Characterization and quantification of phenolic compounds and antioxidant properties of Salvia species growing in different habitats

Industrial Crops and Products 49 (2013) 904–914

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Characterization and quantification of phenolic compounds and antioxidant properties of Salvia species growing in different habitats Mouna Ben Farhat a,∗ , Ahmed Landoulsi a , Rym Chaouch-Hamada a,b , Jose A. Sotomayor c , María J. Jordán c a

Laboratoire de Biochimie et Biologie moléculaire, Faculté des Sciences de Bizerte, Zarzouna 7021, Tunisia Institut Préparatoire aux Etudes d’Ingénieurs de Bizerte, Route Menzel Abderrahman, Zarzouna 7021, Tunisia c Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Departamento de Recursos Naturales y Desarrollo Rural, C./Mayor s/n, 30150 La Alberca (Murcia), Spain b

a r t i c l e

i n f o

Article history: Received 5 February 2013 Received in revised form 15 June 2013 Accepted 29 June 2013 Keywords: Antioxidant activities Methanolic extracts Phenolic compounds Rosmarinic acid Salvia species Total phenolic contents

a b s t r a c t Few literature data are available on the antioxidants of Salvia verbenaca, Salvia aegyptiaca and Salvia argentea. Such lack of information prevents exploitation of these potential pool of compounds with strong antioxidant activity. So, in the present study, phenolic contents and compositions and antioxidant activities of methanolic extracts of four Salvia species, growing in various habitats, were determined. The total phenolic contents estimated spectrophotometrically ranged from (67.67–72.02 mg GAE/g DW) for S. argentea extracts to (112.93–161.37 mg GAE/g DW) for Salvia officinalis samples. The HPLC analysis detected the presence of several phenolic acids, particularly the caffeic acid derivatives along with flavonoids and phenolic diterpenes. Rosmarinic acid was found to be the most abundant compound in all analyzed extracts and showed its highest values in S. officinalis samples (13,680.22–18,378.00 ␮g/g DW). Varying degrees of antioxidant and radical-scavenging efficacity of different sage extracts were illustrated. S. officinalis methanolic extracts had the strongest antioxidant capacity as evaluated by DPPH (10.08–3.37 ␮g/mL), ABTS (644.85–766.30 ␮M TE/mg) and FRAP (178.65–197.33 mM Fe(II)/mg) assays. Significant correlations between total phenolics, the most representative phenolic compounds, particularly rosmarinic acid and the antioxidant assays were verified. Variations of total phenolic contents and compositions and antioxidant activities between Salvia samples grown at different regions were significant. Salvias aerial part extracts could be valuable as an effective and safe source of functional food materials such as natural antioxidants. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The genus Salvia is one of the most widespread and important aromatic and medicinal genera of the Lamiaceae family. An unusually large number of useful secondary metabolites such as terpenoid compounds and phenolic derivatives (Roby et al., 2013), have been isolated from the genus, which features prominently in the pharmacopoeias of many countries throughout the world. Sages has been credited with a long list of medicinal uses such as antihydrotic, spasmolytic, antiseptic, anti-inflammatory and in the treatment of mental and nervous conditions (Baricevic and Bartol, 2000). Moreover, the genus Salvia gained its commercial interest, not only for use in therapy, but also as a spice to flavour meats, such as sausage and poultry (Gali-Muhtasib et al., 2000).

∗ Corresponding author. Tel.: +216 98 912 176; fax: +216 72 590 566. E-mail address: [email protected] (M.B. Farhat). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.06.047

Antioxidants have great importance in terms of reducing oxidative stress which could cause damage to biological molecules (Bektas et al., 2005). The antioxidant activity of Salvia species extracts has been related to their total phenolic content, hence plant extracts rich in polyphenols (mainly phenolic acids and flavonoids) usually have a higher antioxidant capacity (Roby et al., 2013; Tosun et al., 2009). Researches of antioxidant substances in foods and medicinal natural sources have gained increased interest. Indeed, the use of plant materials containing phenolic constituents in lipids and lipid-containing foods is of great importance since they minimize rancidity, retard the formation of toxic oxidation products, maintain nutritional quality and increase the shelf life of food products (Maisuthisakul et al., 2007). Additionally, antioxidant compounds play a crucial role in the treatment of various pathologies related to degenerative disorders by acting as free radical scavengers, thus decreasing the extent of oxidative damage (Rauter et al., 2012). Many phenolic compounds have been shown to exert anticancer or anticarcinogenic/antimutagenic activity to a greater or lesser extent (Jaberian et al., 2013).

M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 904–914

A variety of Salvia species were reported to have promising radical scavenging abilities (Ben Farhat et al., 2009, 2013; Kamatou et al., 2010; Tepe et al., 2007) and many Salvia species are of commercial interest to the pharmaceutical and food industries as a source of natural antioxidants. The phytochemical analysis of several members of the genus showed the presence of many phenolic compounds belonging mainly to the classes of phenolic acids, phenolic glycosides, phenolic diterpenes, flavonoids, anthocyanins and coumarins (Cuvelier et al., 1994; Lu and Foo, 2002). The research into the determination of natural antioxidant sources is very important to promote public health. Several Salvia species have shown to be a rich source of polyphenols, however, as far as our literature survey could ascertain, establishment of phenolic composition and antioxidant capacities, along with their variation according to collection sites in Salvia verbenaca, Salvia aegyptiaca and Salvia argentea species have not been previously published. Therefore, the main aim of this research project was to screen above mentioned Salvia species in comparison with Salvia officinalis, with respect to their phenolic contents and antioxidant activity and the evaluation of their variation in coherence with the growing habitats. 2. Materials and methods 2.1. Chemicals, solvents, reagents 2,2-Diphenyl-1-picrylhydrazyl (DPPH• ), 2,2 -azinobis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt [ABTS(NH4 )2 ], 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox), potassium persulfate, the Folin–Ciocalteu reagent, gallic acid and high-purity standards were purchased from Sigma–Aldrich (Madrid, Spain). Methanol, acetonitrile, petroleum ether, formic acid, ethanol, glacial acetic acid, hydrochloric acid, anhydrous sodium carbonate, FeCl3 ·6H2 O and sodium acetate were supplied from Scharlau Chemie S.A. (Sentmenat, Spain). 2,4,6Tripyridyl-s-triazine (TPTZ) was obtained from Fluka (Madrid, Spain). Methanol was of HPLC grade and other reagents were of analytical grade. 2.2. Plant material Salvia aerial parts were randomly collected from different regions in north and centre of Tunisia at flowering period (March, April, May, June and July 2008). S. officinalis plant material was obtained from cultivated individuals and those of S. verbenaca, S. aegyptiaca and S. argentea were provided by wild plants. A voucher specimen was deposited at the Herbarium of the Laboratory of Biochemistry and Molecular Biology at the Faculty of Sciences of Bizerte. Details of collection sites and voucher specimens are illustrated in Table 1. 2.3. Preparation of the plant extracts Plant material was dried at room temperature until it reached a constant weight and then finely ground to pass a 2-mm sieve. 0.5 g of dried samples were firstly homogenized with 30 mL of petroleum ether under magnetic stirring for 5 min and taken to dryness at room temperature. Secondly, they were extracted using 150 mL of methanol in a Soxhlet extractor (B-811) (Büchi, Flawil, Switzerland), for 2 h under a nitrogen atmosphere. Methanolic extracts were taken to dryness at 40 ◦ C under vacuum conditions in an evaporator system (Syncore Polyvap R-96) (Büchi, Flawil, Switzerland). The residue was re-dissolved in methanol and made up to 5 mL (Jordán et al., 2009). The concentration of the extracts was expressed in terms of mg of dry methanolic extract weight per

