In Vitro Toxicity, Genotoxicity and Antigenotoxicity of Nigella Sativa Extracts from Different Geographic Locations

South African Journal of Botany 126 (2019) 132–141

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South African Journal of Botany

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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 recommended 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). Actually, the plant is of uttermost importance and exten- 2013). The positive controls used were 0.4 mg mL−1 4-NQO in tests sively consumed in the Muslim world as it has been mentioned in “Pro- without S9mix and 0.8 mg mL−1 benzo(α)pyreneB(α)P with S9mix. phetic Hadit,” as a “natural remedy for all the diseases except death” The tested extract concentrations ranged from 0 to 18 mg/mL. (Ijaz et al., 2017). It is also identified as the curative black cumin in the Genotoxicity and toxicity measurements were performed using a Holy Bible, and is described as the Melanthion of Hippocrates and microplate luminometer that enables online measurements of emitted Dioscorides and as the Gith of Pliny (Atta-ur-Rahman et al., 1985). It is light (e.g., every 5 min over a period of 4 h = 48 rounds). Data were therefore not surprising that research is being conducted on the biolog- transferred into an Excel macrosheet and the signal to noise ratio (S/ ical, beneficial, but also potential adverse health effects of this important N), i.e., the light production of exposed bacteria divided by the light pro- plant. duction of non-exposed bacteria, was calculated for each measurement. S/N was calculated for the recN2-4 and pr1 strain separately, as well as 2. Materials and methods the ratio between the maximum S/N values of the recN2-4 and pr1strains. All calculations occurred automatically and were based on 2.1. Plant material measurements between 60 and 240 min of incubation. Based on experimental grounds a compound is considered genotoxic when the maxi- Nigella sativa seeds from plants growing at three different locations mum S/N (recN2-4)/max S/N (pr1) (=rec/pr1) is greater than 1.5. in Morocco were obtained from local suppliers. The three regions Substances are considered toxic if there is a substantially decrease in were: Erfoud (South East; 31°26′10″N 4°13′58″W), Settat (Centre- the light emission resulting in the S/N ratio decreases below 0.8 in West; 33°0′N7°37′W), and Fkih ben Salah (Mid atlas; 32°30′00″N Cytox strain. In this case, we cannot use the result for genotoxicity as- 06°32′00″W) (Fig. 1). sessment of the test compound (extract). The test concentration The seeds were washed and dried and then ground to obtain a fine shoud then be reduced. powder which was homogenized and dissolved in phosphate buffered Antimutagenicity properties were evaluated as for the Ames assay saline (PBS, pH = 7.4; Haq et al., 1995). Stock solutions of 180, 80 and (see below). 134 T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141

