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South African Journal of Bota

Antimutagenic activity of Myrtus communis L. using theSalmonella microsome assay

N. Hayder a, I. Skandrani a, S. Kilani a, I. Bouhlel a, A. Abdelwahed a, R. Ben Ammar a,A. Mahmoud a, K. Ghedira a, L. Chekir-Ghedira a,b,⁎

a Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03, Faculté de Pharmacie de Monastir, Rue Avicenne, 5000 Monastir, Tunisiab Laboratoire de Biologie Moléculaire et Cellulaire, Faculté de Médecine Dentaire de Monastir, Rue Avicenne, 5000 Monastir, Tunisia

Received 31 May 2007; received in revised form 29 August 2007; accepted 2 October 2007

Abstract

The mutagenic and antimutagenic activities of hexane, chloroform, ethyl acetate and methanol extracts from leaves of Myrtus communis, wereinvestigated by the Salmonella typhimurium assay. The different extracts showed no mutagenicity when tested with Salmonella typhimuriumstrains TA98 and TA100 either with or without metabolic system (S9). On the other hand, each of the tested extracts exhibited a significantprotective effect against the mutagenicity induced by aflatoxin B1 (AFB1) in Salmonella typhimurium TA100 and TA98 assay systems, andagainst the mutagenicity induced by sodium azide in TA100 and TA1535 assay system. Ethyl acetate and methanol extracts showed the highestlevel of protection towards the direct mutagen, sodium azide, and indirect mutagen AFB1.© 2007 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Antimutagenicity; Mutagenicity; Myrtus communis; Salmonella typhimurium assay

1. Introduction

Since ancient times, several diseases have been treated byadministration of plant extracts based on traditional medicine(Pezzuto, 1997). Myrtus communis (Myrtaceae) a perennialshrub, widely distributed in the Mediterranean area has been usedtraditionally as an antiseptic and disinfectant drug. The essentialoil obtained from the leaves ismainly used in the treatment of lungdisorders (Gauthier et al., 1989) and has been found to possessantibacterial (Chevolleau et al., 1993; Hayder et al., 2003), anti-louse (Lauk et al., 1996) and antioxidant activities (Romani et al.,2004).

Many plant species have been demonstrated to contain in largeamounts endogenous substances with mutagenic (Zani et al., 1991)and antimutagenic properties (Calomme et al., 1996; Choi et al.,1997; Lee et al., 2000; Park et al., 2004; Kilani et al., 2005; Bouhlelet al., 2007; Ben Mansour et al., 2007; Ben Ammar et al., 2007).

⁎ Corresponding author. Laboratoire de Biologie Moléculaire et Cellulaire,Faculté de Médecine Dentaire de Monastir, Rue Avicenne, 5000 Monastir,Tunisia. Tel.: +216 97 316282; fax: +216 73 461150.

E-mail addresses: leila.chekir@laposte.net, l_chekir@yahoo.fr(L. Chekir-Ghedira).

0254-6299/$ - see front matter © 2007 SAAB. Published by Elsevier B.V. All righdoi:10.1016/j.sajb.2007.10.001

In this study, we report the antimutagenic effect of extractsisolated from the leaves of Myrtus communis collected frommountainous regions in Tunisia.

2. Materials and methods

2.1. Plant materials

M. communis var italica was collected from the NationalPark of Boukornine in the north east of Tunisia, in November1998. Identification was carried out by Pr. Chaieb (Departmentof Botany, Faculty of Sciences. University of Sfax), accordingto the flora of Tunisia (Pottier-Alapetite, 1978).

A voucher specimen has been kept in our laboratory forfuture reference (M.c.11-98). The leaves were shade dried,powdered and stored in a tightly closed container for furtheruse.

2.2. Plants extracts

The powdered leaves (200 g) were extracted by Soxhletapparatus for 6 h with successively 1.5 l of each of the followingsolvents, hexane, chloroform, ethyl acetate and methanol to

ts reserved.

122 N. Hayder et al. / South African Journal of Botany 74 (2008) 121–125

obtain the respective extracts. These extracts, with differentpolarities, were concentrated to dryness and the residues werekept at 4 °C. Then, they were resuspended in Dimethyl-Sulfoxide (DMSO). The yields of the extracts were respectively4.6, 0.6, 2.4 and 28.66%.

