Environmental and Molecular Mutagenesis 48:58^66 (2007)
Antigenotoxic Activities of Crude Extracts From Acacia salicina Leaves He¤di B. Mansour,1 Jihed Boubaker,1 Ine's Bouhlel,1 Amor Mahmoud,1 Ste¤phane Bernillon,2 Jemni B. Chibani,3 Kamel Ghedira,1 and Leila Chekir-Ghedira1,4* 1
Unite´ de Pharmacognosie/Biologie Mole´culaire ‘‘899/UR/07-038’’. Faculte´ de Pharmacie de Monastir. Rue Avicenne, 5000 Monastir, Tunisie 2 Equipe de recherche en physicochimie et en Biotechnologie (ERPCB-EA3914), IUT-UFR, Universite´ de Caen-Basse Normandie, France 3 Laboratoire de Biochimie et Biologie Mole´culaire, Faculte´ de Pharmacie de Monastir, Rue Avicenne, 5000 Monastir, Tunisie 4 Laboratoire de Biologie Mole´culaire et Cellulaire, Faculte´ de Me´decine Dentaire de Monastir, Rue Avicenne, 5000 Monastir, Tunisie For centuries, plants have been used in traditional medicines and there has been recent interest in the chemopreventive properties of compounds derived from plants. In the present study, we investigated the effects of extracts of Acacia salicina leaves on the genotoxicity of benzo[a]pyrene (B(a)P) and nifuroxazide in the SOS Chromotest. Aqueous, total oligomers flavonoids (TOF)-enriched, petroleum ether, chloroform, ethyl acetate, and methanol extracts were prepared from powdered Acacia leaves, and characterized qualitatively for the presence of tannins, flavonoids, and sterols. All the extracts significantly decreased the genotoxicity induced by 1 lg B(a)P (þS9) and 10 lg nifuroxazide (S9). The TOF-enriched and methanol extracts decreased the SOS response induced by B(a)P to a greater extent, whereas the TOF-enriched and the ethyl acetate extracts exhib-
ited increased activity against the SOS response produced by nifuroxazide. In addition, the aqueous, ethyl acetate, and methanol extracts showed increased activity in scavenging the 1,1-diphenyl2-picrylhydrazyl (DPPH) free radical, while 100– 300 lg/ml of all the test extracts were active in inhibiting O 2 production in a xanthine/xanthine oxidase system. In contrast, only the petroleum ether extract was effective at inhibiting nitroblue tetrazolium reduction by the superoxide radical in a nonenzymatic O 2 -generating system. The present study indicates that extracts of A. salicina leaves are a significant source of compounds with antigenotoxic and antioxidant activity (most likely phenolic compounds and sterols), and thus may be useful for chemoprevention. Environ. Mol. Mutagen. 48:58–66, 2007. C 2006 Wiley-Liss, Inc. V
Key words: Acacia salicina; SOS chromotest; antigenotoxic effect; antioxidative activity; radical scavenging activity; superoxide anion
INTRODUCTION Since ancient times, traditional medicine has used plant extracts for the treatment of diseases. Historically, plants and microorganisms have provided the pharmaceutical industry with important components of new medications. Thus, traditionally-used medicinal plants are a potential source of chemotherapeutic drugs. However, the continued use of medicinal plants would be supported by scientific evidence of their safety and effectiveness. One of the potential uses of plant-derived compounds is as antimutagenic agents [Calomme et al., 1996] and antioxidants [Yagi et al., 2002]. Such compounds may be useful in preventing cancer and other mutation-related C 2006 Wiley-Liss, Inc. V
diseases by fortifying physiological defence mechanisms, or by acting as protective factors [De Flora, 1998]. According to [Lee et al., 2003], the free-radical theory of ageing postulates that damage caused by the reactions of
*Correspondence to: Leila Chekir-Ghedira, Department of Cellular and Molecular Biology, Faculty of Dental Medicine, Rue Avice`nne, 5000 Monastir, Tunisie. E-mail:
[email protected] Received 24 July 2006; provisionally accepted 26 August 2006; and in final form 3 November 2006 DOI 10.1002/em.20265 Published online 18 December 2006 in Wiley InterScience (www.interscience. wiley.com).
