Phytochemical composition and in vitro antioxidant activity of by-products of Salvia verbenaca L. growing wild in different habitats

Industrial Crops and Products 49 (2013) 373–379

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Phytochemical composition and in vitro antioxidant activity of by-products of Salvia verbenaca L. growing wild 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, 7021 Zarzouna, Tunisia Institut Préparatoire aux Etudes d’Ingénieurs de Bizerte, Route Menzel Abderrahman, 7021 Zarzouna, 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 1 March 2013 Received in revised form 9 May 2013 Accepted 14 May 2013 Keywords: Antioxidant activity By-products Phenolic profile Rosmarinic acid Salvia verbenaca Total phenolic content

a b s t r a c t The phenolic contents and corresponding antioxidant activities of post-distilled Salvia verbenaca growing wild in different habitats were evaluated. Total phenolic content varied from 55.03 mg GAE/g DW to 136.33 mg GAE/g DW. Rosmarinic acid (349.60–2560.37 ␮g/g) was the dominant phenolic compound of all analyzed samples. The DPPH and ABTS scavenging powers and FRAP assay showed that post-distilled aerial parts collected in Sers, Touiref and Beja were the most potent radical-scavengers and reducing agents. Results revealed remarkable significant (p < 0.05) variations in total and individual phenolic contents as well as in antioxidant activity across S. verbenaca different methanolic extracts. In addition, our study verified that the methanolic fraction had strong antioxidant activities which were significantly (p < 0.05) correlated with several identified phenolics and total phenolic content. These results supported the significant potential to use S. verbenaca by-products as a source of natural antioxidants. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Actually, interest has increased considerably in the research of naturally occurring antioxidants for use in foods or medicinal materials as an alternative to synthetic antioxidants, which are being restricted due to their possible toxicity (Namiki, 1990). In addition, a particular attention is dedicated to by-products resulting from essential oil production, i.e., distillate residues of aromatic plants, which constitute a potential pool of compounds with strong antioxidant activity. Polyphenols possess ideal structural chemistry for free radical-scavenging activity, and they have been shown to be more effective antioxidants in vitro than tocopherols and ascorbate (Rao et al., 2010). Antioxidant properties of polyphenols arise from their high reactivity as hydrogen or electron donors, and from the ability of the polyphenol derived radical to stabilize and delocalise the unpaired electron (chainbreaking function) and from their ability to chelate transition metal ions (Rice-Evans et al., 1997). Among the various medicinal and culinary herbs, some species are of particular interest because they may be used for the production of raw materials or preparations containing phytochemicals with significant antioxidant capacities and health

∗ 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.2013.05.006

benefits (Exarchou et al., 2002). Several Salvia species contain a wide variety of polyphenols that have antioxidant activities (Lu and Foo, 1999, 2001, 2002). Salvia verbenaca L. (Lamiaceae) is a perennial herbaceous plant, more than likely indigenous to the Mediterranean countries and the Canary Islands and has spread into Europe and Asia (Codd, 1985). Like other Salvia species of commercial use, namely, Salvia officinalis L., Salvia fruticosa Miller, Salvia lavandulaefolia Vahl. and Salvia sclarea L., S. verbenaca is cultivated in several countries mainly to obtain dried leaves to be used as raw material in herbal medicine (Baser, 2000). The literature survey revealed limited work carried out on polyphenolic compounds and biological activities of S. verbenaca. Generally, phenolic contents are highly influenced by abiotic environmental factors (temperature, moisture, climatic conditions, elevation. . .), biotic effects (human disturbance, herbivores pressure, competing plants. . .), post-harvesting treatments (dryness, distillation. . .), methods of extraction, as well as the genetic outfits (Jordán et al., 2009; Liu et al., 2010; Munné-Bosch et al., 2001; Nieto et al., 2011; Parejo et al., 2002; Sellami et al., 2009). Despite the great potential of some Salvia species as valuable sources of bioactive molecules, there is a lack of satisfactory knowledge about some members of this genus such as the species S. verbenaca. To the best of our knowledge, there is no report carried out on the quantification of phenolics along with the evaluation of antioxidant activity of S. verbanaca by-products. Therefore,

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this study was undertaken to investigate and compare phenolic contents and profiles and antioxidant activities of S. verbanaca post-distilled plants collected in different habitats. The possible relationship between phenolic contents, individual phenolic components and antioxidant activity was assessed.

curve of concentrations ranging from 25 to 300 mg/L of gallic acid. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of dry plant material weight.

