Abstract
Hypericum (Hypericaceae) species are extensively used in several fields such as traditional medicine, food and crop protection. Despite its usage in many fields, the identification of the genotoxic potential of this herb is still incomplete. In this study, we evaluated genotoxic effects of the petroleum ether, hexane, ethyl acetate, and methanol extract of Hypericum lysimachioides Boiss. var. lysimachioides by Ames Salmonella/microsome test and SOS chromotest. The mutagenic activity of Hypericum lysimachioides var. lysimachioides extracts was investigated by using Salmonella typhimurium strains TA98 and TA100 and also the SOS chromotest with Escherichia coli PQ37 strain, with or without S9 metabolic activation. In this initial report we demonstrated that all extracts of H. lysimachioides var. lysimachioides showed significant mutagenic activity on both strains of Salmonella either with or without S9 mixture. No mutagenicity was found in the SOS chromotest either with or without S9 mixture. These results indicate a significant mutagenicity of the petroleum ether, hexane, ethyl acetate and methanol extracts of Hypericum lysimachioides var. lysimachioides in vitro. It can be suggested that quercetin and flavonol or their synergistic effects may be main mutagenic agents in the photopharmaceuticals Hypericum lysimachioides var. lysimachioides extract.
Introduction
Environmental substances such as synthetic chemicals and daily foods contain a high number of genotoxic agents. Recently, some authors have reported that various degenerative diseases, such as cancer, neurodegenerative, reproductive diseases and cardiovascular disorders, are associated with exposure to these genotoxic substances (CitationAruoma, 1994; CitationDe Flora et al., 1996). These substances consist of many compounds, whose origins have proved to have considerable pharmacological activity. There are undesirable properties of some compounds, such as mutagenicity, carcinogenicity and toxicity. Also, the medicinal use of some drugs of vegetable origin is only possible after modification of their chemical structures, frequently resulting in string pharmacological activity and lower toxicity and mutagenicity (CitationSimoes et al., 1999).
In many parts of the world, thousands of species of endemic higher plants are used in traditional medicine. Also, there are many studies showing considerable interest in mutagenicity and antimutagenicity of medical plants used by traditional herbalists. The plants used commonly in traditional medicine have been considered to be benign and not to have any side effects due to their long use and being natural in origin. This concept is largely circumstantial and it is important to determine toxicology of plant extracts, especially those which are used frequently over long periods (CitationElgorashi et al., 2002; CitationPopat et al., 2001).
Hypericum (Hypericaceae) is one of the plants used traditionally in medicine, crop protection, and flavoring, as well as fragrance in food (CitationIsman et al., 2001; CitationDaferera et al., 2003). This genus is represented in the flora of Turkey by 77 species (CitationRobson, 1975). The leaves of Hypericum species, when held to the light, reveal translucent dots, giving the impression that the leaf is perforated. Therefore, this species known as “binbirdelikotu” or “kantaron” in Turkish (CitationBaytop, 1984; CitationSaya et al., 2001).
Chemical compositions of these extracts are well documented, and they include hypericin, hyperforin, quercetin, quercitrin, xanthon, pyrone, and phloroglucinol (CitationUmek et al., 1999; CitationVerotta et al., 2000; CitationConstantine & Karchesy, 1998; CitationChung et al., 1999; CitationDecosterd et al., 1991; CitationFerraz et al., 2001; CitationKikuchi et al., 1985; CitationTrifuniviç et al., 1998).
Hypericin, a major bioactive component of Hypericum species, is widely used in neurological disorders and depression (CitationOkpanyi et al., 1990). Recently, hypericin has been shown to have antitumor activity (CitationVandenbogaerde & Witte, 1995; CitationKamuhabwa et al., 2000), highly active against HIV (CitationTakahashi et al., 1989; CitationMeruelo et al., 1988), and exhibits a number of pharmacological effects. Antimicrobial and antioxidant activities of Hypericum lysimachioides Boiss. var. lysimachioides, Hypericum triquetrifolium Turra. and Hypericum scabroides Robson & Poulter were also reported (CitationToker et al., 2006; CitationHakimoğlu et al., 2007; CitationKızıl et al., 2008).
