The correlation between antimutagenic activity and total phenolic content of extracts of 31 plant species with high antioxidant activity

Plants have formed the basis of many traditional medicine systems throughout the world for centuries and continue to provide mankind with new remedies. Numerous useful drugs have been developed from lead compounds discovered from medicinal plants. Up to this day, this strategy remains an important route to new pharmaceuticals [1]. Medicinal plants are important in health care systems of developing countries for primary health care needs. The popularity of medicinal plants is connected with their easy access, claimed therapeutic efficacy based on local knowledge and expertise amongst the local communities as well as affordability [2]. Plants contain many metabolites with various bioactivities including antioxidant, anti-inflammatory and anticancer activities [3]. Many bioactive compounds from plants are antioxidants and have been shown to have possible health effects, partly due to their antioxidative properties [4].

Many mutagens and carcinogens act by generating reactive oxygen species (ROS) and ROS are widely recognised for playing a harmful role in living systems by inducing oxidative damage to cell structures and biomolecules such as lipids, nucleic acids and proteins [5]. DNA mutation is a crucial step in carcinogenesis, and elevated levels of oxidative DNA lesions have been noted in many tumours, strongly implicating such damage in the aetiology of cancer. Oxidative DNA base lesions are mutagenic, thus the prevention of oxidative DNA lesions is important to limit mutagenesis, cytostasis, and cytotoxicity and may contribute to prevention of mutation-related diseases [6, 7]. For this study, 4-nitroquinoline 1-oxide (4-NQO) was the mutagen of choice. 4-NQO is a quinoline derivative and tumorigenic compound used in the assessment of the efficacy of drugs and procedures in the prevention of cancer as it produces all stages of carcinogenesis [8, 9]. 4-NQO induces potential intracellular oxidative stress [10]. It can undergo redox cycling and generates reactive oxygen species such as the superoxide radical and hydrogen peroxide [11].

The mechanisms and the types of active compounds involved in the protective effects of plants against mutations have not been clearly identified. A common factor in the pathogenesis of chronic degenerative diseases is the involvement of oxidative stress. Plant compounds may reduce oxidative stress, thereby reducing the risk of diseases [12]. To minimize the detrimental genotoxic effects of mutagens caused by exposure to free radicals, chemical compounds, air pollutants or metabolic processes, the use of natural antimutagens is a good alternative. Antimutagens that complement DNA repair systems and those that have antioxidant properties may be found in plants [13]. It is certainly worth investigating what place these compounds have in the prevention of mutations.

Recently the role of phenolics in the prevention of free radical-mediated diseases has become more important. Due to their antimutagenic/anticarcinogenic activities, phenolic compounds (simple phenols, phenolic acids, naphthoquinones, xanthones, stilbenes, flavonoids, lignans, lignins and condensed tannins) have a major role in the chemoprevention of cancer [14]. In an attempt to correlate antioxidant activity and antimutagenic activity, we investigated the potential antimutagenic effects of 31 plant extracts with good antioxidant activities and high total phenolic content. These plant species may have applications as probes for development of antimutagenic agents of natural origin. Kaur et al. [14] reviewed studies that investigated antimutagenic and anticarcinogenic potential of polyphenols and concluded that polyphenolic compounds have a major place in the chemoprotection of cancer and as a result, it is certainly worth investigating what role these compounds have in the prevention of cancer.

The TLC-based antioxidant assay is a fast and simple technique to determine the presence of free radical scavenging compounds in crude plant extracts. Moreover, DPPH is not specific to any particular class of antioxidants, and thus provides the overall antioxidant capacity of the sample [24]. Almost all the plant extracts assayed in this study contained antioxidant compounds. Of the 120 plant species examined, 117 (97.5%) had antioxidant compounds shown as fast reacting bands with high intensity yellow colour on the TLC chromatograms The three species that did not have any antioxidant activity based on this assay were: Maerua rosmarinoides, Baphia racemosa and Abutilon sonneratianum. The different intensity of the spots suggests that the extracts contain antioxidant compounds with different activities [25]. Extracts from 31 plant species that were the most active from this initial screening were selected for further study (Table 1).

All the selected 31 plant extracts effectively reduced the DPPH free radical with EC₅₀ values ranging from 1.20 ± 0.22 to 19.06 ± 1.50 μg/ml (Table 2). The radical scavenging properties of the extracts indicate the antioxidant potential of the extracts. Out of 31 extracts assayed for quantitative antioxidant activity, 17 (54.8%) had activities higher than that of L-ascorbic acid (vitamin C). The higher antioxidant activities of the plant extracts is be due to the presence of more than 11 different antioxidant compounds present in some of the crude extracts (Fig. 1).

Polyphenolic compounds may constitute the main class of natural antioxidants present in plants, food and beverages [26]. The total phenolic content of 31 extracts ranged from 5.17 ± 0.97 to 18.65 ± 3.86 mgGAE/g plant extract. The total phenolic content of the plant extracts correlated well with the respective antioxidative activity of the plant extracts (Fig. 2). Good correlation was found between the gallic acid equivalent/mg and the logarithm of EC₅₀ values (R²˃ 0.9447). Phenolic constituents react with active oxygen radicals such as hydroxyl radical, superoxide anion radical and lipid peroxyl radical [27, 28]. These compounds have a broad spectrum of chemical and biological activities including radical scavenging properties.

The results for the mutagenic effects of 31 plant leaf extracts in the Ames test (S. typhimurium TA98 and TA100) are summarised in Table 3. The tester strains used in this study were selected because they are sensitive and detect a large proportion of known bacterial mutagens and are most commonly used routinely within the pharmaceutical industry [29]. Only one plant extract; Halleria lucida (#3) was mutagenic in TA98 (Table 3). The mutagenicity of Halleria lucida may be due to histidine and histidine related precursors present in plant extracts which often interfere with the Ames test by increasing the number of spontaneous revertants thus resulting in false-positive results [30]. In some cases, the number of colonies in test sample (eg. sample 21) is lower than the negative control. For all the extracts tested in the current experiments, the density of background bacterial lawn was compared to that of the negative control (after 48 h) and had no visible differences, indicating a lack of toxicity to the bacteria at the concentration tested. It is a known phenomenon that higher plants often produce antimicrobial agents and these can kill the tester strain [31]. Although the bacterial lawn was present, the low numbers of revertant colonies in S. typhimurium TA100 may indicate toxicity since the tester strain TA100 is more sensitive to toxic substances than strain TA98 [32]. An alternative explanation could be that the plant extracts induced a mild stress resulting in activation of DNA repair mechanisms. This reduces the number of revertants even in ‘control’ cultures (where no DNA damage was induced). In other words, normally, spontaneous mutations occur in control cultures because there was some DNA damage that was not important enough to activate DNA repair mechanisms. Now, with the plant extracts there is (also) no induced DNA damage (control level of revertants/mutations) but DNA repair activation occurred, repairing the spontaneous mutations.

The background levels as well as positive control values were in all cases within the normal limits found in our laboratory and in accordance with literature data [33]. The absence of mutagenic response by plant extracts against Salmonella typhimurium bacterial strains in the Ames test is a positive step in the safe use of plants in traditional medicine [34]. An extensive database has shown that many chemicals that are positive in the Ames test also have mutagenic activity in other tests [20]. Moreover, the proportion of carcinogens identified as mutagens by the Ames test ranges from about 50 to 90% [33].

