An in vitro Study on the DNA Damaging Effects of Phytochemicals Partially Isolated from an Extract of Glinus lotoides Jemal Demma, 1,2 Hesham El-Seedi, 3,4 Ephrem Engidawork, 2 Teshome Leta Aboye, 4,5 Ulf Göransson 4 and Björn Hellman 1 * 1 Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, Box 591, 751 24 Uppsala, Sweden 2 Department of Pharmacology, School of Pharmacy, Addis Ababa University, P O Box 1176, Addis Ababa, Ethiopia 3 Department of Chemistry, Faculty of Science, El-Menoufia University, 32512 Shebin El-Kom, Egypt 4 Department of Medicinal Chemistry, Division of Pharmacognosy, Uppsala University, Box 574, 751 23 Uppsala, Sweden 5 Department of Pharmaceutical Chemistry, School of Pharmacy, Addis Ababa University, P O Box 1176, Addis Ababa, Ethiopia An extract of Glinus lotoides, a medicinal plant used in Africa and Asia for various therapeutic purposes, was recently shown to cause DNA damage in vitro. To further explore the potential genotoxicity of this plant, frac- tionation of the crude extract was performed using reverse phase solid-phase extraction and a stepwise gradi- ent elution of methanol in water. Four fractions were collected and subsequently analysed for their DNA damaging effects in mouse lymphoma cells using an alkaline version of the comet assay. To identify potential genotoxic and non-genotoxic principles, each fraction was then subjected to liquid chromatography coupled to mass spectrometry, LC-MS/MS. 1D and 2D nuclear magnetic resonance analyses were used to confirm the identity of some saponins. Although fractions containing a mixture of flavonoids and oleanane-type saponins or oleanane-type saponins alone produced no DNA damage, those containing hopane-type saponins exhibited a pronounced DNA damaging effect without affecting the viability of the cells. To conclude, even if this study presents evidence that hopane-type of saponins are endowed with a DNA damaging ability, further studies are needed before individual saponins can be cited as a culprit for the previously reported genotoxicity of the crude extract of G. lotoides. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: Glinus lotoides; flavonoids; saponins; comet assay; mouse lymphoma cells. Supporting information may be found in the online version of this article (Supplementary Material) INTRODUCTION Glinus lotoides L. (Molluginaceae), commonly known as lotus sweetjuice, is widely found in tropical and subtropical regions of the World. In Ethiopia, where the species is known as ‘metere’, seeds of this plant have been used as an anthelmintic, laxative, antimicrobial and antidiabetic drug (Pankhurst, 1965; Kloos et al., 1978; Endale et al., 1997, 1998). In India, it is used as a traditional medicine against diarrhoea and bilious attacks, as a purgative and for curing boils, wounds and pains (Biswas et al., 2005). Methanolic extracts of G. lotoides have also been reported to have antitumor activity against Dalton’s ascetic lym- phoma in mice (Kavimani et al., 1999). Moreover, Abdel- Hameed et al. (2008) have recently shown potential antimicrobial activity of methanolic extract of the plant, and Sathiyanarayanan et al. (2006) have also reported anticholesterolemic, hepatoprotective and antioxidant activities of an ethanolic extract in rats. Previous phytochemical works on G. lotoides have indicated the presence of a number of oleanane- and hopane-type triterpenoid saponins (>20 saponins) and flavonoids (mainly C-glycosidal flavones) (Barua et al., 1976; Abegaz and Tecle, 1980; Hamed et al., 1996, 2005; Hamed and El-Emary, 1999; Endale et al., 2005a, 2005b). Further chemical analysis revealed that the crude extract of the seeds contained as much as 21.3% flavonoids and 25.4% saponins, and that these constitu- ents contribute to the pharmacological activities of the seeds (Endale et al., 2005a). Numerous studies have reported various effects of these two classes of com- pounds. Saponins are known to have not only foaming and emulsifying properties (Price et al., 1987), but also a diverse range of biological and pharmacological effects such as antimicrobial, insecticidal and molluscicidal activ- ities (Sindambiwe et al., 1998; Attele et al., 1999; Sparg et al., 2004), as well as haemolytic properties (Oda et al., 2000). Saponins have wide applications in beverages and confectionery, as well as in cosmetics (Price et al., 1987; Petit et al., 1995; Uematsu et al., 2000) and pharmaceutical products (Sparg et al., 2004). Flavonoids have also been reported to have multiple biological effects, including anti-inflammatory, antiviral, anti-allergic, antioxidant and free radical-scavenging abilities (Bors and Saran, 1987; Middleton and Kandaswami, 1992; Gil et al., 1994; Sahu and Gray, 1996; Duthie et al., 1997). Currently, medicinal applications of the seeds of G. lotoides have initiated much interest, and the crude extract has now been formulated in tablet form to be used for the claimed therapeutic purposes in Ethiopia * Correspondence to: Björn Hellman, Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden. E-mail: bjorn.hellman@farmbio.uu.se PHYTOTHERAPY RESEARCH Phytother. Res. 27: 507–514 (2013) Published online 31 May 2012 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.4744 Copyright © 2012 John Wiley & Sons, Ltd. Received 26 October 2011 Revised 22 March 2012 Accepted 02 May 2012