IN-VITRO MUTAGENIC AND CYTOTOXIC ACTIVITY OF NIGELLA SATIVA SEED EXTRACT BY AMES AND MTT ASSAY

IN-VITRO MUTAGENIC AND CYTOTOXIC ACTIVITY OF NIGELLA SATIVA SEED EXTRACT BY AMES AND MTT ASSAY

B. Durga ^*, M. V. Dass Prakash and A. Julius

Department of Biochemistry ¹, Prince Shri Venkateshwara Arts and Science College, