Antioxidant activity, total phenolic and flavonoid ...

Industrial Crops and Products 51 (2013) 171–177

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Antioxidant activity, total phenolic and flavonoid content variation among Tunisian natural populations of Rhus tripartita (Ucria) Grande and Rhus pentaphylla Desf. Chetoui Itidel, Messaoud Chokri, Boussaid Mohamed, Zaouali Yosr ∗ Department of Biology, Laboratory of Plant Biotechnology, National Institute of Applied Science and Technology, B.P. 676, 1080 Tunixs Cedex, Tunisia

a r t i c l e

i n f o

Article history: Received 21 May 2013 Received in revised form 31 August 2013 Accepted 3 September 2013 Keywords: Antioxidant activity Phenolic contents Flavonoids Rhus tripartita R. pentaphylla

a b s t r a c t The antioxidant activity, total phenolic and flavonoid contents of methanolic extracts from different plant parts of Tunisian Rhus tripartita (Ucria) and R. pentaphylla Desf. populations were determined. The total phenolic content in stem cortex was higher than that of the other plant parts with the highest content in R. tripartita. Flavonoids were more represented in leaf and fruit extracts of R. pentaphylla. Antioxidant activity, determined by DPPH and FRAP assays, was high. The highest activity was found in stem cortex extract from both species with the lowest in R. pentaphylla. A significant correlation between antioxidant activity and phenolic content was revealed. PCAs performed on compound contents or antioxidant activities of all extracts did not reveal clear groupings of populations according to their bioclimatic zone. The global divergence among populations seems to be linked to altitudes and geographic distances. Based on their phenolic contents and antioxidant activity several populations from each species could be used as starting material to develop sustainable production. Conservation strategies of the populations should be made appropriately within their bioclimatic zones in respect to geographic location. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Due to their high contents in phenols, flavonoids and other phytochemicals, Rhus species are widely used in both modern and traditional medicine. The extracts showed antimalarial (Ahmed et al., 2001), antimicrobial (McCutcheon et al., 1992), antitumorigenic (Lee et al., 2004; Choi et al., 2012), antioxidant (Lee et al., 2002; Rima et al., 2011; Olchowik et al., 2012), antiviral (Lin et al., 1999), hypoglycaemic (Giancarlo et al., 2006), leukopenic (Yang and Du, 2003), atherosclerosis (Zargham and Zargham, 2008) and anticonvulsant (Ojewole, 2008) properties. The most studied Mediterranean Rhus species was R. coriaria L. (Kandan and Sökmen, 2004; Kosar et al., 2007; Chakraborty et al., 2009; Mohammadi et al., 2010; Moazeni and Mohseni, 2012). In Tunisia, the genus Rhus is represented by two species: Rhus tripartita (Ucria) Grande [=R. tripartitum (Ucria) D.C. = R. oxyacanthoides Dum. Cours. = R. oxyacantha Shousb. Ex. Cav.] and R. pentaphylla (Pottier-Alapetite, 1979; Le Floc’h and Boulous, 2008). R. pentaphylla spreads in the north and the center of the country, and is relieved by R. tripartita from the center to the far southern

∗ Corresponding author. Tel.: +216 71703829x929; fax: +216 71704329. E-mail addresses: [email protected] (B. Mohamed), [email protected] (Z. Yosr). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.09.002

region. The two species may be sympatric in several regions. They grow mainly on eroded substrates, under a rainfall ranging from 100 to 600 mm/year and at altitudes ranging from 10 to 500 m. The populations of both species are widely scattered, presumably remnant sites of previously more continuously distributed species. The overharvesting of roots and other plant parts for traditional medicine dyeing and tanning (Le Floc’h, 1983) had led to a high death rate of trees and a decreasing of the size of populations (Le Houerou, 1969). R. pentaphylla is being lost at a higher rate than R. tripartita. Its habitat has been mainly cleared for agricultural and coal mining operations. Recent work on R. tripartita reported that root cortex extract, rich in proanthocyanidines with the presence of (+) catechin, (−) epicatechin-3-ogallate, proanthocyanidic oligomers and polymers, showed high antioxidant activity and prevented DDTinduced thymocytes death in rat (Tebourbi et al., 2006). A new biflavonoid masazinoflavonone and the isobiflavonoid calodenone have been isolated from methanolic aerial part extract of this species (Mahjoub et al., 2006) which showed also antibacterial, antifungical (Abbassi and Hani, 2011) and anti-inflammatory activities. Rhus pentaphylla seed, leaf and root aquous extracts rich in tannins, flanonoids and coumarins showed substantial antibutyrylcholinesterasic activity that could be used for the treatment of Alzeimer’s disease (Ben Mansour et al., 2011).

