Evaluation of antioxidant and antigenotoxic activity of two flavonoids from Rhamnus alaternus L. (Rhamnaceae): Kaempferol 3-O-β-isorhamninoside and rhamnocitrin 3-O-β-isorhamninoside

Food and Chemical Toxicology 49 (2011) 1167–1173

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Evaluation of antioxidant and antigenotoxic activity of two flavonoids from Rhamnus alaternus L. (Rhamnaceae): Kaempferol 3-O-b-isorhamninoside and rhamnocitrin 3-O-b-isorhamninoside Wissem Bhouri a,b, Mohamed Ben Sghaier a,b, Soumaya Kilani a,b, Ines Bouhlel a,b, Marie-Geneviève Dijoux-Franca c, Kamel Ghedira b, Leila Chekir Ghedira a,b,⇑ a

Laboratoire de Biologie Cellulaire et Moléculaire, Faculté de Medecine Dentaire Monastir, Rue Avicenne, Monastir 5000, Tunisia Unité de pharmacognosie, 99/UR/07, Faculté de Pharmacie, Monastir Rue, Avicenne 5000, Tunisia c Laboratoire de Botanique, Pharmacognosie et Phytothérapie, UMR CNRS 5557 Ecologie Microbienne, Faculté de Pharmacie Université Claude Bernard, Bâtiment Nétien-8 Avenue Rockefeller 69373, Lyon cedex 08, France b

a r t i c l e

i n f o

Article history: Received 28 October 2010 Accepted 15 February 2011 Available online 19 February 2011 Keywords: K3O-ir R3O-ir Genotoxicity Antigenotoxicity Antioxidant capacity

a b s t r a c t The antioxidant activity of kaempferol 3-O-b-isorhamninoside (K3O-ir) and rhamnocitrin 3-O-b-isorhamninoside (R3O-ir), isolated from the leaves of Rhamnus alaternus L., was determined by the ability of each compound to inhibit NBT photoreduction and to scavenge the free radical ABTS+. Genotoxic and antigenotoxic activities were assessed using the SOS chromotest. At a concentration of 150 lg/assay the two compounds showed the most potent inhibitory activity against superoxide anion by respectively 80.4% and 85.6%. K3O-ir was a very potent radical scavenger with an IC50 value of 18.75 lg/ml. Moreover, these two compounds exhibit an inhibitory activity against genotoxicity induced by nitrofurantoine and aflatoxine B1 using the SOS chromotest bacterial assay system in the presence of Escherichia coli PQ37 strain. In this study, we have also evaluated correlation between antigenotoxic and antioxidant effects of K3O-ir and R3O-ir. The highest correlation was showed with R3O-ir (r = 0.999). Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Medicinal and spice plants, which are well known for their pharmacological activity, contain many substances that exhibit radicalscavenging properties. Among these substances, polyphenolic compounds are abundant in foods of plant origin. The application of such bioactive plant components may increase the stability of foods and, at the same time, improve their health properties associated with anti-cancer, antiallergic and anti-inflammatory activities of polyphenols in the human body (Moure et al., 2001; Rice-Evans et al., 1996). Polyphenols are believed to possess the ideal chemical structure for scavenging free radicals. It has been demonstrated in vitro assays that polyphenols are more active than vitamins E and C, commonaly used antioxidants (Rice-Evans et al., 1997). Probably the most important natural polyphenols are flavonoids because of their broad spectrum of chemical and biological activities, including antioxidant and free radical scavenging properties (Kähkönen et al., 1999). ⇑ Corresponding author at: Laboratoire de Biologie Cellulaire et Moléculaire, Faculté de Medecine Dentaire Monastir, Rue Avicenne, Monastir 5000, Tunisia. E-mail address: [email protected] (L.C. Ghedira). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.02.011

The genus Rhamnus (Rhamnaceae), which is encountered both in temperate and in tropical countries, includes well-known medicinal species possessing various biological properties (Mai et al., 2001). Generally, Rhamnus species contain anthraquinones such as emodin (Wei et al., 1992) or chrysophanol (Alemayu et al., 1993; Abegaz and Peter, 1995) , their reduced forms or their glycosides (Abegaz and Peter, 1995), while some others contain flavonoids (Coskun et al., 1990; Lin and Wei, 1994; Marzouk et al., 1999). Rhamnus alaternus (Rhamnaceae) is a small tree located principally in the north of Tunisia, where it is known as ‘‘Oud El-khir’’. Traditionally, it has been used as a digestive, diuretic, laxative, hypotensive and for the treatment of hepatic and dermatological complications (Boukef, 2001). Previous studies have shown potent antioxidant, free radical scavenging, antimutagenic and antigenotoxic activities of crude extracts from R. alaternus (Ben Ammar et al., 2007a,b; Ben Ammar et al., 2008a,b; Chevolleau et al., 1992; Ben Ammar et al., 2005). In our studies on the elucidation of the antioxidant, genotoxic and antigenotoxic effects of flavonoids, two triglycoside flavonoids isolated from the leaves of R. alaternus i.e. the K3O-ir and R3O-ir were tested.