2.3. Ames assay absorption of NR occurs. Absorption of non-treated cells was given a 100% value to which data from exposed cells were compared. Sodium The potential mutagenic and antimutagenic effects of the Nigella Dodecyl Sulfate (SDS) in concentrations of 0.42, 0.35, 0.28, 0.21, 0.14, sativa seed extracts were determined using the Ames test with Salmo- and 0.07 mM was used as a positive control. nella typhimurium strain TA 98 and TA 100 as described by Maron and Ames (1983).Briefly, 100 μL of bacterial stock was incubated in 20 mL of Oxoid Nutrient broth for 16 h at 37°C on a rotary shaker. Of this over- 2.5. Comet assay in C3A cells night culture, 0.1 mL was added to 2.0 mL of top agar (containing 0.05 M Histidine/ Biotin) together with 0.1 mL test solution and 0.5 mL phos- The alkaline comet assay (Singh et al., 1988; Tice et al., 2000; Olive phate buffer (or 4% rat liver S9mix). To determine mutagenicity, the and Banáth, 2006) was used to investigate genotoxicity in human C3A test solution contained 50 μL test sample and 50 μL solvent control. cells. The cells were seeded and incubated in a standard growth me- The positive control in the test with S9mix was 1 μg/plate 2- dium for 24 h. Then they were exposed with the test substance in con- aminoanthracene (2AA). In the test without S9mix, the positive controls centrations of 2, 4, 9 mg/mL for the next 24 h. The test concentration were 0.2 μg/plate 4-nitroquinoline oxide (4-NQO) for TA98, and 5 μg/ was based on the result of a preliminary neutral red uptake test plate sodium azide (SA) for TA100 respectively. (NRU). Ethyl methanesulfonate (EMS, 0.25 M) was used as positive con- To determine antimutagenicity, the test solution contained 50 μLtest trol. After exposure, cells were harvested, re-suspended in 150 μL ice sample and 50 μL positive control. The top agar mixture was poured cold PBS and kept on ice to prevent further DNA damage. Then, 50 μL over the surface of a minimal agar plate and incubated for 48 h at of cell suspension was mixed with agarose and was loaded on the pre- 37 °C. After incubation the numbers of revertant colonies (mutants) in coated slide. After complete hardening, slides were immersed in ice each plate were counted. A compound was considered a mutagen cold lysing buffer (2.5 M NaCl; 100 mM EDTA; 10 mM TRIS; 1 v% Triton when the number of colonies in one or both strains was doubled com- X-100 and 10 v% DMSO, pH 10) for at least 1 h. An horizontal electro- pared to that found in the negative (solvent) control plates. The test ex- phoresis tray was filled with the denaturation buffer at pH 13 tract was diluted to the final concentration of 9–4–1 mg/plate. The (temperature b 17 °C) and connected to a circulating pump to homoge- negative (solvent) control was PBS, as extracts were dissolved in this nize and distribute the buffer evenly. Slides were kept in the electropho- solvent. resis tray for 40 min to allow DNA unwinding. After unwinding, the Antimutagenicity was expressed as the percentage of inhibition of single-strand DNA was electrophoresed for 20 min. The voltage was mutagenicity induced by the positive control. It was calculated using set to 1 V/m and the current was adjusted to 300 mA. Then, slides the formula: were quickly removed from the tray and washed three times with neu- tralization buffer (TRIS buffer, pH 7.5). Finally, the slides were put into Inhibitionð%Þ¼ ð1 – T=MÞ100 ice cold ethanol for 10 min in the fridge and dried overnight at room temperature in the dark. To visualize DNA damage, DNA was stained Here T is the number of revertants per plate in the presence of mu- with GelRed and Vectashield. The analysis was done automatically tagen and the test solution and M is the number of revertants per with a fluorescent microscope (AxioImager. Z2) linked to an image plate in the positive control. All cultures were prepared in triplicate (ex- analysis system (Metafer4, version 3.8.5; Metasystems, Altlussheim, cept for the solvent control where five replicates were used). Absence of Germany). Two slides were prepared for each exposure condition. The toxicity was confirmed when a background layer of bacterial growth percentage of DNA in the comet tail was used as a measurement of was observed. DNA damage (Verschaeve et al., 2013). It was assumed that an extract had no antimutagenic potency when it reduced the mutagenicity of the positive control by less than 25%. An inhibition of mutagenicity by 25–40% was qualified as weak 2.6. The micronucleus test in C3A cells antimutagenic. An extract was considered strong antimutagenic when the inhibition was greater than 40% (Ong et al., 1986). The cytokinesis-block micronucleus (CBMN) assay was performed according to standard procedures (e.g., Fenech, 2000). The cell prepara- 2.4. The Neutral Red Uptake (NRU) test in human C3A cells tion and exposures were the same as for the comet assay, but the positive control was 15 μg/mL Methyl methanesulfonate (MMS). After 24 h We performed the Neutral Red Uptake (NRU) test on human C3A exposure, the excess media was discarded and 30 μL cytochalasin B cells. These are a clonal derivative of Hep G2 liver cancer cells that we (CytB) and 1970 mL FBS were added to each well. The plate was again previously already used because they have nitrogen metabolizing activ- incubated for 24 h. Then, cells were harvested, followed by a hypothonic ity comparable to perfused rat livers and largely conserved phase I and II shock with 0.075 M KCl at 35 °C and fixation with ice cold fixator (3 biotransformation capabilities (e.g., Cappoen et al., 2012; Edziri et al., Methanol:1 Acetic acid). Finally, the cell suspension was spread on 2013; Akremi et al., 2016; Makhuvele et al., 2018). three slides for each condition and dried overnight. After staining with The NRU in vitro cytotoxicity test was performed according to stan- DAPI stain in Vectashield, cells were scored to the presence of dard procedures described by Repetto et al. (2008). Extracts were tested micronuclei using the Metafer 4 software. The objective was to detect in the concentration range of 0 up to 18 mg/mL. Human C3A cells were at least 2000 cells for each condition. Results were analyzed as described plated in 96 well plates (40.000 cells per well) and incubated in by Fenech et al. (2003). Dulbecco's Modified Eagle Medium (DMEM) + 10% Fetal Bovine To examine the cytotoxicity of a test compound, the Cytokinesis-

