The antibacterial activity of the three tested A. salicina leaf extracts was evaluated on five pathogenic bacteria. Our results showed that these extracts exhibited various levels of antibacterial effect against all the tested bacterial strains. Minimum Inhibitory Concentration (MICs) values ranged from 0.0625 to over 10 mg/ml, and Minimum Bactericidal Concentration (MBCs) values ranged from 0.125 to more than 10 mg/ml. Generally, TOF extract displayed a strong activity against both Gram-negative and Gram-positive bacteria. The result of the antimicrobial activity is presented in Table .
Antibacterial activity of Acacia salicina extracts, expressed as Minimum Inhibitory Concentration (MIC) and as Minimum Bactericidal Concentration (MBC)
Staphylococcus aureus was the most susceptible bacterial species, followed by Salmonella typhimurium, then Salmonella enteritidis and Enterococcus faecalis and finally Escherichia coli, with MIC values of 0.062, 0.125, 0.250, 0.250 and > 10 mg/ml respectively. Compared to ampicillin, used as a positive control against S. aureus (0.225 mg/ml), the tested TOF extract was twice more active with MBC value of 0.125 mg/ml. E. coli was found to be the least sensitive strain to A. salicina extracts.
Compared to the other extracts, TOF extract was the most active one against all the tested bacterial strains. Its biological efficiency is probably related to the high amounts of flavonoids and polyphenolic compounds, in its chemical composition. We previously reported, that A. salicina
extracts, particularly TOF extract, contains flavonoidic, polyphenolic and coumarinic compounds [12
]. These families of compounds are reported to play a role in the prevention of colonisation by parasites, bacteria and fungi [13
Our results indicate that Gram-positive bacteria are more sensitive to the antimicrobial effect of A. salicina
extracts than Gram-negative ones. It is interesting to note that A. salicina
extracts exhibited an antimicrobial activity, particularly towards organisms of interest to the medical field such as Staphylococci
. In fact, Salmonella
remains a primary cause of food poisoning worldwide, and massive outbreaks have been reported in recent years. The centre for disease control and prevention estimated that approximately 1.4 million cases of salmonellosis were annually reported in the United States [14
]. The European Union reported more than 100.000 cases of salmonellosis [15
]. In Tunisia, between 1978 and 1993, 1022 Salmonella
strains were isolated: 578 in hospitals and 444 from the environment [16
]. Some pathogenic Salmonella
serotypes adapted to man, such as S. typhimurium
, usually cause severe diseases such as enteric fever in humans. However, some pathogenic Salmonella
serotypes, such as S. enteritidis
or S. typhimurium
, can infect a wide range of hosts and are termed ubiquitous. Likewise, foodborne illness resulting from the consumption of food contaminated with pathogenic bacteria, has been a vital concern to public health. Salmonella
spp. and E. coli
accounted for the largest number of outbreak cases and deaths.
Mutagenic and antimutagenic activities
In experiments, prior to the mutagenicity study, it was ascertained that the different extracts added to the inductor bacteria do not influence their viability.
According to Ames et al. [19
] and Marques et al. [20
] a compound is classified as a mutagen if it is able to increase at least twice the number of revertants compared to spontaneous revertants. Based on this, most mutagenicity assays conducted with extracts were negative. None of the tested extracts produced a significant increase of his+
revertant number of both Salmonella typhimurium
TA 102 and TA 98 strains, in the absence of the S9 metabolizing system. In fact, mutation frequencies obtained with various concentrations of the tested samples do not change significantly when compared to spontaneous mutation frequencies. However, in the presence of S9, a mutagenic effect is observed with all the tested extracts, in the presence of S. typhimurium
TA98 strain, at the two highest tested doses, and only with aqueous extract at the highest tested dose in the S. typhimurium
TA 102 assay system. The result of antimutagenic activity in S. typhimurium
TA98 assay system, is reported in Table .
Effects of extracts from A. salicina on the mutagenicity induced by NOPD (10 μg/plate) and B(a)P (7.5 μg/plate) in S. typhimurium TA98 assay system respectively in the absence and in the presence of S9
The result of mutagenic activity in S. typhimurium TA 98 and TA102 assays systems is reported in Table .
Mutagenic activity of extracts from A. salicina by the S. typhimurium TA98 and TA102 assay systems in the presence and absence of the metabolic activation system (S9)
Besides, we envisaged the study of the antimutagenic activity of the same extracts toward four different mutagens, having diverse chemical structures and mode of actions, by using the Ames assay.