1168

W. Bhouri et al. / Food and Chemical Toxicology 49 (2011) 1167–1173

2. Materials and methods 2.1. Chemicals Ampicillin; 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); the mutagens aflatoxin B1(AFB1) and nitrofurantoine (NF). Potassium persulfate (K2S2O8), and 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid) (Trolox) from Aldrich (Steinheim, Germany). 3-Nitrotetrazolium blue chloride (NBT), Riboflavine, superoxide dismutase (SOD) and Dimethyl sulfoxide (DMSO) was obtained from Fluka (Steinheim, Germany). S9 Fraction was purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA). 2.2. Extraction method Dried and powdered leaves (100 g) of R. alaternus were first defatted with petroleum ether (1 L), then extracted with chloroform (1 L), ethyl acetate (1 L), and methanol (1 L) using a Soxhlet apparatus (6 h). Four different extracts were obtained. They were concentrated to dryness and kept at 4 °C in the absence of light. Among these extracts, only the Soxhlet methanolic extract was fractioned and purified in this study. Additionally, in order to obtain a total oligomer flavonoid (TOF) enriched extract, the powdered leaves were macerated in water:acetone mixture (1:2) for 24 h, under continuous stirring. The extract was filtered and the acetone was evaporated under low pressure, to obtain an aqueous phase. The phlobaphenes were removed by precipitation with an excess of NaCl at 5 °C for 24 h. The supernatant was extracted with ethyl acetate, concentrated and precipitated in an excess of chloroform. The precipitate was then separated and TOF extract yielded. 2.3. Fractionation and isolation methods K3O-ir was directly obtained by fractionation of the TOF extract on a silica gel column with EtOAc:MeOH:H2O (100:15:13) solvent system as eluent. The methanolic extract (6 g) was fractionated by vacuum liquid chromatography (VLC) on a silica gel column eluted with CH2Cl2:MeOH with a gradual increase in of the MeOH content and eight fractions were collected. Fractions 5, 6 and 7 were rechromatographed over a silica gel column using an EtOAc:MeOH:H2O (100:15:13) solvent system, to give seven subfractions. The subfraction 5 was rechromatographed on a C18 gel column using an H2O:MeOH (70:30–0:100) gradient solvent system to afford R3O-ir. K3O-ir and R3O-ir were identified by analysis of the negative fast atom bombardment mass spectra (FAB-MS), 1H NMR (Nuclear magnetic resonance) spectroscopy and 13C NMR spectroscopy Fig. 1 (Ben Ammar et al., 2009). 2.4. Bacterial tester strain Escherichia coli PQ 37 strain was kindly provided by Prof. M. Quillardet (Institut Pasteur, Paris, France). The complete genotype, as well as strain construction details can be found in Quillardet and Hofnung (1985). Frozen permanent copies of the tester strain were prepared and stored at 80 °C. 2.5. SOS chromotest The SOS chromotest was employed to determine the effect of K3O-ir and R3O-ir on the genotoxicity of B[a]P: indirect acting mutagen, and nitrofurantoine (direct acting mutagen) induced genotoxicity. The SOS chromotest with Escherichia coli PQ37 strain was performed according to the procedure described by Quillardet and Hofnung (1985). Three doses of each compound (1, 5, and 10 lg/assay) were

6' R1 7 6

A 5

9 10

C 4

'5

B 4''

3

O

8

OH

2'

2

H

3 O-Gal(6-1) Rha (3-1) Rha

OH

O

R1 kaempferol 3-O-isorhamninoside rhamnocitrin-3-O-isorhamninoside

OH OCH3

Fig. 1. Chemical structures of compounds.

prepared and tested in triplicate with and without an exogenous metabolic activation system (S9). Positive controls were prepared by exposure of the bacteria to either AFB1 or NF. After 2 h of incubation at 37 °C, with shaking, two sets of 300 ll samples were used for assaying b-galactosidase (b-gal) and alkaline phosphatase (AP) activities. In this assay, the b-galactosidase synthesis (lacZ gene) is dependent on sfiA activation and is used to measure the induction of SOS repair system. The activity of the constitutive enzyme alkaline phosphatase was used as a measure of protein synthesis and toxicity. Enzyme activities were assessed spectrophotometrically. The SOS induction factor (IF) in treated cells was obtained by comparing b-galactosidase/alkaline phosphatase ratio in treated and untreated cells (Kevekordes et al., 1999). For the evaluation of the protective effect of K3O-ir and R3O-ir on the induction of the SOS response by NF (in the absence of the S9 activation preparation) and AFB1 (in the presence of the S9 activation preparation), 10 ll of NF solution (10 lg/assay) or AFB1 solution (5 lg/assay) were added into tubes with 10 ll of the compound tested concentrations. Antigenotoxicity was expressed as percentage inhibition of genotoxicity induced by either NF or AFB1 according to the formula:

ð%ÞInhibition ¼ 100  ðIF1  IF0 Þ=ðIF2  IF0 Þ  100 where IF1 is the induction factor in the presence of the tested compound and the genotoxin, IF2 the induction factor in the absence of the tested compound and in the presence of the genotoxin, and IF0 is the induction factor of the negative control. Data were collected as a mean ± S.D. of three experiments.

