Keywords

Nigella sativa, Alternative medicine, AMES test, Cytotoxicity.

Introduction

The plant Nigella sativa is well known for its therapeutic effect against various diseases/conditions such as, Parkinson, urease, infertility, overweight, memory impairment, lung cancer, diabetes etc [1-7]. Thymoquinone, dithymquinone, thymohydroquinone, and thymol are the main active compounds responsible for the therapeutic effects of Nigella sativa seeds. Variable doses of extracts of Nigella sativa are being recommended by the medical practitioner around South Asian, Middle Eastern and European countries for a range of medical conditions. However, limited attention is given to the related dose dependent toxicity.

Few earlier studies have reported adversity of uses of Nigella sativa as a therapeutic agent. A recent study evaluated the potential role of Nigella sativa seed oil on oxidative stress and revealed that it was able to elevate the concentration of glutathione, lactate dehydrogenase, hepatic succinate dehydrogenase and attenuate the level of malondialdehyde, superoxide dismutase, etc [8]. Another study reported that cultured rat cortical neurons when exposed to different times and concentrations of Nigella sativa dry methanolic extract, it did not induce any toxicity. However, the same study also advocates evidently that it modulated amino acid release in cultured neurons, for example, secretion of glutamate, aspartate, and glycine were decreased [9]. In addition, few studies have noticed increased phagocytic activity of peritoneal macrophages, hepatotoxicity and nephrotoxicity following Nigella sativa administration [10-13]. Thymoquinone, which has been shown to exert anti-inflammatory, anti-oxidant and anti-neoplastic effects both in vitro and in vivo appear to have multiple physiological side-effects, including cell death [14]. From the above studies it is apparent that despite its medicinal properties, it also has potential for serious side-effects if taken at higher doses.

We assume that prolonged traditional herbal usage cannot be guaranteed for its safety, because, it is difficult to monitor or to detect the collective multifactorial delayed effects arising from long-term use. In this study we have attempted to estimate cytotoxicity of ethanolic extract of Nigella sativa seed (NSEte) in Wistar albino rats and through reverse mutation test in strains of Salmonella typhimurium.

MATERIALS AND METHODS

Test materials

Fresh seeds of Nigella sativa, were procured commercially (voucher no.21, dated 04.02.2012), authenticated in the Department of Botany, University of Ranchi, Ranchi, India. The seeds were shaded, dried and powdered. The powdered seeds (500 g) was extracted with petroleum ether (60-80°C) to remove lipids. It was then filtered and the residue was extracted with 95% ethanol by soxhlet extraction. The extract obtained was evaporated and concentrated on a water bath at atmospheric pressure to a semisolid condition, which was further dried in an oven at 30°C on a shallow dish to constant weight to remove the solvent completely (yield, 22.78%).

Animals

Adult Wistar albino rats, approximately three months old, weighing 180–200 g, were used in this investigation. The animals were maintained in individual polypropylene cages with a 12:12-h light: dark schedule. The temperature in the animal house during the study period was maintained at 23 ± 2ºC, and the relative humidity ranged between 32-70%. The feeding schedule consisted of two rat pellet meals a day, and water was provided ad libitum. Daily intake of food and water were quantified precisely. The animals were maintained under veterinary supervision in accordance with the Guidelines for Care and Use of Animals in Scientific Research15. The experimental protocol has the approval of the Institutional Animal Ethical Committee (IAEC). (Z/23/3.12.2011).

Experimental design

The animals were divided into four groups, ten animals in each, Group I, served as control, were treated orally with olive oil at 10 ml/kg body weight each day for 90 days, Group II, Group III and Group IV were treated orally with the NSEte, at a standardized dose of 5, 50 and 500 mg/kg body weight, respectively, each day for 90 days.

Euthanization

After the last scheduled dose of extract was administered animals were euthanized by cardiac puncture under ether anesthesia.