Environmental and Molecular Mutagenesis. DOI 10.1002/em Antigenotoxicity of Acacia
free radicals, called reactive oxygen species (ROS), is responsible for the ageing process, and that this damage may be mitigated by the action of antioxidants. The hypothesis is that the use of antioxidants that scavenge ROS provides biological resistance to free radicals, retards the process of ageing, and decreases the risk of age-associated degenerative diseases, such as cancer, cardiovascular disease, immune system decline, and brain dysfunction [Finkel and Holbrook, 2000]. In view of drawbacks encountered when using synthetic compounds for treating humans, there has been growing interest in the use of plant preparations as antioxidative and chemopreventive agents. Acacia (Fabaceaes) is an evergreen tree that is a native of Australia, but now is widely distributed in the Mediterranean area. Acacia species are frequently used for the treatment of various illnesses because of their reputed pharmacological effects; published information indicates that Acacia has hypoglycemic effects [Wadood et al., 1989], anti-inflammatory activity [Dafallah and Al-Mustapha, 1996], cestocial activity [Ghosh et al., 1996], activity against platelet aggregation [Shah et al., 1997], spasmogenic and vasoconstrictor activities [Amos et al., 1999], antihypertensive and antispasmodic activities [Gilani et al., 1999], and inhibitory effects against hepatitis C virus [Hussein et al., 2000]. In Tunisian traditional medicine, the uses of Acacia differ according to the species and according to the region of the country. Based on information gathered from traditional healers, herbalists, and inhabitants from rural south Tunisia, Acacia salicina is frequently used in such diverse applications as in the treatment of inflammatory diseases, as ‘‘fibrifuge’’ to treat cancer, and to promote human fertility. In south Tunisia, infusions made from fresh or dried leaves are taken orally, or alternatively, chopped fresh leaves are applied directly to inflamed sores. Traditional medical uses of Acacia in north Tunisia are somewhat different; in this region, Acacia is used for the treatment of diarrhea and rheumatism. In many instances, neither the medicinal nor the adverse effects of plants used in traditional medical practices have been documented rigorously. In vitro screening tests indicate that some plants that are used in traditional medicine are mutagenic [Elgorashi et al., 2003], indicating that their use may pose a long-term risk to patients. As part of our studies on potential chemopreventive agents, we have evaluated the genotoxic, antigenotoxic, antiradical, and antioxidant effects of extracts from A. salicina collected from Medenine in the south of Tunisia. MATERIALS AND METHODS Chemicals Benzo[a]pyrene (B(a)P), nifuroxazide, xanthine, xanthine oxidase (XOD), and riboflavin were purchased from Sigma (St. Louis, MO). O-Nitrophenylb-D-galactopyranoside (ONPG) and p-nitrophenylphosphate (PNPP) were
59
purchased from Merck (Darmstadt, Germany). 1,1-diphenyl-2-picryl-hydrazyl (DPPH), allopurinol, a-tocopherol, nitroblue-tetrazolium (NBT), n-butanol, methanol, digitonin, and NH4Fe(SO4) 12H2O were purchased from Aldrich (St. Louis, MO). Diphenylborinic acid 2-aminoethyl ester was purchased from Acros Organics (Ceel, Belgium).
Plant Materials A. salicina was collected from the Arid Region Institute (IRA) situated in the south east of Tunisia in October, 2003. Botanical identification was carried out by Pr. Chaib (Department of Botany, Faculty of Sciences of Sfax). A voucher specimen (AS-10.03) has been deposited in the Laboratory of Pharmacognosy, Faculty of Pharmacy of Monastir, for future reference. The leaves were shade-dried, powdered, and stored in a tightly closed container.
Extraction Procedure An aqueous extract was prepared from the powdered leaves by boiling in water for 15–20 min. The extract was filtered and lyophilized, and the residue was dissolved in water. To obtain an extract enriched in total oligomers flavonoids (TOF), we macerated the powdered leaves in 1:2 (v:v) water/acetone for 24 hr with continuous stirring. The extract was filtered and the acetone was evaporated under low pressure to obtain an aqueous solution. The tannins were partially removed by precipitation with an excess of NaCl for 24 hr at 58C, and recovery of the supernatant. This latter fraction was extracted with ethyl acetate, followed by concentration and precipitation with an excess of chloroform. The precipitate was separated and yielded the TOF-enriched extract [Ghedira et al., 1991]. Petroleum ether, chloroform, ethyl acetate, and methanol extracts were obtained from the powdered leaves with a soxhlet apparatus (6-hr extraction). This resulted in four extracts of different polarities. The extracts were concentrated to dryness and kept at 48C.
Preliminary Phytochemical Analysis The various extracts were screened for the presence of tannins, flavonoids, and sterols by using the methods previously described by Sokmen et al. [2005] and Tona et al. [2004]. Two milligrams of each extract were dissolved in 2 ml of the appropriate solvent. The identification of major chemical groups was carried out by thin layer chromatography (TLC) on silica gel 60 F254 Merck (layer thickness, 0.25 mm), as follows. For flavonoids, the TLC was developed in n-butanol/acetic acid/ water (4:1:5), and the spots were visualized with 1% aluminium chloride in methanol under UV (366 nm). Steroids were identified with the Libermann-Burchard reagent after separation on TLC using n-hexane/ CH2Cl2 (1:9) as the mobile phase. A range of colors was produced after heating sprayed plates for 10 min at 1008C. The test for tannins was carried out with FeCl3. Each class of tannins produced a specific color.
Quantitative Analysis of Extracts Flavonoids were quantified by using the method described by [Dohou et al., 2003]. Twenty milligrams of each extract were dissolved separately in 2 ml of 80% methanol and sonicated (30 sec, 100%) with a Sonics vibra-cell ultrasonic processor (Bioblock Scientific, Illkirch, France). After addition of 100 ll of diphenylborinic acid 2-aminoethyl ester (1% (w/v) in methanol) to each solution, the absorbance of flavonoids was determined spectrophotometrically at 404 nm and compared to a quercetin standard (0.05 mg/ml). The percentage of total flavonoids was then calculated in quercetin equivalents according to the following formula: F ¼ ð0:05 Aext =Aq Þ 100=Cext , where Aext and Aq were the absorbance of the extract and of quercetin, respectively, and Cext was the extract concentration (10 mg/ml).
Environmental and Molecular Mutagenesis. DOI 10.1002/em 60
Mansour et al.