905

g of dry plant weight. The extracts were kept in vials at −80 ◦ C until their corresponding analysis. 2.4. Determination of total phenolic contents Colorimetric quantification of total polyphenol was determined as described by Singleton and Rossi (1965). Briefly, 15 ␮L of methanolic extracts were added to 1185 ␮L of distilled water and 75 ␮L of Folin–Ciocalteu reagent. A vigorous stirring was performed and 225 ␮L of a solution of sodium carbonate (20%) were added. After 2 h of incubation, the absorbance of the resulting blue-coloured solution was measured at 765 nm and 25 ◦ C with a Shimadzu (UV-2401PC, Japan) spectrophotometer. Quantitative measurements were performed, based on a standard calibration curve of concentrations ranging from 25 to 300 mg/L of gallic acid. The total phenolic content was expressed as gallic acid equivalents (GAEs) in milligrams per gram of dry plant material weight. 2.5. HPLC analysis HPLC analysis was performed on a reverse phase ZORBAX SBC18 column (4.6 mm × 250 mm, 5 ␮m pore size, Hewlett Packard, USA) using a guard column (ZORBAX SB-C18 4.6 mm × 125 mm, 5 ␮m pore size, Hewlett Packard, USA) at ambient temperature, according to a method adapted from Zheng and Wang (2001). Extracts were passed through a 0.45 ␮m filter (Millipore SAS, Molsheim, France) and 20 ␮L were injected in a Hewlett Packard (Germany) system equipped with a G1311A quaternary pump and G1315A photodiode array UV–vis detector. The mobile phase was acetonitrile (A) and acidified water containing 5% formic acid (B). The gradient was as follows: 0 min, 5% A; 10 min, 15% A; 30 min, 25% A; 35 min, 30% A; 50 min, 55% A; 55 min, 90% A; 57 min, 100% A and then held for 10 min before returning to the initial conditions. The flow rate was 1.0 mL/min and the wavelengths of detection were set at 280 and 330 nm. The identification of the phenolic components was made by comparison of retention times and spectra with those of commercially available standard compounds. For the purpose of quantifying, linear regression models were determined using standard dilution techniques. Phenolic compound contents were expressed in ␮g per g of dry plant material weight. 2.6. DPPH• radical-scavenging activity DPPH• quenching ability of sage extracts was measured according to Brand-Williams et al. (1995). Briefly, a 500 ␮L of methanolic extracts at different concentrations were added to 1 mL of DPPH• methanolic solution (0.1 mM). Decolorations were measured using a Shimadzu (UV-2401PC, Japan) spectrophotometer at 517 nm after incubation for 20 min at room temperature in the dark. Absorbance was measured against a blank of 500 ␮L of sample plus 1 mL of methanol and corresponds to the extract ability to reduce the radical DPPH• to the yellow-coloured diphenylpicrylhydrazine. The absorbance of the control consisting of 500 ␮L of methanol and 1 mL of DPPH• solution was measured daily against a blank of 1.5 mL of methanol. Measurements were performed in triplicate. The percentage activity for the DPPH• was calculated according to:



% decoloration = 1 −

 absorbance sample  absorbance control

× 100

The antiradical activity was expressed as IC50 , the antiradical dose required to cause a 50% of inhibition. Concentrations are expressed as micrograms of dry plant methanolic extract per one millilitre of methanol.

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M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 904–914

Table 1 Voucher specimen and collection sites eco-geographical characteristics of four Salvia species. N◦

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Code

OK OS OB OR VT VB VN VC VA VR AE AC AG RS RM

Collection sites

Kelibia Soliman Bou Arada Sers Tunis Bir Mroua Enfida Chott Meriem Bou Arada Rass Zebib Enfida Chott Meriem Ghraba Sers Makther

Species

S. officinalis

S. verbenaca

S. aegyptiaca

S. argentea

Bioclimatic stage

Sub-humid Semi-arid superior Semi-arid moderate Semi-arid moderate Higher semi-arid Sub-humid Lower semi-arid Lower semi-arid Semi-arid moderate Sub-humid Lower semi-arid Lower semi-arid Higher arid Semi-arid moderate Higher semi-arid