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The aims of this study were to (i) assess the antioxidant activity and phenol contents of Tunisian R. tripartita and R. pentaphylla plant extract from leaves, fruits and stem cortex and compare their distribution within and between populations; (ii) identify some populations with organs, other than roots, high in polyphenol contents and antioxidant capacity. To the best of our knowledge, no studies taking into account the within and among populations variation of polyphenols for the two species has been carried out. Studying the within and among populations phenolic variation may help the improvement and the preservation strategies of the two taxa. 2. Material and methods 2.1. Plant material Leaves, stem cortex and fruits were harvested from the same female plants growing in nine natural populations (Table 1). Seven and two populations of R. tripartita and R. pentaphylla, respectively were considered. The populations belong to the semi-arid and the upper arid climates (Emberger, 1966). Plant parts were taken from ten individuals sampled at random in each population between the end of February and the beginning of March 2009. To avoid bias due to the development stage of organs, materials were taken from 3 years old branches; leaves with the same size from the same zone and fruits at full mature stage. Samples were air-dried at 30 ◦ C in a shady place at room temperature for 10 days. Vouchers specimens are deposited in the herbarium of the National Institute of Applied Science and Technology (Tunis).

2.5.1. Free radical-scavenging assay The free radical-scavenging activity of extracts was evaluated with the DPPH assay (Brand-Williams et al., 1995) based on the measurement of the reducing ability of antioxidants toward the DPPH radical. One milliliter of diluted extract was added to 3 ml of the methanolic DPPH solution (4 × 10−5 M). The mixture was then shaken and allowed to stand at room temperature in the dark. After 60 min, the decrease in absorbance was measured at 517 nm against a blank (methanol solution). A mixture consisting of 1 ml of methanol and 3 ml of DPPH solution was used as the control. The antiradical activity (three replicates per treatment) was expressed as SC50 (mg ml−1 ), the concentration required to scavenge 50% of free radicals. A lower SC50 value corresponds to a higher antioxidant activity. The ability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect (%) =

A − A 0 1 A0

× 100,

where A0 is the absorbance of the control at 60 min, and A1 is the absorbance of the sample at 60 min. 2.5.2. FRAP assay The ferric reducing ability was assessed according to Benzie and Strain (1996). The FRAP reagent contained 2.5 ml of 10 mM of 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ) solution in 40 mM HCl plus 2.5 ml of 20 mM FeCl3 and 25 ml of 0.3 M acetate buffer, pH 3.6. 900 ␮l FRAP reagent was mixed with 90 ␮l distilled water and 30 ␮l of diluted extracts (1:10 v/v) and warmed to 37 ◦ C in a water bath. A standard curve was prepared using different concentrations of FeSO4 ·7H2 O (200–2000 ␮mol/l). Results were expressed in mmol Fe2+ /l of plant extract. Trolox and BHT were used as reference compounds.

2.2. Samples preparation Phenolic extracts from each organ were obtained by magnetic stirring of 2.5 g of dry matter powder with 25 ml of methanolic solvent for 12 h, then filtered through a Whatman No. 4 filter paper, evaporated under vacuum to dryness and kept for 24 h at 4 ◦ C until assayed, within one week of preparation. 2.3. Total phenolic content Total phenolic content was determined using the Folin–Ciocalteu reagent, following Singleton’s method slightly modified (Dewanto et al., 2002). 0.5 ml of extract at appropriate dilutions was added to 2.5 ml of Folin–Ciocalteu reagent and 2 ml of Na2 CO3 solution. The absorbance was measured at 760 nm, after incubation for 90 min in dark. The total phenolic content was expressed as mg Gallic acid equivalents per gram of dry weight (mg GAE g−1 DW) through the calibration curve with Gallic acid. 2.4. Total flavonoid contents Total flavonoids were expressed as mg Rutin equivalents per gram of extract (mg RE g−1 DW) (Miliauskas et al., 2004). Flavonoids were measured by mixing 1 ml of appropriate diluted methanolic extract with 1 ml of 2% AlCl3 methanolic solution. After incubation at room temperature for 15 min, the absorbance was measured at 430 nm.