Chromosomal aberration

Animals were injected with 0.2 ml of 0.5% colchicines to arrest the cells at metaphase. Bone marrow was aspirated from femur in a test tube and cell suspension was prepared. Cells were then treated with KCl solution (0.56%) and fixed in Carnoy’s fixative. Cells were then stained with 5% Giemsa and observed under 100x oil immersion. A total of 500 metaphase arrested cells were evaluated for aberrant chromosomes. Likewise, 500 bone marrow cells were also examined for mitotic activity by scoring the number of cell in mitosis. Cytotoxic activity was estimated by the mitotic index, calculated by dividing cells to the total numbers of cell counted. For positive control ten Wistar albino rats were used. Positive control animals were orally administered with 50 mg/kg body weight of acrylamide (Himedia Laboratories, Pvt. Ltd., Mumbai) for 5 days.

Micronucleus test

Bone marrow cells were collected in fetal bovine serum (FBS) and after washing smeared on to glass slide. Further, slides were stained with May-Gruenwald and Giemsa stain according to Schimid [16]. A total of 500 erythrocytes were examined for micronuclei. For positive control ten Wistar albino rats were used. Mitomycine C (3 μg/g body weight) was injected before 48 h of euthanization for positive control.

Reverse mutation assay

The test strains used in this study were Salmonella typhimurium TA100, TA98 and TA97a (Bruce Ames Laboratory, Molecular and Cell Biology, University of California) (Table 1). Each test strain was incubated overnight in 2.5% Nutrient broth at 37°C with shaking at 100 strokes/min, in dark. Rat liver was used to prepare S9, treated with Phenobarbital (Sigma-Aldrich, MO, USA) and 5, 6-benzoflavone (Sigma- Aldrich, MO, USA). The S9 metabolic activation mixture (S9 mix) was used for the bacterial reverse mutation test and survival test as described by Maron and Ames [17] with slight modification. S9 mix was prepared by adding 0.1 ml of S9, 8 mmol of MgCl2, 33 mmol of KCl, 5 mmol of D-glucose-6- phosphate, 4 mmol of NADPH, 4 mmol of NADH and 100 mmol of sodium phosphate (pH 7.4). Top agar consisting of 0.05 mmol histidine, 0.6% NaCl and 0.6% agar (Himedia Laboratories, Pvt. Ltd., Mumbai) was sterilized by an autoclave.

Table 1. Salmonella typhimurium strains used in this experiment

Reverse mutation test was conducted according to the standard protocol (with slight modification) as described by Yahagi et al. [18] and Mortelmans and Zeiger [19]. Briefly, separate test tubes containing 0, 0.1, 1 and 10 g of NSEte, 500 μl of buffer (0.1 M sodium phosphate buffer, pH 7.4) with or without S9 mix and 100 μl of test strain suspension was incubated for 20 min at 37°C with shaking at 120 strokes/min in dark. Further, 2 ml of top agar was added to the test tube and mixed, the mixture was then poured onto the surface of a plate containing minimal glucose agar medium (VBE medium (Vogel Bonner E), 0.5% Glucose, 1.5% Agar). Plates containing 20 ml of minimal glucose agar medium was incubated for 48 h at 37°C and the reverted colonies were counted. Experiments were repeated thrice for each dose to calculate an average value. The positive controls for the culture without S9 mix was Sodium Azide (20 μg/plate) (Sigma-Aldrich, MO, USA) and for cultures with S9 mix was 2- aminoanthracene (20 μg/plate) (Sigma- Aldrich, MO, USA) for all test strains.

Survival experiments

To determine the cytotoxic effects of NSEte, the pre-incubation assay mixture in the bacterial reverse mutation test was diluted in 0.9% NaCl. This suspension contained 2.0 × 10³ cells/ml. An aliquot (100 μl) of this suspension was plated on nutrient agar (0.8% nutrient broth, 0.5% NaCl, and 1.5% agar). The plates were then incubated at 37°C for 24 h and survival fractions were calculated as percentage survival compared to the negative group. All experiments were performed in triplicate.

Statistical analysis

Significant differences between treatment and the control group in the micronucleus assay were assessed by Dunnet’s multiple comparison tests. Oneway ANOVA and Tukey’s HSD post hoc analysis was used for positive results of the assay. Values are expressed as mean ± standard deviation (sd), and P<0.05 was considered statistically significant.