2.7. ABTS•+ radical cation decoloration assay The ABTS•+ cation radical-scavenging activity of extracts was determined according to Re et al. (1999). ABTS•+ radical cation was produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 16 h before use. A working solution was diluted with ethanol to an absorbance of 0.70 (±0.02) nm (constant initial absorbance value used for standard and samples) at 734 nm and 30 ◦ C. An aliquot (15 ␮L) of each sample (with appropriate dilution) or Trolox standard was mixed with the working solution (1.5 mL) of ABTS•+ , and the decrease of absorbance was measured after 6 min at 734 nm using a Shimadzu (UV-2401PC, Japan) spectrophotometer. Measurements were performed in triplicate. The ABTS•+ scavenging rate was calculated, to express the antioxidant ability of the sample and results were expressed in terms of Trolox equivalent antioxidant capacity (TEAC, ␮M of Trolox equivalents per mg of dry plant methanolic extract). 2.8. Ferric reducing antioxidant power (FRAP) The capacity of plant extracts to reduce ferric ions was assessed by the method described by Benzie and Strain (1996). The FRAP reagent was freshly prepared from 300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) made up in 40 mM HCl and 20 mM FeCl3 ·6H2 O solution. All three solutions were mixed together in the ratio of 10:1:1 (v/v/v). An aliquot of 40 ␮L of each sample (with appropriate dilution) was added to 1.2 mL of FRAP reagent. The absorption of the reaction mixture was measured at 593 nm after 2 min incubation at 37 ◦ C. Measurements were performed in triplicate. Fresh working solutions of known Fe (II) concentrations (FeSO4 ·7H2 O) of (0–2 mM) were used for calibration. The antioxidant capacity based on the ability to reduce ferric ions of samples was calculated from the linear calibration curve and expressed as mM of FeSO4 equivalents per mg of dry plant methanolic extract. 2.9. Statistical analysis All data were reported as means ± standard deviation of at least three experiments. Analysis of variance was performed by ANOVA procedure and significant differences were calculated according to Duncan’s multiple range tests. Differences at p < 0.05 were considered statistically significant. A principal component analysis was performed in order to discriminate Salvia species and their collection sites on the basis of the phenolic composition. Correlation

Soil pH

7.66 8.02 8.15 7.15 7.94 7.95 7.96 8.05 8.15 7.50 7.96 8.05 7.50 7.60 7.32

Geographical location

Voucher specimen

Longitude (N)

Latitude (E)

Altitude (m)

36◦ 51 36◦ 41 36◦ 21 36◦ 04 36◦ 49 36◦ 47 36◦ 02 35◦ 53 36◦ 20 37◦ 16 36◦ 02 35◦ 53 34◦ 59 35◦ 55 36◦ 00

11◦ 05 10◦ 29 9◦ 37 9◦ 01 10◦ 08 10◦ 37 10◦ 24 10◦ 35 09◦ 39 10◦ 04 10◦ 24 10◦ 35 10◦ 44 09◦ 11 09◦ 16

17 16 252 487 67 86 10 8 252 14 10 8 98 862 725

SO 2008-121 SO 2008-122 SO 2008-123 SO 2008-124 SV 2008-125 SV 2008-126 SV 2008-127 SV 2008-128 SV 2008-129 SV 2008-130 SA 2008-135 SA 2008-136 SA 2008-137 SR 2008-138 SR 2008-139

analysis of phenolic compounds, total phenolics identified by HPLC and total phenolics determined by the Folin–Ciocalteu method versus different antioxidant assays was performed. Results were processed by computer programs Excel and STATISTICA software version 5.1. 3. Results and discussion 3.1. Determination of total phenolic content Determined total phenolic amounts as estimated by the Folin–Ciocalteu colorimetric method revealed that Salvia species exhibited high and largely variable contents depending on the species and the collection site (Fig. 1). Samples of S. officinalis displayed the highest phenolic contents, particularly the plants harvested in Bou Arada (158.79 mg GAE/g DW) and Soliman (161.37 mg GAE/g DW). On the other hand, S. argentea exhibited the lowest levels of phenolics (67.67, 72.02 mg GAE/g DW). Similarly, several Salvia species, such as S. officinalis (166 mg GAE/g DW) cultivated in Finland (Dorman et al., 2003), S. africana-caerulea (115 mg GAE/g DW) and S. disermas (69.0 mg GAE/g DW) (Kamatou et al., 2010) showed total phenolic amounts included in the range obtained for sages of the current study. However, postdistilled S. argentea revealed much lower contents of 41.47 and 48.90 mg GAE/g DW (Ben Farhat et al., 2013). Alike, other Salvia species demonstrated lower levels compared with results of the present investigation, namely S. verbenaca (4.27 mg GAE/g DW) sampled in Egypt (Khalil et al., 2007), S. fruticosa Miller (41.58–44.60 mg GAE/g DW) collected in Turkey (Dincer et al., 2012) and S. gilliesi (33.2 mg GAE/g DW) (Borneo et al., 2009). Differences in total phenolic contents between collection sites within each species were statistically significant (p < 0.05), except for S. argentea. The variations may be attributed to growth conditions (Lamien-Meda et al., 2010) and to genotypes, which influence the accumulation of phenolic compounds by synthesizing different quantities and/or types of phenolics (Shahidi and Naczk, 1995). As previously reported, literature point out that the total phenolic content measured by the Folin–Ciocalteu procedure does not give a full picture of the quality or quantity of the phenolic constituents in the extracts (Katsube et al., 2004; Wu et al., 2004). In fact, the method is predictable due to the weak selectivity of the Folin–Ciocalteu reagent, as it reacts positively with different antioxidant compounds (phenolic and non phenolic substances) (Que et al., 2006). Moreover, various phenolic compounds respond differently in this assay, depending on the number of their phenolic groups (Singleton and Rossi, 1965).

Table 2 Extract yields and phenolic profiles of four Salvia species methanolic extracts. Identified compounds

Contents (␮g/g of dry plant material weight)

S. officinalis

Phenolic diterpenes Carnosic acid Carnosol Methyl carnosate Flavonoids Luteolin-7-Oglucoside Apigenin-7glucoside Luteolin Apigenin Genkwanin Naringin Hesperidin Naringenin Cirsiliol Cirsimaritin Salvigenin Total Extract yields (mg/g DW)

OS n=6

OB n=6

OR n=6

VC n=3

VR n=3

VA n=3

VB n=3

VN n=3

VT n=3

695.04 ± 18.21 a 312.43 ± 2.53 d 18,378.00 ± 393.26 a 18.82 ± 1.39 b 122.31 ± 2.65 c

522.34 ± 7.35 b 593.32 ± 11.45 b 17,930.58 ± 320.20 b 29.74 ± 1.05 a 412.60 ± 7.13 a

236.49 ± 4.76 c 703.29 ± 17.74 a 17,456.90 ± 319.11 c 14.49 ± 2.41 c 121.15 ± 2.16 c

