1. Introduction
It has been known that toxic components are present in many plants including vegetables [1] [2] [3] . Besides several reports on the cytotoxicity of some plants, several investigations have revealed that many plants used as food or in traditional medicine have mutagenic effects in in vitro assays [4] [5] [6] [7] .
Some plants that expressed cytotoxic and mutagenic activities had shown correlation with the incidence of tumours and cancers [8] . Cancer is one of the major causes of morbidity and mortality. While all the factors that contribute to its onset are not fully known, it is clear that there are chemical agents that can induce cancer. These agents are called carcinogens. Most carcinogens induce cancer because they are mutagens.
The most definitive way to detect carcinogens is to inoculate a sample into animals and monitor for the development of tumours. This process is expensive, time consuming, and cumbersome. Therefore, a rapid, economical screening method is used to distinguish between compounds that might be carcinogenic and those that are likely to be proven harmless. The Ames test is a screening assay for carcinogens that uses bacteria to detect chemical mutagens. It is based on the premise that most carcinogens induce cancer because they are mutagens. The Salmonella mutagenicity test was designed to detect chemically induced mutagenesis [9] . If these agents are shown to be mutagenic for bacteria, they may also alter DNA in eukaryotic cells. The test is used world-wide as an initial screen to determine the mutagenic potential of chemicals and drugs and had been shown to correlate well with carcinogenicity test using rodent [10] [11] .
Regulation of herbal products in many countries does not require mutagenicity testing although many herbal plants have been reported to be mutagenic. However, it has been reported that some polyherbal products which contains traditionally used herbs were found to be cytotoxic [12] and thus they might be mutagenic. Thus, the aim of this study was to evaluate the mutagenicity potential of some medicinal herbal preparations sold over-the-counter.
2. Materials and Methods
2.1. Herbal Samples
Twelves polyherbal preparations were purchased from retail shops. The products were packaged in either plastic bottles or plastic envelope, appropriately labelled with information on herbal composition and were at least 6 months before the expiration date. All products were in fine powder form approximately of 12 mesh particle size. Table 1 shows the formulation of the polyherbal preparations and their intended uses.
2.2. Herbal Extracts
Since all of the herbal preparations were in fine powder form, it could be extracted directly with some solvents. There is no universal solvent to extract all plant constituents. However, methanol has been reported to be able to extract a wide range of plant constituents and combination of methanol with chloroform had been used in extraction of metabolites, DNAs, RNAs and proteins from plant simultaneously [13] . The herbal samples in this study were extracted according to the method described by [14] . Sample (20 g) was mixed with 75 ml of methanol/chloroform (1:1) in a screw-capped bottle and stirred with magnetic bar on a stirrer plate for 24 h. The extract was filtered through Whatman No. 1 filter paper and evaporated to dryness using vacuum evaporator at 40˚C. A portion of the dried extract was reconstituted in dimethyl sulfoxide (DMSO) to 250 µg/ml.
2.3. Mutagenicity Test
The most definitive way to detect carcinogens is to inoculate the test sample into
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Table 1. Herbal formulations and intended usage of some polyherbal preparations.
aPlant parts used: n.i, not indicated; Radix, the root; Rhizoma, rhizome or a creeping horizontal stem generally bearing roots on its underside; Flos, the flowers, Fructus, the fruit or berry; Semen, the seed usually removed from the fruit and may or may not contain the seed coat; Herba, the aerial parts or the aboveground parts of plants which may include the flower, leaf, and the stem; Cordex, the bark collected from the root, stem, or branches; Concha; Spina.
animals such as rodent and monitor for the development of tumours. This process is expensive, time consuming, and cumbersome. The bacterial Salmonella mutagenicity test has been used world-wide as an initial screen to determine the mutagenic potential of chemicals and drugs and had been shown to correlate well with carcinogenicity test using rodent [10] [11] .
2.3.1. Muta-Chromoplate Kit
A commercial kit, the Muta-Chromplate (Environmental Biodetection Products Incorporation, EBPI, Ontario, Canada), was used to evaluate the mutagenicity of the herbal extracts. This test kit was based on the validated Ames bacterial reverse-mutation test [9] but was performed entirely in liquid culture.
2.3.2. Chemicals
The following chemicals were provided by EBPI: Davis-Mingioli salt (5.5 times concentrated), D-glucose (40%, w/v), bromocressol purple (2 mg/ml), D-biotin (0.1 mg/ml), and L-histidine (0.1 mg/ml). A standard mutagen provided by the manufacturer was sodium azide (NaN3, 0.5 µg/100 µl). All chemicals were kept at 2˚C ± 1˚C prior to use.
2.3.3. Preparation of Reagent Mixture
The solutions provided by EBPI were mixed aseptically in a sterile bottle as follows: Davis-Mingioli salt, 21.62 ml; D-glucose, 4.75 ml; bromocressol purple, 2.38 ml; D-biotin, 1.19 ml; and L-histidine, 0.06 ml.
2.3.4. Test Bacterial Strain
Salmonella typhimurium TA100 were purchased from Environmental Biodetection Products Incorporation (EBPI, Ontario, Canada). The bacterium was maintained on Nutrient agar at 4˚C. The bacteria was streaked for single colonies on Nutrient agar plate and incubated at 37˚C for 48 h. Several single colonies were picked using inoculating loop and inoculated into Nutrient broth and incubated at 37˚C for 18 h before the test was carried out.