Serum (FBS) for 24 h at 37 °C and 5% CO2. Next, extracts were added Block Proliferation Index (CBPI) was calculated by determining the in different concentrations for another 24 h. Cells were then washed number of mononucleated, binucleated and multinucleated cells with phosphate buffer saline (PBS) after which 200 μL0.05mg/mLneu- among 500 cells. Cells were visualized after staining with Acridine or- tral red solution was added. After 3 h, cells were again washed in PBS to ange (0.1%) followed by washing three times with Sorensen buffer, remove the remaining dye. Addition of 200 μL ethanol/acetic acid (50/1) pH 6.8 (0.026 M KH2PO4 and 0.015 M Na2HPO4.2H2O). If the CBPI resulted in the release of the dye from the cells that were placed on a index is equal to 1 this means that all cells are mononucleated and stirring plate until a homogenous color was formed (approximately hence that there is 100% cytotoxicity. The CBPI is given by 1 h). The optical density (OD) was measured with a spectrophotometer. CBPI = (M1 + 2 ∗M2 + 3 ∗Mm)/N. Where: M1, M2, Mm represent

The OD620 measured as a reference value was subtracted from the OD540 the number of respectively mononucleated, binucleated and multinu- which is the optical density at the wavelength at which maximal cleated cells. N is the number of cell scored (= 500). T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141 135

Fig. 2. Vitotox test results of Moroccan N. sativa in three different regions (Erfoud, Fkih ben Salah, and Settat). Figure a, c, e are results of genotoxic tests. Figure b, d, f are antigenotoxic tests.

3. Results Vitotox results for cultures that were simultaneously exposed to the extracts and positive controls are summarized in Fig. 2b, d and f. Con- 3.1. Vitotox test comitant exposure to the plant extract was in the given concentrations of 0 up to 18 mg/mL. Overall it can be seen that the plant extract A summary of the Vitotox test results is presented in Fig. 2.Itcan lowered the genotoxicity of the mutagen but important reduction be seen that the sample from Erfoud (Fig. 2a) was not genotoxic as of genotoxicity at the higher concentrations is misleading as these S/N ratio's never exceeded a value of 1.5. Concentrations of 9 and concentrations were also toxic (especially Fkih ben Salah and to a some- 18 mg/mL were toxic as S/N in the cytox strain were considerably what lower extent Settat). With S9, toxicity is lower but also the lower than 0.8. The signal to noise ratio in the genox strain also antigenotoxic effect is rather low. The extract concentration of showed values well below 1. Addition of S9 greatly removed the tox- 9 mg/mL showed the most important antigenotoxic effect. Overall icity and there was also no sign of genotoxicity (S/N in genox and antigenotoxicity was not very explicit. cytox strains were all approximately 1). The positive controls (resp. 4-NQO and B(α)P) were clearly genotoxic as values above 1.5 were 3.2. Ames test reached in the genox strain and no interference with light production was observed in the cytox strain (S/N ~ 1). The sample from Settat Results of the Ames test on TA98 and TA100 performed on Moroccan (Fig. 2c) and Fkih ben Salah (Fig. 2e) also showed no genotoxicity Nigella sativa extracts are presented in Table 1 where data are expressed but most concentrations in cultures without S9 showed important as fold response compared to the negative controls. The number of re- toxicity, especially the sample from Fkih ben Salah (S/N bb0.8 in vertants per plate was in the negative controls always in accordance cytox and genox strain). Addition of S9 again drastically reduced with the literature (Maron and Ames, 1983) and our own historical con- the toxic response. trol data. Positive controls (0.2 μg/plate 4-NQO and 5 μg/plate SA in 136 T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141

