Elsevier

Journal of Ethnopharmacology

Volume 122, Issue 1, 25 February 2009, Pages 136-142
Journal of Ethnopharmacology

Potential genotoxicity of plant extracts used in Ethiopian traditional medicine

https://doi.org/10.1016/j.jep.2008.12.013Get rights and content

Abstract

Aim of the study

Although traditional herbal medicines are widely used in Ethiopia, no information is available on their potential genotoxicity. In the present study, hydroalcoholic extracts of Glinus lotoides, Plumbago zeylanica, Rumex steudelii and Thymus schimperi were evaluated for their DNA damaging effects using the comet assay.

Material and methods

Mouse lymphoma L5178Y cells were exposed to different concentrations of the extracts for 3 h with and without metabolic activation (S9-mix) using 4-nitroquinoline-N-oxide and benzo(a)pyrene as positive controls, and vehicles as negative controls.

Results

In the absence of S9, all extracts were found to induce significant DNA damage without affecting the cell viability. T. schimperi and R. steudelii were the most potent DNA-damaging extracts, and G. lotoides and P. zeylanica the least potent. The addition of S9 had different effects on the DNA damage induced by the extracts: it lowered the DNA damaging effect of P. zeylanica, did not affect the DNA damaging effect of T. schimperi, and increased the DNA damaging effects of R. steudelii and G. lotoides.

Conclusion

The findings of the present study suggest that all extracts evaluated have a genotoxic potential in vitro which needs to be substantiated by further studies.

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.

References (55)

  • A. Endale et al.

    Quantitative determination of the group of flavonoids and saponins from the extracts of the seeds of Glinus lotoides and tablet formulation thereof by high-performance liquid chromatography

    Journal of Chromatography A

    (2005)
  • S.M. Galloway et al.

    DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens

    Mutation Research

    (1998)
  • T. Gebre-Mariam et al.

    Antiviral activities of some Ethiopian medicinal plants used for the treatment of dermatological disorders

    Journal of Ethnopharmacology

    (2006)
  • E. Gebrie et al.

    Phytochemical screening and pharmacological evaluations for the antifertility effect of the methanolic root extract of Rumex steudelii

    Journal of Ethnopharmacology

    (2005)
  • A.I. Hamed et al.

    Triterpenoidal saponins from Glinus lotoides Var Dictamnoides

    Phytochemistry

    (1999)
  • A. Hartmann et al.

    The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay)

    Toxicology Letters

    (1997)
  • B. Havsteen

    Commentary: flavonoids, a class of natural products of high pharmacological potency

    Biochemical Pharmacology

    (1983)
  • B. Hellman et al.

    The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay

    Mutation Research

    (1995)
  • L.S. Hoyos et al.

    Evaluation of the genotoxic effects of a folk medicine. Petiveria alliacea (anamu)

    Mutation Research

    (1992)
  • T.S. Kumaravel et al.

    Relaible Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals

    Mutation Research

    (2006)
  • G.B. Marini-Bettolo et al.

    Plant screening by chemical and chromatographic procedures under field conditions

    Journal of Chromatography

    (1981)
  • S.O. Mueller et al.

    Characterization of the genotoxicity of anthraquinones in mammalian cells

    Biochimica et Biophysica Acta

    (1999)
  • S.O. Mueller et al.

    Occurrence of emodin, chrysophanol and physcion in vegetables, herbs and liquors. Genotoxicity and antigenotoxicity of the anthraquinones and of the whole plants

    Food and Chemical Toxicology

    (1999)
  • M. Romero-Jimenez et al.

    Genotoxicity and antigenotoxicity of some traditional medicinal herbs

    Mutation Research

    (2005)
  • N.P. Singh et al.

    A simple technique for quantitation of low levels of DNA damage in individual cells

    Experimental Cell Research

    (1988)
  • Y.R. Sohni et al.

    Bacterial mutagenicity of eight medicinal herbs from Zimbabwe

    Mutation Research

    (1994)
  • D. Abebe et al.

    Medicinal Plants and Enigmatic Health Practices of Northern Ethiopia

    (1993)
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