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Anti-genotoxic and free-radical scavenging activities of extracts from (Tunisian) Myrtus communis
Mutation Research 564 (2004) 89–95
Short communication
Anti-genotoxic and free-radical scavenging activities of extracts from (Tunisian) Myrtus communis N. Haydera,c , A. Abdelwaheda,c , S. Kilania,c , R. Ben Ammara,b , A. Mahmoudb , K. Ghedirab , L. Chekir-Ghediraa,c,∗ a
c
Laboratoire de Biochimie et de Biologie Mol´eculaire, Facult´e de Pharmacie de Monastir, Rue Avicenne, 5000 Monastir, Tunisie b Laboratoire de Pharmacognosie, Facult´ e de Pharmacie de Monastir, Rue Avicenne, 5000 Monastir, Tunisie Laboratoire de Biologie Mol´eculaire et Cellulaire, Facult´e de M´edecine Dentaire de Monastir, Rue Avicenne, 5000 Monastir, Tunisie Received 8 January 2004; received in revised form 3 June 2004; accepted 11 August 2004
Abstract The effect of extracts from leaves of Myrtus communis on the SOS reponse induced by Aflatoxin B1 (AFB1) and Nifuroxazide was investigated in a bacterial assay system, i.e. the SOS chromotest with Escherichia coli PQ37. Aqueous extract, the total flavonoids oligomer fraction (TOF), hexane, chloroform, ethyl acetate and methanol extracts and essential oil obtained from M. communis significantly decreased the SOS response induced by AFB1 (10 g/assay) and Nifuroxazide (20 g/assay). Ethyl acetate and methanol extracts showed the strongest inhibition of the induction of the SOS response by the indirectly genotoxic AFB1. The methanol and aqueous extracts exhibited the highest level of protection towards the SOS-induced response by the directly genotoxic Nifuroxazide. In addition to anti-genotoxic activity, the aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed an important free-radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. These results suggest the future utilization of these extracts as additives in chemoprevention studies. © 2004 Elsevier B.V. All rights reserved. Keywords: Myrtus communis; SOS response; Anti-genotoxic effect; Free-radical scavenging
1. Introduction In recent years there has been increasing interest in anti-mutagenesis [1] and anti-oxidant activity [2] of ∗ Corresponding author. Tel.: +216 97 316282; fax: +216 73 461150. E-mail address: [email protected] (L. Chekir-Ghedira).
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.08.001
compounds of plant origin. Such compounds may be useful in preventing cancer and other mutation-related diseases, which can be pursued by avoiding exposures to recognized mutagens and/or carcinogens, by fortifying physiological defense mechanisms, or by favoring the intake of protective factors [3]. In view of several drawbacks of synthetic compounds for the human organism, examination of preparations of plant origin for
90
N. Hayder et al. / Mutation Research 564 (2004) 89–95
this purpose has received increasing attention. Myrtus communis (Myrtaceae) is a perennial shrub, widely distributed in the Mediterranean area. In folk medicine, the leaves are used as a mouthwash for the treatment of candidiasis [4]. The essential oil obtained from the leaves is used mainly in the treatment of lung disorders [4]. In our studies on the elucidation of anti-genotoxic principles, the anti-radical and anti-genotoxic effects of extracts from M. communis collected from mountainous regions in Tunisia were observed.
Essential oil was extracted by steam distillation (European pharmacopoeia, 1997) of dried leaves of M. communis. Hexane, chloroform, ethyl acetate and methanol extracts were obtained by Soxhlet extraction (6 h). These four types of extract, with different polarities, were concentrated to dryness and the residue was kept at 4 ◦ C. 2.3. Preliminary phytochemical analysis
2. Materials and methods
Plant materials were screened for the presence of tannins, flavonoids and coumarins by using the methods previously described by Tona et al. [7].
2.1. Plant materials and chemicals
2.4. Activation mixture
M. communis var italica was collected from the National Park of Boukornine situated in the north-east of Tunisia in November 1998. Identification was carried out by Pr. Chaieb (Department of Botany, Faculty of Sciences, University of Sfax), according to the flora of Tunisia [5]. A voucher specimen has been kept in our laboratory for future reference. The leaves were shade dried, powdered and stored in a tightly closed container for further use. Aflatoxin B1 (AFB) and Nifuroxazide were purchased from Sigma, USA. 1,1-Diphenyl-2picrylhydrazyl (DPPH) was from Aldrich, USA.
The S9 microsome fraction was prepared from the livers of rats treated with Aroclor 1254 [8]. The composition of the activation mixture was (per 10 ml of S9 mix): salt solution (1.65 M KCl + 0.4 M MgCl2 ·6H2 O), 0.2 ml; G6P (1M), 0.05 ml; NADP (0.1 M), 0.15 ml; Tris buffer (tris(hydroxymethyl)aminomethane) (0.4 M pH 7.4), 2.5 ml; Luria broth medium, 6.1 ml; S9, 1 ml.