Table 1 did not reduce the DNA damage seen by EMS alone. On the contrary, Results from the Ames test performed on Moroccan N. sativa extracts. Nd = Not done. we saw a statistically significant increase in DNA damage for all extracts Data are expressed as fold response compared to the negative controls. which was most important for the extracts from Fkih bin Salah, followed Plant species Tested extract -S9 +S9 by Erfoud and Settat. Co-genotoxicity of the latter was only present at concentration (mg/plate) TA 98 TA 100 TA 98 TA 100 the highest extract concentration which was borderline toxic according to the NRU test. Nigella sativa 9 1.36 0.92 1.52 1.27 (Erfoud) 4 1.08 0.91 1.24 1.08 1 1.06 0.96 1.13 1.10 3.5. Micronucleus test Nigella sativa 9 nd 1.06 nd 1.23 (Fkih ben Salah) 4 nd 0.97 nd 1.17 1 nd 1.06 nd 1.22 As indicated above the NRU test showed toxicity of the plant extracts Nigella sativa 9 1.04 1.25 1.60 1.46 at the concentration of 9 mg/mL, especially the sample from Settat, and (Settat) 4 1.32 1.17 1.42 1.23 to a lesser extent Erfoud. According to CPBI values toxicity at this 1 1.23 1.00 1.32 1.06 concentration was also evident and present in the extracts from all locations. Calculated % cytostasis values were indeed all higher than 70% (Table 3). Lower concentrations were not toxic (data not shown). The extract from Erfoud and Fkih ben Salah induced significantly higher cultures without S9 and 1 μg/plate 2AA with S9) always gave the ex- number of micronuclei than those found in the negative control but this pected results validating the study (not shown). As shown, revertant was obvious only at the toxic concentration of 9 mg/mL. Lower concen- colonies in cultures exposed to the extracts were never twice as high trations showed no increased frequencies of micronuclei, except the as in the negative (solvent) control cultures. The extracts were therefore sample of Erfoud (2 mg/mL), but there was no dose-effect relationship, not mutagenic. as no effect was found at 4 mg/mL. The extracts also did not show antimutagenic properties in the Ames In cells exposed to extract and mutagen (MMS) the extract from fi assay as the percentage inhibition never exceeded 25% (except Settat, 1 Erfoud signi cantly induced more micronuclei than MMS alone. The mg/plate; % inhibition = 25.2%). Overall a positive % inhibition corre- sample from Erfoud therefore showed co-genotoxicity except at the sponding to antimutagenicity was negligible or inexistent. On the con- toxic extract concentration of 9 mg/mL. Extracts from Fkih ben Salah fi trary, in some cases a negative % inhibition was found indicating a co- and Settat inversely exhibited antigenotoxicity by signi cantly reducing mutagenic response. In cultures without S9 this was moderately so for the number of scored micronuclei at the highest non-toxic concentra- the Settat and Fkih ben Salah samples in TA100, and in cultures with tion (Fig. 5). S9 in TA98 where the negative % inhibition was high (Settat) to very high (Erfoud). This might also be the case for Fkih ben Salah where 4. Discussion the test could not be done by shortage of material. Thus, in the Ames assay Nigella sativa sometimes showed co-mutagenicity rather than Environmental variations can influence the production and accumu- antimutagenicity (Table 2). lation of secondary metabolites such as alkaloids, flavonoids, and terpenoids, which assist in coping with abiotic stress such as increased solar 3.3. Neutral Red Uptake test (NRU) on C3A cells radiation, rainfall, drought or soil nutrition conditions (Liu et al., 2015), or with biotic factors like soil microbial communities (Nihorimbere The NRU test revealed that concentrations higher than 9 mg/mL re- et al., 2011). Flavonoids, a class of natural compounds found in many duce cell viability of human C3A cells to less than 70%. Hence, this and plants, including the seeds of N. sativa, for example possess protective higher concentrations were toxic (Fig. 3). Our cytogenetic investigations functions during drought stress (Ramakrishna and Ravishankar, 2011). (comet and micronucleus tests) were therefore conducted at concentra- Soil contamination with toxic metals is another example of causes of tions up to 9 mg/mL. physiological changes in plants that may include, among others, inacti- vation of certain enzymes and blocking functional groups of metaboli- 3.4. Comet assay cally important molecules (Okem, 2015). Many studies have shown that plants grown in area polluted by heavy traffic tend to accumulate As shown in Fig. 4 the extract from Settat (4 mg/mL) and Erfoud (2 more heavy metals than those in residential area (Barthwal et al., and 4 mg/mL) showed some DNA damaging capacity but this was low 2008). Contaminated soils were shown to stimulate plant defense and far from the effect produced by EMS which considerably increased systems by producing more active compounds to strengthen metal- thepercentageofDNAinthecomettail.ExposureofcellstoEMSobvi- detoxification capacity (Emamverdian et al., 2015). Environmental ously resulted in genotoxicity, whereas co-exposure with plant extract variations are thus responsible for chemical and physiological changes