2.6. Data analysis All assays were performed on ten individuals per population with three replications. Results were reported as means ± standard deviation. Two-way analyses of variance (ANOVA) with organ and population or organ and species effects, were performed for the parameters total phenolic and flavonoid contents as well as for averages of DPPH scavenging activity and ferric reducing antioxidant power. Duncan’s multiple range test was used to compare mean values among populations or species. All analyses were performed using SAS program (SAS, 1990, Institute Inc., Cary, NC). The correlation between antioxidant capacity (determined by DPPH or FRAP assays) and total phenolic or flavonoids contents was tested by Mantel’s test (Mantel, 1967). The global variation of the compound contents or antioxidant capacities among populations was assessed using Principal Component Analyses (PCAs) (Princomp procedure, SAS program). 2 = Squared Mahalanobis distance (D2 ) (Hebrant, 1974) [D(i,j)

(xi − xj ) cov−1 (xi − xj ), where cov−1 = inverse variance − covariance individual matrices; xi , xj being, respectively, the vector of means of ith and the jth population] among populations based on: (i) all phenol contents, (ii) geographical distance, and (iii) altitudes of sites were calculated. The correlation of phenol content matrix with those of geographical distances or altitudes was estimated by Mantel’s test using the program TFPGA 1.3 (Miller, 1997).

3. Results and discussion

2.5. Antioxidant activity

3.1. Polyphenolic contents

The antioxidant activity was assessed by 1,1-diphenyl-2picrylhydrazyl (DPPH) and ferric reducing ability (FRAP) systems.

The two-way analyses of variance showed that averages of total phenolic and flavonoid contents varied significantly


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Table 1 Location and main ecological traits of the populations of Rhus tripartita and R. pentaphylla. Species

R. tripartitia

R. pentaphylla

Code of population

Locality

1 2 3 4 5 6 7

Bir Cheouich Sidi Neji Sbikha Ain Cherichera El Bharna El Karma Bouhedma Dj. Mountain

8 9

Chott Mariem Msaken

Bioclimatic zonea MSA

UA

LSA

Latitude

Longitude

Altitude (m)

Rainfall mm/year

Soil

35◦ 44 N 36◦ 19 N 35◦ 53 N 35◦ 59 N 35◦ 38 N 34◦ 24 N 34◦ 29 N

10◦ 19 E 9◦ 50 E 10◦ 03 E 9◦ 81 E 9◦ 51 E 7◦ 55 E 9◦ 29 E

120 120 75 250 500 500 200

350–550 350–550 250–400 250–400 250–400 250–400 150–200

Silty, loam very stoney

35◦ 54 N 35◦ 44 N

10◦ 33 E 10◦ 34 E

10 50

400–500 400–500

Silty, loam very stoney Calcareous with rock outcrops Calcareous with rock outcrops Calcareous with rock outcrops Calcareous with rock outcrops very stony Deep silty sandy loam Silty loam very stony

MSA = mean semi arid, UA = upper arid, LSA = lower semi arid. a Bioclimatic zones were defined according to Emberger’s (1966) pluviothermic coefficient. Q2 = 2000P/(M2 − m2) where P is the mean annual rainfall (mm). M is the average maximum temperature (K) in the warmest month (June) and m (K) is the mean minimum temperature in the coldest month (February).