2.2. Extraction method The powdered leaves were extracted with boiling water for 15–20 min. After filtration, the extracts were filtered and lyophilised (aqueous extract). The residues were dissolved in water and filter-sterilized through a 45 m millipore filter. In order to obtain an extract enriched in total flavonoid oligomers (TOF), the powdered leaves were macerated in a water/acetone mixture (1:2), during 24 h with continuous stirring. The extract was filtered and the acetone was evaporated under low pressure in order to obtain an aqueous phase. Tannins were removed by precipitation with an excess of NaCl during 24 h at 5 ◦ C, and the supernatant was recovered. The latter was extracted with ethyl acetate, concentrated and precipitated with an excess of chloroform. The precipitate was separated and yielded the TOF extract.
2.5. Anti-genotoxicity assay Inhibition of bacterial genotoxicity was tested in Escherichia coli strain PQ37, which was kindly provided by Dr. Quillardet, (Institut Pasteur, Paris, France). The procedure described by Quillardet and Hofnung [6] was followed. The extracts was dissolved in dimethylsulfoxide and tested in triplicate, with and without exogenous metabolic activation. As a measure of anti-genotoxicity, the SOS-inducing potency (SOSIP) was calculated from the linear part of the induction factor–dose-response curve. The induction factor (IF) was calculated as the ratio of Rc /R0 , where Rc is equal to -gal activity/AP activity determined for the test compound at concentration c, and R0 is equal to -gal activity/AP activity in the absence of the test compound. The -gal and AP activities were calculated according to the method recommended by Quillardet and Hofnung [6]. Anti-genotoxicity was expressed as percentage inhibition of genotoxicity according to the formula: inhibition (%) = 100 − (IF1 − IF0 /IF2 − IF0 ) × 100, where IF1 is the induction factor in the presence of the test compound, IF2 the induction factor in the absence
N. Hayder et al. / Mutation Research 564 (2004) 89–95
of the test compound and IF0 the induction factor of the negative control. Data were collected with a mean ± standard deviation of three independent experiments, and analysed for statistical significance using the Dunett test. 2.6. Radical-scavenging activity on DPPH The free-radical scavenging capacity of the compounds tested was determined with 1,1-diphenyl-2picryl-hydrazyl. An aliquot of each tested compound at various concentrations (100, 30, 10, 3 or 1 mg/ml in ethanol) was mixed with 23.6 g/ml of DPPH solution in ethanol. After incubation of the mixture for 30 min, the absorbance of the remaining DPPH was determined colorimetrically at 517 nm. The scavenging activities were expressed as a percentage of the absorbance of the control DPPH solution [2]. The results are expressed as mean of at least three independent experiments. Results were expressed as percentage activity. Mean inhibiting concentrations IC50 were calculated by use of the Litchifield & Wilcoxon test [9].
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3.2. Anti-genotoxicity assay As shown in Tables 2 and 3, all extracts from M. communis were effective in reducing the IF induced by Aflatoxin B1 (10 g per assay with S9; indirect-acting genotoxicant), as well as by Nifuroxazide (20 g per assay without S9: direct-acting genotoxicant). The aqueous extract, the TOF, and the ethyl acetate and methanol extracts were more effectively antigenotoxic than the hexane and chloroform extracts: at a concentration of 600 g per assay these extracts significantly decreased the SOSIP of AFB1 by respectively 96%, 95%, 99% and 99%. At the same concentration of extracts, the SOSIP of Nifuroxazide decreased about respectively 96%, 95%, 94% and 96%. However, the hexane and chloroform extracts showed a weaker antigenotoxic activity when 600 g per assay of each were added to the assay system. The SOSIP of Nifuroxazide was decreased only by, respectively, 57% and 60% and that of AFB1 decreased by 73%. The essential oil showed weak anti-genotoxic activity. The SOSIP of Nifuroxazide decreased by 63% when 400 g was added to the assay system, while the SOSIP of AFB1 decreased by only 17% under these conditions (Tables 2 and 3).
3. Results
3.3. Radical-scavenging activity on DPPH
3.1. Phytochemical study
DPPH is a molecule containing a stable free radical. In the presence of an electron-donating anti-oxidant, the purple color typical of the free DPPH radical diminishes in intensity, a change that can be followed spectrophotometrically at 517 nm. This simple test provides information on the ability of a compound to donate an electron, on the number of electrons a given molecule can donate, and on the mechanism of anti-oxidant action. The radical-scavenging activities of the extracts, measured as decolorizing activity following the trap-
The results of our assay on the tested crude extracts are shown in Table 1. The aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed the presence of various quantities of flavonoids, coumarins and tannins. In contrast, the chloroform extract contained just a small quantity of flavonoids. Previous phytochemical screening of M. communis [10,11] had also demonstrated the presence of flavonoids, tannins and coumarins. Table 1 Phytochemical screening of extracts from M. communis Extract
Aqueous extract
TOF extract
Hexane extract
Chloroform extract
Ethyl acetate extract
Methanol extract
Tannins Flavonoids Coumarins
++ ++ ++
++ ++++ ++++
– – –
– + –
++ ++ ++
++ ++++ ++
–: Absent; +: low quantity; ++: high quantity; ++++: very high quantity.