Quercetin is another component of Hypericum which was determined to be mutagenic in numerous studies. In one study of quercetin it was found to be mutagenic without S9 mix activation, and with S9 mix it increased significantly (CitationBjeldanesi & Chang, 1997). Quercetin was also indicated to have comutagenic activity on 2-acetylaminofluorene (AAF) (CitationOgawa et al., 1987). The 3-O-acetyl ester of flavonol, quercetin, was mutagenic with S9 mix. Three quercetin glycosides were mutagenic after pre-incubation with hesperidinase, a crude extract of Aspergillus niger (CitationNagao et al., 1981). Furthermore, quercetin enhanced the mutagenicity of the tricyclic aromatic amines (aminofluorene, aminoanthracene, and aminophenanthrene) and their acetamides by 1.2–5.9-fold (CitationAdam et al., 1990).
Animals fed with H. perforatum flowers for 2 weeks showed significant signs of toxicity, including erythema, edema of the portion of the body exposed to light, alopecia, and changes in blood chemistry. Mutagenic activity in a Salmonella/microsome test was attributed to flavonols in one study and to quercitin in another, but other genotoxicity assays were negative. Adverse reactions to Hypericum extract in the clinical treatment of depression include skin rashes and itching, dizziness, constipation, fatigue, anxiety, and tiredness (CitationBjeldanesi & Chang, 1997; CitationOkpanyi et al., 1989). Ethanol, chloroform, and hexane extracts of H. perforatum significantly reduced growth in all three cell lines (CitationSchmitt et al., 2006). The hexane fraction of Hypericum ascyron L. and the n-BuOH fraction of Hypericum japonicum Thunb. both showed appreciable antibacterial activities with minimum inhibitory concentration (CitationMu et al., 2006).
The bacterial tests are commonly employed as an initial screening for genotoxic activity. Two of these bacterial tests are Salmonella/microsome (Ames test) and SOS chromotest, which have been recommended by various workers as a valid indicator of mutagenicity/genotoxicity of various substances present in the environment. The Salmonella/microsome mutagenicity test is an in vitro test detecting point mutation on Salmonella typhimurium strains. The SOS chromotest shows primary DNA damage on Escherichia coli PQ37.
Although Hypericum lysimachioides Bois var. lysimachioides usage is widespread, the identification of the genotoxic potential of this herb is still incomplete. Therefore, this study was performed to investigate the mutagenic activity of the petroleum ether, ethyl acetate, hexane and methanol extracts of H. lysimachioides var. lysimachioides by using the Salmonella/microsome assay and SOS chromotest, either with or without metabolic activation.
Materials and methods
The strains S. typhimurium TA98 and TA100 were purchased from the Salmonella Genetic Stock Centre (University of Calgary, Canada), and their genetic backgrounds were controlled as described (CitationAmes et al., 1973; CitationLevin et al., 1982) every 6 months.
The strains S. typhimurium TA98 and TA100 were used with and without addition of rat hepatic metabolic fraction S9 mix (CitationMaron & Ames, 1983).
The bacteria strain E. coli PQ37 was obtained from M. Hofnung (Institute Pasteur, Paris). Its genotype was controlled as described by CitationQuillardet and Hofnung (1985).
Collection of plant material
H. lysimachioides var. lysimachioides was collected from the south-eastern Anatolia region in June 2005. A voucher specimen has been deposited in the Herbarium of the Department of Biology, Faculty of Science and Arts, Dicle University (DUF-2514). It was identified by A. Selçuk Ertekin from the same institution.
Preparation of extracts
Aerial parts (stems, leaves and flowers) were dried for 10 days at room temperature. The dried and finely powdered bark material was extracted by adding solvents in increasing order of polarity, namely petroleum ether, hexane, ethyl acetate, and methanol. The obtained extracts were kept in dark glass bottles at 4°C until use.