2. Materials and methods

HPLC was performed on a reverse phase Zorbax SB-C18 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 was 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 micrograms per gram of dry plant material weight.

2.1. Plant material Aerial parts from 40 randomized individual plants of S. verbenaca L. were collected as homogenous samples from each population of 10 Tunisian locations. Plant material was harvested at the flowering stage in March and April 2008. Details of collection sites are provided in Table 1. A voucher specimen was deposited at the Herbarium of the Laboratory of Biochemistry and Molecular Biology at the Faculty of Sciences of Bizerte under the numbers (SV 2008-125, SV 2008-126, SV 2008-127, SV 2008-128, SV 2008-129, SV 2008-130, SV 2008-131, SV 2008-132, SV 2008-133, SV 2008134) respectively for Tunis, Bir Mroua, Enfida, Chott Meriem, Bou Arada, Rass Zebib, Beja, Touiref, Sers and Hancha. 2.2. Chemicals 2,2-Diphenyl-1-picrylhydrazyl (DPPH• ), 2,2 -azinobis (3acid) diammonium salt ethylbenzothiazoline-6-sulfonic [ABTS(NH4 )2 ], 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox), potassium persulfate 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). 2,4,6-Tripyridyl-s-triazine (TPTZ) was obtained from Fluka (Madrid, Spain). Methanol and was of HPLC grade and other reagents were of analytical grade. 2.3. Extraction of polyphenolic compounds Distilled plant material was dried in an oven at 35 ◦ C until it reached a constant weight and then finely ground to pass a 2 mm sieve. For the extraction, dried samples of 0.5 g were first homogenized with 30 mL of petroleum ether under magnetic stirring for 5 min and taken to dryness at room temperature. Second, 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 redissolved in methanol and made up to 5 mL (Jordán et al., 2009). The methanolic extract yield was expressed in terms of milligrams of dry methanolic extract weight per gram of dry plant weight. The extracts were kept in vials at −80 ◦ C until their corresponding analysis. Two extracts were prepared for each sample. 2.4. Determination of total phenolic contents Based on the procedure of Singleton and Rossi (1965), total phenolic content was estimated by the Folin–Ciocalteau colorimetric method, using gallic acid as a standard phenolic compound. Briefly, 15 ␮L of methanolic extracts were added to 1185 ␮L of distilled water and 75 ␮L of Folin–Ciocalteau 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-colored 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

2.5. HPLC analysis

2.6. DPPH• radical-scavenging activity The method described by Brand-Williams et al. (1995) was adopted to determine the ability of the methanolic extracts to scavenge DPPH• free radicals. Briefly, 500 ␮L of methanolic extracts at different concentrations (2.5 ␮L/mL to 45 ␮L/mL) 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. 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 × 100 Absorbance control

The results were expressed as the inhibitory concentration of the extract necessary to decrease 50% (IC50 ) of the DPPH• absorbance. Concentrations are expressed in micrograms of dry plant methanolic extract per milliliter of methanol. 2.7. ABTS•+ radical cation decoloration assay The ABTS free radical-scavenging activity of each sample was determined according to the method described by 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,

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Table 1 Collection sites of Salvia verbenaca and their eco-geographical characteristics. N

Collection sites

Bioclimatic stage

Temperature (◦ C/year)

Rainfall (mm/year)

Soil pH

1 2 3 4 5 6 7 8 9 10

Tunis Bir Mroua Enfida Chott Meriem Bou Arada Rass Zebib Beja Touiref Sers Hancha

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

18.5 19.0 18.5 18.0 17.8 18.0 18.0 17.0 16.8 18.6

470 500 350 300 450 650 450 500 700 250

7.94 7.95 7.96 8.05 8.15 7.50 7.50 7.60 7.32 7.50

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 ability to reduce ferric ions was measured using the method described by Benzie and Strain (1996). The FRAP reagent was freshly prepared from 300 mM acetate buffer, pH 3.6, 10 mM 2,4,6tripyridyl-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 millimolar of FeSO4 equivalents per milligram of dry plant methanolic extract. 2.9. Statistical analysis All data were reported as mean ± standard deviation of independent replicates. Data were analyzed by an analysis of variance (p < 0.05) and the means were separated by Duncan’s multiplerange test (ANOVA procedure). Pearson’s correlation coefficients were calculated. Results were processed by computer programs Excel and STATISTICA version 5.1.