between organs, populations and species. The organ × population or organ × species (except for total phenolic contents) interactions were significant (data not shown). 3.1.1. Variation among species For both species, the highest total phenolic content was recorded in stem cortex extracts (Table 2). R. tripartita contains the highest amounts (141.79 mg EAG g−1 DW versus 108.83 mg EAG g−1 DW in R. pentaphylla). Contents in leaves and fruits ranged from 64.13 to 75.16 mg EAG g−1 DW for R. pentaphylla, and from 71.16 to 76.88 mg EAG g−1 DW for R. tripartita. Whatever the species considered, flavonoid yields in leaves showed the highest amount (62.91 and 58.91 mg ER g−1 DW in R. pentaphylla and R. tripartita, respectively). The proportion found in stem cortex and fruits was significantly low (9.66–9.96 to 5.88–10.41 mg ER g−1 DW); the total phenolic contents obtained from aerial parts of R. tripartita were similar to those detected in root cortex (Tebourbi et al., 2006). Thus, the use of these organs could replace that of roots, and avoid the death of plants. However, comparison between the polyphenolic compositions of the sets of organs should be carried out to guide appropriate material use. 3.1.2. Variation within species In R. tripartita, the total phenolic contents from leaves ranged from 45.00 to 102.06 mg EAG g−1 DW, the highest level of variation was recorded between the populations 6 and 3, belonging to the upper bioclimatic zone (Table 2). Stem cortex (219.48 mg EAG g−1 DW) and fruit (120.75 mg EAG g−1 DW) contents were significantly higher in the population 1 from the mean semi-arid climate. Flavonoids were more represented in leaves (73.99 mg ER g−1 DW), stem cortex (12.73 mg ER g−1 DW) and fruits extracts (11.93 mg ER g−1 DW) taken respectively from the populations 3, 7 and 1 (from different bioclimates). A high polyphenolic variation among to the two geographically close R. pentaphylla populations 9 and 8 (27 km each from another) was also revealed. The highest amounts of the two sets of components were noted for the population 8. For both populations, extracts from stem cortex were rich in total phenols (69.15–156.44 mg EGA g−1 DW) and those from leaves in flavonoids (56.08–71.11 mg ER g−1 DW). Phenolic compounds are intermediates of phenylpropanoid pathway. Their production is regulated by differential expression of basic genes and highly ordered process (Mamti et al., 2006; Chang et al., 2009) with respect to plant development stage and ecological factors (Dutta et al., 2005). The differential accumulation of total phenols in stem cortex, and that of flavonoids in leaves for the two tested Rhus species should be linked to differential cytological and physiological activities within organs. The decreasing of

total phenols from stem cortex to fruits and leaves suggests a close interaction between these organs and different process of biosynthesis/degradation, and transport involved in the distribution of these compounds at the plant level (Castillo et al., 1997; Hudaib et al., 2002).

3.1.3. Divergence among all populations based on the combined analysis of total phenolic and flavonoid contents The scatter plot, obtained by PCA (Fig. 1A), based on total phenolic and flavonoid contents of the three plant parts for all populations allowed three population groups (I–III) along axes 1 (77.1% of the inertia) and 2 (19.16% of the variation). The populations clustered without evident relationships to species and bioclimatic zone. Leaf (TPLv) and stem cortex (TPSc) total phenolic contents, and leaf flavonoid content (FLv) separate group I from group II. The latter, including the three populations 4, 5 and 6 of R. tripartita and the population 9 of R. pentaphylla, differed from group I, formed by mixed R. pentaphylla (population 8) and R. tripartita (populations 1, 2, 3) populations by low organ polyphenol content. The group III, constituted of the R. tripartita population 7 (upper arid bioclimate), showed low fruit total phenol contents (TPFr). Mahalanobis distance matrix based on contents of phenols was significantly correlated with matrices of geographical distances (Mantel’s test, r = 0.349, P = 0.016) and altitudes of sites (Mantel’s test, r = 0.347, P = 0.018). The within-species polyphenolic differences recorded seem to support the occurrence of both selective forces and high genetic variability among populations. The divergence between the two R. pentaphylla populations occurring over a short distance (27 km) and growing under similar bioclimates, substrates and altitudes cannot result only from local environmental factors. It could also result from genetic drift due to a low migration rate of genes via seed or pollen dispersal leading to the increasing of variation among these populations. Environmental factors such rainfall and temperature also seem unlikely strong enough to induce the high variation recorded between the R. tripartita populations 3, 4, 5, 6 and 7 growing in the same bioclimatic zone. Isolation per distance and altitudes, are likely to be of great influence on the among populations variation. The high variation, within bioclimates could be presumably due to genetic factors rather to environmental effect. The dioecy of the two species imposes an outcrossing mating system and the frequency of males and females (the sex-ratio) within populations may influence the phenolic compositions of individuals (Gouyon et al., 1986). Thus, studies including mapping of male and female within populations, and the cultivation of the populations in the same environmental conditions were needed to verify these hypotheses. Further, it is important to assess the extent of seed migration among populations and its effects upon

