Chemical investigation of different crude extracts from Teucrium ramosissimum leaves. Correlation with their antigenotoxic and antioxidant properties

Food and Chemical Toxicology 49 (2011) 191–201

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Chemical investigation of different crude extracts from Teucrium ramosissimum leaves. Correlation with their antigenotoxic and antioxidant properties M. Ben Sghaier a, W. Bhouri a, A. Neffati a, J. Boubaker a, I. Skandrani a, I. Bouhlel a, S. Kilani a, L. Chekir-Ghedira a,b,⇑, K. Ghedira a a b

Department of Pharmacognosy, Faculty of Pharmacy, Rue Avicenne, Monastir 5000, Tunisia Department of Cellular and Molecular Biology, Faculty of Dental Medicine, Rue Avicenne, Monastir 5000, Tunisia

a r t i c l e

i n f o

Article history: Received 25 May 2010 Accepted 13 October 2010

Keywords: Teucrium ramosissimum SOS chromotest Antioxidant capacity Xanthine oxidase activity NBT/Riboflavine

a b s t r a c t The effect of extracts obtained from Teucrium ramosissimum leaves on genotoxicity and SOS response induced by aflatoxin B1 (0.5 lg/assay) as well as nitrofurantoin (5 lg/assay) was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixture. However, all the extracts, particularly the total oligomers flavonoids (TOF) extract significantly decreased the genotoxicity induced by aflatoxin B1 and nitrofurantoin. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non-enzymatic (NBT/Riboflavine assay) systems. TOF extract was the most effective one in inhibiting both xanthine oxidase activity and NBT reduction. Our findings emphasize the potential of T. ramosissimum to prevent mutations and also its antioxidant effect. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Free radicals may be defined as any chemical species that are capable of existing with one or more unpaired outer shell electrons. They are extremely reactive and generally highly unstable (Martinez-Cayuela, 1995). Reactive oxygen species, such as superoxide radical ðO 2 Þ, hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2), are of the greatest biological significance (Martinez-Cayuela, 1995; Schoneich, 1999). They are extremely reactive and potentially damaging transient chemical species. In addition to exogenous sources of free radicals, such as ionizing radiation, tobacco smoke, pesticides, pollutants, and some medications, they are produced continuously in all cells, as metabolic byproducts by a number of intracellular systems: small cytoplasmic molecules, cytoplasmic proteins, membrane enzymes, peroxisomes, mitochondrial electron transport systems, and microsomic electron transport systems (Martinez-Cayuela, 1995). All cellular components, proteins, polyunsaturated fatty acids, nucleic acids and carbohydrates, are prominent biological targets of reactive oxygen species, giving rise to metabolic and cellular disturbances (Martinez-Cayuela, 1995). Fortunately, within biological systems, there are enzymatic systems and chemical scavengers: ⇑ Corresponding author. Address: Université de Monastir, Unité de ‘‘Pharmacognosie/Biologie Moléculaire”, Faculté de Medecine Dentaire, Rue Avicenne, Monastir 5000, Tunisia. Fax: +216 73 461 150. E-mail address: [email protected] (L. Chekir-Ghedira). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.10.016

dietary antioxidants (a-tocopherol, b-carotene, ascorbic acid, glutathione and uric acid), some hormones (estrogen, angiotensin) and endogenous enzymes (superoxide dismutase, glutathione peroxidase and catalase). All of them are able to remove oxygen free radicals formed in cells and thus protect against oxidative damage (Halliwell and Gutteridge, 1990; Martinez-Cayuela, 1995). Tissue damage resulting from the imbalance between reactive oxygen species generating and scavenging systems (oxidative stress) has been implicated in the pathology of a number of disorders, such as atherosclerosis, ischemia–reperfusion injury, cancer, malaria, diabetes, inflammatory joint disease, asthma, cardiovascular diseases, cataracts, immune system decline, and could play a role in neurodegenerative diseases and ageing processes (Florence, 1995; Nakagami et al., 1995; Martinez-Cayuela, 1995; Schoneich, 1999; Young and Woodside, 2001). In recent years, there has been a considerable interest in finding natural antioxidants from plant materials. The antioxidant phytochemicals from plants, particularly flavonoids and other polyphenols, have been reported to inhibit the propagation of free radical reactions, to protect the human body from disease (Kinsella et al., 1993; Terao and Piskula, 1997), and to retard lipid oxidative rancidity (Duthie, 1993). In addition, the use of synthetic antioxidants has been questioned because of their toxicity (Valentao et al., 2002). Therefore, there have been numerous researches on these bioresources to seek for potential natural and possibly economic and effective antioxidants to replace the synthetic ones.

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The main aim of this research project was to screen the species of Teucrium ramosissimum, with respect to their total polyphenol and flavonoid contents, antigenotoxic and antioxidant activities, as potential source of natural antioxidants. The relationship between phenolic content and antioxidant activity was also statistically investigated. 2. Materials and methods

A known volume of each extract was placed in a 10 ml volumetric flask to estimate flavonoid content according to the method of Zhishen et al. (1999). Distilled water was added to make the volume 5 ml, then 0.3 ml NaNO2 (1:20, w/v) was added to this dilution. Three milliliters of AlCl3 (1:10, w/v) were added 5 min later. After 6 min, 2 ml of 1 N NaOH were added and the total absorbance was measured at 510 nm (Kumar and Chattopadhyay, 2007). Quercetin (0.05 mg/ml) was used as standard for constructing a calibration curve. Flavonoid content was expressed according to the following formula:

% Flavonoids ¼ ð½ðDOextract  0:05Þ=DOQuercetin =Extract concentrationÞ  100

2.1. Plant material The aerial part of T. ramosissimum was collected in January 2005 from the mountainous region of Gafsa in Southeast Tunisia. The plant was identified by Pr. Mohamed Chaieb (Department of Botany, Faculty of Sciences, University of Sfax, Tunisia) according to the Flora of Tunisia (Pottier-Alaptite, 1978). A voucher specimen (Tr-02-05) was deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, University of Monastir in Tunisia, for future reference. The leaves were shade-dried, powdered, and stored in a tightly closed container for further use. 2.2. Preparation of plant extracts In order to obtain an extract enriched in total oligomers flavonoids (TOF), the powdered leaves were macerated in water–acetone mixture (1:2), during 24 h with continuous stirring. The extract was filtered, and the acetone was evaporated under low pressure in order to obtain an aqueous phase. Tannins were partially removed by precipitation with an excess of NaCl during 24 h at 5 °C, and the supernatant was recovered. The latter was extracted with ethyl acetate, concentrated, and precipitated with an excess of chloroform. The precipitate was separated and yielded the TOF extract, which was dissolved in water (Ghedira et al., 1991). Petroleum ether and ethyl acetate extracts were obtained by Soxhlet extraction (6 h) using 100 g of the powdered leaves and 1 l of solvent. These two types of extract, with different polarities, were concentrated to dryness and each residue was kept at 4 °C. Then, the extracts were resuspended in dimethylsulfoxide (DMSO). In the present study, three extracts were investigated. The doses of extracts we tested in the SOS chromotest assay, were (500, 250 and 50 lg/assay) for the petroleum ether extract, (250, 50 and 25 lg/assay) for the ethyl acetate extract and (50, 25 and 12.5 lg/assay) for the TOF extract, whereas the doses tested in xanthine/ xanthine oxidase enzymatic assay (300, 150 and 50 lg/ml) and inhibition of NBT reduction (3000, 1000, 300 and 100 lg/ml) are in accordance with previous investigations (Hayder et al., 2003, 2004, 2005; Ben Ammar et al., 2005; Kilani et al., 2005a,b; Bouhlel et al., 2006; Ben Mansour et al., 2007), where a number of preliminary dose-finding tests involving a number of plant extracts were conducted. This means that the doses were suitable for testing the majority of the extracts, however, not necessarily all. Therefore, some extracts may be toxic at one ore more of the applied doses.

2.5. Determination of tannin content According to Nwabueze (2007), extraction of tannins was achieved by dissolving 5 g of extract in 50 ml of distilled water in a conical flask, allowing the mixture to stand 30 min with shaking of the flask at 10 min intervals and then centrifuging it at 5000g to obtain a supernatant (tannin extract). The extract was diluted to 100 ml in a standard flask using distilled water. Five milliliters of the diluted extract and 5 ml of standard tannic acid (0.01 g/l) were measured into different 50 ml volumetric flasks. One milliliter of Folin–Denis reagent was added to each flask, followed by 2.5 ml of saturated sodium carbonate solution. The solutions were made up to the 50 ml mark with distilled water and incubated at room temperature (20–30 °C) for 90 min. The absorption of these solutions were measured against a blank (containing 5 ml of distilled water in place of extract or standard tannic acid solution) in a Spectronic Genesys 10s, Thermo Electron Corp. (Madison, WI, USA) spectrophotometer at 760 nm wavelength. Tannin content (tannic acid equivalents) was calculated in triplicate, using the following formula:

% Tannins ¼ ½ðDOextract =e  1Þ=Extract concentration  100 where e is the molar extinction (=3.27 l g1cm1) and l = 1 cm.

acid

% Sterols ¼ ðPsteroids =P extract Þ  100

Plant material were screened for the presence of tannins, flavonoids, coumarins and sterols, using the methods previously described by Tona et al. (1998, 2004). Two milligrams of each extract were separately dissolved in 2 ml of the adequate solvent. The detection of major chemical groups was carried out by thin-layer chromatography (TLC) on silica gel 60 F254 from Merck (Dramstadt, Germany) (layer thickness, 0.25) as follows: for flavonoids, TLC was developed in n-butanol/acetic acid/water 4:1:5 (top layer), then spot were visualized with 1% AlCl3 solution in methanol under ultraviolet (UV) 366 nm. Coumarins were detected under UV (366 nm) thanks to their blue fluorescence, which becomes intense after spraying 10% potassium hydroxide solution in ethanol. Steroids were detected with Libermann–Burchard as a reagent using n-hexane/CH2Cl2 1:9 as a mobile phase. A range of colors are produced after heating sprayed plates for 10 min at 100 °C. The test for tannins was carried out with FeCl3. Each class of tannins gave a specific coloration.

2.7. Bacterial tester strains

% Polyphenols ¼ ð½ðDOextract  0:2Þ=DOGallic acid =Extract concentrationÞ  100

of tannic

Twenty milligrams of each extract dissolved in 500 ll of acetone was mixed with 250 ll of a digitonin solution (2% in the alcohol 78%) and heated to 60 °C for reaching half of volume. After cooling to room temperature for 15 min, the precipitate was separated on a weighed filter (M0). Then, the filter was washed 10 times with water, 10 times with alcohol 80%, once with acetone, once with alcohol 80%, and finally once with anhydrous ether, the filter was dried for 3 h at 80 °C. After cooling, the filter was weighed (Mf). Sterol content was expressed according to the following formula:

where Psteroids = (Mf  M0)  0.25.

The polyphenol content of T. ramosissimum extracts was quantified by the Folin–Ciocalteau’s reagent and was expressed as gallic acid equivalents (Yuan et al., 2005). Aliquots of test samples (100 ll) were mixed with 2 ml of 2% Na2CO3 and incubated at room temperature for 2 min. After the addition of 100 ll 50% Folin–Ciocalteau’s phenol reagent, the reaction tube was further incubated for 30 min at room temperature, and finally, absorbance was read at 720 nm. Gallic acid (0.2 mg/ml) was used as a standard. Polyphenol content was expressed according to the following formula:

(l g1 cm1)

2.6. Determination of total sterol content

2.3. Preliminary phytochemical analysis

2.4. Determination of total polyphenol and flavonoid contents

coefficient

Escherchia coli PQ37 has the genotype F thr leu his4 pyr D thr galE galK lacU169 Srp300:: Tn::10 rpoB rpsl uvrA rfa trp::Muc+ sfiA::Mud (Apy lac) and the construction details of this strain were described by Quillardet and Hofnung. Frozen permanent copies of the tester strain were prepared and stored at 80 °C. 2.8. Metabolic activation The S9 microsome fraction is prepared from the livers of rats treated with Aroclor 1254 (Maron and Ames, 1983). For SOS chromotest assay, the composition of S9 mix is the following: 1.65 M potassium chloride (KCl); 0.4 M magnesium chloride hexahydrate (MgCl26H2O); 1 M glucose-6-phosphate (G6P); 0.1 M nicotinamide adenine dinucleotide phosphate (NADP); 0.4 M pH 7.4 Tris-buffer; Luria broth medium at a concentration of 0.61 ml/ml mix; S9 fraction at a concentration of 0.1 ml/ml of mix. The S9 fraction was stored at 80 °C. 2.9. The SOS chromotest assay The SOS chromotest employs the error-prone DNA repair pathway of E. coli PQ37, also known as the SOS response, a complex regulatory network that is induced by DNA-damaging substances (Walker, 1987). The test involves incubation of the bacteria with the sample under investigation and subsequent determination of b-galactosidase (b-gal) activity (i.e., the level of SOS induction). Alkaline phosphatase (Ap) activity is also measured, as a toxicity control.