Geographical location Longitude (N)

Latitude (E)

Altitude (m)

36◦ 49 36◦ 47 36◦ 02 35◦ 53 36◦ 20 37◦ 16 36◦ 41 36◦ 15 36◦ 00 35◦ 07

10◦ 08 10◦ 37 10◦ 24 10◦ 35 09◦ 39 10◦ 04 09◦ 10 08◦ 33 09◦ 07 10◦ 44

67 86 10 8 252 14 284 447 487 52

2007). Therefore, the estimation of phenolic contents of plants permitted their valorization as a source of natural antioxidants. Nevertheless, it should be taken into consideration that the FolinCiocalteau reagent is used to obtain a crude estimate of the amount of polyphenolics present in an extract and it is extremely important to point out that the colometric method does not provide a specific assay for phenolic compounds as it reacts positively with many easily oxidizable non-phenolic compounds (Que et al., 2006). 3.2. Determination of extract yield and polyphenolic composition The extract yields and data obtained from HPLC analysis of S. verbenaca methanolic extracts are given in Table 2. Extract yields of post-distilled plant material rose from 55.09 mg/g DW in plants harvested in Enfida to 119.33 mg/g DW in those collected in Bir Mroua. Previous findings showed that non-distilled material of S. verbenaca growing in Turkey yielded a higher level of 175.40 mg/g DW (Tepe, 2008). As it can be seen in Table 2, the identified compounds from different S. verbenaca methanolic extracts can be grouped into three classes of phenolics, namely six phenolic acids (p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric, ferulic acid and rosmarinic acid), three phenolic diterpenes (carnosic acid, methyl carnosate and carnosol) and seven flavonoids (naringenin, luteolin, cirsiliol, apigenin, genkwanin, naringin and hesperidin). p-Hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, rosmarinic acid, luteolin, cirsiliol, apigenin and genkwanin have been previously isolated from S. verbenaca collected in Spain, Egypt and Turkey (Adzet et al., 1988; Khalil et al., 2007; Saleh and Sabri, 1980; Tepe, 2008). Among the identified phenolic compounds, rosmarinic acid was present in the largest amounts

3. Results and discussion 3.1. Determination of total phenolic content The total phenolic content was analyzed spectrophotometrically using gallic acid as a standard and results are given in Fig. 1. The phenolic amounts ranged from the lowest values of 55.03 mg GAE/g DW (Enfida) and 55.49 mg GAE/g DW (Hancha) to the highest level of 136.33 mg GAE/g DW exhibited by extracts of Sers. Compared with our results, S. verbenaca grown in Egypt illustrated a poor content of polyphenolics (4.27 mg GAE/g DW) (Khalil et al., 2007). The differences in total phenolic content between S. verbenaca collection sites was statistically significant (p < 0.05). The variation may be attributed to the different 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). The interests of phenolics are increasing in the food industry since they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food (Aneta et al.,

Fig. 1. Phenolic contents (mg GAE/g DW) of Salvia verbenaca methanolic extracts obtained from different growing regions. Bars sharing the same small letter did not share significant differences at p < 0.05 (Duncan test).

376

Table 2 Extract yields and content of phenolic compounds of Salvia verbenaca methanolic extracts. Identified compounds

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

Phenolic acids p-Hydroxybenzoïc acid Vanillic acid Caffeic acid p-Coumaric acid Ferulic acid Rosmarinic acid Phenolic diterpenes Carnosic acid Carnosol Methyl carnosate Flavonoids Naringenin Luteolin Cirsiliol Apigenin Genkwanin Naringin Hesperidin Total identified Extract yieldm (mg/g DW)

Bir Mroua n=6

173.69 ± 1.90d 28.49 81.48 57.18 43.57 1065.78

± ± ± ± ±

1.70b 11.83cd 1.07e 5.48bc 173.51c

65.83 ± 9.68de 22.58 ± 3.40e 286.37 ± 11.64d 472.03 9.96 75.44 3.65 3.29 34.82 39.37 2434.82 75.18

