In Vitro Toxicity, Genotoxicity and Antigenotoxicity of Nigella Sativa Extracts from Different Geographic Locations
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South African Journal of Botany 126 (2019) 132–141 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb In vitro toxicity, genotoxicity and antigenotoxicity of Nigella sativa extracts from different geographic locations T. Nguyen a,b,H.Talbic,A.Hilalic,d,R.Anthonissena,A.Maesa, L. Verschaeve a,e,⁎ a Sciensano, Risk and Health Impact Assessment Service, Brussels, Belgium b Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium c Pharmacological Laboratory of the National Agency of Medicinal and Aromatic Plants, Taounate, Morocco d Higher Institute of Health Sciences, Hassan I University, Settat, Morocco e University of Antwerp, Department of Biomedical Sciences, Antwerp, Belgium article info abstract Article history: Nigella sativa or black cumin is used as a spice and as a natural remedy against a great variety of illnesses. Received 12 December 2018 However, plants growing at different locations, i.e., coping with different environmental stress conditions, may Received in revised form 18 February 2019 have different contents of specialized compounds include alkaloids, flavonoids, and terpenoids. Therefore their Accepted 19 February 2019 properties and biological effects may be different. Available online 7 March 2019 We used the neutral red uptake test, the bacterial Vitotox and Ames assays and the micronucleus and comet tests Edited by Kannan RR Rengasamy in human C3A cells to investigate the in vitro cytotoxicity, genotoxicity and antigenotoxicity of aqueous seed extracts of this plant originating from three different regions in Morocco. Keywords: Nigella sativa seed extracts showed different degrees of cytotoxicity depending on the location where the test Nigella sativa samples came from. Overall they were not genotoxic but some indications of both antigenotoxicity and Cytotoxicity co-genotoxicity were found. In this the results are somewhat in contradiction with previous reports. Genotoxicity Actually, our results show that many factors may influence the outcome of an investigation. The location of the Antigenotoxicity samples, but also the applied test system and other experimental conditions are examples of factors that should Seed extracts be taken into account when evaluating the biological properties and potentials for nutritional and medicinal ap- Geographic location plication in humans. This obviously complicates these kind of investigations and assessment and shows that it may be time to develop and agree on common protocols and procedures rather than performing experiments solely based on a laboratory's capacity, availabilities and opportunities. © 2019 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction modern medicines already originate from plants. Examples are taxol which is extracted from the Yew tree (Taxus baccata; Bharadwaj and Despite the fact that we now have medicines against virtually any Yu, 2004; Brito et al., 2008), quinine from the bark of the quinine tree disease or health problem there still is a continuous search for new (Cinchoa pubescens; WHO, 2014), aspirine from the leaves of the willow medical preparations. One of the reasons is that existing treatments tree (Jones, 2015; Jeffreys, 2008) or atropine which comes among are not always accurate enough, alleviating the symptoms but not cur- others from leaf and roots of the nightshade (Atropa belladonna; ing the disease. Another reason is that existing medicinal preparations McLendon and Bhimji, 2018). The search for new medicines from are sometimes very costly and therefore not accessible to everybody. plant origin is a continuous effort and should go along with investiga- They also often have unwanted side-effects and may lose their efficacy tions of their potential adverse health effects. It is indeed imperative with time. that a plant constituent with a given interesting medicinal property is Medicinal plants which are used since ancient times and proved ac- without danger with respect to other biological endpoints, or at least curate against particular illnesses are one of the important sources of that the risks are known. A medical drug or preparation may for exam- new lead compounds with potential medicinal applications. Plants in ple not be mutagenic or carcinogenic, unless this is the desired property general are so diverse that many still need to reveal their properties (as may be the case for anticancer medicines). A Chinese tea which was and potencies in health care systems. As a matter of fact many of our very popular in the 1990s and which contained a combination of three herbs, Aristolochia fangchi, Stephania tetranda and Magnolia officinalis, appeared to