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M. Ben Sghaier et al. / Food and Chemical Toxicology 49 (2011) 191–201 2.9.1. Antigenotoxic assay The SOS chromotest was employed to determine the effect of the T. ramosissimum extracts on aflatoxin B1 (AFB1: indirect acting mutagen) and nifuroxazide (direct acting mutagen) induced genotoxicity. Genotoxicity and antigenotoxicity assays were performed according to the procedure outlined by Quillardet and Hofnung (1985). Exponential-phase culture of E. coli PQ37 was grown at 37 °C in Luria broth medium (1% bactotryptone, 0.5% yeast extract and 1% NaCl) plus 20 lg/ml ampicillin and diluted 1:9 into fresh medium; 100 ll aliquots were distributed into glass test tube containing various doses of T. ramosissimum extracts in a 0.6 ml finale volume. The extracts were dissolved in DMSO or distilled water and tested in triplicate, with or without exogenous metabolic activation. A positive control was prepared by exposure of the bacteria to either nifuroxazide or AFB1. After 2 h of incubation at 37 °C, with shaking, 300 ll samples were used for assay of b-galactosidase (b-gal) and alkaline phosphatase (Ap) activities. In this assay, b-galactosidase synthesis (lac Z gene) is dependent on sfiA activation and is used as a measure of SOS repair system induction. The activity of the constitutive enzyme alkaline phosphatase was used as a measure of protein synthesis and toxicity. These enzyme activities were assayed in a Helios Alpha, Spectronic Unicam (Cambridge, England) spectrophotometer. The SOS induction factor (IF) was calculated as the ratio of Rc/R0, where R0 and Rc are equal to (b-gal) activity/alkaline phosphatase (Ap) activity determined, respectively, in the absence and in the presence of the test compound at a concentration c. The IF in treated cells was obtained by comparing b-galactosidase and alkaline phosphatase activity in treated and untreated cells. The result was considered positive when the IF for b-galactosidase activity was >2. For the evaluation of the effect of T. ramosissimum extracts on of the SOS response induced by nifuroxazide (in the absence of S9 mix activation mixture) and aflatoxin B1 (AFB1) (in the presence of the S9 activation mixture), 10 ll of nifuroxazide solution (5 lg/assay) or AFB1 solution (0.5 lg/assay) were added into tubes with 10 ll of the tested concentration of each extract. Antigenotoxicity was expressed as percentage inhibition of genotoxicity induced by either nifuroxazide or AFB1 according to the formula:

Inhibition ð%Þ ¼ ½100  ðIF1  IF0 Þ=ðIF2  IF0 Þ  100 where IF1 is the induction factor in the presence of both test compound and mutagen, IF2 the induction factor in the absence of the test compound and in the presence of mutagen and IF0 the induction factor of the untreated cells. 2.10. Evaluation of xanthine oxidase inhibition and superoxide radical scavenging activity Both the inhibition of XOD activity and the superoxide anion scavenging activity were assessed in vitro in three assays. The inhibition of XOD activity was measured according to the increase in absorbance at 290 nm as proposed by Cimanga et al. (1999); while the superoxide anion scavenging activity was detected spectrophotometrically by the nitrite method described by Oyangagui (1984), with some modifications introduced by Russo et al. (2005). Briefly, the assay mixture consisted of 100 ll of the test extract (50, 150 and 300 lg/ml), 200 ll xanthine (X) (final concentration 50 lM) as the substrate, hydroxylamine (final concentration 0.2 mM), 200 ll EDTA (0.1 mM) and 300 ll distilled water. The reaction was initiated by adding 200 ll XOD (11 mU/ml) dissolved in phosphate buffer (KH2PO4 20.8 mM, pH 7.5). The assay mixture was incubated at 37 °C for 30 min. Before measurement of the uric acid production at 290 nm, the reaction was stopped by adding 0.1 ml of HCl (0.5 M). The absorbance was measured spectrophotometrically against a blank solution prepared as described above but replacing XOD with buffer solution. Another control solution, without the tested extract, was prepared in the same manner as the assay mixture to measure the total uric acid production (100%). The latter was calculated from the differential absorbance. To detect the superoxide scavenging activity, 2 ml of the coloring reagent consisting of sulphanilic acid solution (300 lg/ml), N-(1-naphtyl) ethylenediamine dihydrochloride (5 lg/ml) and acetic acid (16.7%, v/v) were added. This mixture was allowed to stand for 30 min at room temperature and the absorbance was measured at 550 nm on a Helios Alpha, Spectronic Unicam (Cambridge, England) spectrophotometer. For both inhibition of XOD and superoxide anion scavenging effect, allopurinol was used as positive control. The dose–effect curve for each tested extract was linearized by regression analysis and used to derive the IC50 values. Values are presented as mean ± standard deviation of three determinations. 2.11. Superoxide radical scavenging activity The inhibition of NBT reduction by photochemically generated O 2 was used to determine the superoxide anion scavenging activity of the extracts by using the methods previously described by Siddhurrajir et al. (2002). Quercetin was used as a positive control. The degree of the scavenging was calculated by the following equation:

Scavenging ð%Þ ¼ ½ODcontrol  ODsample =ODcontrol   100 The reference substance and the samples were assayed at 10, 3, 1, 0.3 and 0.1 mg/ml concentrations with three repetitions. 2.12. Statistical analysis Data were collected and expressed as the mean ± standard deviation of three independent experiments and analyzed for statistical significance from control. The data were tested for statistical differences by one-way ANOVA followed by Duncan’s multiple comparison tests using STATISTICA (Version 6.0, Statsoft Inc.). The criterion for significance was set at p < 0.05. The IC50 values and the correlation coefficients between studies parameters were demonstrated by linear regression analysis.