As shown in Table , all the extracts prepared from A. salicina
were effective in reducing the mutagenicity induced by NOPD (10 μg/plate without S9) and MMS (2.5 mg/plate), a directacting mutagens, in respectively S. typhimurium
TA 98 and TA 102 assay systems. All the tested extracts showed a clear dose-dependent response. Compared to methanolic and aqueous extracts, TOF extract was more effective against both NOPD and MMS direct mutagens. It reduces their mutagenicity by 99.4% and 93.5% respectively, at the highest tested dose (500 μg/plate). The possible mechanism of the potent protection of all the tested extracts against direct mutagens such as NOPD and MMS could be explained by the induction of the oxidative defence system and/or DNA repair enzymes, which are required to protect against oxidative-like mutations. Teel et al. [21
] reported that the antimutagenic/anticarcinogenic activity of plants may be due to the interaction of the compounds with target DNA tissue, which, in turn, blocks the site(s) of DNA to electropholic attack by reactive mutagenic moieties. In our case, we speculate that the protective effect of the extracts against the tested mutagens is probably exerted by three different ways; firstly, the plant extracts may adsorb the mutagen in a way similar to the carcinogen adsorption which has been associated with chemical component; secondly, extracts could induce
DNA glicosylase enzymes which are capable of repairing alkylating DNA bases, and finally the reductive ability of the samples assessed in this study suggests that extracts were able to donate electrons to free radicals, making the radicals stable and unreactive [22
Methanolic, aqueous and TOF extracts reduced the mutagenicity caused by the indirect mutagen B(a)P (7.5 μg/plate), a metabolically activated genotoxin, using the S. typhimurium
TA 98 strain, in a dose dependent manner (Table ). Our results, revealed that TOF extract was the more potent inhibitor of frame-shift mutations, due to the lack of a base pair in the GC-pair regions of gene D, induced by B(a)P in the S. typhimurium
TA 98 assay system. When we tested at doses of 50, 250 and 500 μg/plate, all extracts, mixed with 2-AA, showed a toxic effect on S. typhimurium
TA102 cell viability. This effect could be due to the formation of a complex between the mutagenic agent (2-AA) and extract components, inducing cell death at high concentration [22
]. Therefore, decreased extract concentrations to 5, 10 and 25 μg/plate, did not influence bacterial viability. The tested extracts revealed an inhibitory effect against the mutagenicity induced by the 2-AA by, respectively, 32.6%, 44.6% and 32.6% at the lowest tested dose (5 μg/plate). The result of antimutagenic activity in S. typhimurium
TA102 assay system, is reported in Table .
Effects of extracts from A. salicina on the mutagenicity induced by MMS and 2-AA in S. typhimurium TA102 assay system respectively in the absence and in the presence of S9
The inhibitory effects of A. salicina
leaf extracts on the mutagenicity of both direct mutagens (NOPD, MMS) and metabolically activated mutagens (B(a)P, 2-AA), may be ascribed to flavonoid and tannin contents of TOF, methanolic and aqueous extracts [12
]. We cannot, however, exclude the possibility of other compounds, with antimutagenic properties, participating to the antimutagenic effect of A. salicina
extracts. The results of our experiments are consistent with the known antioxidant activities of flavonoids [23
] and tannins [24
]. Flavonoids are the most likely candidates, among the known compounds, to be present in the TOF-enriched, methanolic and aqueous extracts, involved in the antimutagenic activity of A. salicina
extracts and in preventing oxidative lesions [25
]. In this study, we used S. typhimurium
TA102 strain, which is generally selected for specific detection of the oxidative damages [26
]. Thus we suppose that the constituents A. salicina
extracts should inhibit free radicals and ROS, produced by oxidation and redox-cycling of both B(a)P and 2- AA, and through reducing the activity of enzymes involved in B(a)P and 2-AA metabolisation. The extracts may both inhibit microsomal activation and directly protect DNA from the electrophilic B(a)P epoxide; 7,8-dihydroxy, 9,10-epoxy-7,8,9,10- tetrahydrobenzo[a
]pyrene, a putative ultimate carcinogenic metabolite [27
]. They may also protect DNA from the electrophilic N
-hydroxy-2-aminoanthracene, a metabolite of 2-AA that interacts with DNA [28
] as well as from other intermediates of the two aforementioned mutagens. In fact, several metabolic intermediates and ROS formed during microsomal enzyme activation are also capable of breaking DNA strands. The antioxidant activity expressed by A. salicina
extracts may provide a common mechanism for inhibiting the mutagenicity of both B(a)P and 2-AA. However, the toxic effects obtained with the highest doses (500, 250 and 50 μg/plate) against S. typhimurium
TA102 strain, when extracts were combined with mutagens, can be explained by the presence of molecules in these extracts which form, with the control mutagen, complexes with high bactericidal effect. In the presence of lower doses, probably, a weak number of molecules should react with 2-AA metabolites giving minor bactericidal complexes. No lethal effect, against S. typhimurium
TA 102 strain is then detected.
We also noticed that methanolic, aqueous and TOF-enriched extracts were more efficient in reducing B(a)P mutagenicity than 2-AA mutagenicity. Curiously, these extracts exhibited both mutagenic and antimutagenic activities. We hypothesize that the presence of reactive intermediates resulting from both B(a)P and the tested extracts could result in their mutual neutralization (antagonist effects). These intermediates may form complexes preventing their penetration through the bacterial cell wall and thus inhibiting their mutagenic effects [29
The chemical analysis of leaf extracts from A. salicina
, harvested from the south east of Tunisia [12
] revealed important differences from those obtained from A. salicina
collected from the centre of Tunisia [10
]. These differences may explain the different biological activities revealed by the two A. salicina
On the other hand, our study is in accordance with results reported by Mansour et al. [12
] as far as we confirmed the antigenotoxic effect of A. salicina
extracts described by these authors, who used a different prokaryotic assay i.e. the SOS chromotest in the presence of E. Coli
PQ37, described by Quillardet and Hofnung [30
]. However, some differences arised from evaluating O2
scavenging capacity when comparing the antioxydant results of the present study and those reported by Ben Mansour et al.
]. They revealed no scavenging effects against superoxyde anion. This could be explained by the different antioxydant assays used in each study. In fact, Ben Mansour et al.
carried out a nonenzymatic O2 .
generating system to evaluate scavenging effects of A. salicina
leaf extracts. Yet, we used in the present study the enzymatic X/XOD superoxide generating assay system to evaluate O2 .
scavenging activity of A. salicina