Condensed tannins were quantified according to the method developed by [Porter et al., 1986]. Solutions (1 g/l) of each extract were sonicated (30 sec, 100%), distributed in glass tubes, and sealed with a Teflon-lined screw cap. To each tube was added 2.5 ml of n-butanol-HCl (95:5, v/v) and 100 ll of a 2% (w/v) ferric reagent (NH4Fe (SO4) 12H2O). The solutions were capped, thoroughly mixed, and suspended in a constant-level water bath at 958C for 40 min. The solutions were cooled and the visible spectrum was determined at 540 nm. The percentage of total condensed tannins was then calculated in cyanidol (standard) equivalents according to the following formula:
DPPH Radical-Scavenging Activity The free-radical scavenging capacity of the extracts was determined with DPPH. Ethanol solutions were prepared containing 100, 30, 10, 3, and 1 lg/ml of the extracts and 23.6 lg/ml of DPPH. After incubation for 30 min at ambient temperature, the absorbance of the remaining DPPH was determined at 517 nm. Radical scavenging activity was measured as the decrease in absorbance of the samples vs. a DPPH standard solution [Yagi et al., 2002]. Results were expressed as ‘‘percentage inhibition’’ (%) of the DPPH and the mean 50% inhibiting concentration (IC50). % is defined by the formula
T ¼ ½ðA540nm = 2 lÞ 3 1=Cext 3 100; where l ¼ 1 cm, [ ¼ 42,390 l/mol/cm, and Cext is extract concentration. Sterols were quantified by a method developed in our laboratory. Liu et al. [1999] reported that digitonin reacts specifically with steroids. On the basis of this observation, we evaluated sterols in the extracts by their reaction with digitonin. Twenty milligrams of each extract were dissolved separately in 5 ml of pure acetone and distributed in glass tubes. After addition of 2.5 ml of 2% (w/v) digitonin in methanol at 808C, the tubes were incubated in a water bath at 608C until a reduction to half volume. The remaining solutions were cooled at room temperature and filtered with a Durieux filter N8111 (Bioblock Scientific). After washing several times with boiling water, then successively with 808C methanol, acetone, and finally with anhydrous ether, the filter was dried for 3 hr at 808C. The weight of the residue was used to quantify supplemented sterols. The percentage of total sterols was then calculated according to the following formula: S ¼ ðPst =Pext Þ 3 100; where Pst is the weight of sterols and Pext is the extract weight (20 mg).
Activation Mixture The S9 microsome fraction was prepared from the liver of rats treated with Arochlor 1254 [Maron and Ames, 1983]. Ten milliliters of S9 mix contained 0.2 ml salt solution (1.65 M KCl, 0.4 M MgCl2 6H2O), 0.05 ml 1 M glucose-6-phosphate, 0.15 ml 0.1 M NADP, 2.5 ml 0.4 M Tris buffer (pH 7.4), 6.1 ml Luria broth, and 1 ml S9 fraction.
Antigenotoxicity Assay Inhibition of bacterial genotoxicity was evaluated in the SOS Chromotest using Escherichia coli PQ 37 (kindly provided by Pr. Quillardet, Institut Pasteur, Paris, France) and the procedure described by [Quillardet and Hofnung, 1985]. The extracts were dissolved in dimethylsulfoxide and tested in triplicate, with (B(a)P assays) and without (nifuroxazide assays) exogenous metabolic activation. The SOS-induction potency (SOSIP) was calculated from the linear part of the induction factor (IF) dose-response as a measure of genotoxicity. The IF was calculated as the ratio of Rc/R0, where Rc is the b-galactosidase (b-gal) activity/alkaline phosphatase (AP) activity determined for the test compound at concentration c, and R0 is the b-gal activity/AP activity in the absence of the test compound. The b-gal and AP activities were calculated according to the method recommended by Quillardet and Hofnung [1985]. Antigenotoxicty was expressed as the percentage inhibition of genotoxicity according to the following formula: Inhibition ð%Þ ¼ 100 ðIF1 IF0 =IF2 IF0 Þ 3 100, where IF1 is the IF in the presence of both the test compound and the mutagen, IF2 is the IF in the absence of the test compound and in the presence of the mutagen, and IF0 is the IF of the negative control. Data were expressed as the mean 6 standard deviation of results from three independent experiments.
ð%Þ ¼ ½ðODcontrol ODsample Þ=ODcontrol 3 100, where ODcontrol is the initial absorbance and ODsample the value for the test sample after incubation [Lee et al., 2003]. IC50 was defined as the concentration (in lg/ml) of substrate that caused 50% loss of DPPH activity (color) and it was calculated by using the Litchfield and Wilcoxon test [Galati et al., 2001; Sokmen et al., 2005]. The results were expressed as the mean of data from at least three independent experiments.
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. The reaction was carried out at room temperature under fluorescent lighting (20 W, 20 cm). The standard incubation mixture contained 6.5 mM N,N,N0 ,N0 -tetramethylethylenediamine (TMEDA), 4 lM riboflavin, 96 lM NBT, and 51.5 mM potassium phosphate buffer (pH 7.4). After 6 min incubation, the reaction was stopped by switching the light off and adding 0.05 ml SOD (1 mg/ml). For each extract concentration, one 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 extract components. For the estimation of the superoxide-driven reduction of NBT, the absorbance of the control sample was subtracted from that of standard reaction mixture. The plant extracts and the reference substance (quercetin) were assayed at different concentrations with three repetitions. IC50 values (concentration required to inhibit NBT reduction by 50%) were calculated from dose-inhibition curves [Kostyuk et al., 2004; Sokmen et al., 2005].