222.24 ± 11.23 c 442.03 ± 7.23 c 13,680.22 ± 101.77 d 16.00 ± 0.80 c 298.91 ± 8.27 b

61.27 ± 0.53 E 223.03 ± 4.08 A 9576.23 ± 160.06 B nd 111.18 ± 2.78 D

84.68 ± 0.56 D 188.82 ± 2.23 D 5429.69 ± 112.50 E nd 182.85 ± 1.94 C

90.55 ± 0.52 C 207.37 ± 4.02 B 5730.05 ± 91.95 D nd 193.43 ± 11.92 C

162.10 ± 0.93 A 224.81 ± 2.04 A 12,838.88 ± 33.98 A nd 417.10 ± 5.23 A

87.63 ± 4.54 CD 184.39 ± 0.71 D 4432.59 ± 71.28 F nd 46.44 ± 0.50 E

103.38 ± 1.14 B 195.36 ± 0.73 C 6379.19 ± 25.12 C nd 255.78 ± 6.18 B

nd nd nd

nd nd nd

nd nd nd

nd nd nd

9.48 ± 0.20 D 37.91 ± 0.13 E nd

30.12 ± 0.58 B 61.68 ± 0.41 D nd

29.37 ± 0.45 B 71.33 ± 0.91 C nd

33.98 ± 0.88 A 138.68 ± 0.52 A nd

1.23 ± 0.08 E 10.50 ± 0.44 F nd

10.82 ± 0.44 C 83.69 ± 0.27 B nd

5147.83 ± 54.14 b 5947.03 ± 173.45 a 6464.21 ± 530.22 b

6001.75 ± 390.12 a 5855.20 ± 28.43 a 5406.25 ± 7.35 c

3278.30 ± 227.59 d 5167.70 ± 414.49 b 7174.00 ± 73.27 a

4296.28 ± 2.47 c 5045.42 ± 318.10 b 4816.59 ± 199.40 d

98.73 ± 4.48 A 41.60 ± 0.37 AB 575.21 ± 4.74 C

96.88 ± 3.65 A 38.92 ± 1.56 C 341.60 ± 9.20 D

97.29 ± 0.36 A 42.02 ± 0.92 AB 278.48 ± 2.24 E

95.15 ± 1.07 A 43.75 ± 2.58 A 2.64 ± 0.07 F

83.70 ± 2.55 B 27.16 ± 0.88 D 645.53 ± 12.50 B

96.64 ± 0.41 A 40.26 ± 0.77 BC 752.38 ± 3.59 A

386.63 ± 0.39 b

661.04 ± 65.60 a

411.48 ± 3.97 b

442.54 ± 59.93 b

nd

nd

nd

nd

nd

nd

210.01 ± 0.70 b

913.90 ± 166.89 a

315.48 ± 0.24 b

808.62 ± 130.44 a

nd

nd

nd

nd

nd

nd

66.44 ± 1.90 a 64.14 ± 1.83 c 22.94 ± 0.20 a 857.92 ± 8.41 a nd nd nd nd nd

48.40 ± 0.14 b 77.51 ± 4.22 a 25.60 ± 4.58 ab 817.69 ± 5.61 b nd nd nd nd nd

21.41 ± 0.54 d 55.77 ± 6.65 d 21.57 ± 0.80 b 677.88 ± 39.70 c nd nd nd nd nd

27.84 ± 0.23 c 69.86 ± 0.33 b 22.02 ± 0.74 b 485.77 ± 32.41 d nd nd nd nd nd

26.26 ± 0.96 C 10.49 ± 0.44 C 14.44 ± 1.23 B 642.93 ± 7.76 C 457.85 ± 11.88 A 1054.71 ± 13.42 C 61.58 ± 1.27 C nd nd

19.49 ± 0.32 D 7.17 ± 0.26 D 8.10 ± 0.23 E 750.49 ± 9.40 B 141.58 ± 1.98 F 696.96 ± 7.04 E 83.32 ± 0.73 B nd nd

45.04 ± 2.19 B 11.74 ± 0.07 A 11.72 ± 0.74 C 876.36 ± 25.17 A 243.03 ± 7.07 D 912.60 ± 9.28 D 62.38 ± 2.65 C nd nd

31.05 ± 1.76 A 16.03 ± 0.33 B 16.61 ± 0.65 A 606.20 ± 6.61 C 322.21 ± 10.62 B 1806.17 ± 40.16 A 108.13 ± 2.86 A nd nd

13.53 ± 0.23 E 6.45 ± 0.07 E 12.17 ± 0.83 C 447.54 ± 41.93 E 298.86 ± 10.24 C 350.16 ± 1.12 F 35.20 ± 0.80 D nd nd

25.01 ± 0.22 D 7.42 ± 0.50 C 10.00 ± 0.06 D 550.06 ± 25.77 D 213.86 ± 10.10 E 1415.37 ± 4.64 B 81.33 ± 0.99 B nd nd

38,693.75 ± 167.63 b 232.63 ± 3.63 ab

39,295.92 ± 98.65 a 251.73 ± 17.55 a

35,655.89 ± 429.45 c 207.48 ± 9.73 c

30,674.34 ± 151.92 d 211.79 ± 10.88 bc

13,002.90 ± 196.20 B 182.96 ± 1.85 C

8162.34 ± 93.04 E 166.90 ± 1.45 D

8902.76 ± 98.53 D 244.83 ± 4.15 A

16,863.48 ± 59.48 A 243.92 ± 4.02 A

6683.06 ± 106.52 F 120.84 ± 3.30 E

10,220.54 ± 4.56 C 222.88 ± 2.51 B

M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 904–914

Phenolic acids Caffeic acid Ferulic acid Rosmarinic acid Gallic acid p-Hydroxybenzoic acid Vanillic acid p-Coumaric acid Chlorogenic acid

S. verbenaca

OK n=6

907

908

Table 2 (Continued) Identified compounds

Contents (␮g/g of dry plant material weight) S. argentea

S. aegyptiaca AE n=3

RS n=3

RM n=3

Phenolic acids Caffeic acid Ferulic acid Rosmarinic acid Gallic acid p-Hydroxybenzoic acid Vanillic acid p-Coumaric acid Chlorogenic acid