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N. Hayder et al. / Mutation Research 564 (2004) 89–95
Table 2 Effect of extracts on genotoxicity induced by Aflatoxin B1 (10 g/assay) -gal (U)
Ap (U)
IF
10
8.6 ± 0.001
8.6 ± 0.003
7.19
0
Aqueous extract
0 50 300 600
2.06 ± 0.001 5.3 ± 0.005 3.12 ± 0.002 2.52 ± 0.001
14.8 ± 0.001 10.2 ± 0.001 14 ± 0.01 14 ± 0.001
1 3.73* 1.60* 1.29*
0 67 93 96
TOF extract
0 50 300 600
2.06 ± 0.001 6.62 ± 0.001 6 ± 0.005 4.5 ± 0
14.8 ± 0.001 12.26 ± 0.001 12.93 ± 0.001 13.66 ± 0.005
1 3.88* 3.33* 1.3*
0 53 62 95
Essential oil
0 40 100 400
2.06 ± 0.001 8.6 ± 0 8.5 ± 0 7.55 ± 0.004
14.8 ± 0.001 9 ± 0.01 8.6 ± 0.001 8.86 ± 0.003
1 6.8 7.18 6.13
0 6 0 17
Hexane extract
0 50 300 600
2.06 ± 0.001 2.57 ± 0.001 3.07 ± 0.001 3.85 ± 0.001
14.8 ± 0.001 4.7 ± 0.001 7.85 ± 0.001 10.26 ± 0.001
1 3.87* 2.81* 2.69*
0 54 71 73
Chloroform extract
0 50 300 600
2.06 ± 0.001 3.87 ± 0.001 4.45 ± 0.1 4.55 ± 0.05
14.8 ± 0.001 11.06 ± 0.001 12 ± 0 12.13 ± 0.001
1 2.51* 2.66* 2.65*
0 76 73 73
Ethyl acetate extract
0 50 300 600
2.06 ± 0.001 3.02 ± 0.001 2.82 ± 0 3.35 ± 0.001
14.8 ± 0.001 18 ± 26 18 ± 66 17 ± 66
1 1.36* 1.08* 1.06*
0 94 99 99
Methanol extract
0 50 300 600
2.06 ± 0.001 3.02 ± 0.001 2.6 ± 0.001 2.77 ± 0.002
14.8 ± 0.001 7.73 ± 0 17.6 ± 0.004 18.66 ± 0
1 1.22* 1.06* 1.06*
0 94 99 99
Extract Aflatoxin B1
∗
Dose (g/assay)
Inhibition of genotoxicity (%)
P < 0.05.
ping of the unpaired electron of DPPH, are shown in Table 4. The aqueous extract is a very potent radical scavenger with a percentage decrease versus the absorbance of DPPH standard solution of 99% at a concentration of 100 g/ml and an IC50 value of 1.9 g/ml, which makes it more active than ␣-tocopherol. The TOF, and the ethyl acetate and methanol extracts showed scavenging activities with a percentage decrease versus the absorbance of the DPPH standard solution of 76.6%, 92.3% and 94.6% at a concentration of 100 g/ml and IC50 values of 3, 5.2 and 6.5 g/ml, respectively. These results indicate that the aqueous extract, the TOF, and the ethyl acetate and methanol extracts show considerable anti-oxidant activity, as mea-
sured by their capacity to scavenge the stable free radical DPPH.
4. Discussion Aqueous, methanol and ethyl acetate extracts from M. communis exhibit a strong binding activity toward the free radical DPPH. In the SOS chromotest, the same extracts strongly inhibited the genotoxicity of both AFB1 and Nifuroxazide. The inhibitory effect of these extracts on the genotoxicity of AFB1 may be attributed to flavonoids [1], coumarins [12] and/or tannins [13]. This is supported by the weak anti-genotoxic
N. Hayder et al. / Mutation Research 564 (2004) 89–95
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Table 3 Effect of extracts on genotoxicity induced by Nifuroxazide (20 g/assay) -gal (U)
Ap (U)
IF
20
20.5 ± 0.01
10.68 ± 0.005
10.66
Aqueous extract
0 50 300 600
1.98 ± 0.001 9.7 ± 0.001 1.78 ± 0.001 2.71 ± 0.001
10.84 ± 0.001 10.72 ± 0.05 10.92 ± 0.01 10.92 ± 0.003
1 5.02* 3.51* 1.37*
0 52 74 96
TOF extract
0 50 300 600
1.98 ± 0.001 10.25 ± 0.003 3.82 ± 0 3.06 ± 0.006
10.84 ± 0.001 10.92 ± 0.003 10.96 ± 0 11.08 ± 0.006
1 5.2* 1.93* 1.53*
0 57 90 95
Essential oil
0 40 100 400
1.98 ± 0.001 18.15 ± 0.005 14.5 ± 0.003 9.1 ± 0.001
10.84 ± 0.001 11 ± 0.005 10.92 ± 0.003 10.84 ± 0.001
1 9.16 7.37 4.61*
0 16 34 63
Hexane extract
0 50 300 600
1.98 ± 0.001 15 ± 0 10.2 ± 0 10 ± 0.001
10.84 ± 0.001 10.88 ± 0 11 ± 0.002 10.8 ± 0.005
1 7.61* 5.15* 5.14*
0 32 57 57
Chloroform extract
0 50 300 600
1.98 ± 0.001 11 ± 0.01 10.2 ± 0.002 9.7 ± 0.001
10.84 ± 0.001 11.04 ± 0.001 10.8 ± 0.01 11.08 ± 0
1 5.53* 5.24* 4.86*
0 53 56 60
Ethyl acetate extract
0 50 300 600
1.98 ± 0.001 6.45 ± 0.005 4.13 ± 0.001 3.05 ± 0.1
10.84 ± 0.001 10.92 ± 0.01 11.04 ± 0.001 11 ± 0.001
1 3.28* 2.07* 1.54*
0 76 89 94
Methanol extract
0 50 300 600
1.98 ± 0.001 5.06 ± 0.002 3.35 ± 0.001 2.73 ± 0
10.84 ± 0.001 11.04 ± 0.002 10.92 ± 0.003 10.92 ± 0.001
1 2.54* 1.70* 1.39*
0 84 93 96
Extract Nifuroxazide
∗
Dose (g/assay)
Inhibition of genotoxicity (%) 0
P < 0.05.