Inhibition of Xanthine Oxidase Activity The inhibition of XOD-generated superoxide formation was evaluated by measuring the UV absorbance of uric acid at 295 nm as proposed by Cimanga et al. [2001]. The assay mixture consisted of a 100 ll solution of test compound, a 200 ll solution of xanthine (X) (final concentration, 50 lM) and hydroxylamine (final concentration, 0.2 mM), 200 ll 0.1 mM EDTA, and 300 ll distilled water. The reaction was initiated by adding 200 ll XOD (final concentration, 11 mU/mL) dissolved in 0.2 M phosphate buffer (pH 7.5). Following incubation at 378C for 30 min, the reaction was stopped by adding 100 ll 0.58 M HCl. The absorbance then was measured against a blank solution prepared in the same way as described above, but replacing XOD by buffer solution (no production of uric acid). A control solution without the test compound was prepared in the same manner as the assay mixture to measure the total uric acid production. Uric acid production was calculated from the differential absorbance. Allopurinol was used as a positive control for inhibition.
Statistical Analysis Data were expressed as the mean 6 standard deviation of three independent experiments. Data were analyzed for statistical significance using Dunnett’s test.
Environmental and Molecular Mutagenesis. DOI 10.1002/em Antigenotoxicity of Acacia
61
TABLE I. Quantitative Phytochemical Screening (%) of Extracts from A. salicina
Tannins Flavonoids Sterols
Aqueous extract
TOF extract
Petroleum ether extract
Chloroform extract
Ethyl acetate extract
Methanol extract
7.2 9.9 0
8.1 14.0 0
0 0 12.5
0 0 5
1.9 2.2 2
7.5 10.3 0
TABLE II. Effect of Extracts on the Genotoxicity of Benzo[a]pyrene (B(a)P; 1 lg/assay, + S9) in the SOS Chromotest Test article NC 1 lg B(a)P 1 lg B(a)P þ aqueous extract 1 lg B(a)P þ TOF-enriched extract 1 lg B(a)P þ petroleum ether extract 1 lg B(a)P þ chloroform extract 1 lg B(a)P þ ethyl acetate extract 1 lg B(a)P þ methanol extract
Dose extract (lg/assay) 0 0 200 50 10 10 5 1 500 200 50 500 200 50 50 10 5 200 50 10
b-gal(U) 2.46 6.31 2.75 3.13 3.24 2.13 2.44 2.77 3.12 4.84 6.2 3.44 3.68 2.93 1.72 3.18 3.31 2.24 2.68 3.15
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
AP (U)
0.01 10.6960.02 0.001 4.34 6 0.003 0.03 4.34 6 0.01 0.01 4.34 6 0.01 0.01 2.97 6 0.01 0.02 7.04 6 0.02 0.03 4.34 6 0.03 0.01 4.78 6 0.05 0.02 4.56 6 0.01 0.01 5.43 6 0.05 0.001 6.39 6 0.02 0.01 7.6 6 0.01 0.01 5.91 6 0.01 0.03 4.34 6 0.01 0.02 3.87 6 0.02 0.02 3.91 6 0.01 0.01 3.91 6 0.01 0.03 6.17 6 0.01 0.01 6.13 6 0.01 0.01 5.95 6 0.02
IF
% inhibition of genotoxicity
1.00 6.32 2.72* 3.13* 4.74 1.31* 2.45* 2.52* 3.00* 3.87 4.21 1.96* 2.70* 2.93* 2.83* 3.53* 3.68* 1.60* 1.90* 2.32*
0 0 68 60 30 94 73 72 63 46 40 82 68 64 82 52 50 89 83 75
b-gal, b galactosidase; AP, alkaline phosphatase; IF, induction factor; NC, negative control. All assays conducted with S9. * Significantly different from the B(a)P-only value, P < 0.05.
RESULTS Phytochemical Analysis The results of our analysis of the crude extracts are shown in Table I. The aqueous and methanol extracts contained similar amounts of flavonoids (9.9% and 10.3%, respectively) and tannins (7.2 and 7.5%, respectively), whereas the TOF extract contained relatively high quantities of both flavonoids (14.0%) and tannins (8.1%). The ethyl acetate extract had lower amounts of flavonoids and tannins (2.2 and 1.9%, respectively), while there were no flavonoids or tannins detected in either the petroleum ether or chloroform extracts. Only the chloroform, petroleum ether, and ethyl acetate extracts contained sterols, with the petroleum ether extract having the highest sterol concentration (12.5%). Antigenotoxicity Assay In a series of SOS Chromotest assays on the extracts by themselves, the extracts had no effect on the viability
of the tester strain (Tables II and III for test concentrations). In addition, most genotoxicity assays conducted with the extracts by themselves were negative. None of the extracts produced a significant increase in the IF in the absence of S9, whereas significant genotoxicity in the presence of S9 was observed only with the highest test doses (500 lg/assay) of the petroleum ether extract (IF ¼ 6.94) and the chloroform extract (IF ¼ 5.98) (data not shown). As shown in Tables II and III, all the extracts prepared from A. salicina were effective in reducing the IF induced by B(a)P (1 lg/assay with S9), a metabolically activated genotoxin, as well as the IF induced by nifuroxazide (10 lg/assay without S9), a direct-acting genotoxin. The chloroform, methanol, ethyl acetate, and TOFenriched extracts were more effective inhibitors of B[a]P and nifuroxazide genotoxicity than either the petroleum ether or aqueous extracts. The chloroform, methanol, ethyl acetate, and TOF-enriched extracts, at concentrations of 500, 200, 50, and 10 lg/assay, respectively, decreased the SOSIP of B(a)P by 82, 89, 82, and 94%. At
Environmental and Molecular Mutagenesis. DOI 10.1002/em 62
Mansour et al.