95.56 ± 4.84 b 519.01 ± 11.84 a 5843.58 ± 124.28 a nd 446.77 ± 36.09 a nd nd nd

117.64 ± 2.71 a 439.03 ± 3.77 b 6060.01 ± 146.14 a nd 250.51 ± 2.18 b nd nd nd

62.49 ± 0.19 A 287.70 ± 3.95 A 4815.52 ± 79.24 A 15.41 ± 0.15 A 147.76 ± 7.84 A nd nd 60.48 ± 0.39 B

46.80 ± 0.33 B 251.97 ± 4.10 B 3806.58 ± 51.20 B 13.21 ± 0.55 B 74.33 ± 1.64 B nd nd 63.47 ± 0.57 A

Phenolic diterpenes Carnosic acid Carnosol Methyl carnosate

82.75 ± 1.24 a 36.69 ± 0.64 a 5215.99 ± 121.45 a

77.41 ± 1.65 b 35.01 ± 1.13 a 1394.75 ± 20.31 b

55.41 ± 0.84 A nd 1983.33 ± 28.93 A

42.45 ± 1.02 B nd 1614.90 ± 9.08 B

Flavonoids Luteolin-7-O-glucoside Apigenin-7-glucoside Luteolin Apigenin Genkwanin Naringin Hesperidin Naringenin Cirsiliol Cirsimaritin Salvigenin

911.86 ± 14.08 a 1103.57 ± 3.46 a 17.98 ± 0.84 a 23.68 ± 0.28 a 13.77 ± 0.08 a 190.78 ± 6.27 b nd nd nd 28.89 ± 0.31 a 313.26 ± 11.54 a

818.70 ± 13.19 b 858.84 ± 22.19 b 17.24 ± 0.09 a 23.53 ± 0.25 a 13.93 ± 0.24 a 438.23 ± 18.30 a nd nd nd 28.02 ± 0.94 a 9.99 ± 0.12 b

55.93 ± 1.76 B 37.90 ± 1.26 A 18.22 ± 1.25 B 6.23 ± 0.49 A nd 47.71 ± 0.26 A nd 889.60 ± 25.87 A nd nd nd

79.22 ± 1.97 A 18.09 ± 0.34 B 20.84 ± 0.34 A 5.64 ± 0.65 A nd 42.18 ± 0.79 B nd 667.10 ± 5.74 B nd nd nd

Total Extract yields (mg/g DW)

14,844.13 ± 219.54 a 126.31 ± 2.56 b

10,582.85 ± 177.95 b 145.68 ± 2.27 a

8483.71 ± 78.75 A 102.75 ± 1.66 B

6746.79 ± 51.73 B 123.34 ± 3.06 a

Values followed by the same letter did not share significant differences at 5% (Duncan test) within the same species, nd: not detected.

M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 904–914

AC n=3

M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 904–914

909

Fig. 1. Total phenolic contents (mg GAE/g DW) of (a): S. officinalis, (b): S. verbenaca, (c): S. aegyptiaca and (d): S. argentea collected in different growing regions. Bars sharing the same small letter did not share significant differences at p < 0.05 (Duncan test).

3.2. Contents of polyphenolic compounds Methanolic extract yields estimated on the basis of the dry weight of different Salvias are shown in Table 1. It was observed that the extraction yield varied according to the species and its collection site. Results showed the highest extract yields for S. officinalis (207.48–251.73 mg/g DW) followed by S. verbenaca (120.84–244.83 mg/g DW), S. aegyptiaca (126.31, 145.68 mg/g DW) and S. argentea (102.75, 123.34 mg/g DW) (Table 2). These values agree rather well with a previous published data notifying a similar extract yield for S. verbenaca (175.4 mg/g DW) collected in Turkey (Tepe, 2008). To the best of our knowledge, the qualitative-quantitative analysis of S. verbenaca, S. aegyptiaca and S. argentea polyphenolics and the variation of the individual phenolic compounds according to the collection site have not yet been published. So, in the current study HPLC analysis of methanolic extracts of the species mentioned above along with results related to S. officinalis are presented in Table 2 and representative chromatograms are shown in Fig. 2. The HPLC analysis led to the assessement of total identified phenolic amounts ranging from (6746.79–8483.71 ␮g/g DW) in S. argentea samples to (30,674.34–39,295.92 ␮g/g DW) in those of S. officinalis. The results permitted the identification of fourteen phenolic compounds in S. officinalis and S. argentea methanolic extracts, fifteen compounds in S. aegyptiaca methanolic extracts and sixteen compounds in S. verbenaca methanolic extracts. The identified phenolics belonged to three representative classes of constituents, namely phenolic acids, flavonoids and abietane diterpenoids, as previously reported in Salvia species (Matkowski et al., 2008). Among the identified phenolics, nine compounds are common to the four analyzed species, namely caffeic acid, ferulic acid, rosmarinic acid, p-hydroxybenzoic acid, carnosic acid, methyl carnosate, luteolin, apigenin and naringin. Nevertheless, as shown in Table 2, vanillic and p-coumaric acids, hesperidin and cirsiliol

were exclusively identified in S. verbenaca extracts. Similarly, cirsimaritin and salvigenin were only detected in S. aegyptiaca extracts and chlorogenic acid in S. argentea samples. In agreement with literature data (Adzet et al., 1988; Ben Farhat et al., 2009, 2013; Dincer et al., 2012; Lu and Foo, 2002; Nikolova et al., 2006; Roby et al., 2013; Santos-Gomes et al., 2002), the phenolic compounds reported in this study were previously identified in several Salvia species. All analyzed samples were characterized by a clear prevalence of rosmarinic acid. This caffeic acid dimer is generally the most abundant phenolic compound in Lamiaceae (Kim and Lee, 2004). Rosmarinic acid has many biological activities such as inhibiting the HIV-1, antitumor, antihepatitis and protecting the liver, inhibiting the blood clots, anti-inflammation and antioxidant (Tepe, 2008). This caffeic acid derivative common in many plants and very often present in our diet is a strong radical scavenger and has been reported to be more effective than Trolox (Lu and Foo, 2001). Different levels of rosmarinic acid were detected in our Salvia species and the highest values marked S. officinalis samples, especially Kelibia extracts (18,378.00 ␮g/g DW). Approximatively four times lower phenolic amounts were recorded for S. argentea methanolic extracts (3806.58, 4815.52 ␮g/g DW). According to the literature, S. officinalis revealed a content in rosmarinic acid in the range of 6000–47,000 ␮g/g DW (Lamien-Meda et al., 2010), plants of S. argentea submitted to a preliminary distillation process demonstrated a content of 613.25 and 941.81 ␮g/g DW (Ben Farhat et al., 2013), S. verbenaca was characterized by a level of 26,120 ␮g/g DW (Tepe, 2008) and concentrations of 6811, 10,861, 12,864 and 25,627 ␮g/g DW were detected respectively in extracts of Thymus vulgaris, Ocimum basilicum, Rosmarinus officinalis and Origanum vulgare (Shan et al., 2005). As well established in literature, caffeic acid plays a central role in the biochemistry of Lamiaceae (Lamien-Meda et al., 2010; Lu and Foo, 2002) and constitute the building block