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Table 2 Averages of total phenolic (mg EAG g−1 DW) and flavonoid (mg ER g−1 DW) contents according to organs and populations for each species. Species

Rhus tripartita

Rhus pentaphylla

Pop.a code

Total phenols

Flavonoids

Leaf

Stem cortex

Fruit

Leaf

Stem cortex

Fruit

1 2 3 4 5 6 7

99.93 ± 5.70a 69.33 ± 5.72b 102.06 ± 5.31a 58.88 ± 5.72b 69.17 ± 5.36b 45 ± 5.72c 69.23 ± 2.79b

219.48 ± 17.23a 150.48 ± 17.04cd 187.04 ± 16.92b 124.31 ± 16.95ed 115.63 ± 16.91e 76.94 ± 17.24f 174.88 ± 13.95bc

120.75 ± 5.62a 106.81 ± 5.69a 109.43 ± 5.6a 50.72 ± 5.62bc 65.96 ± 5.22b 39.16 ± 5.70c 10.46 ± 2.78d

65.02 ± 1.52a 65.70 ± 1.62a 73.99 ± 1.03a 50.70 ± 1.61b 51.46 ± 1.62b 52.64 ± 1.63b 67.33 ± 0.79a

7.64 ± 0.82cd 11.43 ± 0.82ab 8.95 ± 0.76bc 5.63 ± 0.80d 11.46 ± 0.76ab 10.82 ± 0.80ab 12.73 ± 0.81a

11.93 ± 0.70a 10.30 ± 0.78ab 10.36 ± 0.70ab 8.75 ± 0.63b 10.11 ± 0.79ab 9.79 ± 0.85ab 5.12 ± 0.88c

Mean

71.16b

141.79a

76.88b

58.91a

9.66b

9.96b

8 9

83.17 ± 5.66a 48.26 ± 5.72b

156.44 ± 17.13a 69.15 ± 17.01b

143.82 ± 5.72a 31.47 ± 5.65b

71.11 ± 1.60a 56.08 ± 1.42b

7.15 ± 0.81a 4.82 ± 0.83b

14.28 ± 0.80a 7.93 ± 0.82b

Mean

64.13b

108.83a

75.16b

62.91a

5.88c

10.41b

Numbers followed by the same letter in column between populations and in lines for means between organs for each species are not significant at P > 0.05 (Duncan’s multiple range test). a Pop. code: see Table 1.

their chemical divergence. The high differentiation among several neighboring populations (e.g. 4–6 and 8–9, 20–27 km distant) could indicate that seeds do not disperse far. The preferential grouping of R. pentaphylla populations with those of R. tripartita could not easily interpreted based only on quantitative phenolic differences. It may suggest closely genetic relationships between the two taxa. The investigation of the polyphenolic compositions of the two species over their complete distribution areas, mainly in their sympatric geographic range could help to improve this hypothesis. 3.2. Antioxidant activity 3.2.1. Variation among species The highest scavenging activity expressed as SC50 was found for stem cortex extracts (5.32 and 7.03 ␮g/ml) without significant differences among species (Table 3). R. tripartita fruit (25.33 ␮g/ml) and R. pentaphylla leaf (18.33 ␮g/ml) extracts have the lowest antioxidant activity. FRAP values differed significantly among species for the three sets of extracts (13.81 versus 9.38, 15.03 versus 13.67 and 10.90 versus 10.52 mmol/l for leaves, stem cortex and fruits, respectively). Investigations on antioxidant activities in Rhus

species extracts estimated by different methods (e.g. FRAP, DPPH, lipid peroxidation, ␤-carotene bleaching, ascorbic acid oxidation) were numerous and underline a substantial activity. However, existing results are primarily limited to several species such as R. verniciflua Stokes (Kang et al., 2002; Olchowik et al., 2012), R. chinensis (Diakpo and Yao, 2010), R. coriaria (Candan and Sokmen, 2004; Kosar et al., 2007; Mohammadi et al., 2010; Pourahmad et al., 2010), R. hirta (Wu et al., 2012) and R. tyhina (Kossah et al., 2011). Findings reported a large variation of antioxidant activities among plant parts. Physiological (e.g. developmental stage), environmental (e.g. season, temperature, light, soil) and genetic factors determine the variation of the antioxidant activity of these compounds. Our results confirm those of Mahjoub et al. (2010) and Ben Mansour et al. (2011) reporting substantial antioxidant activity variation among pant parts from the two species. 3.2.2. Variation within species The antioxidant capacity between populations of R. tripartita, despite its high level of variation, did not show statistical differences for leaf (from 12.99 ␮g/ml for the population 2 to 19.72 ␮g/ml for the population 7) and fruit extracts (from15.95 ␮g/ml in the

Fig. 1. Principal Component Analysis performed on phenolic and flavonoid contents (A) and antioxidant activity (B) for the nine populations of the two species. Plot according to axes 1–2.


175

Table 3 Total antioxidant capacity determined by DPPH and FRAP test systems for methanolic extracts from populations of Rhus tripartita and Rhus pentaphylla. Species

Pop. codea

(DPPH SC50 )

FRAP (mmol/l)

Leaf

Stem cortex

Fruit

Leaf

Stem cortex

Fruit

1 2 3 4 5 6 7

13.89 ± 0.07a 12.99 ± 0.06a 17.01 ± 0.07a 17.29 ± 0.15a 15.64 ± 0.09a 15.24 ± 0.06a 19.72 ± 0.06 a

3.56 ± 0.02bc 4.13 ± 0.02bc 8.09 ± 0.04ab 2.89 ± 0.02bc 11.08 ± 0.06a 2.73 ± 0.03bc 1.63 ± 0.01cc

22.83 ± 0.08a 30.38 ± 0.24a 25.79 ± 0.13a 15.95 ± 0.13a 21.22 ± 0.11a 32.45 ± 0.14a 35.47 ± 0.61aa

16.04 ± 2.81ab 15.05 ± 2.83abc 14.38 ± 2.39bc 12.06 ± 2.76de 13.43 ± 2.82cd 11.67 ± 2.83e 16.37 ± 2.74a

16.67 ± 2.82b 14.91 ± 2.84cd 16.31 ± 2.82bc 15.20 ± 2.82bcd 12.47 ± 2.81e 14.53 ± 2.21d 18.32 ± 2.79a

16.73 ± 2.83a 9.99 ± 2.83bc 14.48 ± 2.82a 7.26 ± 2.82d 10.48 ± 2.80bc 8.47 ± 2.82cd 11.96 ± 2.87b

Mean

15.74b

5.32c

25.33a

13.81b

15.03a

10.90b

Rhus pentaphylla

8 9 Mean

7.47 ± 0.05a 27.38 ± 0.23b 18.33a

5.62 ± 0.04a 8.21 ± 0.05a 7.03c

15.09 ± 0.10a 11.52 ± 0.08a 12.90b

10.55 ±2.81a 8.41 ± 2.83b 9.38b

15.90 ± 2.83a 11.81 ± 2.83b 13.67a

16.85 ± 2.84a 6.49 ± 2.83b 10.52b

Standard

Trolox BHT

Rhus tripartita

4.10 ± 0.02 21.22 ± 0.31

Numbers followed by the same letter in column between populations and in lines for means between organs in each species are not significant at P > 0.05 (Duncan’s multiple range test). a Pop code: see Table 1.

population 4 and 35.47 ␮g/ml in the population 7). Concerning stem cortex extracts, the highest activity was noted for the population 7 (1.63 ␮g/ml), and the lowest for the population 5 (11.08 ␮g/ml). The only difference between SC50 values among R. pentaphylla populations was noted for leaf extracts. The latter, from the population 8, showed the lowest SC50 (7.47 ␮g/ml). Compared to Trolox (SC50 = 4.10 ␮g/ml) and BHT (SC50 = 21.22 ␮g/ml), Rhus extracts from both species exhibited the highest antioxidant activity. The level of antioxidant activity variation, estimated by the FRAP method was more important than that noticed based on DPPH assay. For R. tripartita, the highest FRAP values were observed for leaf (16.37 mmol/l) and stem cortex extracts (18.32 mmol/l) of the population 7, and for fruit extracts (16.73 mmol/l) of the population 1. Regarding R. pentaphylla, the highest activities (from 10.55 mmol/l in leaves to 16.85 mmol/l in fruits) were noted for all plant parts from the population 8. The antioxidant activities were differently correlated with the two sets of phenolic compounds. All FRAP values were correlated to total phenols of all organs (r ranged from 0.699 to 0.782, P < 0.01) and flavonoids of fruits (r = 0.492, P < 0.01). SC50 was correlated only with total phenolic (r = −0.379, P < 0.01) contents of leaves. Thus, compared to total phenolic compounds, the contribution of flavonoids in antioxidant activity seems to be low and organ dependent (Soong and Barlow, 2004; Fratianni et al., 2007; Maisuthisakul et al., 2007). Our findings on the correlation between antioxidant activity and polyphenolic contents were consistent with previous studies (Djeridane et al., 2006; Kosar et al., 2007; Wu et al., 2012).

3.2.3. Divergence among populations based on antioxidant activities The PCA plot performed on antioxidant activities measured by both FRAP and DPPH assays, showed a degree of association among populations (Fig. 1B). Axis 1 (74.56% of the total variation) allowed a separation of populations into three groups (I–III) according to the level of their fruit antioxidant activity expressed as SC50 (SC50 Fr). The group I, formed by the southern population 7 of R. tripartita, exhibiting the highest IC50 of fruits (corresponds to the lowest antioxidant activity), was clearly separated from the group II formed by the central distributed populations 1, 2, 3, 4, 5 and 6 of R. tripartita, and the population 8 of R. pentaphylla with intermediate antioxidant activity. Axis 2 (17.39% of the inertia) was

correlated negatively with the antioxidant activity of stem cortex, expressed as FRAP (FRAP Sc), and positively with that of leaf extract expressed as SC50 (SC50 Lv). It separates the population 9 of R. pentaphylla (group III) from the remnant populations. This population is characterized by the highest antioxidant activity of fruit extracts and the lowest antioxidant activity of leaf and stem cortex extracts. The aggregation of populations of both species in distinct groups according to polyphenolic contents and antioxidant activities indicates the possibility of selecting high material (i.e. populations, clones) for large scale production. The populations 7, 3 and 1 of Rhus tripartia and the population 8 of R. pentaphylla exhibiting high total phenolic contents in stem cortex associated with high antioxidant activities could be used as starting material to develop industrial and medicinal uses.

4. Variation of phenol contents and implication for conservation of the populations The large variation of total phenolic and flavonoid contents and antioxidant activities among populations provides guidelines for the ex situ and in situ conservation strategies of the two species. All populations have decreased in size in the last decades as a result of habitat destruction due to multiple anthropic pressures. Most populations are small and scattered in isolate patches throughout their distribution area. If such destruction activities continue without protection measures, the genetic diversity and the abundance of species will be endangered. Thus, protection measures should be implemented. All populations should be protected because of their high polyphenolic variation even at a low scale space. The best way to conserve populations is to protect habitats in areas where they are living. Reducing anthropic pressures (i.e. harvesting practices) in most degraded populations may lead to the increasing of their size (i.e. regeneration via seeds). The conservation in situ should be made appropriately according to geographical distance among populations within the same bioclimatic zone. The populations 1, 3 and 7 of R. tripartita and the population 8 of R. pentaphylla exhibiting high amounts of polyphenols in stem cortex and leaves should first be preserved. They might be important sources of polyphenols and could serve as starting material for clonal propagation (i.e. cutting, in vitro culture) by selecting the best phenolic plants. The ex situ conservation (e.g. in field collections) should be based on the collection and propagation of vegetative parts from both male and female individuals within and among populations in each bioclimatic zone.

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The collection of seeds should be less important than that of cuttings. Seeds give males or females and plants required several years to reach the reproductive stage. The content and quality of polyphenols in plants determine their industrial use. Thus, breeding and plant propagation programs should be implemented. Identifying female and male genotypes from the populations of the two species, and their cultivation in adequate proportion in commercial orchards may ensure sustainable production. 5. Conclusions Phenolic extracts from different parts of Rhus species, have attracted a great deal of scientific attention because of their health benefits. Studies showed a large variation in phenolic composition and antioxidant activity depending on species, phonological stages, plant parts, environmental and genetic factors. Our study, on Tunisian R. tripartita and R. pentaphylla showed that extracts from the two taxa exhibited substantial polyphenolic contents and high antioxidant activity, suggesting their use in medicinal and industrial (i.e. tanning and dyeing skins) fields. For each species, the amounts of phenols and their antioxidant activity are dependent on the plant part and the population. The highest total phenolic contents were detected in stem cortex extracts from the populations 1, 3 and 7 of R. tripartita. Flavonoids were mainly concentrated in leaves (i.e. populations 1 and 3 of R. tripartita and 8 of R. pentaphylla). The use of the three plant aerial parts could replace that of roots extensively used in Tunisia, to avoid the death of trees and the decreasing size of the populations. The two populations 7 (R. tripartita) and 8 (R. pentaphylla) with high antioxidant activity could be implemented in improvement programs. The in situ conservation of the populations as well as the propagation of individual in fields of collection should be made urgently because of the high level of destruction of habitats. Regarding the importance of the variability in polyphenolic contents according to populations, the chemical studies should be performed jointly to those related to the within and among populations genetic diversity to draw up efficient improvement and conservation programs of the two species. Conflict of interest The authors declare that there are no conflicts of interest. References Abbassi, F., Hani, H., 2011. In vitro antibacterial and antifungal activities of Rhus tripartitum used as antidiarrhoeal in Tunisian folk medicine. Nat. Prod. Res. 26, 2215–2218. Ahmed, M.S., Galal, A.M., Ross, S.A., Ferreira, D., El Sohly, M.A., Ibrahim, A.R.S., Mossa, J.S., El-Feraly, F.S., 2001. A weakly antimalarial biflavanone from Rhus retinorrhoea. Phytochemistry 58, 599–602. Ben Mansour, H., Yatouji, S., Mbarek, S., Houas, I., Delai, A., Dridi, D., 2011. Correlation between antibutyrylcholinesterasic and antioxidant activities of three aqueous extracts from Tunisian Rhus pentaphyllum. Ann. Clin. Microbiol. Antimicrob. 32, 1–10. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Leb. Wissenchaft und Techn. 28, 25–30. Candan, F., Sokmen, A., 2004. Effects of Rhus coriaria L. (Anacardiaceae) on lipid peroxidation and free radical scavenging activity. Phytother. Res. 18, 84–86. Castillo, J., Benavente-Garcia, O., Sabater, F., Marin, F., Ortuno, A., Del Rio, J.A., 1997. Flavanone neohesperidosides distribution in Citrus aurantium stems. Evidence for a possible translocation process in relation with biosynthesis in leaves and fruits. Phytochemistry 1, 77–84. Chakraborty, A., Ferka, F., Simi, T., Brantnerb, A., Dusinská, M., Kundid, M., 2009. DNA-protective effects of sumac (Rhus coriaria L.), a common spice: results of human and animal studies. Mutat. Res. 661, 10–17. Chang, J., Lu, O.J., He, G., 2009. Regulation of polyphenols accumulation by combined expression silencing key enzymes of phenylpropanoid pathway. Acta Biochim. Biophys. Sin. 41 (2), 123–130.

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