TABLE III. Effect of Extracts on the Genotoxicity of Nifuroxazide (10 lg/assay, –S9) in the SOS Chromotest Test article NC 10 lg nifuroxazide 10 lg nifuroxazide þ aqueous extract 10 lg nifuroxazide þ TOF-enriched extract 10 lg nifuroxazide þ petroleum ether extract 10 lg nifuroxazide þ chloroform extract 10 lg nifuroxazide þ ethyl acetate extract 10 lg nifuroxazide þ methanol extract
Dose extract (lg/assay) 0 10 200 50 10 10 5 1 500 200 50 500 200 50 50 10 5 200 50 10
b-gal (U) 2.22 14 4.17 3.48 3.55 2.44 3 2.62 7.11 8 8.44 4.88 6.22 6.88 7.11 7.55 7.55 6.77 7.55 8
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
0.01 0.01 0.04 0.02 0.04 0.03 0.02 0.03 0.05 0.02 0.04 0.03 0.04 0.02 0.04 0.05 0.02 0.05 0.03 0.04
AP (U)
IF
% inhibition of genotoxicity
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
1.00 6.48 2.56* 2.90* 2.96* 1.25* 1.28* 2.38* 2.92* 3.06* 4.39 2.26* 4.32 5.74 2.23* 4.24 5.29 3.11* 5.22 5.55
0 0 72 65 64 96 95 75 67 64 42 77 39 14 79 45 25 62 23 17
8.22 8 5.95 4.46 4.43 7.22 8.68 4.1 9 9.68 7.11 8 5.33 4.44 11.85 6.59 5.27 8 5.35 5.33
0.01 0.01 0.03 0.04 0.03 0.03 0.03 0.04 0.03 0.04 0.03 0.03 0.04 0.03 0.03 0.03 0.02 0.02 0.04 0.02
b-gal, b galactosidase; AP, alkaline phosphatase; IF, induction factor; NC, negative control. All assays conducted in the absence of S9 activation. * Significantly different from the nifuroxazide-only value, P < 0.05.
the same concentration of each of the extracts, the SOSIP of nifuroxazide was decreased by 77, 62, 79, and 96%. By comparison, 200 and 500 lg/assay of the aqueous and petroleum extracts decreased the SOSIP of B(a)P by about 68 and 62%, respectively, and the SOSIP of nifuroxazide by about 72 and 67%.
indicate that the methanol, ethyl acetate, and aqueous extracts showed the greatest DPPH antiradical activities. The TOF-enriched and petroleum ether extracts had lower but significant antiradical activity, while the chloroform extract exhibited weak DPPH radical-scavenging activity. Effects on Superoxide Anion Generating Systems
DPPH Radical-Scavenging Activity DPPH is a molecule containing a stable free radical. In the presence of an antioxidant that can donate an electron to DPPH, the purple color typical of the free DPPH radical decays, a change that can be followed spectrophotometrically at 517 nm. This simple test can provide information on the ability of a compound to donate an electron, the number of electrons a given molecule can donate, and the mechanism of antioxidant action. The radical-scavenging activities of the extracts measured as decolorizing activity following the trapping of the unpaired electron of DPPH are shown in Table IV. The methanol and ethyl acetate extracts were very potent radical scavengers, with a percentage decrease vs. the absorbance of the DPPH standard solution of 87 and 73%, respectively, at a concentration of 100 lg/ml, and IC50 values of 2.34 and 2.61 lg/ml. These values were slightly greater than that of the positive control, 3 lg/ml a-tocopherol. Aqueous extract (100 lg/ml), the TOFenriched extract, and the petroleum ether and chloroform extracts had scavenging activities of 68, 74.5, 64, and 47%, respectively, and these extracts had IC50 values of 9.44, 24, 29, and >100 lg/ml, respectively. These results
The superoxide radical (O 2 ) 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 (HO2) that reacts with polyunsaturated fatty acids. Finally, superoxide may react with NO to generate peroxynitrite, which is known to be toxic towards DNA, lipids, and proteins. The NBT assay is based on the capacity of the extracts to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) in the presence of riboflavin. Under these conditions, NBT can be unevenly reduced in the presence of the O 2 radical to a tetrazoinyl radical that can dismute to the formazan. 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. Only the petroleum ether extract had activity in this assay. This extract produced a 67% decrease in NBT photoreduction at a concentration of 10 mg/ml and an IC50 value of 3.3 mg/ml. The petroleum ether extract was
Environmental and Molecular Mutagenesis. DOI 10.1002/em Antigenotoxicity of Acacia TABLE IV. DPPH Free-Radical Scavenging Activity of Extracts from A. salicina Leaves Extract
Concentration (lg/ml)
% Inhibition
IC50 (lg/ml)
Aqueous extract
1 3 10 30 100
40.45 43.43 52.93 64.54 68.1
6 6 6 6 6
2.3 0.9 2.1 1.8 0.6
TOF extract
1 3 10 30 100
22 40.1 45.45 62.72 74.5
6 6 6 6 6
1.3 1.4 1.2 0.8 1.1
Methanol extract
1 3 10 30 100