3. Results and discussion 3.1. Phytochemical study and metabolite content of T. ramosissimum extracts Phenolic compounds are very important plant constituents because they exhibit antioxidant activity by inactivating lipid free radicals or preventing decomposition of hydroperoxides into free radicals (Pokorny, 2001). Flavonoids are phenolic compounds, which are very effective antioxidants (Yanishlieva-Maslarova, 2001). The Folin–Ciocalteu method is a rapid and widely-used assay, to investigate the total phenolic content but it is known that different phenolic compounds have different responses in the Folin–Ciocalteu method (Kahkonen et al., 1999). Therefore, in this work, the total polyphenol content of the extracts was expressed as gallic acid equivalents (Capecka et al., 2005) following confirmation of linearity of the response of the assay using the extract. The total flavonoid content of the T. ramosissimum extracts was determined by the method of Zhishen et al. (1999). Table 1 shows the percent yield of tested extracts. The highest (5.79%) of PE extract and the lowest (0.128%) of TOF extract. The different tested extracts showed the presence of various quantities of tannins, coumarins, sterols and particularly, flavonoids. The total polyphenol, flavonoid, tannin and sterol contents are reported in Table 2. The ethyl acetate is more enriched in flavonoid and total polyphenolic compounds than TOF extract. In fact, the flavonoid and polyphenolic contents in 1 mg of EA extract were, respectively, equivalent to 835 lg of quercetin and 306.66 lg of gallic acid. Yet the flavonoid and polyphenolic contents in 1 mg of TOF extract were, respectively, equivalents to 662.5 lg of quercetin and 315 lg of gallic acid. The percentage of sterol in PE and EA extracts was, respectively, 18.6% and 12%. Moreover, the tannin content in EA extract was equivalent 123.05 lg of tannic acid. 3.2. Genotoxicity assay In a series of experiments preceding the antigenotoxic study, it was ascertained that the different amounts of extracts added to the bacterial tester strain do not influence its viability. The results of

Table 1 Phytochemical screening of extracts from T. ramosissimum aerial parts. Extracts

Yield (%, w/w)

Tannins

Flavonoids

Coumarins

Sterols

PE EA TOF

5.79 2.6 0.128

 ++ +

 ++++ ++++

 ++ ++

++ + 

 = not detectable; + = low quantities; ++ = average quantities; ++++ = high quantities. PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids.

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Table 2 Quantitative polyphenol, flavonoid, tannins and sterol contents of extracts from T. ramosissimum aerial parts. Metabolites

Extracts

Total polyphenols (gallic acid equivalentsa) Flavonoids (quercetin equivalentsa) Tannins (tannic acid equivalentsa) Sterols (%)

PE

EA

TOF

–

306.66 ± 8

315 ± 9

– – 18.6 ± 3

835 ± 11 123.05 ± 7 12 ± 1.5

662.5 ± 13 – –

PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids. a Means of three experiments.

genotoxicity with and without the metabolic activation system are reported in Tables 3 and 4. Experiments realized with the tested extracts revealed no genotoxicity induction insofar as the induction factor is not higher than 1.5 except with EA extract (IF = 1.54) which marginally genotoxic at 250 lg/assay with metabolic activation. In fact, according to Kevekordes et al. (1998) compounds are classified as non-genotoxic if the IF remains inferior to 1.5, as marginally genotoxic if the IF ranges between 1.5 and 2 and as genotoxic if the IF exceeds 2. 3.3. Antigenotoxicity assay Doses of 5 lg/assay of nitrofurantoin (directly acting mutagen) and 0.5 lg/assay of AFB1 (indirectly acting mutagen) were chosen for the antigenotoxicity studies, since these doses were not toxic. They induced a significant response of the SOS system and give the maximum of mutagenicity for both nitrofurantoin and AFB1. The inhibitory effects of the tested extracts on the mutagenicity induced by AFB1 using the SOS chromotest are illustrated by Table 6. Increased concentrations of PE, EA and TOF extracts, decreased AFB1-induced genotoxicity. Indeed, the highest inhibition percentages of genotoxicity obtained with the above mentioned extracts were, respectively, 92.16% (at a concentration of 12.5 lg/assay) for the TOF extract, 91.2% (at a concentration of 500 lg/assay) for the PE extract and 91.06% (at a concentration of 25 lg/assay) for the EA extract. A lower but even highly significant inhibition percentage of AFB1-induced genotoxicity was obtained with the EA extract at a concentration of 25 lg/assay (70.98%). Excepting the TOF extract, which reduced AFB1 genotoxicity in an inverse dose–response manner, the two remaining extracts induced a decrease of the mutagen genotoxicity as a function of extract concentration.