Table 2 Results from the Ames test performed on Moroccan N. sativa extracts in combination with a chemical mutagen. Used mutagens were SA, 1 μg/plate (TA98, -S9), 2AA 0.5 μg/plate (TA98, + S9), SA 1 μg/plate (TA100, -S9), and 2AA 0.5 μg/plate (TA100, +S9). Given data were fold response values and % inhibition.

Plant species Tested extract -S9 +S9 concentration TA 98 Antimutagenicity TA Antimutagenicity TA 98 Antimutagenicity TA 100 Antimutagenicity (mg/plate) (%) 100 (%) (%) (%)

Nigella sativa- Erfoud 9 0.93 6.9 1.16 −15.7 1.72 −72.1 0.81 18.6 4 0.97 3.2 1.12 −11.9 1.46 −46.3 0.78 22.4 1 0.95 5.1 1.16 −16.3 1.11 −11.5 0.93 7.2 Nigella sativa-Fkih ben 9 1.28 −28.2 0.88 12.5 Salah 4 1.18 −18.4 1.06 −5.0 1 1.11 −10.6 1.11 −10.8 Nigella sativa- Settat 9 1.13 −12.8 1.31 −30.5 1.47 −46.9 0.91 9.4 4 1.14 −13.9 1.27 −26.7 1.31 −30.5 1.03 −2.8 1 1.09 −8.8 1.23 −23.4 1.02 −2.4 0.75 25.2

Antimutagenicity (% inhibition N 25) or comutagenicity (b -25) are given in bold. T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141 137

Fig. 3. NRU test results expressed as percentage viability (%) of C3A cells exposed to aqueous extracts of N. sativa from three different regions in Morocco at different concentrations and exposed to both extracts and EMS 0.5 M (a, b, c) and NI50 determination curves for the positive controls SDS (d). in plants which may potentially also influence the plant's potential isochemical and fersialitic. The climate is arid to semi-arid with a dry adverse or beneficial effects for humans (Sampaio et al., 2016). season from April to October and a wet season from November to With respect to N. sativa, many studies already showed that environ- March. The annual rainfall is 300 mm, the average temperature is 18 mental factors generated important changes in seed yield and oil con- °C with a maximum in August of 38 °C and a minimum in January of tent. Particularly, black cumin yields appeared to be strongly 3.5 °C. There is no particular air pollution, but Fkih ben Salah was yet depended on water scarcity and soil gypsum content (Al-Kayssi et al., shown to be more polluted than other cities due to landfill activities 2011), with regions with high rainfall having higher seed yields than and increasing pollutant load generated by leachates (Merzouki et al., regions where rainfall was less important (Nimet et al., 2015). Also, in- 2015; Merzouki et al., 2016). creased nitrogen applications resulted in a consistent decrease in Physiological changes due to different environmental stimuli the content of Mn, Zn, and Ni in seed oil (Ashraf et al., 2006). In this may, as indicated above, possibly result in different toxic, mutagenic paper we report on further investigations of N. sativa seed properties and/or antimutagenic properties. They were the subject of our present obtained at different locations. We thereby focussed on it's in vitro tox- investigation. icity, genotoxicity and antigenotoxicity. Seeds were therefore obtained In vitro cytotoxicity studies were conducted with the NRU test and from three different regions in Morocco; Erfoud, Settat and Fkih ben showed that the extract from Erfoud was the most toxic, followed by Salah. Settat and Fkih ben Salah. With respect to genotoxicity/antigenotoxicity Erfoud is a town that lies in an oasis in the Sahara desert and has vir- some investigations were already conducted before on extracts from N. tually no precipitation during the year. Its average rainfall/year is sativa or its principal constituents. In a preliminary investigation some less than 200 mm. The maximum temperatures can rise to more of us found that aqueous seed extracts (from the Settat region) at a con- than 40 °C while the nights are very cold. The annual temperature is centration of ~ 0.2 mg/mL in culture (10 mg/mL stock solution) induced 20.8 °C. The region of Erfoud has a calcareous brown soil on limestone micronuclei in human blood lymphocytes. However at increasing con- or shale substrate and is not particularly subjected to air pollution or centrations a decrease in cell proliferation kinetics, cell viability and soil contamination. The Settat region is in the northeast of Morocco micronucleus frequencies was found as a result of toxicity (Talbi et al., and closest to the Atlantic Ocean. It has a raw mineral isohumic, 2014). The genotoxicity at the lower concentrations was seen as an in- fersialitic and hydromorphic soil. The climate is semi-arid with an aver- dication of the potential anticancer properties of this herbal prepara- age annual rainfall of 372 mm and an average annual temperature of 18 tion. In two further studies it was found that Nigella sativa L. seed °C. There is no particular air pollution but some locations are irrigated extract (now from Erfoud) decreased the frequency of micronuclei in- with wastewater resulting in a soil contaminated with heavy metals duced by Methotrexate (MTX), increased cell proliferation kinetics and other contaminants (Meftah El Kadmiri et al., 2006; Glouib et al., (Talbi et al., 2016), and also reduced MTX-induced chromosome aberra- 2007). The soil at Fkih ben Salah is mainly of calci-magnesium type, tions and sister chromatid exchanges (Talbi et al., 2017). Here 138 T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141