activities obtained with chloroform and hexane extracts reported in this study, in so far as these two extracts contain neither flavonoids nor tannins and coumarins, as shown by chemical analysis. We cannot however, exclude the possibility that other compounds with antigenotoxic properties participate in the inhibitory effect. The results of our experiments confirm the known anti-oxidant activities of flavonoids [14,15] and tannins [16]. Flavonoids are the most likely candidates among the compounds known to be present in the aqueous extract, the TOF, and the ethyl acetate and methanol extracts, for providing the anti-mutagenic effect and preventing oxidative lesions [17].
The aqueous, methanol and ethyl acetate extracts showed significant anti-genotoxic activity in the assays with Nifuroxazide and AFB. This suggests that these extracts inhibit microsomal enzyme activation or that they directly protect DNA strands from the electrophilic metabolite of the mutagens. However, the inhibition of genotoxicity and mutagenesis is often complex, acting through multiple mechanisms [18]. The inhibition of the P450 mono-oxygenase system is known to play a role in the anti-mutagenic effects of some plant extracts [19] on the mutagenicity of AFB1 and, as we suspect also, in the anti-mutagenic activity of the extracts of M. communis. In summary, M. com-
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Table 4 Anti-oxidant activities on scavenging the DPPH free radical of extracts from M. communis Extract
Concentration (g/ml)
Inhibition (%)a
Aqueous extract
1 3 10 30 100
41 ± 2.1 57 ± 1 95.6 ± 0.9 97 ± 1.1 99 ± 0.6
1.9
TOF extract
1 3 10 30 100
23.3 ± 1.8 50 ± 1.5 53.3 ± 1.2 60 ± 0.9 76.6 ± 1.2
3
Methanol extract
1 3 10 30 100
13.3 ± 3 20 ± 1.3 90 ± 2.4 94 ± 2.1 94.6 ± 1.1
6.5
Ethyl acetate extract
1 3 10 30 100
27.7 ± 1.3 42 ± 2.5 72 ± 3 91.3 ± 2.1 92.3 ± 1.2
5.2
Essential oil
1 3 10 30 100
0 0 0 0 16.6 ± 2
< 100
Hexane extract
1 3 10 30 100
0 0 0 0 33.3 ± 2.1
< 100
Chloroform extract
1 3 10 30 100
0 0 0 0 20 ± 2
< 100
␣-Tocophenol
1 3 10 30 100
30 ± 2.1 50 ± 1.3 97.3 ± 1.8 98 ± 1.3 98.7 ± 2.2
and to determine the exact mechanism of action, are required.
IC50 (g/ml)
3.1
a Inhibition of absorbance at 517 nm relative to that of standard DPPH solution.
munis extracts and their components could be suitable anti-genotoxic, anti-mutagenic and perhaps anticarcinogenic agents, but further studies to fractionate the active extracts, to identify the active compounds
References [1] M. Calomm, L. Pieters, A. Vlietink, D.V. Berghe, Inhibition of bacterial mutagenesis flavono¨ıds, Planta Medica 62 (1996) 222–226. [2] A. Yagi, A. Kabash, N. Okamura, H. Haraguchi, S.M. Moustafa, T.I. Khalifa, Antioxidant, free radical scavenging and antiinflammatory effects of aloesin derivatives in Aloe vera, Planta Medica 68 (2002) 957–960. [3] S. De Flora, Mechanisms of inhibitors of mutagenesis and carcinogenesis, Mut. Res. 420 (1998) 151–158. [4] L. Bezanger-Bequese, M. Pinkas, M. Torch, A. Maloine (Eds.), Les plantes dans la th´erapeutique moderne, Paris, 1975. [5] G. Pottier-Alaptit, Flore de la Tunisie, Imprimerie officielle de la r´epublique tunisienne, Tunisie, 1997. [6] P.Q. Quillardet, M. Hofnung, The SOS chromotest colorimetric bacterial assay for genotoxins: procedures, Mut. Res. 147 (1985) 65–78. [7] L. Tona, K. Kambu, N. Ngimbi, K. Cimanga, A.J. Vlietnick, Antiamoebic and phytochemical screening of some Congolese medical plants, J. Ethnopharmacol. 61 (1998) 57–65. [8] D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mut. Res. 113 (1983) 173–215. [9] E.M. Galati, N. Miceli, M.F. Taviano, R. Sanogo, E. Raneri, Anti-inflamatory and antioxidant activity of Ageratum conyzoides, Pharm. Biol. 39 (2001) 336–339. [10] A.M. Diaz, A. Abeger, Myrtus communis, composicion quimica y actividad biologica de sus extractos, Una revision, Fitoterapia 8 (1987) 167–174. [11] J. Hinou, N. Lakkas, S. Philianos, Les constituants polyph´enoliques de Myrtus communis L, Plante M´edicinales et Phytot´erapies 22 (1988) 98–103. [12] K.T. Lee, I.C. Sohn, H.J. Park, D.W. Kim, G.O. Jung, K.Y. Park, Essential moiety of antimutagenic and cytotoxic activity of heeder agenin monodesmosides and bidesmosides isolated from the stem bark of Kalopanox pictus, Planta Medica 66 (2000) 329–332. [13] O. Schimmer, M. Lindenbaum, Tannins antimutagenic properties in the herb of Alchemilla species and Potentilla anserine, Planta Medica 47 (1992) 466–467. [14] J. Vaya, S. Mohmod, A. Goldblum, M. Aviram, N. Volkavor, A. Shaalam, R. Musa, S. Tamir, Inhibition of LDL oxidation by flavono¨ıds in relation to their structure and calculated enthalpy, Phytochemistry 62 (2003) 89–99. [15] K.