Using nitrofurantoin as mutagen, the induction of SOS response was also affected by the three tested extracts. As shown in Table 5, at a concentration of 50 and 25 lg/assay, the IF of nitrofurantoin decreased by 79.38%, 77.66% and 83.04% in the presence of PE, EA and TOF extracts, respectively. This study showed that, towards nitrofurantoin-induced mutagenicity, TOF extract was the most effective antimutagen while EA extract was the weakest one. We can also notice that all tested T. ramosissimum extracts induced a decrease of nitrofurantoin genotoxicity in an inverse dose-dependent manner, except the TOF extract for which concentrations did not follow the inhibition effect. The SOS chromotest has been recommended for routine use in environmental applications requiring the assessment of genotoxic activity (Helma et al., 1996). This assay was widely used particularly for testing genotoxic and antigenotoxic potentials of extracts from medicinal plants (De Carvalho et al., 2003; Hayder et al., 2004; Shon et al., 2004; Ben Ammar et al., 2005, 2007; Ben Mansour et al., 2007; Abdelwahed et al., 2007; Skandrani et al., 2007). In our study, none of the tested extracts exhibit genotoxic effect. Indeed, in the SOS chromotest, a genotoxic extract must have an IF > 2 (Kevekordes et al., 1998). This suggest that DNA does not seem to be relevant target for T. ramosissimum tested extracts and they did not produce DNA lesions that block DNA synthesis, leading to the SOS system. The absence of genotoxicity is not a characteristic of all natural products in use, since other medicinal plants, assayed with the SOS chromotest, in the presence or not of the S9 preparation, have resulted positive for genotoxicity (De Carvalho et al., 2003). On the other hand, antigenotoxic properties elicited by plant species have a full range of prospective applications in human healthcare. Herbal remedies and phytotherapy drugs containing active principles are currently developed to protect against electrophile (e.g., free radical) attack to DNA and its widespread outcomes such as ageing and cancer. The antigenotoxic study of the T. ramosissimum extracts evaluated by the SOS chromotest towards the indirect mutagen AFB1 and the direct mutagen nitrofurantoin, revealed significant antigenotoxic effects of these extracts, particularly TOF extract. These results indicate that these active extracts may be able to interact and neutralize electrophiles such as nitrofurantoin or may inhibit microsomal activation of AFB1 to electrophilic metabolites. T. ramosissimum extracts may act, as described for others polyphenols such as flavonoids, by inhibiting mutation or initiation caused by inhibition of pro-mutagen activation and trap the electrophiles by chemical reaction or conjuga-

Table 3 Evaluation of genotoxicity of different extracts from T. ramosissimum by the SOS chromotest with Escherichia coli PQ37 strain in the absence of the exogenous metabolic activation system (S9). Extracts

Doses (lg/assay)

b-gal (U)

NC PC (nitrofurantoin)

0 5

5.85 ± 0.0225 56.31 ± 0.036

13.32 ± 0.0075 12.83 ± 0.028

1 9.97*

PE

50 250 500

4.5 ± 0.005 3.77 ± 0.011 2.77 ± 0.009

21.55 ± 0.024 17.15 ± 0.017 9.9 ± 0.011

0.88* 0.92* 1.18*

EA

25 50 250

1.83 ± 0.003 2.67 ± 0.013 1.94 ± 0.025

TOF

12.5 25 50

3.756 ± 0.018 4.263 ± 0.0085 3.236 ± 0.0055

AP (U)

8.06 ± 0.017 7.12 ± 0.0045 5.03 ± 0.023 13.125 ± 0.0085 15.075 ± 0.0005 16.825 ± 0.0005

IF

0.34* 0.58* 0.59* 1.211* 1.194* 0.813*

NC, negative control; PC, positive control; b-gal (U), b-galactosidase units; AP (U), alkaline phosphates units; IF, induction factor; PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids. * Significant difference between the control and treated groups, p < 0.05, n = 3 in each group.

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M. Ben Sghaier et al. / Food and Chemical Toxicology 49 (2011) 191–201 Table 4 Evaluation of genotoxicity of different extracts from T. ramosissimum by the SOS chromotest with Escherichia coli PQ37 strain in the presence of the exogenous metabolic activation system (S9). Extracts

Doses (lg/assay)

b-gal (U)

AP (U)

IF

NC PC (nitrofurantoin)

0 5

4.98 ± 0.012 51.1 ± 0.0235

21.64 ± 0.015 11.76 ± 0.008

1 18.87*

PE

50 250 500

3.6 ± 0.0265 3.16 ± 0.015 3.14 ± 0.0035

19.58 ± 0.002 20.79 ± 0.0255 23.23 ± 0.026

0.79* 0.66* 0.59*

EA

25 50 250

3.13 ± 0.0065 3.58 ± 0.002 4.02 ± 0.0065

10.45 ± 0.005 11.55 ± 0.001 11.85 ± 0.022

1.36* 1.4* 1.54*

TOF

12.5 25 50

3.65 ± 0.011 3.69 ± 0.0035 4.9 ± 0.0885

21.82 ± 0.065 18.7 ± 0.059 18.07 ± 0.0015

0.72* 0.85* 1.17*

NC, negative control; PC, positive control; b-gal (U), b-galactosidase units; AP (U), alkaline phosphates units; IF, induction factor; PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids. * Significant difference between the control and treated groups, p < 0.05, n = 3 in each group.

Table 5 Effect of extracts on genotoxicity induced by aflatoxin B1 (0.5 lg/assay) in the presence of an exogenous metabolic activation system (S9). Extracts

Dose (lg/assay)

b-gal (U)

AP (U)

IF

% of inhibition

NC PC (aflatoxin B1)

0 0.5

2.36 ± 0.09 26.8 ± 0.56

9.6 ± 0.65 8.68 ± 0.14

1 12.35

– –

PE

50 250 500

3.5 ± 0.36 3.81 ± 0.01 2.56 ± 0.07

5.36 ± 0.3 6.07 ± 00 5.1 ± 0.250.03

2.6 2.48 2

85.9 ± 1.24* 86.96 ± 2.48* 91.2 ± 0.35*

EA

25 50 250

6.45 ± 0.08 3.33 ± 0.01 4.36 ± 0.02

5.5 ± 0.04 6.15 ± 0.26 8.18±

3.89 1.93 1.89

70.98 ± 0.233* 90.66 ± 0.07* 91.06 ± 0.46*

TOF

12.5 25 50

2.84 ± 0.01 3.15 ± 0.01 5.79 ± 0.29

5.62 ± 0.08 5.28 ± 0.13 5.75 ± 0.17

1.78 2.14 3.59

92.16 ± 0.35* 88.55 ± 0.28* 73.99±2.77*

NC, negative control; PC, positive control; b-gal (U), b-galactosidase units; AP (U), alkaline phosphates units; IF, induction factor; PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids. * Significant difference between the control and treated groups, p < 0.05, n = 3 in each group.