Aliquots were prepared from the dried crude extracts and dissolved in 10% DMSO to give an initial concentration of 1, 0.5 and 0.05 mg/mL for mutagenic screening by using the Ames test as described by CitationMaron and Ames (1983) and SOS chromotest as described by CitationQuillardet and Hofnung (1985).
Salmonella mutagenicity assay
The potential mutagenic effects of Hypericum extracts were evaluated on two S. typhimurium strains TA98 and TA100. S. typhimurium TA98 and TA100 were used to identify reverse mutations from histidine dependence to histidine independence by the plate incorporation method of CitationAmes et al. (1973). The plate incorporation assay was performed according to CitationMaron and Ames (1983), by adding 0.1 mL of the overnight bacterial culture and 0.1 mL of crude Hypericum extracts at different concentrations to the test tubes. In the case of metabolic activation, 0.5 mL S9 mixture was supplemented. After incubation of the culture for 24 h at 37°C, 2.5 mL of top agar was added to each tube and then plated on minimal agar. Histidin revertants were counted after 72 h of incubation at 37°C on a colony counter.
The S9 mixture is mammalian liver tissue that is used in the Salmonella/microsome test to provide a first approximation of mammalian metabolism. S9 mix, used routinely for general mutagenesis screening, was prepared from livers of male rat according to CitationMaron and Ames (1983). All steps were performed at 4°C with cold and sterile solutions and glassware. S9 fraction was distributed in aliquots in small sterile tubes and stored at -80°C. The crude Hypericum extracts were prepared freshly for each experiment by dissolving them in DMSO. The positive controls used were 1 μg/plate NaN3 for TA100, 1.5 μg/plate 2-AF for TA98. Compounds were tested by using five concentrations depending on the samples tested. Three plates each in two separate experiments were used for each concentration tested and for positive and negative controls.
SOS chromotest
The genotype of E. coli PQ37 strain was described by CitationQuillardet and Hofnung (1985). In this assay, the lacZ gene of the tester strain E. coli PQ37 is fused to the bacterial sfiA SOS operon. Thus, lacZ is concomitantly expressed in the case of bacterial SOS response; photometrically determined β-galactosidase induction is indicative of the extent of SOS induction and bacterial genotoxicity. Bacterial alkaline phosphatase activity serves to determine the range of bacterial cytotoxicity; the ratio of bg/ap activity is termed as induction factor IF and indicates the extent of SOS induction of a compound tested.
This assay was performed according to CitationQuillardet and Hofnung (1985) and following adaptation of CitationMarzin et al. (1986). In brief, 100 μL of a 13 h overnight culture of E. coli PQ37 was diluted with 5.1 mL La-medium (L-medium + ampicillin) and incubated until a transmission of 40% at 600 nm was reached (corresponding to 200 × 106 cfu/mL). This culture was diluted as 1:40 with L-medium, resulting in 5 × 106 cfu/mL. Test substances and cultures were incubated for 2.5 h at 37°C, and after incubation, the mixtures were divided into two series: One for β-galactosidase activity measurement (an induction assay) and the other for alkaline phosphatase activity (a control of protein synthesis). β-galactosidase activity was determined after addition of 2.7 mL of B buffer and 600 μL ONPG (ortho-nitrophenyl-β-D-galactopyranoside) as substrate. Constitutive alkaline phosphatase activity was determined after addition of 2.7 mL of P buffer and 600 μL PNPP (p-nitrophenyl phosphate) as substrate. All mixtures were incubated at 37°C for 25 min in water bath, and then photometrical determinations were performed at 405 nm.
Calculations of test results
The means and standard deviations were calculated. Data obtained in improved Salmonella/microsome test were subjected to ANOVAs. Data obtained in tests were calculated and expressed in percentage frequency and the significance at different dose levels was tested by application of ANOVA test. Differences between the groups were considered to be statistically significant at P < 0.05. All data were analyzed by using SPSS 13.0.