40.6 63.8 69.1 75.15 87.28
6 6 6 6 6
1.2 2.2 2.3 1.3 0.7
2.34
Ethyl acetate extract
1 3 10 30 100
43.63 57.38 66.66 69.7 72.60
6 6 6 6 6
2.2 1.4 0.9 1.4 1.1
2.61
Petroleum ether extract
1 3 10 30 100
36.6 43 47 51.51 64
6 6 6 6 6
1.6 1.5 1.4 2.3 0.5
29
Chloroform extract
1 3 10 30 100
0 0 19.39 6 1.3 37.17 6 0.8 47.17 6 1.3
>100
a-Tocopherol (positive control)
1
30 6 2.1
3
3 10 30 100
50 97.3 98 98.7
6 6 6 6
9.44
24
1.3 1.8 1.3 2.2
more active than the positive control, quercetin, in the assay (Fig. 1). Results of the XOD-inhibition assay suggest that all the test extracts decreased the XOD-generated superoxide radical in a concentration-dependent manner (Fig. 2). The ethyl acetate, chloroform, and TOF-enriched extracts were the most potent inhibitors of XOD-activity, with IC50 values of 235, 100, and 270 lg/ml, respectively; the methanol, petroleum ether, and aqueous extracts had somewhat lower inhibitory activity. The positive control, allopurinol, was considerably more potent than any of the test compounds, with an IC50 value of 0.35 lg/ml. DISCUSSION The antigenotoxic properties elicited in this study suggest that A. salicina may have several applications in
63
human health care. Herbal remedies and phytotherapy drugs containing active principles can potentially protect against electrophilic (e.g., free radical) attack of DNA and the outcomes of such attack, such as ageing and cancer. Even for populations that use herbs as traditional medications, encouraging the use of species with chemopreventive activities could be helpful as part of life expectancy improvement strategies: costs are low, herbs usually have little or no toxicity during long-term oral administration, and they are widely available. Two different reactive species were used to evaluate the antioxidant activity of the A. salicina extracts: the DPPH and superoxide radicals. The superoxide anion and other ROS contribute to oxidative stress, and are known contributors to genetic damage, as well as degenerative diseases such as cancer [Sander et al., 2004], Parkinson disease, and heart ischemia [Gonzalez-Avila et al., 2003]. Since the DPPH radical is not biologically relevant, the DPPH assay was performed as a preliminary study to estimate the direct free-radical scavenging abilities of the test extracts. The activity of extracts against the superoxide radical is a semiquantitative test [Baratto et al., 2003], and the enzymatic assay (X/XOD) has more relevance to physiological conditions than the photochemical-NBT assay. The results obtained with the aqueous, methanol, and ethyl acetate extracts revealed relatively strong antiradical activity towards the DPPH free radical. On the other hand, the effects of the TOF-enriched, methanol, ethyl acetate, and aqueous extracts in the XOD-inhibition assay might be caused by inhibition of the enzymatic activity of XOD, and not by direct quenching of the radicals [Nam et al., 2006]. This possibility is supported by the detection of flavonoids and phenolic compounds in these extracts. Several flavonoids and other phenolic compounds are considered antioxidants not only because they act as free-radical scavengers, but also because they inhibit XOD [Cos et al., 1998]. XOD catalyses the oxidation of hypoxanthine and xanthine to uric acid. During the oxidation of xanthine, superoxide radicals and hydrogen peroxide are formed; this enzyme is considered an important biological source of superoxide radicals [Schuldt et al., 2004]. In contrast, the petroleum ether extract seems to exert its action via direct quenching of reactive ROS. This extract displayed the weakest inhibition of XOD; nevertheless, it was the only extract that had activity in the nonenzymatic NBT/riboflavin assay system. In fact, sterols, which are the main constituents of the petroleum ether extract, were previously found to possess significant antioxidant effects [Argolo et al., 2004]. Sterols, however, are the main constituent of the chloroform extract, which was effective in inhibiting XOD but not in scavenging superoxide radicals. We hypothesize that the sterols present in the chloroform extract possess different antioxidant properties than sterols present in the petroleum ether extract, as the molecules in the two extracts have different polarities.
Environmental and Molecular Mutagenesis. DOI 10.1002/em 64
Mansour et al.
Fig. 1. Scavenging effects of petroleum ether extract of A. salicina against photochemically generated superoxide free radicals (O 2 ). *P < 0.05.