RESULTS

Chromosomal aberration in bone marrow cells

Chromosomal aberration per 500 dividing cells was recorded. The incidences of structural abnormalities of chromosomes, in the single chromatid and double chromatid were minimal. The variation was recorded insignificant in all test groups, however Group II and Group IV showed more than one type of aberration such as fragments, rings, and variably damaged chromosomes. The mitotic indices revealed no statistically significant variation. The variation in number of aberration between test groups and positive controls were found highly significant, as shown in Table 2.

Table 2. Frequency of aberrant cells and mitotic indices (MI) of bone marrow following treatment with NSEte (Values are in mean ± SD) (n=10)

Formation of polychromatic and normochromatic erythrocytes

The incidences of micronuclei in polychromatic red blood cells (RBC) of Group I animals were found close to 0.2%, whereas, ratio of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were about 6%. There were no significant variations in number of micronuclei, observed in Group II, Group III and Group IV when compared to control (Table 3). Animals treated with Mitomycine C showed significantly higher percentage of PCEs, NCEs and their respective ratios.

Table 3. Micronucleus record of rats following treatment with NSEte. (A total of 500 erythrocytes were counted) (Values are in percentage) (n=10)

Salmonella reverse mutation assay

Average number of revertant colonies found in negative control showed almost three folds increase for TA100 and TA97a in both the presence and absence of S9 mix when compared to TA98. Incorporation of NSEte showed insignificant mutagenic activity for the applied range of doses in both absence and presence of S9 mix. No significant variation in mutagenic indices were observed for any applied doses and found comparable to negative control. Percentage of survival fractions were mostly recorded 100%, however, TA100 show slight variation in survival rate but considered insignificant for any dose related response (Table 4). Sodium Azide and 2- aminoanthracene incorporated plates were observed with more than 500 colonies, the mutagenic indices were found significantly higher when compared to control and the test groups (Not shown in table).

Table 4. Mutagenicity in Salmonella typhimurium strains with and without metabolic activation following incorporation of NSEte. (Values are in mean ± SD)

DISCUSSION

An earlier study reported that Nigella sativa fixed oil has low toxicity which was evident by its high value of LD50 and almost no morphological changes in the histopathological examination of vital organs20. There are wide ranges of studies which proved its hepato-protective effect as well as its mild hepatotoxicity [21-25]. Overall, there are mixed conclusion over the toxicity of Nigella sativa in the scientific world, it falls in both positive and negative categories. We presume that it is a commercially available supplement, and the consumption in raw form is still quite popular. People usually consume it in very small quantity, therefore, unaware to the unknown effect of its constituents. In the present study we observed no clear sign of cytotoxicity caused by NSEte regardless of the concentration of doses.

The result of chromosomal aberration in this study showed normal chromosomal morphology despite varying doses. Similarly, micronucleus test revealed no adverse activity induced by doses applied in this investigation. In addition, no growth inhibition of bone marrow cell was observed in any of the experimental groups, since no differences were found in the PCE: NCE ratio among the groups.

Lower nontoxic concentrations are important to be preliminarily determined in order to show the real mutagenic effects of the tested compounds [26,27]. Therefore, in this study lower doses were also taken in to account. A measurable increase at one or more concentration in the number of revertant colonies per plate in at least one strain with or without a metabolic activation system, and/or a statistical analysis of an increase over background response can determine the positive result. Our results clearly show that none of the Salmonella strains used in this experiment indicated NSEte induce mutagenesis with or without metabolic activation, thus, revealing the doses under examination are less likely to induce genetic interference.

CONCLUSION

We estimate from the study that the ethanolic extract of Nigella sativa seeds is highly unlikely to produce any cytotoxic activity at doses investigated. However, further studies are required to ascertain its safety at higher doses.

Acknowledgement

The work at our lab is financially support by the University Grant Commission (UGC), New Delhi, India. We are thankful to the Indian Biological Research Consultancy Services (IBRCS), Ranchi, for the technical support.

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