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Fig. 2. HPLC chromatograms of methanolic extracts of different Salvia species with responses at 330 (b1, c1, d1) and 280 nm (b2, c2, d2) overlaid. Peaks of Salvia verbenaca (b1, b2): 1, caffeic acid; 2, p-coumaric acid; 3, ferulic acid; 4, rosmarinic acid; 5, luteolin; 6, cirsiliol; 7, apigenin; 8, genkwanin; 9, p-hydroxybenzoic acid; 10, vanillic acid; 11, naringin; 12, hesperidin; 13, naringenin; 14, carnosol; 15, carnosic acid; 16, methyl carnosate. Peaks of Salvia aegyptiaca (c1, c2): 1, caffeic acid; 2, luteolin-7-Oglucoside; 3, ferulic acid; 4, apigenin-7-glucoside; 5, rosmarinic acid; 6, luteolin; 7, apigenin; 8, cirsimaritin; 9, genkwanin; 10, salvigenin; 11, p-hydroxybenzoic acid; 12, naringin; 13, carnosol; 14, carnosic acid; 15, methyl carnosate. Peaks of Salvia argentea (d1, d2): 1, chlorogenic acid; 2, caffeic acid; 3, luteolin-7-O-glucoside; 4, ferulic acid; 5, apigenin-7-glucoside; 6, rosmarinic acid; 7, luteolin; 8, apigenin; 9, gallic acid; 10, p-hydroxybenzoic acid; 11, naringin; 12, naringenin; 13, carnosic acid; 14, methyl carnosate.

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Fig. 3. Principal component analysis scatter plot of the different Salvia species and their collection sites based on the common phenolic composition. S. officinalis: Kelibia (OK), Soliman (OS), Bou Arada (OB), Sers (OR); S. verbenaca: Chott Meriem (VC), Rass Zebib (VR), Bou Arada (VA), Bir Mroua (VB), Enfida (VN), Tunis (VT); S. aegyptiaca: Chott Meriem (AC), Enfida (AE); S. argentea: Sers (RS), Makther (RM); caffeic acid (CAF), p-hydroxybenzoic acid (BEN), methyl carnosate (MCAR), ferulic acid (FER), rosmarinic acid (ROS), luteolin (LUT), apigenin (API), naringin (NAR), carnosic acid (CAR).

of a variety of metabolites in the genus Salvia (Lu and Foo, 2002). Quantitatively, caffeic acid and its derivatives (caffeic acid, ferulic acid, rosmarinic acid, p-coumaric acid and chlorogenic acid) constitute the major part of the identified phenolics in the methanolic extracts of the analyzed samples. Their amounts were the highest (14,344.49–19,385.48 ␮g/g DW) in S. officinalis followed by lower concentrations in S. verbenaca (4715.11–13,364.46 ␮g/g DW), S. aegyptiaca (6458.15, 6616.68 ␮g/g DW) and S. argentea (4168.82, 5226.19 ␮g/g DW). Large amounts of methyl carnosate (6464.21–7174.00 ␮g/g DW), acid (3278.30–6001.75 ␮g/g DW) and carnosol carnosic (5167.70–5947.03 ␮g/g DW) were illustrated in S. officinalis extracts. It should be noticed that commercially, the quality of a sage extract is highly dependent on the content of rosmarinic acid and diterpenoids, particularly carnosol and carnosic acid (Nakatani, 1997). Thus, S. officinalis extracts, particularly plants cultivated in Kelibia and Soliman, seemed to show the best quality since they demonstrated the highest contents of rosmarinic acid, carnosic acid and carnosol. Identification of individual compounds from group of flavonoids confirms the presence of naringenine in reasonable amounts in S. verbenaca (350.16–1806.17 ␮g/g DW) and S. argentea (667.10, 889.60 ␮g/g DW) methanolic extracts. On the other hand, the major part of flavonoid fraction was represented by luteolin-7-O-glucoside (818.70, 911.86 ␮g/g DW) and apigenin-7-glucoside (858.84, 1103.57 ␮g/g DW) in S. aegyptiaca plants. To analyze the data of quantitative distribution of phenolic compounds across various geographic locations through four Salvia species, principal component analysis (PCA) was carried out. The PCA performed using the amounts of the four Salvia species common phenolic constituents showed that the three first principal components accounted for 78.68% of the total variation. The first axis (PC1), representing 61.84% of the inertia, is mainly correlated to caffeic acid (loading, 0.91), methyl carnosate (loading, 0.83), rosmarinic acid (loading, 0.93), luteolin (loading, 0.72), apigenin (loading, 0.96) and carnosic acid (loading, 0.96). The second axis (PC2) accounted for 17.84% of the total variation and ferulic acid (loading, 0.68) and naringin

