Potential genotoxicity of plant extracts used in Ethiopian traditional medicine
Introduction
The use of plants for the treatment of various ailments is an old practice probably as old as mankind itself. Many of the plants species used for this purpose have been found to contain therapeutic substances which can be extracted and used in preparation of drugs, but the plant itself can also be used either directly or as an extract for medication, a practice that is particularly popular in developing countries (Ishii et al., 1984, Hoyos et al., 1992). It has been estimated that more than 80% of the world's population utilizes plants as their primary source of medicinal agents (Cordell, 1995), largely due to the high cost of Western pharmaceuticals, but also because the traditional medicines are generally more acceptable from a cultural and spiritual perspective. Even in the Western world, the use of herbal medicines is steadily growing with approximately 40% of the population reporting use of herbs to treat medical illness (Bent and Ko, 2004).
Although plant extracts have been used in the treatment of diseases according to knowledge accumulated over centuries, it is also known that many plants synthesize toxic substances, which in nature act as defence against infections, insects and herbivores. Previous studies have also indicated that some substances present in some medicinal plants are potentially toxic and carcinogenic (De Sá Ferreira and Vargas, 1999) and it has also been reported that some traditional medicines may have a genotoxic potential (Sohni et al., 1994, Basaran et al., 1996, Romero-Jimenez et al., 2005). Assessment of the potential genotoxicity of traditional medicines is indeed an important issue as damage to the genetic material may lead to critical mutations and therefore also to an increased risk of cancer and other diseases.
Although little is known about their toxicological profiles, the plant extracts used in the present work have indeed been used for many years in Ethiopia. During recent years their major chemical constituents (potentially bioactive phytochemicals), have also been identified. The seeds of G. lotoides L. (Molluginacae), locally known as ‘Mettere’ are used for various purposes, including treatment of prevalent tapeworm infestation. The taeniacidal activity has been confirmed (Endale et al., 1997, Endale et al., 1998), and major bioactive phytochemicals that have been associated with this plant are different types of saponins and flavonoids (Abegaz and Tecle, 1980, Hamed and El-Elmary, 1999, Endale et al., 1998, Endale et al., 2005, Endale, 2005). Judging from recent single and repeat dose oral studies on rats, the hydroalcoholic extract of the seeds of G. lotoides seem to have low general toxicity (Demma et al., 2007).
The roots of P. zeylanica L. (Plumbaginaceae), locally known as ‘Amira’, are also used to treat various ailments, including malaria and various topical and systemic microbial infections (Abebe and Ayehu, 1993, Ahmad et al., 1998, Olagunju et al., 1999, Gebre-Mariam et al., 2006). The major chemical constituents seem to be plumbagin and various plumbagic acid glycosides and coumarins (Van der Vijver and Lotter, 1971, Kamal et al., 1983, Lie-Chwen et al., 2003). The dried leaves of T. schimperi R. (Lamiaceae), locally known as ‘Tosgne’, are used for the treatment of hypertension, and fungal and bacterial infections (Dagne et al., 1998) and the major components in the essential oil of thyme are various phenols such as thymol and carvacol and their precursors (Asfaw et al., 2000). The root of R. steudelii H. (Polygonaceae), locally known as ‘Tult’, is traditionally used as an antifertility agent, and its antifertility activity has been demonstrated in rats (Desta, 1994, Gebrie et al., 2005a, Gebrie et al., 2005b). Major constituents of the hydroalcoholic root extract include, among others, various saponins, phytosterols and polyphenols (Gebrie et al., 2005a).