2.3. Preliminary phytochemical analysis

Plant materials were screened for the presence of tannins,flavonoids and coumarins, using the methods previouslydescribed by Tona et al. (1998, 2004). Two mg of each extractwas separately dissolved in 2 ml of the adequate solvent. Theidentification of major chemical groups was carried out by ThinLayer Chromatography (TLC) on silica gel 60 F254 Merck(layer thickness 0.25 mm) as follows; for flavonoids, TLC wasdeveloped in n-butanol/acetic acid/water 4:1:5 (top layer), spotswere visualized with 1% aluminum chloride solution inmethanol under UV light 366 nm (Harborne, 1974). Coumarinswere detected under UV (366 nm) thanks to their bluefluorescence which becomes intense after spraying 10%potassium hydroxide solution in ethanol. The test for tanninswas carried out with FeCl3. Each class of tannins gave a specificcoloration.

2.4. Bacterial strains

Salmonella typhimurium strains TA100, TA98 and TA1535which are histidine-requiring mutants, were kindly provided byPr. Quillardet, (Institut Pasteur de Paris, France), and maintainedas described by Maron and Ames (1983). The genotypes of thetest strains were checked routinely for their histidine requirement,deep rough (rfa) character, UV sensitivity (uvr B mutation) andpresence of the R factor. They were stored at −80 °C.

S. typhimurium TA98 is frame shift sensitive strain whichcontains, the hisD3052 mutation, as well as the plasmid pKM101. In addition to encoding ampicilline resistance, this plasmidencodes the muc AB genes products which enhance the SOSmutagenesis (Maron and Ames, 1983). S. typhimurium TA1535and TA100 strains contain the same base-pair substitutionmutation hisG46 (Zeiger et al., 1985).

S. typhimurium TA98, TA100 and TA1535 strains are knownto be more responsive to certain mutagens as AFB1 (for TA98and TA100 strains) and sodium azide (for TA100 and TA1535strains) (Maron and Ames, 1983).

2.5. Mutagens

Aflatoxin B1 (AFB1) was purchased from the Sigmachemical CO., St. Louis, (USA) and dissolved in DMSO.Sodium azide was purchased from the Aldrich chemical CO.,Milwaukee, WI, (USA) and dissolved in distilled water.

2.6. Activation mixture

The S9 microsome fraction is prepared from the livers of ratstreated with Aroclor 1254 (Maron and Ames, 1983). Thecomponents of S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM

G6P, 4mMNADP, 0.1M sodium phosphate buffer, pH 7.4, and S9fraction at a concentration of 0.04 ml/ml of mix. The S9 fractionwas stored at −80 °C.

2.7. Mutagenicity test

The test is based on the plate incorporation method (Maronand Ames, 1983), using S. typhimurium test strains TA100 andTA98, with and without an exogenous metabolic activationsystem, the S9 fraction in S9 mix. The test strains from frozencultures were grown overnight for 12–16 h in oxoid nutrientbroth No. 2. Various concentrations of each extract dissolved inDMSO were added to 2 ml of top agar, supplemented with0.5 mM L-histidine and 0.5 mM D-biotine, mixed with 100 μl ofbacterial culture (approximate cell density 2–5×108 cells/ml)and then poured on to a plate containing minimum agarmedium. The plates were incubated at 37 °C for 48 h and thehis+ revertant colonies were counted. The influence ofmetabolic activation was tested by adding 500 μl of S9 mixture.Negative and positive control cultures gave number ofrevertants per plate that were within the normal limits foundin the laboratory. The criteria for a positive response were adoubling for TA98 and TA100 strains and a tripling for TA1535strain, of the relevant solvent control value (Maron and Ames,1983; Marques et al., 2003). Data were collected with a mean±standard deviation of three plates (n=3).

2.8. Antimutagenicity test

A modified plate incorporation procedure (Lee et al., 2000)was employed to determine the effect of all extracts on AFB1and sodium azide-induced mutagenicity. In brief, 0.5 ml of S9mixture for indirect mutagen AFB1 and 0.5 ml of phosphatebuffer for direct mutagen sodium azide was distributed insterilized capped tubes in an ice bath, then 0.1 ml of testcompounds (50 μl of mutagen and/or 50 μl of test compound)and 0.1 ml of bacterial culture (prepared as described inmutagenicity test) were added. After vortexing gently andpreincubating at 37 °C for 45 min, 2 ml of top agarsupplemented with 0.5 mM L-histidine and D-biotine wereadded to each tube and vortexed for 3 s. The resulting entire wasoverlaid on the minimal agar plate. The plates were incubated at37 °C for 48 h and the revertant bacterial colonies on each platewere counted. The inhibition rate of mutagenicity (%) wascalculated relative to the number of revertant colonies in thecontrol group treated with the corresponding mutagen, by thefollowing equation:

Inhibition rate kð Þ¼ ½1� ððnumber of revertants on test plates

�number of spontaneous revertantsÞ=ðnumber of revertants on mutagen control plates