(loading, −0.65) mainly contributes to its definition. Results of PCA demonstrated a wide range of compositions (Fig. 3). On the one hand, the PC1 opposited S. officinalis, characterized by the highest contents of caffeic acid (222.24–695.04 ␮g/g DW), methyl carnosate (4816.59–7174.00 ␮g/g DW), rosmarinic acid (13680.22–18378.00 ␮g/g DW), luteolin (21.41–66.44 ␮g/g DW), (55.77–77.51 ␮g/g DW) and carnosic acid apigenin (3278.30–6001.75 ␮g/g DW), to the remaining species. On the other hand, the PC2 showed a contrast between samples of S. argentea and S. aegyptiaca, revealing the lowest concentrations of naringin (190.78, 438.23 ␮g/g DW), and those of S. verbenaca, illustrating the lowest concentrations of ferulic acid (184.39–224.81 ␮g/g DW). Despite similar phenolic profile were observed within each studied Salvia species, the estimated concentrations of the majority of the identified compounds varied significantly (p < 0.05) according to the collection site (Table 2). These results point out that the differences could be attributed to the effect of environmental conditions, as previously reported for sage and rosemary (Areias et al., 2000; Ben Farhat et al., 2009), but also to genotypic effects (Bas¸kan et al., 2007; Lamien-Meda et al., 2010). In addition, Santos-Gomes et al. (2002) and Bas¸kan et al. (2007) reported that sage extract composition is influenced by the instability of some of the most effective antioxidant compounds such as carnosol and carnosic acid depending on temperature, light, oxygen and solvent used in extraction. Such large variations in the amounts of individual phenolic compounds could contribute to sustainable valorization of the studied Salvia species as bio-resources and as a reference for manufacturers to select the best species and the adequate collection site according to their needs. 3.3. Antioxidant capacity Evaluation of radical scavenging properties and antioxidant abilities is relevant with plants claimed to have medicinal applications and to be useful as food additives. So, in the present study, the antioxidant capacity of four Salvia species from different growing habitats was evaluated using three assays. DPPH, ABTS and FRAP

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Table 3 Antioxidant capacity of Salvia species methanolic extracts. Species

Collection site

DPPH (IC50 , ␮g/mL)

S. officinalis

OK OS OB OR VR VB VT VA VN VC AC AE RS RM

7.32 3.37 10.08 8.69 26.73 23.00 25.99 28.75 36.28 25.20 22.62 21.13 33.85 77.07

S. verbenaca

S. aegyptiaca S. argentea

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 c 0.04 d 0.04 a 0.02 b 0.03 c 0.46 f 0.01 d 0.01 b 0.01 a 0.04 e 0.04 a 0.02 b 0.05 b 0.09 a

ABTS (␮M TE/mg) 734.61 766.30 691.82 644.85 187.77 205.27 194.46 184.88 165.28 201.05 318.09 311.81 173.38 141.23

± ± ± ± ± ± ± ± ± ± ± ± ± ±

FRAP (mM Fe(II)/mg)

9.98 b 2.89 a 10.28 c 4.37 d 1.57 cd 0.29 a 5.27 c 1.40 d 10.80 e 1.45 b 58.39 a 29.57 a 5.93 a 0.54 b

197.33 189.88 186.04 178.65 121.15 159.25 135.59 127.13 106.50 134.52 164.09 149.24 105.33 81.56

± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.43 a 1.84 ab 1.35 ab 12.19 b 0.19 d 2.89 a 6.02 b 0.98 c 2.20 e 0.19 b 0.64 a 6.79 b 0.67 a 2.53 b

Values followed by the same small letter did not share significant differences at 5% (Duncan test).

are common antioxidant tests used for the characterization of plant extracts. In fact, the results of a single-assay can give only a reductive suggestion of the antioxidant properties of extracts towards food matrices. Moreover, the chemical complexity of extracts, often a mixture of dozens of compounds with differences in functional groups, polarity and chemical behaviour, could lead to scattered results, depending on the test employed. Therefore, an approach with multiple assays in screening work is highly advisable (Tepe et al., 2007). Results of antioxidant capacities of Salvia species are shown in Table 3. The three used tests revealed the highest antioxidant activities in S. officinalis extracts and the lowest in those of S. argentea. Except the extracts of S. aegyptiaca collection sites, a significant (p < 0.05) variation of the antioxidant activity was detected in samples collected in different habitats within each species. S. officinalis cultivated in Soliman revealed the greatest free radical-scavenging activity in DPPH (3.37 ␮g/mL) and ABTS (766.30 ␮M TE/mg) systems and that cultivated in Kelibia in the FRAP (197.33 mM Fe(II)/mg) assay. In parallel, methanolic extracts of Bir Mroua were the most active S. verbenaca samples in the DPPH (23.00 ␮g/mL), ABTS (205.27 ␮M TE/mg) and FRAP (159.25 mM Fe(II)/mg) tests. Similarly, the three tests attributed the highest antioxidant power to the samples growing wild in Chott Meriem for the species S. aegyptiaca. Also, comparing the two collection sites of S. argentea, extracts of Sers were more active, particularly the DPPH assay illustrated an IC50 for Sers (33.85 ␮g/mL) as less than the half of the value detected for Makther (77.07 ␮g/mL). S. argentea antioxidant capacity was much higher than that of post-distilled plant material of the same species evaluated by DPPH (69.35–90.68 ␮g/mL), ABTS (97.94–104.04 ␮M TE/mg) and FRAP (78.47–83.09 mM Fe(II)/mg) assays (Ben Farhat et al., 2013). In this context, Kamatou et al. (2010) classified several Salvia species into plants with good (IC50 < 30 ␮g/mL), moderate (30 < IC50 < 80 ␮g/mL) and poor (IC50 > 80 ␮g/mL) antioxidant activity using the DPPH assay. According to Kamatou et al. (2010) classification our S. officinalis, S. verbenaca and S. aegyptiaca samples were marked by a good free radical-scavenging activity and those of S. argentea and S. verbenaca growing in Enfida by a moderate antioxidant activity. The current investigation reinforce the importance of S. officinalis, S. verbenaca, S. aegyptiaca and S. argentea for their traditional use as food condiments and medicinal plants and emphasize their potential further use as functional food ingredients and therapeutic agents. It provides also a basis to select the species and the collection sites with the best antioxidant properties.