Fig. 2. Inhibition of xanthine oxidase activity by extracts of A. salicina leaves. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
In the SOS Chromotest, all the test extracts strongly inhibited the genotoxicity of both B(a)P and nifuroxazide. The inhibitory effect of these extracts on the genotoxicity of B(a)P and nifuroxazide may be due to the flavonoids [Calomme et al., 1996] and tannins [Lee et al., 2003; Baratto et al., 2003] detected in the TOF-enriched, methanol, and aqueous extracts, and to the sterols [Argolo et al., 2004] detected in the petroleum ether and chloroform extracts. The protective effect of the ethyl acetate extract may be due to the activity of several of the above-cited compounds. We cannot, however, exclude the possibility that other compounds with antigenotoxic properties participate in the inhibitory effects of the A. salicina extracts. The results of our experiments are consistent with the known antioxidant activities of flavonoids [Vaya et al., 2003; Bouaziz et al., 2005], tannins [Yokozozawa et al, 1998; Zoran et al., 2005], and sterols [Argolo et al., 2004]. Flavonoids are the most likely candidates among the compounds known to be present in the TOF-enriched, metha-
nol, ethyl acetate, and aqueous extracts for providing the antigenotoxic effects and preventing oxidative lesions [Edenharder and Grunhage, 2003; Park et al., 2004]. Sterols, which are the main constituents of the chloroform and petroleum ether extracts, and which are described as possessing significant antioxidant activity [Argolo et al., 2004], are likely candidates for providing the antigenotoxic effects of these preparations. It is possible that these compounds inhibit the free radicals and ROS produced by oxidation and redox-cycling of B(a)P, and through reducing the activity of enzymes involved in B(a)P metabolism. Because all the test extracts showed significant antigenotoxicity in assays with both the direct-acting nifuroxazide and the metabolically activated B(a)P, the extracts may both inhibit microsomal activation and directly protect DNA from the electrophilic BaP epoxide, 7,8dihydroxy 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, a putative ultimate carcinogenic metabolite [Harris et al.,
Environmental and Molecular Mutagenesis. DOI 10.1002/em Antigenotoxicity of Acacia
1985], and other intermediates of the mutagen. In fact several metabolic intermediates and ROS formed during microsomal enzyme activation also are capable of breaking DNA strands. Antioxidant activity expressed by A. salicina extracts may provide a common mechanism for inhibiting the genotoxicity of both B(a)P and nifuroxazide. Curiously, the chloroform and petroleum ether extracts exhibited both genotoxic and antigenotoxic activities at a concentration of 500 lg of extract/plate. We hypothesize that the presence of reactive intermediates resulting from both B(a)P and the chloroform or petroleum ether extract could result in their mutual neutralization. These intermediates may form a complex that prevents them from penetrating through the bacterial cell wall and producing genotoxic effects. In summary, A. salicina extracts appear to contain compounds with antioxidant and chemoprotective properties. However, further studies are required to fractionate the active extracts, to identify the active compounds, and to determine their exact mechanism of action.
ACKNOWLEDGMENTS The authors are grateful to Prof. M. Ghoul, Laboratoire des Bioproce´de´s et Agro-Alimentaire, Institut National de Polytechnique de Lorraine (Nancy, France), for providing xanthine/xanthine oxidase and to Prof. Bacha, Director of Recherche Laboratoire des Substances Biologiquement Compattibles, Faculte´ de Me´decine Dentaire de Monastir (Tunisie), for allowing us the use his UV spectrophotometer. The authors thank Dr. Robert Heflich for the critical reading of this manuscript.
REFERENCES Amos S, Akah PA, Odukwe CJ, Gamaniel KS, Wambede C. 1999. The pharmacological effects of an aqueous extract from Acacia nilotica seeds. Phytother Res 13:683–685. Argolo ACC, Sant’Ana AEF, Pletsch M, Coelho LCBB. 2004. Antioxidant activity of leaf extracts from Bauhinia monandra. Bioresour Technol 95:29–233. Baratto MC, 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. Bouaziz M, Grayer RJ, Simmonds MS, Damak M, Sayadi S. 2005. Identification and antioxidant potential of flavonoids and low molecular weight phenols in olive cultivar chemlali growing in Tunisia. J Agric Food Chem 53:236–241. Calomme M, Pieters L, Vlietink A, Berghe DV. 1996. Inhibition of bacterial mutagenesis flavonoı¨ds. Planta Med 62:222–226. Cimanga K, Ying L, De Bruyne T, Apers S, Cos P, Hermans N, Bakana P, Tona L, Kambu P, Kalenda DT, Pieters L, Vanden Berghe D, Vlietinck AJ. 2001. Radical scavenging and xanthine oxidase inhibitory activity of phenolic compounds from Bridelia ferruginea stem bark. Pharm Pharmacol 53:757–761.