2.3.5. Mutagenicity Assay
Reagent mixture, herbal extract, sterile distilled water and standard mutagen were mixed in 4 treatment bottles at the amount indicated in Table 2. An overnight culture broth of S. typhimurium (5 µl) was inoculated into the bottles and mixed thoroughly with vortex mixer. The content of each bottle was poured into a multi-channel reagent boat and 200 µl aliquots of the mixture were dispensed into all wells of a 96-well microtitration plate using a multi-channel pipette. The plates were placed in a plastic bag to prevent evaporation and then incubated in an incubator (Gallenkamp) at 37˚C for 4 days. Each polyherbal extracts, background and standard mutagen were tested in duplicates.
2.3.6. Interpretation of Results and Statistical Analysis
After incubation, the “blank” plate was observed first and the rest of the plates were read only when all wells in the blank plate were purple indicating the assay was not contaminated. The “background”, “standard” and “test” plates were scored visually and all yellow, partially yellow or turbid wells were scored as positive while purple wells were scored negative. Numbers of all positive wells were recorded. The “background” plate (no herbal extract or standard mutagen added) showed the level of spontaneous or background mutation of the test bacteria. The extract was considered toxic to the test strain if all wells in the test plate were purple.
The mutagenicity of the sample was determined by comparing the number of wells scored as positive in the background plate to the number of positive wells in the treatment plate [15] . Statistical differences were determined using the table for analysis of results provided by the manufacturer (EBPI, Canada) based on statistical analysis by [16] .
3. Results
Table 3 shows the results of mutagenicity testing using S. typhimurium strain TA100. Out of 12 samples tested at 250 µg/ml, 5 extracts were found to be
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Table 2. Set-up of the mutagenicity assay.
-, not added.
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Table 3. Mutagenic activity of the polyherbal extracts in the Ames test using S. typhimurium TA100. All extracts were tested at 250 µg/ml.
+, significant increase in the number of positive wells compared to the control; −, no significant effect observed in the number of positive wells compared to the related control (background).
potentially mutagenic. Extracts of “A”, “C” and “G” were significantly mutagenic at p ≤ 0.01 while “E” and “H” were mutagenic at p ≤ 0.05. Extract “H” contained 2 herbs while “A”, “C”, “E” and “G” comprised of 7 - 11 herbs.
At concentration 250 µg/ml, extracts of polyherbal preparation from samples of “B”, “D”, “F”, “I”, “J”, “K” and “L” were not potentially mutagenic in this experimental condition. Chemical constituents in extracts of “D”, “F”, “I” and “B” had no mutagenic activity at all as number of positive wells was almost similar to the control wells (contained growth medium only).
4. Discussions
Seven out of 12 polyherbal preparations were found to be non-mutagenic at test concentration of 250 µg/ml. The mutagenicity of these extracts could not be definitely ruled out until test using higher extract concentration is carried out as mutagenicity was reported to increase with the increase of the extract concentration [17] .
Polyherbal preparations “B”, “D”, “F”, “J” and “L” comprised several different herbs. There were little or no reports of mutagenicity of any of these plants and combination of these herbs as found in the current study was not mutagenic.
Polyherbal preparation “I” comprised of Eurycoma longifolia, Tacca palmate, Zingiberis aromaticae, Zingiberis officinale, and Helminthoctachys zeylanica. Tacca palmate was reported to be mutagenic [18] . However, combination of T. palmate (30%) with four other herbs (70%) rendered the product to be non-mutagenic.
Zingiber minus, Eugenia caryophyllata, Piper nigrum, Illicium verum, Carum copticum, Astragalus membranaceus and Angelica sinensis were plants in the formulation of “G”. There were little or no reports of mutagenicity of any of these plants. Instead of being mutagenic, Astragalus membranaceus was reported to have antimutagenic property [19] and was used as a natural herbal medicine in East Asia for preventing severe side effects of chemotherapy in patients with cancer. However, combination of these plants as in “G” was found to be mutagenic. Similarly, there were little or no reports of mutagenicity of both E. longifolia and Cistanche deserticola which were the component of “H” but combination of these plants was also mutagenic. These results suggest that the mutagenicity of prepations of “G” and “H” could be due to the synergistic effects of various phytochemicals in the polyherbal preparation.
The idea of formulating polyherbal medicine is to take advantage of synergistic effect of various plants chemical constituents that will increase the effectiveness of the medicine. Use of varieties of herbs in a medicinal herbal formulation is thought to be able to minimise toxicity of the formulation [20] . However, in the presence of diverse phytochemicals in a polyherbal formulation, some phytochemicals may activate promutagens amongst the phytochemicals or some weak mutagens may acts synergistically enhancing the mutagenic effect [21] .
Results of this study show that mutagenicity of a polyherbal preparation cannot be deduced from the information of mutagenicity of individual components of the polyherbal preparation and combination of non-mutagenic plants is not necessarily produce a non-mutagenic herbal medicine
5. Conclusion
Combination of traditionally used herbs or non-mutagenic herbs may produce a mutagenic product most probably through synergistic effect of various phytochemicals combination in the polyherbal extract.
Funding
This research work was sponsored by the Public Service Department of Malaysia through the Federal Government Training Scholarship Program.