2.6. Radical-scavenging activity An improved ABTS [2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt] radical cation decolorization assay was used. It involves the direct production of the blue/green ABTS+ chromophore through the reaction between ABTS and potassium persulfate. The addition of antioxidants to the performed radical cation reduces it to ABTS, to an extent and on a timescale, depending on the antioxidant activity, the concentration of the antioxidant, and the duration of the reaction (Re et al., 1999). ABTS was dissolved in water to a final concentration of 7 mM. ABTS+ was produced by the reaction of ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The ABTS+ solution was diluted with ethanol to an absorbance of 0.7(±0.02) at 734 nm. In order to measure the antioxidant activity of tested compounds, 10 ll of samples at various concentrations (0.05, 0.1, 0.15, 0.2 mg/ml) were added to 990 ll of diluted ABTS+ and the absorbance recorded every minute. The kinetic reaction stopped when the absorbance at 734 nm was stable. Each concentration was analyzed in triplicate. The percentage decrease in absorbance at 734 nm was calculated for each point; the antioxidant capacity of the test compound was expressed in percent inhibition (%), and IC50 value was calculated from regression analysis. The antioxidant activity of the tested compounds was compared to that of a standard powerful antioxidant the Trolox. The results are also reported as the Trolox equivalent antioxidant capacity (TEAC), which is the molar concentration of the Trolox giving the same percentage decrease in absorbance of the ABTS+ radical as 0.2 mg/mL of each of the tested compounds, at a specific time point (Van den Berg et al., 2000).

2.7. Superoxide mediated reduction of nitro blue tetrazolium by photochemically reduced riboflavin The test implements two principal reactions (Liochev and Fridovich, 1995): (a) 2 NBTHNBT + NBTH2 (Formazan). (b) NBTH + O2 NBT + O2 (a quasi-equilibrium). When the riboflavin is photochemically activated, it reacts with the NBT to give NBTH (Beauchamp and Fridovich, 1971) that leads to formazan according to reaction (a). In the presence of oxygen, concentrations of radical species are controlled by the quasi equilibrium (b). Thus, superoxide anions appear indirectly when the test is performed under aerobic conditions. 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. The protocol is described as follow: Reduction of NBT was carried out at room temperature (22 °C) under fluorescent lighting (20 watt, 20 cm). The standard incubation mixture (3.5 ml) contained 6 lM riboflavin, in 16 mM phosphate buffer (pH 7.8) and 85 lM NBT. Tested compounds were dissolved in 0.2% of DMSO. After 5 min incubation, the reaction was stopped by switching off the light and the addition of 0.05 ml SOD (1 mg/ml). For each compound concentration, control sample containing 0.05 ml SOD solution, which was added before exposure to fluorescent lighting, was analyzed to rule out the possible direct reduction of NBT, by tested compounds and other reducing agents. For estimation of the superoxide-driven reduction of NBT the absorbance of a control sample was subtracted from that of standard reaction mixture.

1169

W. Bhouri et al. / Food and Chemical Toxicology 49 (2011) 1167–1173 Table 1 Genotoxicity induced by R3O-ir and K3O-ir in the presence and absence of the exogenous metabolic activation mixture (S9). Dose (lg/assay)

PC NC R3O-ir K3O-ir

5 0 10 5 1 10 5 1

+S9

-S9

b-gal (U)

Ap (U)

IF

b-gal (U)

Ap (U)

IF

33 ± 0.006 1.72 ± 0.003 1.05 ± 0.002 1.58 ± 0.001 1.12 ± 0.002 1.63 ± 0.001 1.61 ± 0.002 1.22 ± 0.003

0.47 ± 0.003 0.5 ± 0.002 0.63 ± 0.002 0.89 ± 0.005 0.46 ± 0.01 0.58 ± 0.02 0.58 ± 0.01 0.42 ± 0.3

20.4 1.00 0.48* 0.51* 0.70* 0.81* 0.80* 0.84*

32.26 ± 0.028 2.7 ± 0 1.95 ± 0.008 2.46 ± 0.002 2.17 ± 0.003 2.7 ± 0.025 2.75 ± 0.000 2.21 ± 0.053