-Y. Park, G.-O. Jung, K.-T. Lee, J. Choi, M.-Y. Choi, G.-T. Kim, H.-J. Jung, H.-J. Park, Antimutagenic activity of flavonoids from the heartwood of Rhus verniciflua, J. Ethnopharmacol. 90 (2004) 73–79. [16] T. Yokozozawa, C.P. Chen, E. Dong, T. Tanaka, G.I. Nishioka, Study on the inhibitiory effect of tannins and flavono¨ıds against DPPH radical, Biochem. Pharmacol. 56 (1998) 213–222.
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[19] F. Zani, M.T. Cuzzoni, M. Dglia, S. Benvenuti, P. Mazza, Inhibition of mutagenicity in Salmonella typhimyrium by Glycyrrhiza globra extract, glycyrrhizinic acid, 18␣- and 18-glycyrrhetinic acids, Planta Medica 59 (1993) 502–507.
Short communication
Anti-genotoxic and free-radical scavenging activities of extracts from (Tunisian) Myrtus communis N. Haydera,c , A. Abdelwaheda,c , S. Kilania,c , R. Ben Ammara,b , A. Mahmoudb , K. Ghedirab , L. Chekir-Ghediraa,c,∗ a
c
Laboratoire de Biochimie et de Biologie Mol´eculaire, Facult´e de Pharmacie de Monastir, Rue Avicenne, 5000 Monastir, Tunisie b Laboratoire de Pharmacognosie, Facult´ e de Pharmacie de Monastir, Rue Avicenne, 5000 Monastir, Tunisie Laboratoire de Biologie Mol´eculaire et Cellulaire, Facult´e de M´edecine Dentaire de Monastir, Rue Avicenne, 5000 Monastir, Tunisie Received 8 January 2004; received in revised form 3 June 2004; accepted 11 August 2004
Abstract The effect of extracts from leaves of Myrtus communis on the SOS reponse induced by Aflatoxin B1 (AFB1) and Nifuroxazide was investigated in a bacterial assay system, i.e. the SOS chromotest with Escherichia coli PQ37. Aqueous extract, the total flavonoids oligomer fraction (TOF), hexane, chloroform, ethyl acetate and methanol extracts and essential oil obtained from M. communis significantly decreased the SOS response induced by AFB1 (10 g/assay) and Nifuroxazide (20 g/assay). Ethyl acetate and methanol extracts showed the strongest inhibition of the induction of the SOS response by the indirectly genotoxic AFB1. The methanol and aqueous extracts exhibited the highest level of protection towards the SOS-induced response by the directly genotoxic Nifuroxazide. In addition to anti-genotoxic activity, the aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed an important free-radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. These results suggest the future utilization of these extracts as additives in chemoprevention studies. © 2004 Elsevier B.V. All rights reserved. Keywords: Myrtus communis; SOS response; Anti-genotoxic effect; Free-radical scavenging
1. Introduction In recent years there has been increasing interest in anti-mutagenesis [1] and anti-oxidant activity [2] of ∗ Corresponding author. Tel.: +216 97 316282; fax: +216 73 461150. E-mail address: [email protected] (L. Chekir-Ghedira).
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.08.001
compounds of plant origin. Such compounds may be useful in preventing cancer and other mutation-related diseases, which can be pursued by avoiding exposures to recognized mutagens and/or carcinogens, by fortifying physiological defense mechanisms, or by favoring the intake of protective factors [3]. In view of several drawbacks of synthetic compounds for the human organism, examination of preparations of plant origin for
90
N. Hayder et al. / Mutation Research 564 (2004) 89–95
this purpose has received increasing attention. Myrtus communis (Myrtaceae) is a perennial shrub, widely distributed in the Mediterranean area. In folk medicine, the leaves are used as a mouthwash for the treatment of candidiasis [4]. The essential oil obtained from the leaves is used mainly in the treatment of lung disorders [4]. In our studies on the elucidation of anti-genotoxic principles, the anti-radical and anti-genotoxic effects of extracts from M. communis collected from mountainous regions in Tunisia were observed.