SOS chromotest: Responses of the SOS chromotest were calculated as ratios of β-galactosidase and alkaline phosphatase activities. The genotoxic activity for concentration c may be expressed in the ratio Rc = β/ap, where β represents β-galactosidase, where ap represents alkaline phosphatase activity. The induction factor (IF) for a compound at concentration c is defined as I = Rc/Ro, in which Ro is the spontaneous ratio measured in the blank test (solvent control). Experiments were performed with and without the metabolic activation mixture for each extract tested. In order to ensure the validity of the assay, a positive control was included in each experiment. The positive control used was 4-NQO (1 μg/mL) without S9 mix and B[a]P (30 μg/mL) with S9 mix. Compounds were tested by using four concentrations, depending on the extracts tested.
Results
The petroleum ether extract of H. lysimachioides (3.67 g) was obtained as a yellow powder, the hexane extract (1.31 g), as a yellowish powder, the ethyl acetate extract (2.87 g) as a brown powder, and the methanol extract (24.12 g) as a purple powder. The results of the Hypericum extracts are shown in and . Different concentrations of extracts were evaluated when the assay was carried out with Salmonella, both in the presence and absence of metabolic activation mixture. The revertant clones obtained at each concentration and treatments were scored manually in the Salmonella/microsome test.
Table 1. Salmonella microsome test results of petroleum ether, ethyl acetate, hexane and methanol extracts of H. lysimachoides var. lysimachoides on S. typhimurium strains.
Table 2. The SOS response induction of petroleum ether, ethyl acetate, hexane and methanol extracts of H. lysimachoides var. lysimachoides in E. coli PQ37.
Acute observation, under a magnifying glass, of the bacterial lawn in each culture dish was used to check for possible toxicity of the treatment. Spontaneous reversion of the tester strains to histidin independence was measured routinely in mutagenicity experiments, and expressed as the number of spontaneous revertants per plate.
All tested Hypericum extracts were seen to have positive results with S. typhimurium TA98 and TA100, both in the presence and absence of S9 mix. The mean number of revertants of the substances tested, the reference mutagens and the negative control with their corresponding standard deviations were determined. Each sample was assayed with three replicate plates at each concentration (). The results show their mean values and standard deviations (SD) of them. The concentration of the bacterial suspension was 0.8-2.4 × 109 cfu/mL. Criteria for a positive test result were as follows: at least double in the number of spontaneous revertants (factor > 2) and a dose-response effect compared to the spontaneous mutation frequency.
As illustrated in , all Hypericum extracts were found to have a mutagenic activity in both strains of S. typhimurium TA98 and TA100, in the absence or presence of S9 mix. The petroleum ether, hexane and methanol extracts of H. lysimachioides var. lysimachioides were shown to have a significant mutagenic activity in S. typhimurium TA100, in the presence of S9 mix.
The bacterial SOS chromotest with E. coli PQ37 was used for the assessment of genotoxicity. Responses with positive control included in each experiment of the SOS chromotest were calculated as discussed in the Material and method section. As shown in , Hypericum extracts did not induce the SOS response in the E. coli PQ37 strain with or without S9 mix. The results of samples of Hypericum extracts in SOS chromotest are presented in .
Discussion
The studied plants are orally or topically used in folk medicine for the treatment of various diseases; however, there are no reports on the toxicity of H. lysimachioides var. lysimachioides in the literature. In this initial report, we demonstrated that the evaluated extracts induced frameshift mutations on S. typhimurium TA98, base pair substitution on S. typhimurium TA100, but did not induce oxidative DNA damage in the strain E. coli PQ37.
The Salmonella/microsome mutagenicity test is commonly employed as an initial screening for genotoxic activity, and in particular for point and frame shift mutation inducing activity. An extensive data base has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests (CitationOECD, 1977).