8.28 ± 0.009 11.73 ± 0.013 10.38 ± 0.03 10.33 ± 0.004 9.02.±0.002 11.8 ± 0.048 15.63 ± 0.027 15.23 ± 0.003

16.91 1.00 0.47* 0.60* 0.63* 1.77* 1.33* 1.12*

NC, negative control; PC, positive control Aflatoxine B1(AFB1) in the presence of S9 and nitrofurantoine (NF) in the absence of S9; R3Oir, rhamnocitrin-3-O-isorhamninoside and K3O-ir, kaempferol 3-O-isorhamninoside: tested compound; U, enzymatic unit; b-gal, b-galactosidase); AP, Alkaline phosphatase; IF, induction factor. Values are means ± SD (n = 3). * significant from control (p < 0.05).

2.8. Statistical analysis Data were collected and expressed as mean, standard deviation of three independent experiments, and analyzed for statistical significance using the ANOVA test (SPSS 10.0 for windows). The criterion for significance was set at p < 0.05.

3. Results 3.1. Genotoxic assay In a series of experiments preceding the antigenotoxicity studies, it was ascertained that the different amount of the compounds added to the bacterial indicator does not influence its viability. The results of genotoxicity with and without the metabolic activation system are reported in Table 1. Experiments realized with R3O-ir and K3O-ir, revealed no genotoxicity induction in so far as the induction factor is not higher than 1.5. However, at a dose of 10 lg/assay, K3O-ir showed an IF of 1.77 in the absence of S9 and 0.81 in the presence of S9. According to Kevekordes et al.(1999), the tested compounds are considered as non genotoxic. 3.2. Antigenotoxic assay Doses of 10 lg/assay of NF and 5 lg/assay of AFB1 were chosen for the antigenotoxicity study, since these doses were not toxic and

induced a significant SOS response. As shown in Fig. 2 (a) and (b) tested molecules were effective in reducing the IF induced by the AFB1 indirectly acting genotoxic (5 lg per assay with microsomal activation and an IF of 43), as well as by the directly acting genotoxic, NF (10 lg per assay without microsomal activation and an IF of 21). R3O-ir was a more effective antigenotoxic than K3O-ir. In fact, at the concentrations of 1, 5 and 10 lg/assay R3O-ir significantly decreases the IF of AFB1 by 92.81%, 95.21% and 96.64% respectively. At the same concentrations, this compound decreases the IF of NF by 31%, 51% and 60.52% respectively. K3Oir showed a weaker antigenotoxic activity than R3O-ir. In fact, 1, 5 and 10 lg/assay decreases the IF of AFB1 by 90.26%, 84.43% and 88.86% respectively, and the IF of NF by 11%, 20 % and 32 % respectively.

3.3. Radical-scavenging activities The free radical scavenging capacity of both R3O-ir and K3O-ir was evaluated by means of the ABTS assay (Fig 3 a, b). For each concentration (0.05, 0.1, 0.15 and 0.2 mg/ml) we calculated the scavenging percentage of ABTS free radical. The first 5 min corresponds to the ‘‘fast’’ scavenging activity, while the final point of stabilization time (20 min) is known as the ‘‘total’’ scavenging activity. The magnitude of scavenging also

Fig. 2. (a) Antigenotoxic effect of rhamnocitrin-3-O-isorhamninoside (R3O-ir) and kaempferol 3-O-isorhamninoside (K3O-ir) on genotoxicity induced by Aflatoxine B1 (5 lg/ assay) in E. coli PQ37 using the SOS chromotest assay. (b) Antigenotoxicic effect of rhamnocitrin-3-O-isorhamninoside (R3O-ir) and kaempferol 3-O-isorhamninoside (K3O-ir) on genotoxicity induced by Nitrofurantoine (10 lg/assay) in E. coli PQ37 using the SOS chromotest assay. Results are represented by the means ± SD of n = 3. ⁄Significant from control (p < 0.05).

1170

W. Bhouri et al. / Food and Chemical Toxicology 49 (2011) 1167–1173

Fig. 3. (a) Kaempferol 3-O-isorhamninoside concentrations and time-dependent ABTS free radical scavenging activity. (b) Rhamnocitrin-3-O-isorhamninoside concentrations and time-dependent ABTS free radical scavenging activity.

3.4. Superoxide radical scavenging activity Table 2 Antioxidant activity of kaempferol 3-O-isorhamninoside (K3O-ir) and rhamnocitrin3-O-isorhamninoside (R3O-ir) towards superoxide anion generated by the nonenzymatic system NBT/Riboflavine.

K3O-ir

R3O-ir

a

Doses (lg/assay)

Inhibition of superoxide anion (%)a

Superoxide anion scavenging activity IC50 (lg/ml)

50 100 150 50 100 150

70,49 ± 0.008 77,80 ± 0.02 80,4 ± 0.02 60,5 ± 0.002 73,9 ± 0.04 85,6 ± 0.009

18.75

The assay was based on the capacity of tested flavonoids to inhibit the formation of formazan in comparison to the NBT/riboflavin reference signal. The decrease of purple color, typical to formazan, was followed spectrophotometrically at 560 nm. As shown in Table 2 K3O-ir and R3O-ir produced an 80.4% and 85.6% decrease in NBT photo reduction, respectively, at a dose of 150 lg/assay. However K3O-ir was more potent superoxide scavenger with an IC50 value of 18.75 lg/ml than R3O-ir (IC50 = 22.5 lg/ml).

22.5

3.5. Correlation analysis

Values are means ± SD (n = 3).

depends on the concentration assayed. Indeed, the percentages of scavenging at stabilization time of K3O-ir (97%) and of R3O-ir (72%) were higher than those obtained at 5 min incubation, at a sample concentration of 0.2 mg/mL. However when the tested compounds concentration is only 0.05 mg/mL the percentage of scavenging at stabilization time drops to approximately 34%. Fifty percent inhibition of ABTS radical formation was estimated by regression analysis to be approximately 0.12 mg/ml for K3O-ir and 0.15 mg/ml for R3O-ir.The TEAC of tested compounds was also calculated when referring to TEAC values, K3O-ir seems to be the more potent antioxidant with TEAC value of 3 mM than R3O-ir (TEAC = 1.75 mM).

It is informative to examine the relation between the anti-genotoxic and antioxidant capacities measured by the different tested methods. Tested compounds with superoxide anion scavenging activity were effective in inhibiting antigenotoxic activity. Indeed a high correlation between the antigenotoxic effect and superoxide radical scavenging activity of R3O-ir and K3O-ir was detected with a coefficient correlation values of 0.988 (Fig. 4a) and 0.800 respectively. However, in contrast to the superoxide anion scavenging activity, the linear correlation was rather low (r = 0.601) when comparing the antigenotoxic activity and ABTS+ scavenging capacity of K3O-ir (Fig 4b). In contrast, the antigenotoxic activity of R3Oir was well correlated with its ABTS+ scavenging capacity (r = 0.999) (Fig 5).

Fig. 4. (a) Correlation between antigenotoxic activity percentage of rhamnocitrin-3-O-isorhamninoside (R3O-ir) and its superoxide anion activity. (b): Correlation between antigenotoxic activity percentage of kaempferol 3-O-isorhamninoside (K3O-ir) and its ABTS+ scavenging capacity.

W. Bhouri et al. / Food and Chemical Toxicology 49 (2011) 1167–1173

1171

Fig. 5. Correlation between antigenotoxic activity percentage of rhamnocitrin-3-O-isorhamninoside (R3O-ir) and its ABTS+ scavenging capacity.

4. Discussion It is well-known that the polyphenolic components of higher plants may act as antioxidants or as agents of other mechanisms contributing to anticarcinogenic or cardioprotective action. The flavonoids constitute a large class of compounds, ubiquitous in plants, containing a number of phenolic hydroxyl groups attached to ring structures, conferring the antioxidant activity (Block,1992; Block and Langseth,1994). In this area R. alaternus L. leaves are characterized by the presence of flavonols. The present study has demonstrated that the K3O-ir and R3Oir, two compounds isolated from leaves of R. alaternus L. exhibited radical scavenging and antioxidant activities. In fact, ABTS assay was performed as a preliminary study to estimate the direct free radical scavenging ability of flavonoids (Cao et al., 1999). On the whole, our data showed that R. alaternus L. leaf derivatives were more effective scavengers, than the known antioxidant Trolox, against ABTS radical by the capacity of antioxidant species to donate electrons or hydrogen atoms to inactivate this radical cation. In the ABTS decolonization assay, potential activity was noted at 0.15 and 0.2 mg/mL for tested compounds. This can be explained by the fact that the two tested compounds are flavonoids rich in hydroxyl groups; however flavonoids are known to stabilize reactive oxygen species by chelating radicals. Because of the high reactivity of the hydroxyl group of the flavonoids, radicals are made inactive (Nijveldt et al., 2001). On the other hand, antioxidant capacity, of K3O-ir and R3O-ir were also evaluated by their abilities to scavenge O2 with NBT/riboflavin assay system. Likewise, results indicated that K3O-ir (CI50 = 18.75 lg/ml) was more effective O2 scavenger than R3O-ir (CI50 = 22.5 lg/ml). When comparing antioxidant activities of our tested compounds to their corresponding aglycones, reported by Edenharder and Grunhage (2003), Rice-Evans et al. (1996), we confirmed their observation, according to which, glycosylation of flavonols may improve their antioxidant capacity. In fact, kaempferol structure with its 2,3-double bond in conjugation with 4-oxo function, responsible for electron delocalization from the B ring, and its 3,5 and 7-OH functions together with the 4-oxo group, meets the criteria of potent radical scavenger as reported by Edenharder and Grunhage (2003) The same authors reported that methylation of 7-hydroxy function, reduced scavenging abilities of flavonols and we believe also for rhamnocitrin which corresponds to 7-OH methylated kaempferol structure. These authors reported that addition of sugar moiety may improve the antioxidant activity of the aglycone. This is in accordance with our results, in fact we revealed a radical scavenging potency of K3O-ir with a TEAC value of 3 mM, which

is higher than that reported by Rice-Evans et al. (1996) for the aglycone part (kaempferol; TEAC value = 1.34 mM.) This observation joins the results reported by Hayder et al. (2008) stipulating that glycosylated myricetin improves antioxidant capacity of the corresponding aglycone. Concerning R3O-ir we revealed a significant (TEAC = 1.75 mM) but lower radical scavenging capacity than K3O-ir, (TEAC = 3 mM). Methylation of the 7-OH function of K3O-ir, clearly reduced scavenging ability against ABTS+, this is substantiated by the studies of Edenharder and Grunhage (2003) and Ben Ammar et al. (2009) who reported that methylation of the 7-OH function of flavonols reduced their scavenging ability. Moreover, R3O-ir showing the lowest antioxidant potency, only satisfies the requirement of 5-hydroxyl substitution and the 2,3-double bond in conjugation with a 4-oxo function (flavonol structure). In order to complete this study, genotoxic and antigenotoxic activities of the tested compounds were evaluated using the SOS chromotest. The results obtained in this bacterial test, showed the absence of genotoxic effect of both K3O-ir and R3O-ir. The SOS chromotest demonstrates that K3O-ir and R3O-ir reduced strongly both AFB1 and NF mutagenicity. This is in accordance with antimutagenic activity of flavonoids reported previously (Yagi et al., 2002; Kilani et al., 2005; Calomme et al., 1996; Edenharder and Grunhage, 2003). Indeed kaempferol was investigated by Edenharder and Grunhage (2003) for its mutagenic/antimutagenic activities. This component exhibited no mutagenicity but a moderate antimutagenic effect, when using the Ames assay. In our study we revealed no genotoxicity but a weak to moderate antigenotoxicity of the corresponding glycoside, in the absence of a metabolizing extract. This is in accordance with the observations of Edenharder and Grunhage (2003) who reported that glycosylation at C-3 has no major influence on glycosylated flavonoid antimutagenic activity. However, we observed a high antigenotoxic effect of the same glycosylated flavonol, in the presence of a metabolizing extract. We can ascribe the high antigenotoxicity of metabolized flavonol glycoside, to different modifications induced by metabolizing enzymes on the structure of the tested compounds. Nonetheless, our results are contrary to those of Edenharder and Grunhage (2003) who reported that the methylation of flavonol 7-OH function reduced their antimutagenic potency. In fact, we found that as well in the presence as in the absence of metabolizing enzymes, R3O-ir exhibited a more potent antimutagenic activity than K3O-ir. Regarding to the antigenotoxic and antioxidant activities of R3O-ir, a strong correlation was reflected by the correlation value obtained from the comparison of the antigenotoxic activity of this compound to its O2 scavenging capacity (r = 0.988) as well as to

1172

W. Bhouri et al. / Food and Chemical Toxicology 49 (2011) 1167–1173

its ABTS+ inhibiting effect (r = 0.999), while K3O-ir antigenotoxicity is weakly correlated to its ABTS+ inhibiting effect (r = 0.601) as well as to its O 2 scavenging capacity (r = 0,8). Although K3O-ir shows higher protective effect against radical attack than R3O-ir, it shows lower antigenotoxicity level compared to R3O-ir. We deduce that antigenotoxic activity can be ascribed not only to antioxidant effect of these molecules but to other additionally mechanisms as DNA repair enzyme induction, as evidenced by Hayder et al. (2008) who reported that, more than antioxidant enzymes, DNA repair enzymes expression was modulated in the presence of glycosylated myricetin, using a microarray system. However, antigenotoxic effect of flavonoids might modulate the genotoxic response of AFB1 and NF by modification of the permeability of bacterial membranes or by some extracellular physical, chemical or enzymatically catalyzed interactions between flavonoids and mutagens. In fact, Kooststra (1994) demonstrated that flavonoids should neutralize free radicals that promote mutations, when they are generated near DNA. Flavonoids can also protect the DNA by interacting directly with the mutagen agents, as in the induced chromosomal aberration by bleomycin alluded by Heo et al. (1994). Nevertheless, the inhibition of mutagenesis is often complex, acting through multiple mechanisms. Edenharder et al. (1997) demonstrated a dual role for flavonoids as far as they not only inhibit membrane-bound cytochrome P-450-dependent monooxygenases, but also inhibit various soluble enzymatic factors suggesting interactions with biological membranes and effects on the expression and fixation of DNA damages. The promising antigenotoxic, antioxidant activities and the absence of genotoxicity of the tested compounds from R. alaternus L., suggested that these compounds are phytopharmaceutical molecules of interest. Antimutagenic 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 aging and cancer (Kinghorn et al., 2004). Conflict of Interest None. Acknowledgments The authors acknowledge the ‘‘Ministère Tunisien de l’Enseignement Supérieur, de la Recherche Scientifique’’ and the ‘‘Ministère Français des Affaires Etrangères (Action Intégrée de Coopération Inter Universitaire Franco-Tunisienne, CMCU 07 G0836 PAR)’’, for the financial support of this study, and also thank Ms. Imen Ghadhab (Pr. of English at the Faculty of Dental Medicine, Tunisia) for English editing. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fct.2011.02.011. References Abegaz, B.M., Peter, M.G., 1995. Emodin and emodinanthrone rhamnoside acetates from fruits of Rhamnus prinoides. Phytochemistry 39, 1411–1414. Alemayu, G., Abegaz, B., Snatzke, G., Duddeck, H., 1993. Bianthrones from Senna longiracemosa. Phytochemistry 32, 1273–1277. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Ben Ammar, R., Bensghair, M., Boubaker, J., Bhouri, W., Naffeti, A., Skandrani, I., Bouhlel, I., Kilani, S., Ghedira, K., Chekir-Ghedira, L., 2008b. Antioxidant activity and inhibition of aflatoxin B1-, nifuroxazide-, and sodium azide-induced

mutagenicity by extracts from Rhamnus alaternus L.. Chem. Biol. Interact. 174, 1–10. Ben Ammar, R., Bhouri, W., Ben Sghaier, M., Boubaker, J., Skandrani, I., Neffati, A., Bouhlel, I., Kilani, S., Mariotte, A.M., Chekir-Ghedira, L., Dijoux-Franca, M.G., Ghedira, K., 2009. Antioxidant and free radical-scavenging properties of three flavonoids isolated from the leaves of Rhamnus alaternus L. (Rhamnaceae): a structure-activity relationship study. Food Chem. 116, 258–264. 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., 2007a. 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., Abdelwahed, A., Hayder, N., Mahmoud, A., ben Chibani, J., Chekir-Ghedira, L., Ghedira, K., 2005. In vitro mutagenicity, antimutagenicity and free radical scavenging activities of Rhamnus alaternus L. (Rhamnaceae) extracts. Pak. J. Biol. Sci. 8, 439–445. Ben Ammar, R., Kilani, S., Bouhlel, I., Ezzi, L., Skandrani, I., Boubaker, J., Bensghair, M., Naffeti, A., Mahmoud, A., Chekir-Ghedira, L., Ghedira, K., 2008a. Antiproliferative, antioxidant and antimutagenic activities of flavonoids enriched extracts from (Tunisian) Rhamnus alaternus L. Combination with the phytochemical composition. Drug Chem. Toxicol. 31, 63–82. Ben Ammar, R., Kilani, S., Bouhlel, I., Skandrani, I., Naffeti, A., Boubaker, J., Ben Sghaier, M., Bhouri, W., Mahmoud, A., Chekir-Ghedira, L., Ghedira, K., 2007b. Antibacterial and cytotoxic activities of extracts from (Tunisian) Rhamnus alaternus (Rhamnaceae). Ann. Microbiol. 57, 453–460. Block, G., Langseth, L., 1994. Antioxidant vitamins and disease prevention. Food Technol. 48, 80–84. Block, G., 1992. A role for antioxidants in reducing cancer risk. Nutr. Rev. 50, 207–213. Boukef, K., 2001. Rhamnus alaternus. Essaydali 81, 34–35. Calomme, M., Pieters, L., Vlietinck, A., Vanden Berghe, D., 1996. Inhibition of bacterial mutagenesis by Citrus flavonoids. Planta Med. 62, 222–226. Cao, G., Sofic, E., Prior, R.L., 1999. Antioxidant and prooxidant behaviour of flavonoids: structure – activity relationships. Free Radical. Biol. Med. 22, 749– 760. Chevolleau, S., Debal, A., Ucciani, E., 1992. Détermination de l’activité antioxydante d’extraits végétaux. Revue Française des Corps Gras. 39, 3–8. Coskun, M., Satake, T., Hori, K., Tanker, M., 1990. Anthraquinone glycosides from Rhamnus libanoticus. Phytochemistry 29, 2018–2020. Edenharder, R., Grunhage, D., 2003. Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroxide in Salmonella typhimurium. Mutat. Res. 540, 1–18. Edenharder, R., Rauscher, R., Platt, K.L., 1997. The inhibition by flavonoids of 2amino-3 methylimidazow4, 5-fxquinoline metabolic activation to a mutagen: a structure–activity relationship study. Mutat. Res. Fund. Mol. Mech. Mut. 379, 21–32. Hayder, N., Bouhlel, I., Skandrani, I., Kadri, M., Steiman, R., Guiraud, P., Mariotte, A.M., Ghedira, K., Dijoux-Franca, M.G., Chekir-Ghedira, L., 2008. In vitro antioxidant and antigenotoxic potentials of myricetin-3-o-galactoside and myricetin-3-o-rhamnoside from Myrtus communis: Modulation of expression of genes involved in cell defence system using cDNA microarray. Toxicol. in vitro 22, 567–581. Heo, H.Y., Lee, S.J., Kwon, C.H., Kin, S.W., Sohn, D.H., Au, W.W., 1994. Anticlastogenic effects of galangin against bleomycininduced chromosomal aberrations in mouse spleen lymphocytes. Mutat. Res. Fund. Mol. Mech. Mut. 311, 225–229. Kähkönen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Heinonen, M., 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47, 3954–3962. Kevekordes, S., Mersch-Sundermann, V., Burghaus, C.M., Spielberger, J., Schmeiser, H.H., Arlt, V.M., Dunkelberg, H., 1999. SOS induction of selected naturally occurring substances in Escherichia coli (SOS chromotest). Mutat. Res. 445, 81– 91. Kilani, S., Abdelwahed, A., Chraief, I., Ben Ammar, R., Hayder, N., Hammami, M., Ghedira, K., Chekir-Ghedira, L., 2005. Chemical composition, antibacterial and antimutagenic activities of essential oil from (Tunisian) Cyperus rotundus. J. Essent Oil Res. 17, 695–700. Kinghorn, A.D., Su, B.N., Jang, D.S., Chang, L.C., Lee, D., Gu, J.Q., Carcach-Blanco, P.J., 2004. Natural inhibition of carcinogenesis. Planta Med. 70, 691–705. Kooststra, M., 1994. Protection from UV-B induced DNA damage by flavonoids. Plant Mol. Biol. 26, 771–774. Lin, C.N., Wei, B.L., 1994. Flavonol and naphthalene glycosides from Rhamnus nakaharai. J. Nat. Prod. 57, 294–297. Liochev, S.I., Fridovich, I., 1995. Superoxide from glucose oxidase or from nitroblue tetrazolium. Arch. Biochem. Biophys. 318, 408–410. Mai, L.P., Guéritte, F., Dumontet, V., Tri, M.V., Hill, B., Thoison, O., Guénard, D., Sévenet, T., 2001. Cytotoxicity of Rhamnosylanthraquinones and Rhamnosylanthrones from Rhamnus nepalensi. J. Nat. Prod. 64, 1162–1168. Marzouk, M.S., El-Toumy, S.A.A., Merfort, I., Nawwar, M.A.M., 1999. Polyphenolic metabolites of Rhamnus disperma. Phytochemistry 52, 943–946. Moure, A., Jose, M., Cruz, D., Franco, Manuel., Domínguez, J., Sineiro, J., Domínguez, H., Núñez, M.J., Parajó, J.C., 2001. Natural antioxidants from residual sources. Food Chem. 72, 145–171. Nijveldt, R.J., van Nood, E., van Hoorn, D.E., Boelens, P.G., van Norren, K., van Leeuwen, P.A., 2001. Flavonoids: a review of probable mechanisms of action and potential applications. Am. J. Clin. Nutr. 74, 418–425.

W. Bhouri et al. / Food and Chemical Toxicology 49 (2011) 1167–1173 Quillardet, P.Q., Hofnung, M., 1985. The SOS chromotest colorimetric bacterial assay for genotoxins: procedures. Mutat. Res. 147, 65–68. Re, P., Pellegrin, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C.A., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933–956. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1997. Antioxidant properties of phenolic compounds. Trends Plant Sci. 2, 152–159.

1173

Van den Berg, R., Haenen, G.R.M.M., Van den Berg, H., Van den Vijgh, W., Bast, A., 2000. The predictive value of the antioxidant capacity of structurally related flavonoids using the Trolox equivalent antioxidant capacity (TEAC) assay. Food Chem. 70, 391–395. Wei, B.L., Lin, C.N., Won, S.J., 1992. Nakahalene and cytotoxic principles of formosan Rhamnus species. J. Nat. Prod. 55, 967–969. Yagi, A., Kabash, A., Okamura, N., Haraguchi, H., Moustafa, S.M., Khalifa, T.I., 2002. Antioxidant, free radical scavenging and anti-inflammatory effects of aloesin derivatives in Aloe vera. Planta Med. 68, 957–960.