Essential oil was extracted by steam distillation (European pharmacopoeia, 1997) of dried leaves of M. communis. Hexane, chloroform, ethyl acetate and methanol extracts were obtained by Soxhlet extraction (6 h). These four types of extract, with different polarities, were concentrated to dryness and the residue was kept at 4 ◦ C. 2.3. Preliminary phytochemical analysis
2. Materials and methods
Plant materials were screened for the presence of tannins, flavonoids and coumarins by using the methods previously described by Tona et al. [7].
2.1. Plant materials and chemicals
2.4. Activation mixture
M. communis var italica was collected from the National Park of Boukornine situated in the north-east of Tunisia in November 1998. Identification was carried out by Pr. Chaieb (Department of Botany, Faculty of Sciences, University of Sfax), according to the flora of Tunisia [5]. A voucher specimen has been kept in our laboratory for future reference. The leaves were shade dried, powdered and stored in a tightly closed container for further use. Aflatoxin B1 (AFB) and Nifuroxazide were purchased from Sigma, USA. 1,1-Diphenyl-2picrylhydrazyl (DPPH) was from Aldrich, USA.
The S9 microsome fraction was prepared from the livers of rats treated with Aroclor 1254 [8]. The composition of the activation mixture was (per 10 ml of S9 mix): salt solution (1.65 M KCl + 0.4 M MgCl2 ·6H2 O), 0.2 ml; G6P (1M), 0.05 ml; NADP (0.1 M), 0.15 ml; Tris buffer (tris(hydroxymethyl)aminomethane) (0.4 M pH 7.4), 2.5 ml; Luria broth medium, 6.1 ml; S9, 1 ml.
2.2. Extraction method The powdered leaves were extracted with boiling water for 15–20 min. After filtration, the extracts were filtered and lyophilised (aqueous extract). The residues were dissolved in water and filter-sterilized through a 45 m millipore filter. In order to obtain an extract enriched in total flavonoid oligomers (TOF), the powdered leaves were macerated in a water/acetone mixture (1:2), during 24 h with continuous stirring. The extract was filtered and the acetone was evaporated under low pressure in order to obtain an aqueous phase. Tannins were removed by precipitation with an excess of NaCl during 24 h at 5 ◦ C, and the supernatant was recovered. The latter was extracted with ethyl acetate, concentrated and precipitated with an excess of chloroform. The precipitate was separated and yielded the TOF extract.
2.5. Anti-genotoxicity assay Inhibition of bacterial genotoxicity was tested in Escherichia coli strain PQ37, which was kindly provided by Dr. Quillardet, (Institut Pasteur, Paris, France). The procedure described by Quillardet and Hofnung [6] was followed. The extracts was dissolved in dimethylsulfoxide and tested in triplicate, with and without exogenous metabolic activation. As a measure of anti-genotoxicity, the SOS-inducing potency (SOSIP) was calculated from the linear part of the induction factor–dose-response curve. The induction factor (IF) was calculated as the ratio of Rc /R0 , where Rc is equal to -gal activity/AP activity determined for the test compound at concentration c, and R0 is equal to -gal activity/AP activity in the absence of the test compound. The -gal and AP activities were calculated according to the method recommended by Quillardet and Hofnung [6]. Anti-genotoxicity was expressed as percentage inhibition of genotoxicity according to the formula: inhibition (%) = 100 − (IF1 − IF0 /IF2 − IF0 ) × 100, where IF1 is the induction factor in the presence of the test compound, IF2 the induction factor in the absence
N. Hayder et al. / Mutation Research 564 (2004) 89–95
of the test compound and IF0 the induction factor of the negative control. Data were collected with a mean ± standard deviation of three independent experiments, and analysed for statistical significance using the Dunett test. 2.6. Radical-scavenging activity on DPPH The free-radical scavenging capacity of the compounds tested was determined with 1,1-diphenyl-2picryl-hydrazyl. An aliquot of each tested compound at various concentrations (100, 30, 10, 3 or 1 mg/ml in ethanol) was mixed with 23.6 g/ml of DPPH solution in ethanol. After incubation of the mixture for 30 min, the absorbance of the remaining DPPH was determined colorimetrically at 517 nm. The scavenging activities were expressed as a percentage of the absorbance of the control DPPH solution [2]. The results are expressed as mean of at least three independent experiments. Results were expressed as percentage activity. Mean inhibiting concentrations IC50 were calculated by use of the Litchifield & Wilcoxon test [9].
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3.2. Anti-genotoxicity assay As shown in Tables 2 and 3, all extracts from M. communis were effective in reducing the IF induced by Aflatoxin B1 (10 g per assay with S9; indirect-acting genotoxicant), as well as by Nifuroxazide (20 g per assay without S9: direct-acting genotoxicant). The aqueous extract, the TOF, and the ethyl acetate and methanol extracts were more effectively antigenotoxic than the hexane and chloroform extracts: at a concentration of 600 g per assay these extracts significantly decreased the SOSIP of AFB1 by respectively 96%, 95%, 99% and 99%. At the same concentration of extracts, the SOSIP of Nifuroxazide decreased about respectively 96%, 95%, 94% and 96%. However, the hexane and chloroform extracts showed a weaker antigenotoxic activity when 600 g per assay of each were added to the assay system. The SOSIP of Nifuroxazide was decreased only by, respectively, 57% and 60% and that of AFB1 decreased by 73%. The essential oil showed weak anti-genotoxic activity. The SOSIP of Nifuroxazide decreased by 63% when 400 g was added to the assay system, while the SOSIP of AFB1 decreased by only 17% under these conditions (Tables 2 and 3).