± ± ± ± ± ± ± ± ±

13.71g 1.93d 5.43c 1.85fg 0.27bc 4.74d 3.34e 67.42f 3.43bcd

Beja n=6

382.79 ± 11.98a

337.72 ± 22.91b

14.51 ± 0.46e 191.19 ± 27.72b 133.78 ± 1.88b 72.89 ± 0.86a 2503.96 ±224.40a

17.18 ± 1.21d 177.36 ± 14.54b 136.69 8.93b 52.11 ± 0.34b 2542.7 ± 167.89a

67.95 ± 3.73d 32.09 ± 1.46bc nd

72.75 ± 2.56cd 36.14 ± 2.21ab 267.40 ± 9.51d

2100.02 ± 79.38b 1402.07 ± 5.17d 21.14 ± 2.03c 14.78 ± 2.54d 53.18 ± 3.15de 47.22 ± 1.70e 13.56 ± 0.51d 18.85 ± 0.99c 2.53 ± 0.57cd 3.82 ± 0.33b 36.79 ± 2.83d 51.80 ± 6.82c 84.48 ± 4.67c 61.72 ± 4.71d 4935.55 ± 104.27d 5919.50 ± 100.08c 119.33 ± 6.88a 65.74 ± 2.10cd

Tunis n=6

Touiref n=6

229.87 ± 8.60c 20.21 97.29 77.65 40.41 1688.01

± ± ± ± ±

0.46c 2.86c 5.67c 3.32cd 63.42b

63.52 ± 15.30de 25.52 ± 7.27de 633.37 ± 11.66b 940.41 13.84 73.16 3.01 2.80 57.30 21.74 4002.25 89.15

± ± ± ± ± ± ± ± ±

22.50e 2.62d 1.72c 0.69fg 0.72cd 3.55bc 3.21f 105.27e 2.89bc

Values are means ± SD of independent replicates. Values followed by the same letter did not share significant differences at 5% (Duncan test). nd: not detected. m Extract yield is expressed in milligrams of methanolic dry extract per gram of dry plant material weight.

Bou Arada n = 10

326.20 ± 22.67b 56.63 231.37 153.71 53.49 2536.82

± ± ± ± ±

0.87a 38.22a 1.27a 3.16b 108.28a

83.07 ± 8.43bc 33.94 ± 4.06ab 436.20 ± 8.55c 2432.55 21.55 124.03 31.29 5.13 65.78 173.98 6806.58 97.87

± ± ± ± ± ± ± ± ±

115.98a 2.52c 1.11b 1.95a 0.87a 5.88b 98a 122.23a 8.90ab

Sers n=6

184.54 ± 4.87d 28.40 89.60 65.75 49.01 1087.53

± ± ± ± ±

0.80b 6.12c 3.20d 5.98bc 93.01c

61.42 ± 7.19de 28.31 ± 4.94d 239.15 ± 4.03f 615.83 31.60 48.40 8.84 2.84 40.91 38.66 2558.51 84.04

± ± ± ± ± ± ± ± ±

9.21f 5.39b 1.82e 1.95e 0.56cd 6.81d 6.77e 49.74f 3.61bcd

383.37 ± 19.21a 28.60 223.65 148.56 77.80 2560.37

± ± ± ± ±

0.40b 3.88a 1.83a 13.02a 62.33a

96.97 ± 6.22a 38.57 ± 2.68ac 220.58 ± 9.84f 2011.57 49.65 155.03 27.09 4.86 81.54 149.97 6257.56 96.14