GENOTOXICITY N.sativa_ARFOUD Anti-GENOTOXICITY Nigella_ARFOUD 40 xxxx 50 xx x

30 40 (median) (median) l

l 30 20

in tai 20 10 x x 10 %DNA %DNAintai 0 0 l l l m S O /m /m / M S S S S S g g M g E M M M M m m m M E E E E D 2 4 9 + + + m M l l l % 5 .5 . m /m /m /m 0 0 .5 g g g 0 m m m 2 4 9

GENOTOXICITY NIGELLA_Fkih ben Salah Anti-GENOTOXICITY Nigella-Fkih ben Salah 40 xxxx 50 xxx xxx x

40 30 30 20 20

10 10 % DNA in tail (median) %DNAintail(median) 0 0 l l l S S S S m S O /m /m / M M M M M S g g g E E E E E M m M + + + D m m 9 M l l l 2 4 m m m m m % 5 / / / .5 .5 . g g g 0 0 0 m m m 2 4 9

GENOTOXICITY NIGELLA_Settat Anti-GENOTOXICITY N.sativa_SETTAT 40 xxxx 60 xxxx

30 40 il (median)

ta 20

20 Ain

N 10

D x %DNAintail(median) % 0 0 l l l m S S S S S O /m /m / M M M M M S g g g E E E M E E m m m M M + + + D 2 4 9 l l l % m m /m /m /m .5 .5 .5 g g g 0 0 0 m m m 2 4 9

Fig. 4. Comet assay results for (anti)genotoxic tests performed in C3A cells of N. sativa extract from three different regions in Morocco: Erfoud, Fkih ben Salah, and Settat. 0.5 M EMS was used as positive control. x = p b .05; xx = p b .01; xxx = p b .01; xxxx = p b .0001. genotoxicity of the extract alone was not investigated. Several other of chromosome deletions and tetraploidy were also reduced. Abdel- studies also reported an antimutagenic (or in general antigenotoxic) Moneim et al. (2017) reported that aqueous seed extracts of Nigella effect of Nigella sativa L. seed extracts. Aboul-Ela (2002) for example in- sativa reduced the chromosome aberration frequency in mouse primary vestigated chromosome aberrations in bone marrow cells of male albino spermatocytes that were treated with the clastogen CCl4.CCl4-induced mice infected with S. mansoni (schistosomiasis) and also conducted an dominant lethals in mice were also not seen any more when the animals in vitro investigation in spleen cells from the mice exposed to the ex- were also treated with an aqueous suspension of the seed extract tracts or thymoquinone (TQ). In both experiments Nigella sativa L. (50 mg/kg body weight). Other protective effects related to mutagen- seed extracts reduced the percentage of chromosome aberrations in induced genotoxicity were, for example, reported for methanol extracts such a way that control levels were obtained again, and the incidence of Nigella sativa root and shoots in the Ames test when TA 1535

Table 3 CBPI values and corresponding cytostasis (%) of all six investigated medicinal plant extracts at the highest tested concentration.