To determine the potential antimutagenic activity of the plant extracts to prevent DNA damage by 4-NQO (positive mutagen/carcinogen), plant extracts were incubated together with 4-NQO. The results are presented in Fig. 3 and Table 4. The percentage inhibition of mutagenic activity of 4-NQO (antimutagenicity) of the plant extracts ranged from 0.8 to 77% in S. typhimurium TA98 (Fig. 3) and from 0.8 to 100% in S. typhimurium TA100 (Fig. 4). In S. typhimurium TA98, eight extracts had more than 25% antimutagenic activity. Only three extracts had 45% and more antimutagenic activity even at the highest concentration tested. in the S. typhimurium TA100 assay, nine plant extracts had more than 25% antimutagenic activity and only seven extracts had 45% and more antimutagenic activity. In the Ames test, the antimutagenic effect is considered moderate when the inhibitory effect is between 25 − 40% and strong when more than 45%. Inhibitory effects of less than 25% are considered weak [35, 36]. Based on the conclusion of Negi and colleagues [35], most of the plant extracts assayed in this study may be considered to have weak or no antimutagenic effects. However, other authors consider a 20% decrease in the number of revertants to score the extract as active [31].

It appears the tested plant extracts have more antimutagenic activity in the S. typhimurium TA100 than in S. typhimurium TA98 assay which may be related the mode and/or mechanism of the antimutagenic effects. Almost 50% of the plant extracts reduced the mutagenic effects of 4-NQO in S. typhimurium TA98 and 77% in S. typhimurium TA100. The remaining plants increased the mutagenic effects of the mutagen 4-NQO, a phenomenon known as co-mutagenicity. A co-mutagenic effect is observed in instances where, when tested alone, plant extracts don’t have any mutagenic effects but in the presence of a positive mutagen, these extracts enhance or increase the mutagenicity of the positive control.

Not all antimutagenic plant extracts had activity in both S. typhimurium TA98 and S. typhimurium TA100. It is possible that these extracts have multiple mechanisms of mutation inhibition/antimutagenesis since they prevent frame-shift mutations detectable in TA98 and base-pair substitutions detectable in TA100. This is one of the many advantages of using the Ames test in antimutagenesis studies as it provides information not only of antimutagenesis but also on possible mode of action (De Flora et al., [37]).

Only extracts of ten plant species had antimutagenic activity in both strains. These extracts may have potential antimutagenic compounds because one of the most important characteristics of antimutagens is their universality, in this case being the inhibition of mutations resulting from both reverse frame shift or base-pair substitutions [35]. The differences in the observed activities in the other plant extracts may be due to compounds contained within the extracts that can only prevent, inhibit and/or reverse frame shift or base-pair substitutions.

Plant extracts with good antimutagenic effects in S. typhimurium TA98 in general had good antioxidant activity and relatively higher phenolic content. Plant extracts with good antimutagenic effects in S. typhimurium TA100 generally had higher antioxidant activity but with lower phenolic content. It is clear that there is a direct correlation between antioxidant activity and antimutagenicity. There is also a direct correlation between antioxidant activity and total phenolic contents of the extracts tested in this study. The relationship between antioxidant activity and total phenolics in all plant extracts is in agreement with the results of other authors. Good correlation was found between the mg GAE/g and the logarithm of EC₅₀ values (R²˃ 0.9447). Polyphenols have been reported to be responsible for the antioxidant activity in plant extracts [23]. Phenolic constituents react with active oxygen radicals such as hydroxyl radical, superoxide anion radical and lipid peroxyl radical [25]. In addition to radical scavenging properties these compounds have a broad spectrum of chemical and biological activities.

It is so much easier to determine antioxidant activity and the number of antioxidant compounds present in extracts than carrying out antimutagenic assays that this approach could be a first step in searching for extracts with compounds that can be used against cancer. Variations in the antioxidant activities of the plant extracts may be largely attributed to differences in the quality and quantity of phenolic compounds and other bioactive compounds present in the extracts. Investigation of extracts of plants that are not mutagenic, cytotoxic and that have antimutagenic activity, may lead to identification of compounds that can prevent broad spectrum human diseases caused by mutations. Based on these findings, several plant extracts that have the desired effects will be further investigated for their antigenotoxic effects in the cytokinesis-block micronucleus/cytome assay and single cell gel electrophoresis/comet assay to further establish their potential to prevent chromosomal aberrations and DNA fragmentation.