3. Results
3.3. Radical-scavenging activity on DPPH
3.1. Phytochemical study
DPPH is a molecule containing a stable free radical. In the presence of an electron-donating anti-oxidant, the purple color typical of the free DPPH radical diminishes in intensity, a change that can be followed spectrophotometrically at 517 nm. This simple test provides information on the ability of a compound to donate an electron, on the number of electrons a given molecule can donate, and on the mechanism of anti-oxidant action. The radical-scavenging activities of the extracts, measured as decolorizing activity following the trap-
The results of our assay on the tested crude extracts are shown in Table 1. The aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed the presence of various quantities of flavonoids, coumarins and tannins. In contrast, the chloroform extract contained just a small quantity of flavonoids. Previous phytochemical screening of M. communis [10,11] had also demonstrated the presence of flavonoids, tannins and coumarins. Table 1 Phytochemical screening of extracts from M. communis Extract
Aqueous extract
TOF extract
Hexane extract
Chloroform extract
Ethyl acetate extract
Methanol extract
Tannins Flavonoids Coumarins
++ ++ ++
++ ++++ ++++
– – –
– + –
++ ++ ++
++ ++++ ++
–: Absent; +: low quantity; ++: high quantity; ++++: very high quantity.
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N. Hayder et al. / Mutation Research 564 (2004) 89–95
Table 2 Effect of extracts on genotoxicity induced by Aflatoxin B1 (10 g/assay) -gal (U)
Ap (U)
IF
10
8.6 ± 0.001
8.6 ± 0.003
7.19
0
Aqueous extract
0 50 300 600
2.06 ± 0.001 5.3 ± 0.005 3.12 ± 0.002 2.52 ± 0.001
14.8 ± 0.001 10.2 ± 0.001 14 ± 0.01 14 ± 0.001
1 3.73* 1.60* 1.29*
0 67 93 96
TOF extract
0 50 300 600
2.06 ± 0.001 6.62 ± 0.001 6 ± 0.005 4.5 ± 0
14.8 ± 0.001 12.26 ± 0.001 12.93 ± 0.001 13.66 ± 0.005
1 3.88* 3.33* 1.3*
0 53 62 95
Essential oil
0 40 100 400
2.06 ± 0.001 8.6 ± 0 8.5 ± 0 7.55 ± 0.004
14.8 ± 0.001 9 ± 0.01 8.6 ± 0.001 8.86 ± 0.003
1 6.8 7.18 6.13
0 6 0 17
Hexane extract
0 50 300 600
2.06 ± 0.001 2.57 ± 0.001 3.07 ± 0.001 3.85 ± 0.001
14.8 ± 0.001 4.7 ± 0.001 7.85 ± 0.001 10.26 ± 0.001
1 3.87* 2.81* 2.69*
0 54 71 73
Chloroform extract
0 50 300 600
2.06 ± 0.001 3.87 ± 0.001 4.45 ± 0.1 4.55 ± 0.05
14.8 ± 0.001 11.06 ± 0.001 12 ± 0 12.13 ± 0.001
1 2.51* 2.66* 2.65*
0 76 73 73
Ethyl acetate extract
0 50 300 600
2.06 ± 0.001 3.02 ± 0.001 2.82 ± 0 3.35 ± 0.001
14.8 ± 0.001 18 ± 26 18 ± 66 17 ± 66
1 1.36* 1.08* 1.06*
0 94 99 99
Methanol extract
0 50 300 600
2.06 ± 0.001 3.02 ± 0.001 2.6 ± 0.001 2.77 ± 0.002
14.8 ± 0.001 7.73 ± 0 17.6 ± 0.004 18.66 ± 0
1 1.22* 1.06* 1.06*
0 94 99 99
Extract Aflatoxin B1
∗
Dose (g/assay)
Inhibition of genotoxicity (%)
P < 0.05.
ping of the unpaired electron of DPPH, are shown in Table 4. The aqueous extract is a very potent radical scavenger with a percentage decrease versus the absorbance of DPPH standard solution of 99% at a concentration of 100 g/ml and an IC50 value of 1.9 g/ml, which makes it more active than ␣-tocopherol. The TOF, and the ethyl acetate and methanol extracts showed scavenging activities with a percentage decrease versus the absorbance of the DPPH standard solution of 76.6%, 92.3% and 94.6% at a concentration of 100 g/ml and IC50 values of 3, 5.2 and 6.5 g/ml, respectively. These results indicate that the aqueous extract, the TOF, and the ethyl acetate and methanol extracts show considerable anti-oxidant activity, as mea-
sured by their capacity to scavenge the stable free radical DPPH.