Table 6 Effect of extracts on genotoxicity induced by nitrofurantoin (5 lg/assay) without an exogenous metabolic activation system (S9). Extracts

Dose (lg/assay)

b-gal (U)

AP (U)

IF

% of inhibition

NC PC (nitrofurantoin)

0 5

4.36 ± 0.14 20.65 ± 0.7

13.06 ± 0.43 11.18 ± 0.79

1 5.6

– –

PE

50 250 500

10.56 ± 0.04 9.98 ± 0.11 9.68 ± 0.45

17.86 ± 1 11.36 ± 0.5 9.21 ± 0.5

1.5 2.2 2.625

79.38 ± 3.72* 50.51 ± 5.04* 32.99 ± 10.96*

EA

25 50 250

8.95 ± 0.24 13.3 ± 0.41 11.47 ± 0.34

13.31 ± 00 16.22 ± 0.13 14.47 ± 0.08

2.03 2.48 2.42

77.6 ± 1.22* 67.82 ± 1.38* 69.13 ± 1.38*

TOF

12.5 25 50

7.64 ± 0.55 6.84 ± 0.04 8.92 ± 0.63

12.22 ± 0.4 11.5 ± 0.21 13.38 ± 0.08

1.89 1.78 2

80.65 ± 0.91* 83.04 ± 0.91* 78.26 ± 3.37*

NC, negative control; PC, positive control; b-gal (U), b-galactosidase units; AP (U), alkaline phosphates units; IF, induction factor; PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids. * Significant difference between the control and treated groups, p < 0.05, n = 3 in each group.

tion; also to exert antioxidant activity or scavenging of reactive oxygen species (De Flora, 1998). In this study, we suspect an eventual correlation between antioxidant and antigenotoxic effects of T. ramosissimum extracts, as suggested for other molecules and/or plant extracts by numerous authors (Avila et al., 2003; Gonzalez et al., 2004). Antioxidant potential expressed by the different extracts may provide a common mechanism for inhibiting the genotoxicity of both AFB1 and nitrofurantoin. However, the inhibi-

tion of mutagenesis is often complex and acts through multiple mechanisms. In the SOS chromotest, extracts from T. ramosissimum inhibited strongly AFB1-induced mutagenicity compared to the results obtained against nitrofurantoin-induced mutagenicity, suggesting that these extracts necessitate metabolic activation. Antigenotoxic activity of the tested extracts may be ascribed to flavonoids (Calomme et al., 1996), tannins (Baratto et al., 2003) and total polyphe-

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nols (Ben Ammar et al., 2008). We cannot, however, exclude the possibility that other compounds with antigenotoxic properties participate in the inhibitory effect of mutagens. 3.4. Xanthine oxidase inhibition and superoxide scavenging activity The superoxide radical ðO 2 Þ is a highly toxic species that is generated by numerous biological and photochemical reactions via the Haber–Weiss reaction, it can generate the hydroxyl radical, which reacts with DNA bases, amino acids, proteins, and polyunsaturated fatty acids, and produces toxic effects. The toxicity of the superoxide radical also could be due to the perhydroxyl intermediate ðHO2 Þ that reacts with polyunsaturated fatty acids. Finally, superoxide may react with nitric oxide to generate peroxynitrite, which is known to be toxic towards DNA, lipids and proteins. The antioxidant activity of T. ramosissimum leaf extracts, was evaluated by the X/XOD enzymatic system. The influence of the T. ramosissimum leaf extracts on XOD activity evaluated by uric acid formation as the final product, and their effect on the superoxide anions ðO 2 Þ enzymatically generated by this system, were evaluated in vitro. The IC50 values of the tested extracts for the inhibition of XOD and as scavengers of superoxide anions ðO 2 Þ are given in Table 7. Both, inhibition of XOD and scavenging of superoxide anions were measured in one assay. Inhibition of XOD involves a decrease in production of uric acid and in superoxide anions which can be followed spectrophotometrically. For each tested extract, two IC50 values (50% inhibitory concentration) were calculated by linear regression analysis: 50% inhibition of XOD activity and 50% reduction of the superoxide level. Fifty percentage inhibition of uric acid production was obtained at IC50 of 29.6 and 30.88 lg/ml with, respectively, EA and TOF extracts. Whereas PE extract did not exceed a 39% inhibition percentage at the highest tested concentration (300 lg/ml). It was concluded that T. ramosissimum extracts were effective inhibitors of XOD. Likewise it appears from the IC50 values of superoxide anions measured in the presence of PE, EA and TOF extracts (respectively, 76.56, 75.88 and 9.62 lg/ml), that TOF extract is the most potent superoxide scavenger and at the same time it was the most enriched in polyphenolic compounds. Whereas petroleum ether extract showed the weakest XOD inhibitory effect and O 2 scavenging activity. The X/XOD assay demonstrated that T. ramosissimum extracts were effective inhibitors of XOD and reduce O 2 generation. Our study showed that TOF and EA extracts are rich in flavonoids. According to the detailed study by Cos et al. (1998) which provides a very important insight to categorize flavonoids into different classes based on their structure and biological activity related to

XOD inhibition and/or O 2 scavenging, flavonoids contained in TOF extracts could be classified into category C which possess both the O 2 scavenging activity as well as XOD inhibitory capacity. While flavonoids contained in EA extracts could be classified into category B which can effectively inhibit XOD activity, but cannot scavenge O 2 radicals. Comparison of the IC50 values of XOD activity and O 2 scavenging capacity, showed that TOF extract have better superoxide scavenging activity than inhibitory effect of XOD. This is in good accordance with previous results described by bouhlel et al. (2008) suggesting that TOF extract from Acacia salicina leaves is better radical scavengers than XOD inhibitor. 3.5. Generation of superoxide anion detected by the non-enzymatic NBT/Riboflavine system The test implements two principal reactions (Liochev and Fridovich, 1995): (a) 2NBTH ? NBT + NBTH2 (Formazan). (b) NBTH þ O2 $ NBT þ O 2 (a quasi-equilibrium). When the riboflavine is photochemically activated, it reacts with the NBT to give NBTH (Beauchamp and Fridovich, 1971) that leads to formazan according to the reaction (a). In presence of oxygen, concentrations of radical species are controlled by the quasiequilibrium (b). Thus, superoxide anions appear indirectly when the test is performed under aerobic conditions.

Fig. 1. Antioxidant activity of T. ramosissimum extracts towards superoxide anion generated by non-enzymatic system NBT/Riboflavine. PE, petroleum ether; EA, ethyl acetate; TOF, total oligomers flavonoids. Symbols represent statistical significance from control (*p < 0.05).