Samples Concentration Genotoxicity test Antigenotoxicity test

CBPI Cytostasis (%) CBPI Cytostasis (%)

N. sativa (Erfoud) 9 mg/mL 1.158 75.2 1.090 85.8 N. sativa (Fkih ben Salah) 9 mg/mL 1.152 76.4 1.084 87.0 N. sativa (Settat) 9 mg/mL 1.178 74.6 1.116 83.5 T. Nguyen et al. / South African Journal of Botany 126 (2019) 132–141 139

xx xxxx xxxx x xx

xxxx xxxx x x

xxxx xxx

Fig. 5. Frequencies of micronuclei observed in C3A cells after treatment with different concentrations of three Moroccan N. sativa extracts alone (ﬁgures at the left) and in conjunction with the positive control MMS (ﬁgures at the right). x = p b .05; xx = p b .01; xxx = p b .01; xxxx = p b .0001.

Salmonella typhimurium bacteria were exposed to the well-known genotoxicity and antigenotoxicity. We indeed demonstrated differences mutagen sodium azide (Bourgou et al., 2007). The same was found for in outcome according to the location from where the Nigella sativa polyherbal aqueous decoctions (including Nigella sativa)and plants and seeds were originating. Overall the extracts were not bleomycin-induced cytogenetic damage in human lymphocytes genotoxic, except in the micronucleus test where the extracts from (Galhena et al., 2017). Ether and ethanol extracts and decoctions were Erfoud and Fkih ben Salah showed increased micronucleus frequencies, found to have antimitotic effects in Allium cepa with the ether extract but not the extract from Settat. This is in contrast with the earlier find- being the most effective in reducing mitosis in root meristem cells (Ali ings of Talbi et al. (2014) who used human peripheral blood lympho- et al., 2007). Another paper investigated the effect of ethanol extracts cytes as the only obvious difference with our study, and that of Salem of Eryngium creticum, Nigella sativa and Teucrium polium in primary (2005) who worked with the MCF-7 mammary tumor cell line. Absence cultures of rat hepatocytes that were exposed to N-methyl-N′-nitro- of genotoxicity was however also found in human lymphocytes by N-nitrosoguanidine (MNNG), which is a directly acting mutagen. The Galhena et al. (2017) who investigated chromosome aberrations, results of this investigation indicated an inhibitory effect of the plant ex- micronuclei and γH2AX foci following exposure to Nigella sativa decoc- tracts on MNNG mutagenicity, while the extracts had no effect on cyto- tions (but not seed extracts that we investigated). Our extract from toxicity indicators such as necrosis and apoptosis (Khadera et al., 2010). Settat showed antigenotoxicity in the micronucleus test but some indi- Linjawi et al. (2015) also found that seed oil of Nigella sativa as well as cations of co-genotoxicity were found in the Ames assay. The latter is in TQ reduced the rate of certain tumor markers (MDA, LDH, ALP and contrast with the data from Bourgou et al. (2007), but these authors AST), inhibited the histopathological alterations and decreased the ex- performed the Ames assay only in strain 1535 which detects base pair pression of Brca1, Brca2, Id-1 and P53 mutations in mammary tissues substitutions and might be not sufficient. It should be remembered of female rats induced by DMBA treatment. In short, literature data that according to OECD guidelines five bacterial strains are recom- especially highlight the antigenotoxic effect of Nigella sativa extracts, mended (OECD, 1997). We used TA98 and TA100 only because of short- regardless their origin. Further investigations on TQ alone gave similar age of material, but they are the most common strains of Salmonella results. Badary et al. (2007), for example, investigated the effect of TQ typhimurium used in the Ames test and detect frameshift mutations on male Swiss albino mice and found that a daily intake of TQ after, (TA98) and base-pair substitutions (TA100). It is known that these before or during exposure to benzo(α)pyrene significantly reduced two strains detect more than 95% of all mutagens (Mortelmans and the frequencies of chromosome aberrations induced by benzo(α) Zeiger, 2000). Co-genotoxicity was also found for the extract from pyrene alone. Danaei and Karami (2017) on the other hand found Fkih ben Salah. Our results are therefore largely in contradiction to that bone marrow cells that were treated with TQ reduced Diazinon most of the published investigations where antigenotoxicity was hematotoxicity, immunotoxicity and genotoxicity although found, yet in other test systems and other extracts. genotoxicity could not be totally prevented. 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