioxidant flavonols and coronary heart disease risk. Lancet, 349, 699. Igea, J.M., Cuesta, J., Cuevas, M., Elias, L.M., Marcos, C., Lazaro, M. & Compaired, J.A. (1991). Adverse reaction to pomegranate ingestion. Allergy, 46, 472–474. Jayaprakasha, G.K., Singh, R.P., & Sakariah, K.K. (2001). Antioxidant activity of grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry, 73, 285–290. Jones, S.M., Zhong, Z., Enomoto, N., Schemmer, P., & Thurman, R.G. (1998). Dietary juniper berry oil minimizes hepatic reperfusion injury in the rat. Hepatology, 28, 1042–1050. Kawamura, Y., Ogawa, Y., Nishimura, T., Kikuchi, Y., Nishikawa, J., Nishihara, T., & Tanamoto, K. (2003). Estrogenic activities of UV stabilizers used in food contact plastics and benzophenone derivatives tested by the yeast two-hybrid assay. Journal of Health Science, 49, 205–212. Kjeldahl, J. (1883). Kjeldahl, Neue Methode zur Bestimmung des Stickstoffs in organischen Ko¨rpern. Zeitschrift fu¨r Analytische Chemie, 23, 366–382. Li, Y.G., Tanner, G., & Larkin, P. (1996). The DMACA-HCl protocol and the threshold proanthocyanidin content for bloat safety in forage legumes. Journal of the Science of Food and Agriculture, 70, 89–101. Miller, D., Wheals, B.B., & Beresford, N. (2001). Estrogenic activity of phenolic additives determined by an in vitro yeast assay. Environmental Health Perspectives, 109, 133–138. Nasri, N., & Triki, S. (2007). Les prote´ines de re´serve du pin pignon (Pinus pinea L.). Comptes Rendus Biologies, 330, 402–409. Nasri, N., Khaldi, A., Fady, B., & Triki, S. (2005). Fatty acids from seed of Pinus pinea L.: composition and population profiling. Phytochemistry, 66, 1729–1735. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. Samoylenko, V., Dunbar, D.C., Gafur, M.A., Khan, S.I., Ross, S.A., Mossa, J.S., . . . , Muhammad, I. (2008). Antiparasitic, nematicidal and antifouling constituents from Juniperus berries. Phytotherapy research, 22, 1570–1576. Sanchez-Lamar, A., Fonseca, G., Fuentes, J.L., Cozzi, R., Cundari, E., Fiore, M., . . ., De Salvia, R. (2008). Assessment of the genotoxic risk of Punica granatum L. (Punicaceae) whole fruit extracts. Journal of Ethnopharmacology, 115, 416–422. Takagi, T., & Itabashi, Y. (1982). Cis-5-OleEnic unusual fatty acids in seed lipids of Gymnospermae and their distribution in triacylglycerols. Lipids, 17, 716–723. Tapiero, H., Tew, K.D., Nguyen, B.G., & Mathe, G. (2002). Polyphenols: do they play a role in the prevention of human pathologies? Biomedicine and Pharmacotherapy, 56, 200–207. Vermerris, W., & Nicholson, R. (2006). Phenolic compound biochemistry. The Netherlands: Springer. Vidakovic, M. (1991). Conifers: Morphology and variation, Graficki Zavod Hrvatske. Wallingford, UK: Zagreb, distributed by C.A.B. International. Wolff, R.L., & Bayard, C.C. (1995). Fatty acid composition of some pine seed oils. Journal of the American Oil Chemists’ Society, 72, 1043–1046. Yong, S.P., Soon, T.J., Seong, G.K., Buk, G.H., Patricia, A.A., Fernando, T., . . . , Gorinstein, S. (2008). Antioxidants and proteins in ethylene-treated kiwifruits. Food Chemistry, 107, 640–648.