Table 7 Inhibition of xanthine oxidase and superoxide anion by T. ramosissimum extracts at the indicated concentrations.a Extracts

Concentrations (lg/ml)

Inhibition of xanthine oxidase activity (%)

IC50 (lg/ml)a

Inhibition of superoxide anion (%)

IC50 (lg/ml)a

PE

300 150 50

38.95 ± 12.72* 37.35 ± 1.7* 31.25 ± 7.72*

–

64.505 ± 1.44* 60.4 ± 13.51* 44.027 ± 0.96*

76.56

EA

300 150 50

95.02 ± 0.56* 91 ± 1.01* 58.55 ± 6.92*

29.6

77.47 ± 3.86* 53.58 ± 4.82* 46.07 ± 6.27*

75.88

TOF

300 150 50

94.32 ± 1.6* 83.52 ± 12.04* 58.9 ± 2.94*

30.88

94.66 ± 0.96* 92 ± 0.96* 71 ± 6.27*

9.62

PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids. (–) 50% of inhibitory effect was not found with the highest tested concentration of extract. a Values expressed as means ± SD (n = 3). * Significant difference between the control and treated groups, p < 0.05, n = 3 in each group.

M. Ben Sghaier et al. / Food and Chemical Toxicology 49 (2011) 191–201

In the presence of an antioxidant that can donate an electron to NBT, the purple color typical of the formazan decays, a change that can be followed spectrophotometrically at 560 nm. TOF extract was the most potent superoxide scavenger in this assay (Fig. 1). This extract produced a 71.34% decrease of NBT photoreduction at a concentration of 10 mg/ml and an IC50 value of 1.53 mg/ml. Whereas EA extract exhibited a relatively lower scavenging activity with an inhibition percentage of 67.13% at a concentration of 10 mg/ml and an IC50 value of 2.72 mg/ml at than TOF extract. The antioxidant activity of the tested extracts may

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be correlated with their phenolic and flavonoid contents revealed in our study. In fact, polyphenols, particularly flavonoids, which are widely distributed in the plant kingdom and are present in considerable amounts in fruits, vegetables, spices, medicinal herbs and beverages have been used to prevent many human diseases, such as diabetes, cancers and coronary heart diseases (Broadhurst et al., 2000). Moreover, flavonoids have been shown to exhibit antioxidative, antiviral, antimicrobial, antiplatelet and antitoxic activities (Middleton and Kandaswami, 1993). The biological activities of

Fig. 2. Correlation between total polyphenol content (a, b), total flavonoid content (c, d) of ethyl acetate and total oligomer flavonoid extract and capacity to inhibit nitrofurantoin (5 lg/assay) induced mutagenicity in E. coli PQ37 assay system in the absence of a metabolic activation (S9) using the SOS chromotest.

Fig. 3. Correlation between total polyphenol content (a, b), total flavonoid content (c, d) of ethyl acetate and total oligomer flavonoid extracts and capacity to inhibit aflatoxin (0.5 lg/assay) induced mutagenicity in E. coli PQ37 assay system in the presence of metabolic activation (S9) using the SOS chromotest.

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these polyphenols in different systems are believed to be due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygens, or decomposing peroxides (Osawa, 1994). The increase in absorption at 560 nm is observed on reduction of NBT by superoxide anion. PE extract showed a pro-oxidant effect and increased the generation of O 2 at a concentration of 10 mg/ml. The pro-oxidant action of plant polyphenols could contribute to cancer chemotherapy as it could cause growth arrest and induce apoptosis in cancerous cells (Galati et al., 2000). 3.6. Correlation of the different activities with the total polyphenol and flavonoid content It is interesting to observe the correlation between the phenolic content and antioxidant activity of plant extracts, since phenolic compounds contribute directly to antioxidant activity (Duh,

1999). In this study, there was a distinct correlation between studied parameters (total polyphenolic content, total flavonoid content, antigenotoxic and antiradical activities) in selected T. ramosissimum plant parts. This correlation was demonstrated by linear regression analysis. With reference to Figs. 2–5, the correlation of the total polyphenol and flavonoid content against the different activities were satisfactory (r > 0.8). Antigenotoxic activity of the tested extracts may be ascribed to flavonoids (r = 0.995, 0.925, 0.99 and 0.989) (Calomme et al., 1996), and total polyphenols (r = 0.936, 0.925, 0.92 and 0.99) (Ben Ammar et al., 2008). Some authors have reported similar correlations between polyphenols and antioxidant activity measured by various methods (Awika et al., 2003; Zheng and Wang, 2001). When the relationship between total phenolic content and total flavonoid content of all extracts was plotted as shown in Fig. 5e and f, the correlation coefficient (r) between these two parameters was about 1 indicating that there is a significant

Fig. 4. Correlation between total polyphenol content and their inhibition of xanthine oxidase (a, b) and inhibition of superoxide anion (c, d), total flavonoid content and their inhibition of xanthine oxidase (e, f) and inhibition of superoxide anion (g, h) of ethyl acetate and total oligomer flavonoid extracts.

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Fig. 5. Correlation between total polyphenol content (a, b), total flavonoid content (c, d) and their superoxide radical scavenging and total polyphenol content and total flavonoid content (e, f) of ethyl acetate and total oligomer flavonoid extracts.

positive relationship between the total phenolic and flavonoid contents of all plant extracts selected in this study. The Folin–Ciocalteu method for the determination of polyphenolic compounds is, such as the methods of antioxidant activity determination, based on redox properties of the compounds. Thus, the values could partially express the antioxidant activity. This is confirmed by the highly significant correlation between the values of Folin–Ciocalteu method and the values of individual methods for antioxidant activity. Our results prove that the content of phenolic compounds and antioxidant activity correlate very well (r = 0.984 for EA extract and r = 0.943 for TOF extract) for the tested T. ramosissimum extracts.