Given the lack of knowledge in combination with their wide use and plethora of ingredients, the major purpose of the present study was to evaluate the potential genotoxicity of the aforementioned plants. The DNA damaging effects of these plant extracts were evaluated in vitro, both in the absence and presence of a metabolic activation system, using cultured mouse lymphoma L5178Y cells and the alkaline version of the comet assay. This technique, which has been used for several years at our department, is a relatively sensitive, inexpensive and rapid technique used world-wide to detect DNA strand breaks, alkali-labile sites, oxidative DNA damage and cross-links in individual cells. The mouse lymphoma L5178Y cells are normally used when evaluating gene mutations, but they have also been recommended to be useful cells when performing the comet assay in vitro (Tice et al., 2000). Rough phytochemical screenings were also performed on the crude extracts employed in the present study using chemical methods to ascertain the presence of claimed bioactive ingredients.
Section snippets
Chemicals and cells
Benzo(a)pyrene (B(a)P; CAS No. 50-32-8) and 4-nitroquinoline-N-oxide (NQNO; CAS No. 56-57-5) were purchased from Sigma, USA. Unless stated otherwise, these and all other chemicals were of analytical grade and double-distilled water was used throughout the experiments. Heterozygous L5178Y TK+/− cells (originally obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC, USA) were generously supplied to us by Dr G. Bolcsfoldi (AstraZeneca R&D, Södertälje, Sweden). To initiate
Results
The results of the preliminary phytochemistry screening are shown in Table 1. As indicated in the table phenolic compounds were found in all plant extracts. Moreover, all plant extracts except T. schimperi contained saponins as well as cardiac glycosides. Whereas flavonoids were found only in G. lotoides, R. steudelii was noted to have both tannins and anthraquinones.
Before initiating the studies on the DNA damaging effects of the four different plant extracts, an initial screening of cell
Discussion
The continuing growth in human exposure to natural products originating from traditional medicines has led to a resurgence of the scientific interest in their biological effects. The assessment of the efficacy and safety profiles of the medicinal plants should be based on scientific evidence-based approaches including, for example, different types of well-established short-term tests when evaluating the genotoxic profile of such plants. The short-term tests for genotoxicity are typically used
Conclusion
The present paper has shown that extracts of G. lotoides, P. zeylanica, R. steudelii and T. schimperi induced significant DNA damage in mouse lymphoma cells without inducing concomitant cytotoxicity, especially when the cells were exposed without metabolic activation. This suggests that components in these extracts might interact directly with the DNA. Studies with crude extracts are appropriate because it is in this form they are used as traditional herbal medicines. However, working with
Acknowledgements
The grant from the Swedish International Development Cooperation Agency/The Department for Research Cooperation (ref. no. 7500723502) is gratefully acknowledged. The authors are indebted to Lena Norgren for excellent technical assistance and to Dr. Asfaw Debella (Ethiopian Health and Nutrition Institute, Dept. of Drug Research) for his help.
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2015, Journal of EthnopharmacologyCitation Excerpt :One of them was R. steudelii, used as an antifertility agent. The results showed that an extract of the roots induced significant DNA damage in mouse lymphoma L5178Y cells without inducing concomitant cytotoxicity, especially when the cells were exposed without metabolic activation (S9-mix) (Demma et al., 2009). BNO 1016 (Sinupret®, Bionorica SE, Neumarkt, Germany) is an extract of a fixed combination of five herbal drugs, among them R. acetosa [Gentian root (Gentianae radix), Primula flower (Primulae flos), Sorrel herb (Rumicis herba), Elder flower (Sambuci flos) and Verbena herb (Verbenae herba), in a ratio of 1:3:3:3:3] that has been developed for the treatment of sinusitis.
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2020, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :After exposure, the cell suspension was split in two parts and immediately diluted in 4 mL of ice cold growth medium, one following the conventional alkaline comet assay protocol, the second following the Flash-comet protocol in order to further reduce inter-assay variability. Our standard procedure for the conventional alkaline comet assay is based on a slightly modified protocol of Singh et al. [2], previously described in great detail with some minor modifications [20,22,23] described in Table 1. First, a mixture of 30 μl of cell suspension in growth medium (approximately 1 × 105 cells/mL) and 210 μl 0.6% low-melting point agarose (Fisher Scientific, UK) was made.