�number of spontaneous revertantsÞÞ� � 100:

Toxicity test for the different levels of samples were alsocarried out, and the sample concentrations employed for

Table 2Mutagenic activity of the Soxhlet extracts from Myrtus communis tested withthe Salmonella typhimurium TA100 and TA 98 assay systems

Treatment Concentrationμg/plate

Revertants per plate±SD

TA 100 TA 98

−S9 +S9 −S9 +S9

Spontaneous – 102±2 102±8 8±3 31±2AFB1 0.3 455±25 359±12Hexane extract 600 103±0 100±3 7±2 31±2

300 106±1 111±3 9±2 31±050 104±2 105±7 8±1 30±3

Chloroform extract 600 100±3 102±8 8±0 39±6300 101±2 105±3 8±1 35±450 101±3 100±8 9±3 34±3

Ethyl acetate extract 600 106±3 103±4 7±2 36±6300 104±1 107±8 8±2 33±350 108±6 100±3 7±1 31±1

Methanol extract 600 105±3 102±4 8±2 38±2300 107±2 100±3 8±0 37±350 108±3 101±0 9±1 34±4

123N. Hayder et al. / South African Journal of Botany 74 (2008) 121–125

antimutagenic test did not show any toxicity to the tester strains.Each dose was tested in triplicate.

3. Results

The preliminary chemical compositions of the tested extractsare shown in Table 1. Methanol extract showed the presence ofhigher quantities of flavonoids and tannins than ethyl acetateextract, whereas they contain almost the same content ofcoumarins. The chloroform extract contains just a weak quantityof flavonoids.

Likewise, previous phytochemical screening of Myrtuscommunis evidenced the presence of flavonoids, tannins andcoumarins (Diaz and Abeger, 1987; Hinou et al., 1988). Nomutagenicity was detected with hexane, chloroform, ethylacetate and methanol extracts, derived from the leaves ofMyrtus communis, when assessed by the Salmonella typhimur-ium assay in the presence of the TA98 and TA100 strains, eitherwith or without the metabolic activation system (S9).

Concentrations of 50, 300 and 600 μg dry weight of eachextract par plate did not increase the revertant number tow-foldabove those obtained in absence of the extract (spontaneousrevertants).

Results of the antimutagenicity assay using AFB1 (0.3 μg/plate with S9) as the mutagenic agent are shown in Table 3.Hexane and chloroform extracts showed a significant but lowerdose-dependent antimutagenic activity, in both Salmonellatyphimurium TA100 and TA98 systems when compared toethyl acetate and methanol extracts. In fact 600 μg/plate of ethylacetate and methanol extracts are able to inhibit totally the AFB1induced mutagenicity in Salmonella typhimurium TA98 assaysystem and by respectively 95 and 93% in TA100 assay system.

Myrtus communis extract antimutagenicity was alsoevaluated against the directly acting sodium azide (1.5 μg/plate) mutagen in both S. typhimurium TA1535 and TA100assay system (Table 4). The results obtained confirm that ethylacetate and methanol extracts are more effective in reducingsodium azide mutagenicity than hexane and chloroformextracts. This difference is more detectable in the presenceof Salmonella typhimurium TA100 assay system.

4. Discussion

Traditional use of plants in alternative medicine frequentlyprovides the basis to select which plant extract it is worthstudying. Extracts of Myrtus communis have been usedextensively in the Mediterranean areas, to treat many types of

Table 1Phytochemical screening of extracts from Myrtus communis

Extract Yield (% W/W) Tannins Flavonoids Coumarins

Hexane extract 4.6 − − −Chloroform extract 0.6 − + −Ethyl acetate extract 2.4 ++ ++ ++Methanol extract 28.66 ++++ ++++ ++

− Absent, + low quantities, ++ high quantities, ++++ very high quantities.

infectious diseases and as antiseptic and anti-inflammatoryagent. These characteristics make them a good therapeuticprospect of study.

Our purpose was to investigate the possible mutagenic andantimutagenic properties of M. communis extracts with theAmes assay. Results obtained with TA98 strain, for frame shiftmutation and TA100 strain for base-pair substituents, showedthat the four tested extracts are not mutagenic, in the Amesassay since even at high concentration it did not induce increasein number of revertants when compared to the number ofspontaneous revertants, either alone or in the presence of S9(Table 2).

The absence of mutagenicity is not a characteristic of allnatural products in use, since other medicinal plants assayedwith the Ames test, with or without the S9, have resultedpositive for mutagenicity (Hyun et al., 1987; Sohni et al., 1995;Anderson et al., 1997).