3.4. Correlation between phenolic compounds, total identified phenolics, total phenolic content and the antioxidant activity It is well known that plant phenolic compounds are the highly effective free radical-scavengers and antioxidants. Cuvelier et al. (1994) and Kamatou et al. (2010) highlighted the fact that Salvias antioxidant properties are essentially attributed to abietane diterpenoids (carnosic acid and carnosol) and caffeic acid derivatives (rosmarinic acid, caffeic acid, ferulic acid, chlorogenic acid, etc.). Moreover, Chen and Ho (1997) specified that the antioxidant activity of polyphenols was related to their chemical structures, particularly to their hydroxyl group (Jaberian et al., 2013) and the presence of a second hydroxyl group in the ortho or para position increase the efficacity of antioxidants such as rosmarinic acid, carnosol and carnosic acid. Therefore, it would be valuable to evaluate the contribution of polyphenolic compounds detected in Salvia species to the total antioxidant capacity through the determination of linear correlation coefficients (Table 4). The results indicated significant correlations between the three tests and caffeic, rosmarinic and carnosic acids, carnosol, methyl carnosate, apigenin, Table 4 Linear correlation coefficients of phenolic compounds and total phenolics versus the antioxidant activity determined by DPPH, ABTS and FRAP.

Caffeic acid Ferulic acid Rosmarinic acid Gallic acid p-Hydroxybenzoic acid Vanillic acid p-Coumaric acid Chlorogenic acid Carnosic acid Carnosol Methyl carnosate Luteolin-7-O-glucoside Apigenin-7-glucoside Luteolin Apigenin Genkwanin Naringin Hesperidin Naringenin Cirsiliol Cirsimaritin Salvigenin Total phenolics identified by HPLC Total phenolic content *

Significant correlation at p < 0.05.

DPPH

ABTS

FRAP

−0.61* −0.51 −0.73* −0.27 −0.19 0.04 0.03 0.73* −0.64* −0.65* −0.56* −0.16 −0.33 −0.44 −0.71* −0.85* −0.64* 0.11 0.35 0.07 −0.07 −0.04 −0.72* −0.71*

0.84* 0.75* 0.90* 0.76* −0.16 −0.45 −0.46 −0.34 0.96* 0.98* 0.92* 0.14 0.34 0.57* 0.98* 0.84* 0.43 −0.54* −0.67* −0.54* −0.06 −0.04 0.98* 0.86*

0.75* 0.68* 0.88* 0.43 0.26 −0.18 −0.13 −0.63* 0.78* 0.80* 0.77* 0.28 0.47 0.55* 0.86* 0.93* 0.54* −0.30 −0.45 −0.25 0.14 0.15 0.89* 0.84*

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genkwanin and total phenolic content determined by HPLC and spectrophotometrically by the Folin–Ciocalteu method. In addition, ferulic acid and luteolin were significantly correlated to ABTS and FRAP assays. Naringin showed a significant correlation with DPPH (r = −0.64) and FRAP (r = 0.54) tests and gallic acid with the ABTS (r = 0.76) assay. Good correlations between phenolics and antioxidant assays were previously reported in several Salvia species (Ben Farhat et al., 2009, 2013; Lamien-Meda et al., 2010; Tepe, 2008; Tepe et al., 2007). It should be mentioned that an absence of significant correlations was detected between several phenolic compounds (p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, luteolin-7-O-glucoside, apigenin-7-glucoside, cirsimaritin, salvigenin) and antioxidant assays (Table 4). A similar result showing a lack of correlation to the widely distributed polyphenols was reported by Ben Farhat et al. (2009, 2013). The synergistic effects of the diversity of major and minor phenolic components of the methanolic extracts of sages should be taken into consideration for total antioxidant activity (Ben Farhat et al., 2009). 4. Conclusions Salvias extracts constitute a rich source of polyphenols and could be used as powerful herbal antioxidants, particularly S. officinalis cultivated in Soliman, since this latter revealed the best antioxidant performance. Sages antioxidant properties should be regarded as an additional health promoting value for use as phytonutrients. In line with the efforts to balance the conservation of biodiversity and encouraging controlled exploitation of plant resources of economic interest, the large variations, illustrated among each Salvia species, could supply a basis for the selection of plants with adequate levels of polyphenolics (yield and quality) and good antioxidant properties that could serve to vegetative propagation. Acknowledgements The authors are deeply grateful to Abderrazak Smaoui (Laboratory of Extremophile Plants, Center of Biotechnology, Borj-Cedria Science and Technology Park, B.P. 901, Hammam-Lif 2050, Tunisia) for botanical identification of plants. This work was supported by the Tunisian Ministry of Higher Education, Scientific Research and Technology. Also we wish to thank the financial support from the European Social Fund. References ˇ Adzet, T., Canigueral, S., Iglesias, J.A., 1988. Chromatographic survey of polyphenols from Salvia species. Biochem. Syst. Ecol. 16, 29–32. Areias, F., Valentão, P., Andrade, P.B., Ferreres, F., Seabra, R.M., 2000. Flavonoids and phenolic acids of sage: influence of some agricultural factors. J. Agric. Food Chem. 48, 6081–6084. Baricevic, D., Bartol, T., 2000. Sage: the genus Salvia. In: Kintzios, S.E. (Ed.), Pharmacology: The Biological/Pharmacological Activity of the Salvia Genus. Harwood Academic Publishers, The Netherlands, pp. 143–184. Bas¸kan, S., Öztekin, N., Bedia Erim, F., 2007. Determination of carnosic acid and rosmarinic acid in sage by capillary electrophoresis. Food Chem. 101, 1748–1752. Bektas, T., Sökmen, M., Akpulat, H.A., Sökmen, A., 2005. In vitro antioxidant activities of the methanol extracts of five Allium species from Turkey. Food Chem. 1, 89–92. Ben Farhat, M., Jordán, M.J., Chaouech-Hamada, R., Landoulsi, A., Sotomayor, J.A., 2009. Variations in essential oil, phenolic compounds and antioxidant activity of Tunisian cultivated Salvia officinalis L. J. Agric. Food Chem. 57, 10349–10356. Ben Farhat, M., Landoulsi, A., Chaouch-Hamada, R., Sotomayor, J.A., Jordán, M.J., 2013. Profiling of essential oils and polyphenolics of Salvia argentea and evaluation of its by-products antioxidant activity. Ind. Crops Prod. 47, 106–112. Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. J. Anal. Biochem. 239, 70–76. Borneo, R., León, A.E., Aguirre, A., Ribotta, P., Cantero, J.J., 2009. Antioxidant capacity of medicinal plants from the province of Córdoba (Argentina) and their in vitro testing in a model food system. Food Chem. 112, 664–670. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensmittel-wissenschaft und Technologie 28, 25–30.

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