be responsible for causing acute kidney failure. The active ⁎ Corresponding author at: Sciensano, Risk and Health Impact Assessment Service, Brussels, Belgium ingredient in Aristolochia was responsible for damaging the body's E-mail address: [email protected] (L. Verschaeve). DNA and triggering tumor growth (Nortier et al., 2000; Nachtergael https://doi.org/10.1016/j.sajb.2019.02.015 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved. T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141 133 et al., 2015). Hypoxis hemerocallidea (known as star flower or yellow star) provides another example. It is often referred to as “the plant that can be used to treat many diseases” (Amusan et al., 2007). It has been used as a traditional medicine for centuries and has been recom- mended for inclusion in the daily diet of HIV patients (Van Wyk, 2015). Although we didn't found it genotoxic (Verschaeve et al., 2013) it is still surrounded by controversy; the plant or its constituents can be toxic and must be used with caution (Mills et al., 2005; Cordier and Steenkamp, 2011; Fasinu et al., 2015). It is recommended to use a safe alternative. It is clear that adverse effects should be prevented as much as possi- ble. This is the reason why a lot of research is now being conducted on, e.g., mutagenicity/genotoxicity of (medicinal) plants and other natural products or their ingredients in order to prevent their abusive use as a medicine, functional food or food additive. As it is assumed that, as mutagens and/or genotoxic compounds are often carcinogens, it is also believed that antimutagens and/or antigenotoxic compounds may protect against cancer (e.g., Verschaeve and Van Staden, 2008). Antigenotoxicity is therefore often part of the evaluation of the potential genetic effects of plant extracts and/or their constituents. However, Fig. 1. Locations where Nigella sativa plant seeds originate from. such screening is labor intensive and may encounter a lot of problems one should be aware of (Verschaeve, 2015). One of the problems is that identical plants may have different properties according to the loca- 10 mg/mL were prepared, filtered through millipore 0.22 μm filter tion where they grow. This is because they are subject to different envi- paper and centrifuged at 4500 rpm for 10 min to remove any insoluble ronmental conditions and hence behave differently (e.g., produce debris. different secondary metabolites) according to the external stimuli they receive (WHO, 2004; Okem, 2015). This means that the results from biological studies of plant extracts may as well depend on the loca- 2.2. Vitotox test tion from where the plants were obtained. If this holds true with respect to their genotoxic/antigenotoxic properties interesting plants may pos- This genotoxicity test is based on Salmonella typhimurium TA104 sibly be misevaluated and lost for further research and applications, just bacteria that contain the lux operon of Vibrio fischeri under transcrip- because of the unlucky location from where they were harvested. tional control of the recN gene, which is part of the SOS system (van In this paper we report on the in vitro toxicity, genotoxicity and der Lelie et al., 1997; Verschaeve et al., 1999; Verschaeve, 2013). Actu- antigenotoxicity of Nigella sativa originating from three different re- ally, two genetically engineered bacterial strains are used in this test gions in Morocco. Nigella sativa or black cumin (not to be confused system; TA104 recN2-4 (genox strain) and TA104pr1 (Cytox strain). with the fennel herb plant Foeniculum vulgare)isindigenoustothe The former strain carries the plasmid containing the luciferase operon Mediterranean region but has been cultivated in other parts of the (luxCDABE), the latter strain constitutively express the lux operon. world including the Arabian Peninsula, northern Africa and parts of Genotoxic compounds activate the recN promoter in the genox strain, Asia. It is used as a spice, especially in Indian and Middle Eastern cui- which results in transcriptional induction of the lux operon followed sines. The oil and seed constituents have shown potential medicinal by enhanced light emission. Cytotoxicity (decreased light emission) or properties in traditional medicine. It is among others used as a natural the non-specific enhancement of light emission by other mechanisms remedy for a number of illnesses including asthma, cough, hyperten- is simultaneously assayed by the Cytox strain. Thus, concomitant use sion, bronchitis, diabetes, headache, eczema, fever, inflammations, and of the Genox and Cytox strains allows the identification of false positive other diseases (Ali and Blunden, 2003; Salem, 2005; Gholamnezhad results or of toxic concentrations (Verschaeve et al., 1999; Verschaeve, et al., 2016).