65
Cos P, Ying L, Calomme M, Hu JP, Cimanga K, Van Poel B, Potier L, Vlientink AJ, Vanden Berghe D. 1998. Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. J Nat Prod 61:71–76. Dafallah AA, Al-Mustapha Z. 1996. Investigation of the anti-inflammatory activity of Acacia nilotica and Hibiscus sabdariffa. Am J Chinese Med 24:263–269. De Flora S. 1998. Mechanisms of inhibitors of mutagenesis and carcinogenesis. Mutat Res 420:151–158. Dohou N, Yamni K, Tahrouch S, Hassani Idrissi LM, Badoc A, Gmira N. 2003. Screening phytochimique d’une ende´mique ibe´ro-Marocaine, Thymelaea lythoroides. Bull Soc Pharm Bordeaux 142:61– 78. Edenharder R, Grunhage D. 2003. Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA 102. Mutat Res 540:1–18. Elgorashi EE, Taylor JLS, Maes A, Van Staden J, De Kimpe N, Verschaeve L. 2003. Screening of medicinal plants used in South African traditional medicine for genotoxic effects. Toxicol Lett 143:195–207. Finkel T, Holbrook NJ. 2000. Oxidative stress and the biology of ageing. Nature 408:239–247. Galati EM, Miceli N, Taviano MF, Sanogo R, Raneri E. 2001. Antiinflamatory and antioxidant activity of Ageratum conyzoides. Pharm Biol 39:336–339. Ghedira K, Chemli R, Richard B, Zeches M, Le Men-Olivier L. 1991. Contribution a` l’e´tude de la pharmacope´e traditionnelle de Tunisie, e´tude des parties ae´riennes d’Ajuga iva. PI Med Phytother 25:100–111. Ghosh NK, Babu SP, Sukul NC, Ito A. 1996. Cestocidal activity of Acacia auriculiformis. J Helmintol 70:171–172. Gilani AH, Shaheen F, Zaman M, Janbaz KH, Shah BH, Akhtar MS. 1999. Studies on hypertensive and antispasmodic activities of methanol extract of Acacia nilotica pods. Phytother Res 13:665– 669. Gonzalez-Avila M, Arriaga-Alba M, De la Garza M, Del Carmen Hernandez-Pretelinc M, Dominguez-Ortiz MA, Fattel-Fazenda S, Vialla-Trevino S. 2003. Antigenotoxic, antimutagenic and ROS scavenging activities of a Rhoeo discolor ethanolic crude extract. Toxicol In Vitro 17:77–83. Harris CC, Vahakangas K, Newman MJ, Trivers GE, Shamsuddin A, Sinopoli N, Mann DL, Wright W. 1985. Detection of benzo[a]pyrene diol epoxide DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers. Proc Natl Acad Sci 82:6672–6676. Hussein G, Miyashiro H, Nakamura N, Hattori M, Kakiuchi N, Shimotohno K. 2000. Inhibitory effects of Sudanese medicinal plant extracts on hepatitis C virus (HCV). Phytother Res 14:510– 516. Kostyuk VA, Potapovich AI, Strigunova EN, Kostyuk TV, Afanas ‘ev IB. 2004. Experimental evidence that flavonoid metal complexes may act as mimics of superoxide dismutase. Arch Biochem Biophys 428:204–208. Lee SE, Shin HT, Hwang HJ, Kim JH. 2003. Antioxidant activity of extracts from Alpinia katsumadai seed. Phytother Res 17:1041– 1047. Liu J, Xiao N, DeFranco DB. 1999. Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking. Methods 19: 403–409. Maron DM, Ames BN. 1983. Revised methods for the Salmonella mutagenicity test. Mutat Res 113:173–215. Nam SH, Choi SP, Kang MY, Koh HJ, Kozukue N, Friedman M. 2006. Antioxidative activities of bran extracts from twenty one pigmented rice cultivars. Food Chem 94:613–620.
Environmental and Molecular Mutagenesis. DOI 10.1002/em 66
Mansour et al.
Park KY, Jung GO, Lee KT, Choi J, Choi MY, Kim GT, Jung HJ, Park HJ. 2004. Antimutagenic activity of flavonoids from the heartwood of Rhus verniciflua. J Ethnopharmacol 90:73–79. Porter LJ, Hrstich LN, Chan BG. 1986. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25:223–230. Quillardet PQ, Hofnung M. 1985. The SOS Chromotest colorimetric bacterial assay for genotoxins: Procedures. Mutat Res 147:65–78. Sander CS, Chang H, Hamm F, Elsner P, Thiele JJ. 2004. Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis. Int J Dermatol 43:326–335. Schuldt EZ, Farias MR, Ribeiro-do-Valle RM, Ckless K. 2004. Comparative study of radical scavenger activities of crude extract and fractions from Cuphea carthagenensis leaves. Phytomedicine 11:523–529. Shah BH, Safdar B, Virani SS, Nawaz Z, Saeed SA, Gilani AH. 1997. The antiplatelet aggregatory activityof Accacia nilotica is due to blockage of calcium influx through membrane calcium channels. Gen Pharmacol 29:251–255. Sokmen M, Angelova M, Krumova E, Pashova S, Ivancheva S, Serkedjieva J. 2005. In vitro antioxidant activity of polyphenol extracts with antiviral proprieties from Geranium sanguinium L. Life Sci 76:2981–2993. Tona L, Cimanga RK, Mesia K, Musuamba CT, De Bruyne T, Apers S, Hernans N, Von Miert S, Pieters L, Totte´ J, Vlietinck AJ. 2004.
In vitro antiplasmodial activity of extracts and fractions from seven medecinal plants used in democratic Republic of Congo. J Ethnopharmacol 93:27–32. Vaya J, Mohmod S, Goldblum A, Aviram M, Volkavor N, Shaalam A, Musa R, Tamir S. 2003. Inhibition of LDL oxidation by flavonoı¨ds in relation to their structure and calculated enthalpy. Phytochemistry 62:89–99. Wadood A, Wadood N, Wahid Shah SA. 1989. Effects of Acacia arabica and Caralluma edulis on blood glucose levels of normal and alloxan diabetic rabbits. J Pak Med Assoc 39:208–212. Yagi A, Kabash A, Okamura N, Hararguchi H, Moustafa SM, Khalifa TI. 2002. Antioxidant, free radical scavenging and anti-inflammatory effects of aloesin derivatives in Aloe vera. Planta Med 68:957–960. Yokozozawa T, Chen CP, Dong E, Tanaka T, Nishioka GI. 1998. Study on the inhibitory effect of tannins and flavonoı¨ds against DPPH radical. Biochem Pharmacol 56:213–222. Zoran M, Dorde M, Nada K. 2005. Polyphenol contents and antioxidant activity of Maydis stigma extracts. Bioresour Technol 96:873– 877.
Accepted by— R. H. Dashwood