± ± ± ± ± ± ± ± ±

74.87c 4.75a 8.71a 7.84b 0.66a 13.42a 8.48b 107.76b 0.71abc

Enfida n=6

Chott Meriem n=6

Hancha n=6

nd

nd

51.18 ± 3.76e

nd 30.79 ± 0.64f nd 32.14 ± 0.34d 349.60 ± 3.25d

nd 60.59 ± 1.63de nd 54.73 ± 3.94abc 1090.09 ± 9.23c

9.59 ± 0.91f 50.77 ± 3.04ef 22.51 ± 0.84f 74.55 ± 16.66a 475.74 ± 7.45d

43.36 ± 1.12f nd nd

87.56 ± 6.53ab 32.64 ± 3.35bc nd

55.47 ± 1.60e nd 1159.73 ± 41.68a

nd 2.68 ± 0.07e nd 2.55 ± 0.12g 1.94 ± 0.16d 21.42 ± 0.94e 26.15 ± 1.77f 510.63 ± 0.79h 55.09 ± 1.87d

nd 15.73 ± 1.37d nd 7.12 ± 1.61ef 3.80 ± 0.92b 46.65 ± 0.04d 42.89 ± 1.07e 1442.70 ± 12.68g 84.73 ± 2.97bcd

254.82 ± 22.14h 51.65 ± 2.42a 57.89 ± 3.81d 23.95 ± 1.00b 2.65 ± 0.12cd 20.26 ± 0.50e 24.19 ± 1.21f 2334.39 ± 37.54f 81.49 ± 1.34bcd

M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 373–379

Rass Zebib n=8

M.B. Farhat et al. / Industrial Crops and Products 49 (2013) 373–379

in all analyzed methanolic extracts. Samples collected in Sers (2560.37 ␮g/g), Beja (2542.79 ␮g/g), Touiref (2536.82 ␮g/g) and Bir Mroua (2503.96 ␮g/g) showed the highest levels of rosmarinic acid however, those collected in Hancha (475.74 ␮g/g) and Enfida (349.60 ␮g/g) were characterized by the lowest levels. Much higher levels of rosmarinic acid were detected in non-treated S. verbenaca, collected in Turkey (Tepe, 2008). This caffeic acid dimer showed many biological activities such as inhibiting the HIV-1, antitumor, antihepatitis and protecting the liver, inhibiting the blood clots and antiinflammation (Tepe, 2008). In addition, the great potential of the rosmarinic acid as a natural antioxidant compound has been demonstrated (Lu and Foo, 1999; Matkowski, 2008). Samples of S. verbenaca were also characterized by substantial levels of naringenin (254.82–2432.55 ␮g/g) and methyl carnosate (220.58–1159.73 ␮g/g). Lower concentrations were detected for the remaining phenolic compounds. In agreement with our findings, it was reported that the majority of the phenolic acids in Salvia species are exclusively caffeic acid derivatives (Lu and Foo, 2002), which occurs in Salvia species predominantly in the dimer form as rosmarinic acid (Cuvelier et al., 1996; Lu and Foo, 1999) and frequently in the monomer form such as caffeic acid (Qian and Li, 1992) and ferulic acid (Cuvelier et al., 1996). It is worth noting that samples collected in Enfida provided a poor content of almost all identified phenolic compounds with a total amount of 510.63 ␮g/g. In addition, Enfida methanolic extracts were marked by the absence of several phenolics, namely p-hydroxybenzoic, vanillic and p-coumaric acids, carnosol, methyl carnosate, naringenin and cirsiliol. Nevertheless, post-distilled plants of Touiref and Sers were the richest in phenolic contents with total identified constituents of 3236.42 ␮g/g and 3310.47 ␮g/g, respectively. The amounts of phenolic compounds varied significantly (p < 0.05) between samples of S. verbenaca collected in different habitats. As previously reported, the variation of phenolic compounds of natural extracts and their antioxidative potential could be attributed to the influence of genetic (Chun et al., 2005) and environmental factors (Areias et al., 2000). It is extremely important to point out, the effect of the distillation process on the concentrations of phenolic compounds. Almela et al. (2006) reported the strong effect of the distillation treatment on the content of three compounds of high antioxidant activity, namely rosmarinic acid, carnosic acid and carnosol of post-distilled Rosmarinus officinalis. In fact, the distillation process could reduce the amounts of hydrophilic phenolic compounds in post-distilled plants (Jordán et al., 2009; Nieto et al., 2011). Linear correlation coefficients were established to explore the trend of association between individual phenolic compounds, total phenolic contents and three environmental characteristics (Table 3). Rosmarinic acid (r = 0.86), methyl carnosate (r = 0.81), naringenin (r = 0.71) and total phenolic contents assessed by the Folin-ciocalteau method (r = 0.85) showed a significant (p < 0.05) positive correlation with the factor altitude. These results indicated the parallel evolution of the above mentioned compounds