3.7. Correlation of the different activities In a comparison of methods used in this study, all the methods showed the capability to determine the antioxidant and antigenotoxic activities of T. ramosissimum extracts. Nevertheless, the three methods are capable of prescreening antioxidant and antigenotoxic activities. As shown in Table 8, a direct correlation between the three methods was demonstrated by linear regression analysis. The strong correlation (r = 1) between the mean values of NBT (%) reduction and inhibition of superoxide anion (X/XO assay) deserves detailed attention. This could be explained from the basic concept that antioxidants are reducing agents. The results suggest

Table 8 Correlation coefficients, ‘‘r”, for relationships between different assays. Extracts

NBT (%) reduction

Inhibition of xanthine oxidase

Inhibition of superoxide anion

Antigenotoxic activity (without S9)

Antigenotoxic activity (with S9)

EA NBT (%) reduction Inhibition of xanthine oxidase Inhibition of superoxide anion

– – –

0.73 – –

0.97 0.6 –

0.99 0.88 0.97

0.99 0.90 0.96

TOF NBT (%) reduction Inhibition of xanthine oxidase Inhibition of superoxide anion

– – –

0.76 – –

1 0.96 –

0.97 0.96 0.88

0.96 0.97 0.90

PE NBT (%) reduction Inhibition of xanthine oxidase Inhibition of superoxide anion

– – –

– – –

– 0.99 –

– 0.98 0.97

– 0.79 0.77

PE extract, petroleum ether extract; EA extract, ethyl acetate extract; TOF extract, total oligomers flavonoids.

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that the reducing ability of polyphenols seems to be an important factor dictating free radical-scavenging capacity of these compounds. In this study, we suspect an eventual correlation (r > 0.77) between antioxidant and antigenotoxic effects of T. ramosissimum extracts. Antioxidant potential expressed by the different extracts may provide a common mechanism for inhibiting the genotoxicity of both AFB1 and nitrofurantoin. However, the inhibition of mutagenesis is often complex and acts through multiple mechanisms. 4. Conclusion The present study has demonstrated that some T. ramosissimum extracts possess potent antioxidant and antigenotoxic activities, which could be derived from compounds such as flavonoids and polyphenols. The antigenotoxic activity could be ascribed, at least in part, to their antioxidant properties but we cannot exclude other additionally mechanisms. These antioxidant and antigenotoxic activities could have contributed, at least partly, to the therapeutic benefits of certain traditional claims. The results presented here could be an additional argument to support the use of this species in the North African tradition medicine. Furthermore, T. ramosissimum extracts could give rise to antimicrobial, anti-inflammatory and antiulcer agents and could be promising candidates for further studies designed to obtain more evidence on their components with potential chemo-preventive activity. Conflict of Interest The authors declare that there are no conflicts of interest. References Abdelwahed, A., Bouhlel, I., Skandrani, I., Valenti, K., Kadri, M., Guiraud, P., Regine Steiman, K., Mariotte, A.M., Ghedira, K., Laporte, L., Dijoux-Franca, M.G., ChekirGhedira, L., 2007. Study of antimutagenic and antioxidant activities of gallic acid and 1,2,3,4,6-pentagalloylglucose from Pistacia lentiscus. Confirmation by microarray expression profiling. Chem. Biol. Interact. 165, 1–13. Avila, M.G., Alba, M.A., de la Garza, Pretelin, M.M.C.H., Ortiz, M.A.D., Fazenda, S.F., Treveino, S.V., 2003. Antigenotoxic, antimutagenic and ROS scavenging activities of a Reo discolor ethanolic crude extract. Toxicol. In Vitro 17, 77–83. Awika, J.M., Rooney, L.W., Wu, X., Prior, R.L., Cisneros-Zevallos, L., 2003. Screening methods to measure antioxidant activity of Sorghum (Sorghum bicolor) and Sorghum products. J. Agric. Food Chem. 51 (23), 6657–6662. Baratto, M.C., Tattini, M., Galardi, C., Pinelli, P., Romani, A., Visioli, F., Basosi, R., Pongi, R., 2003. Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves. Free Radic. Res. 37, 405–412. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Ben Ammar, R., Kilani, S., Abdelwahed, A., Hayder, N., Mahmoud, A., Ben Chibani, J., Chekir-Ghedira, L., Ghedira, K., 2005. In vitro mutagenicity, antimutagenicity, and free radical scavenging activity of Rhamnus alaternus (Rhamnaceae) extracts. Pakistan J. Biol. Sci. 8 (3), 439–445. Ben Ammar, R., Bouhlel, I., Valenti, K., Ben Sghaier, M., Kilani, S., Mariotte, A.M., Dijoux-Franca, M.G., Laporte, F., Ghedira, K., Chekir-Ghedira, L., 2007. Transcriptional response of genes involved in cell defense system in human cells stressed by H2O2 and pre-treated with (Tunisian) Rhamnus alaternus extracts: combination with polyphenolic compounds and classic in vitro assays. Chem. Biol. Interact. 168, 171–183. Ben Ammar, R., Kilani, S., Bouhlel, I., Ezzi, L., Skandrani, I., Boubaker, J., Ben Sghaier, M., Naffeti, A., Mahmoud, A., Chekir-Ghedira, L., Ghedira, K., 2008. Antiproliferative, antioxidant and antimutagenic activities of flavonoidenriched extracts from (Tunisian) Rhamnus alaternus L. Combination with the phytochemical composition. Drug Chem. Toxicol. 31, 63–82. Ben Mansour, B., Boubaker, J., Bouhlel, I., Mahmoud, A., Bernillon, S., BenChibani, J., Ghedira-Chekir, L., 2007. Antigenotoxic activities of crude extracts from Acacia salicina leaves. Environ. Mol. Mutagen. 48, 58–66. Bouhlel, I., Ben Mansour, H., Limem, I., Ben Sghaier, M., Mahmoud, A., Ben Chibani, J., Ghedira-Chekir, L., 2006. Screening of antimutagenicity via antioxidant activity in different extracts from the leaves of Acacia salicina from the center of Tunisia. Environ. Toxicol. Pharmacol. 22, 56–63. Bouhlel, I., Valenti, V., Kilani, S., Skandrani, I., Ben Sghaier, M., Mariotte, A.M., Dijoux-Franca, M.G., Ghedira, K., Hininger-Favier, I., Laporte, L., Chekir-Ghedira,

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