SOS chromotest is a bacterial test for detecting DNA-damaging agents. It is a colorimetric assay based on the detection of primary damage to DNA as measured through the SOS DNA repair system. In this test system, damage of the DNA results in the activation of the SOS system (CitationQuillardet & Hofnung, 1985).
In our study we have investigated the mutagenic effect of Hypericum extracts which were employed to detect the induction of base pair mutations as well as frame shift mutations in the S. typhimurium strains TA98 and TA100 and primary DNA damage in E. coli PQ37.
As it can be seen in , different concentrations tested in the Salmonella/microsome test were found to be significantly (p < 0.01, p < 0.05) mutagenic both in the presence and absence of S9 mix. The petroleum ether, hexane and methanol extracts of H. lysimachioides var. lysimachioides were found to be more effective on S. typhimurium TA100 than TA98 in the presence of S9 mix. Therefore, these extracts can be assumed to be strong mutagens, causing base pair substitution.
Positive results from the Salmonella/microsome test indicate that a substance induces point mutations by base substitutions or frame shifts in the genome of either S. typhimurium and/or E.coli. Negative results, however, indicate that, under the test conditions, the test substance is not mutagenic in the tested species.
As indicated in , Hypericum extracts did not induce SOS response in E. coli PQ37 strain with or without of S9 mix. The reason for the lower sensitivity of the SOS chromotest may be due to relatively short-term contact with the genotoxic agents (or extracts make the presence of SOS inhibiting compounds possible). It is likely that Hypericum extracts make the presence of SOS inhibiting compounds possible.
However, plant extracts exhibiting a positive response and hence a mutagenic effect need to be extensively investigated to determine their possible genotoxicity to humans, as their safe use in traditional medicine is questionable. Screening is required to identify and eradicate the use of all mutagenic plants, since numerous studies have shown that the proportion of carcinogens identified as mutagens by the Ames test ranges from about 50% to 90% (CitationZeiger, 2001). Most of the mutagenicity was exhibited in the presence of metabolic activation in this study.
The short-term bacterial tests used in this study are reliable, quick and easy. These tests are used to screen for possible carcinogens; however, a positive result does not necessarily indicate the substance as being a carcinogen. Also, if a substance screened does not suggest a mutagenic response, it does not necessarily confirm that it is not mutagenic or carcinogenic. It confirms that the substance is not mutagenic to the particular bacterial strain used for the genetic endpoint tested.
From this study and earlier results, we suggest that quercetin and flavanol or their synergistic effect can be the main mutagenic factor (CitationNagao et al., 1981; CitationOgawa et al., 1987; CitationAdam et al., 1990; CitationBjeldanesi & Chang, 1997). The positive results obtained from the Salmonella/microsome test might be checked through further experiments, such as animal tests. These investigations support the view that results from short-term bacterial tests are of very limited transferability to human.
In conclusion, the results from the present study suggest that H. lysimachioides var. lysimachioides extracts are mutagenic in the Salmonella/microsome test but do not induce the SOS response in E. coli PQ37 strain. The genotoxic activity observed in the Salmonella/microsome test might result from several mechanisms, and short-term bacterial test systems are not capable of detecting all possible mechanisms. We think that a test battery consisting of at least both bacteria and mammalian assays should be used in both in vitro and in vivo assays. To test for carcinogenicity, a two-year carcinogenicity test is required to be performed by testing the effect of the mutagenic sample in mice and rats (male and female) (CitationZeiger, 2001).
Acknowledgements
This paper is dedicated to Prof. Dr. Yavuz Ensari, who passed away on 14 August 2000. This work was supported by Dicle University Scientific Research Foundation (DUBAP-03-EF-26). The authors gratefully acknowledge helpful discussion and proofreading of the manuscript by Dr. Murat Kızıl and İbrahim Tunik. The authors also thank Dr. Tahsin Sogut for his valuable contribution to statistical analyses.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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