Table 3 Linear correlation coefficients between the most representative phenolic individual compounds, total phenolic contents and environmental characteristics. Altitude Rosmarinic acid Methyl carnosate Naringenin Total phenolic compounds identified by HPLC Phenolic contents determined spectrophotometrically a

Temperature

Rainfall

a

0.86 0.81a 0.71a −0.01

−0.67 −0.51 −0.42 0.09

0.63a 0.56 0.58 −0.27

0.85a

−0.55

0.55

Significant correlation at p < 0.05.

a

377

Table 4 Antioxidant capacity of Salvia verbenaca methanolic extracts. Collection site

DPPH (IC50 , ␮g/mL)

Rass Zebib Bir Mroua Beja Tunis Touiref Bou Arada Sers Enfida Chott Meriem Hancha

31.19 34.70 26.62 30.34 25.11 33.47 24.47 40.91 28.28 39.85

± ± ± ± ± ± ± ± ± ±

2.25cd 2.43b 0.80ef 2.28cd 2.97ef 4.13bc 1.87f 0.50a 0.16de 3.90a

ABTS (␮M TE/mg) 144.02 139.26 282.17 190.51 287.81 154.97 271.51 134.45 196.72 120.11

± ± ± ± ± ± ± ± ± ±

3.40c 10.59c 6.58a 6.71b 3.65a 6.79bc 4.52a 5.27c 1.61b 6.62c

FRAP (mM Fe(II)/mg) 118.02 109.22 142.07 122.33 131.86 120.53 139.09 101.46 124.27 104.89

± ± ± ± ± ± ± ± ± ±

15.25abc 5.04bc 1.46a 3.70abc 1.05ab 7.53abc 11.23a 1.97c 0.38abc 0.37c

Values are means ± SD of three independent replicates, values followed by the same letter did not share significant differences at 5% (Duncan test).

with regard to increasing elevation of the collection site. A similar result was obtained between rosmarinic acid (r = 0.63) and the annual rainfall. However, a negative significant (p < 0.05) correlation (r = −0.67) characterized the relationship between rosmarinic acid and annual temperature, which confirmed their opposite variation. 3.3. Antioxidant capacity Results of the DPPH and ABTS radical scavenging assays and the FRAP reducing power test are given in Table 4. As can be seen, the effect of the collection site on the antioxidant capacity was significant (p < 0.05). S. verbenaca methanolic extracts scavenging ability of DPPH free radicals is expressed as IC50 values (the concentration reducing 50% of DPPH). The extracts with the highest antioxidant activities were capable of quenching the DPPH free radicals at the lowest concentrations. Methanolic extracts of samples collected in Sers (24.47 ␮g/mL), Touiref (25.11 ␮g/mL) and Beja (26.62 ␮g/mL) showed the best scavenging activity against DPPH, while those of post-distilled plants harvested in Enfida (40.91 ␮g/mL) and Hancha (39.85 ␮g/mL) exerted the weakest DPPH scavenging effect. In earlier studies, Kamatou et al. (2010) reported radical-scavenging activities at different degrees for several Salvia species. The authors recorded a good antioxidant activity (IC50 < 30 ␮g/mL) for S. albicaulis Benth., Salvia aurita L.f, Salvia chamelaeagnea Berg., Salvia muirii L. Bol., Salvia namaensis Schinz, Salvia repens Burch. ex Benth., Salvia runcinata L.f., Salvia schlechteri Briq. and Salvia stenophylla Burch. ex Benth., a moderate activity (IC50 < 80 ␮g/mL) for Salvia africana-caerulea L., Salvia africana-lutea L., Salvia disermas L., Salvia garipensis E.Mey. ex Benth. and Salvia lanceolata Lam. and a poor activity (IC50 > 80 ␮g/mL) for Salvia dolomitica Codd and Salvia radula Benth. Compared to Kamatou et al. (2010) findings, our postdistilled samples are characterized by a good (Sers, Touiref, Beja and Chott Meriem) to a moderate antioxidant activity (Tunis, Rass Zebib, Bou Arada, Bir Mroua, Hancha and Enfida). The ABTS assay is based on the capacity of a sample to scavenge the ABTS radical cation (ABTS•+ ), as compared to a standard antioxidant (Trolox). The antioxidant activity of S. verbenaca ranged from the lowest values of 144.02, 139.26, 134.45, 120.11 ␮M TE/mg attributed, respectively to Rass Zebib, Bir Mroua, Enfida and Hancha to the highest values of 287.81, 282.17 and 271.51 ␮M TE/mg recorded, respectively for Touiref, Beja and Sers. These results revealed an ABTS scavenging activity lower than the activity reported for post-distilled S. officinalis (309.22–346.61 ␮M TE/mg) (Ben Farhat et al., 2009). The FRAP assay is based on the ability of an antioxidant to reduce Fe3+ to Fe2+ in the presence of TPTZ, forming an intense blue Fe2+ –TPTZ complex. The absorbance decrease is proportional to the antioxidant content in the extracts (Benzie and Strain,