4. Discussion Aqueous, methanol and ethyl acetate extracts from M. communis exhibit a strong binding activity toward the free radical DPPH. In the SOS chromotest, the same extracts strongly inhibited the genotoxicity of both AFB1 and Nifuroxazide. The inhibitory effect of these extracts on the genotoxicity of AFB1 may be attributed to flavonoids [1], coumarins [12] and/or tannins [13]. This is supported by the weak anti-genotoxic
N. Hayder et al. / Mutation Research 564 (2004) 89–95
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Table 3 Effect of extracts on genotoxicity induced by Nifuroxazide (20 g/assay) -gal (U)
Ap (U)
IF
20
20.5 ± 0.01
10.68 ± 0.005
10.66
Aqueous extract
0 50 300 600
1.98 ± 0.001 9.7 ± 0.001 1.78 ± 0.001 2.71 ± 0.001
10.84 ± 0.001 10.72 ± 0.05 10.92 ± 0.01 10.92 ± 0.003
1 5.02* 3.51* 1.37*
0 52 74 96
TOF extract
0 50 300 600
1.98 ± 0.001 10.25 ± 0.003 3.82 ± 0 3.06 ± 0.006
10.84 ± 0.001 10.92 ± 0.003 10.96 ± 0 11.08 ± 0.006
1 5.2* 1.93* 1.53*
0 57 90 95
Essential oil
0 40 100 400
1.98 ± 0.001 18.15 ± 0.005 14.5 ± 0.003 9.1 ± 0.001
10.84 ± 0.001 11 ± 0.005 10.92 ± 0.003 10.84 ± 0.001
1 9.16 7.37 4.61*
0 16 34 63
Hexane extract
0 50 300 600
1.98 ± 0.001 15 ± 0 10.2 ± 0 10 ± 0.001
10.84 ± 0.001 10.88 ± 0 11 ± 0.002 10.8 ± 0.005
1 7.61* 5.15* 5.14*
0 32 57 57
Chloroform extract
0 50 300 600
1.98 ± 0.001 11 ± 0.01 10.2 ± 0.002 9.7 ± 0.001
10.84 ± 0.001 11.04 ± 0.001 10.8 ± 0.01 11.08 ± 0
1 5.53* 5.24* 4.86*
0 53 56 60
Ethyl acetate extract
0 50 300 600
1.98 ± 0.001 6.45 ± 0.005 4.13 ± 0.001 3.05 ± 0.1
10.84 ± 0.001 10.92 ± 0.01 11.04 ± 0.001 11 ± 0.001
1 3.28* 2.07* 1.54*
0 76 89 94
Methanol extract
0 50 300 600
1.98 ± 0.001 5.06 ± 0.002 3.35 ± 0.001 2.73 ± 0
10.84 ± 0.001 11.04 ± 0.002 10.92 ± 0.003 10.92 ± 0.001
1 2.54* 1.70* 1.39*
0 84 93 96
Extract Nifuroxazide
∗
Dose (g/assay)
Inhibition of genotoxicity (%) 0
P < 0.05.
activities obtained with chloroform and hexane extracts reported in this study, in so far as these two extracts contain neither flavonoids nor tannins and coumarins, as shown by chemical analysis. We cannot however, exclude the possibility that other compounds with antigenotoxic properties participate in the inhibitory effect. The results of our experiments confirm the known anti-oxidant activities of flavonoids [14,15] and tannins [16]. Flavonoids are the most likely candidates among the compounds known to be present in the aqueous extract, the TOF, and the ethyl acetate and methanol extracts, for providing the anti-mutagenic effect and preventing oxidative lesions [17].
The aqueous, methanol and ethyl acetate extracts showed significant anti-genotoxic activity in the assays with Nifuroxazide and AFB. This suggests that these extracts inhibit microsomal enzyme activation or that they directly protect DNA strands from the electrophilic metabolite of the mutagens. However, the inhibition of genotoxicity and mutagenesis is often complex, acting through multiple mechanisms [18]. The inhibition of the P450 mono-oxygenase system is known to play a role in the anti-mutagenic effects of some plant extracts [19] on the mutagenicity of AFB1 and, as we suspect also, in the anti-mutagenic activity of the extracts of M. communis. In summary, M. com-
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Table 4 Anti-oxidant activities on scavenging the DPPH free radical of extracts from M. communis Extract
Concentration (g/ml)
Inhibition (%)a
Aqueous extract
1 3 10 30 100
41 ± 2.1 57 ± 1 95.6 ± 0.9 97 ± 1.1 99 ± 0.6
1.9
TOF extract
1 3 10 30 100
23.3 ± 1.8 50 ± 1.5 53.3 ± 1.2 60 ± 0.9 76.6 ± 1.2
3
Methanol extract
1 3 10 30 100
13.3 ± 3 20 ± 1.3 90 ± 2.4 94 ± 2.1 94.6 ± 1.1
6.5
Ethyl acetate extract
1 3 10 30 100
27.7 ± 1.3 42 ± 2.5 72 ± 3 91.3 ± 2.1 92.3 ± 1.2
5.2
Essential oil
1 3 10 30 100
0 0 0 0 16.6 ± 2
< 100
Hexane extract
1 3 10 30 100
0 0 0 0 33.3 ± 2.1
< 100
Chloroform extract
1 3 10 30 100
0 0 0 0 20 ± 2
< 100
␣-Tocophenol
1 3 10 30 100
30 ± 2.1 50 ± 1.3 97.3 ± 1.8 98 ± 1.3 98.7 ± 2.2
and to determine the exact mechanism of action, are required.
IC50 (g/ml)
3.1
a Inhibition of absorbance at 517 nm relative to that of standard DPPH solution.
munis extracts and their components could be suitable anti-genotoxic, anti-mutagenic and perhaps anticarcinogenic agents, but further studies to fractionate the active extracts, to identify the active compounds
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