Results of antimutagenic activities showed that ethanol andethyl acetate extracts were highly effective in reducing themutagenicity caused by the indirect mutagen AFB1. Whereaschloroform and hexane extracts exhibited lower antimutagenicactivity against AFB1 (Table 3). Methanol and ethyl acetateextracts showed stronger antimutagenic activity towardssodium azide in TA100 assay system, when compared tochloroform and hexane extracts tested in the same assay system(Table 4).

Methanol and ethyl acetate extracts exhibited almost thesame level of antimutagenicity towards the directly actingmutagen sodium azide in the TA1535 assay system, whencompared to chloroform and hexane extracts.

As far as, methanol and ethyl acetate extracts contain mainlyphenolic compound including tannins, flavonoids and coumar-ins (Table 1), our results confirm the data previously reported onthe antimutagenic properties of these compounds (Calommeet al., 1996; Okuda et al., 1989; Lee et al., 2000).

Table 3Inhibition of the mutagenicity of AFB1 (0.3 μg/plate), induced in the presence of S9, by the soxhlet extracts obtained from Myrtus communis, in Salmonellatyphimurium TA100 and TA98 assay systems

Treatment Concentrationμg/plate

TA 98 TA 100

Number of revertantsper plate±SD

% inhibition ofmutagenesis

Number of revertantsper plate±SD

% inhibition ofmutagenesis

Spontaneous – 39±7 93±10AFB1 0.3 450±12 415±25Hexane extract 50 327±7 29.92 341±13 22.98

300 225±6 54.74 290±7 38.81600 204±10 60 251±6 51

Chloroform extract 50 368±7 19.95 316±6 30.74300 225±10 54.74 282±7 41.30600 205±6 60 271±7 45

Ethyl acetateextract

50 155±8 71.77 164±7 77.95300 109±3 82.96 132±6 87.88600 38±8 100 108±3 95

Methanol extract 50 183±8 64.96 203±8 65.83300 118±3 80.77 131±6 88.19600 39±10 100 116±3 93

124 N. Hayder et al. / South African Journal of Botany 74 (2008) 121–125

Otherwise, the mutagen AFB1 undergoes an epoxidationprocess by hepatic microsomal cytochrome P450 and successiveepoxide hydrolysis and glutathione conjugation. The epoxide andother intermediates substitute for the nitrogens of nucleic acid inDNA strands and cleave DNA strands to cause mutagenicity.Moreover, several metabolic intermediates and reactive oxygenspecies (ROS) formed during the process of microsomal enzymeactivation are also capable of breaking DNA strands. In contrast,sodium azide requires no activation by hepatic microsomalenzymes to damage DNA and induce mutagenicity. Themethanolic and ethyl acetate extracts exhibited significantantimutagenicity towards the sodium azide-induced mutagenicityas well as towards the AFB-1-induced one. This suggests thatthese extracts may inhibit microsomal enzyme activation or that

Table 4Inhibition of the mutagenicity of sodium azide (1.5 μg/plate), without S9, by the soxhand TA1535 assay systems

Treatment Concentrationμg/plate

TA 1535

Number of revertantsper plate±SD

Spontaneous – 14±1Sodium azide 1.5 140±10Hexane extract 50 92±3

300 69±7600 46±4

Chloroform extract 50 89±9300 83±3600 43±7

Ethyl acetate extract 50 66±7300 63±8600 42±9

Methanol extract 50 58±2300 55±3600 40±7

they may directly protect DNA strands from the electrophilicmetabolites of the mutagen. However, the inhibition ofmutagenesis is often complex, acting through multiple mechan-isms (Kada, 1993). The inhibition of the P 450 mono-oxygenasesystem is known to operate in the antimutagenic effects of someplants extracts (Zani et al., 1993) on the mutagen AFB1 and, wesuspect also, in the antimutagenic activity of the extracts ofMyrtus communis. The results suggest that methanolic and ethylacetate extracts have a protective effect against the mutagenicityinduced by both AFB-1 and sodium azide.

These features make Myrtus communis leaves extractspromising candidates for further studies designed to obtainmore evidence on their components with potential chemopre-ventive activity.

let extracts obtained fromMyrtus communis, in Salmonella typhimurium TA100

TA 100

% inhibition ofmutagenesis

Number of revertantsper plate±SD

% inhibition ofmutagenesis

100±10405±2038.09 315±14 29.5056.34 214±3 62.6275 208±4 6542.85 250±10 50.8145.23 213±7 62.9577 201±7 6758.73 160±7 80.3261.11 132±8 89.5078 123±6 9465.07 157±8 81.3167.46 120±5 93.4479 119±9 92

125N. Hayder et al. / South African Journal of Botany 74 (2008) 121–125

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Edited by J Van Staden