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Table 5 Linear correlations coefficients between phenolic individual compounds, total phenolic contents and the antioxidant activity.

p-Hydroxybenzoïc acid Vanillic acid Caffeic acid p-Coumaric acid Ferulic acid Rosmarinic acid Carnosic acid Carnosol Methyl carnosate Naringenin Luteolin Cirsiliol Apigenin Genkwanin Naringin Hesperidin Total phenolic compounds identified by HPLC Phenolic contents determined spectrophotometrically a

DPPH

ABTS

FRAP

−0.74a −0.59a −0.38 −0.76a −0.69a −0.74a −0.79a −0.91a 0.45 −0.83a −0.41 −0.86a −0.60a −0.27 −0.34 −0.11 −0.79a

0.69a 0.52a 0.34 0.71a 0.77a 0.79a 0.71a 0.88a −0.44 0.82a 0.42 0.79a 0.65a 0.37 0.29 0.25 0.82a

0.89a 0.53a 0.70a 0.89a 0.79a 0.90a 0.51a 0.80a −0.57a 0.96a 0.45 0.84a 0.83a 0.59a 0.09 0.26 0.94a

a

a

−0.58

a

0.60

0.79

Significant correlation at p < 0.05.

1996). By-products of S. verbenaca from Beja (142.07 mM Fe(II)/mg) and Sers (139.09 mM Fe(II)/mg) displayed the highest reducing power, however, the weakest activity characterized the samples of Enfida (101.46 mM Fe(II)/mg) and Hancha (104.89 mM Fe(II)/mg). FRAP values detected for post-distilled S. verbenaca are lower than those exhibited by post-distilled S. officinalis (173.42–180.56 mM Fe(II)/mg) (Ben Farhat et al., 2009). Significant differences in DPPH and ABTS scavenging activities and in the ferric reducing power (FRAP) between extracts provided by S. verbenaca plants growing wild in various habitats (Table 4) could be related to differences in polyphenolic composition of analyzed extracts.

reported that the presence of rosmarinic acid, caffeic acid, carnosic acid and derivatives in some Salvia species such as S. aurita, S. chamelaeagnea, S. muirii, S. repens, S. runcinata and S. schlechteri was associated to a good antioxidant activity. It is worth noting that several flavonoids are potent antioxidants, however, some of the identified flavonoids made a rather small contribution to the total antioxidant capacity of S. verbenaca extracts since they did not exhibited significant correlations. Globally, the synergistic effect of the diversity of major and minor constituents present in the methanolic extracts of S. verbenaca should be taken into consideration to account for their total antioxidant activity. In fact, Lu and Foo (2001) reported that most natural antioxidative compounds often work synergistically with each other to produce a broad spectrum of antioxidative properties that create an effective defense system against free radicals. 4. Conclusions The results indicate a large variation in phenolic contents and antioxidant capacities of S. verbenaca growing wild in different locations, the differences are apparently related to the distinct habitats where the plants are grown and their genetic outfits. Post-distilled plants of Sers, Touiref and Beja are characterized by the highest antioxidant capacities and therefore, they could be considered as the promising collection sites that could be exploited as natural antioxidants. These findings highlight the effectiveness of S. verbenaca by-products. 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.

3.4. Correlation between phenolic individual compounds, total phenolic contents and antioxidant activity

References

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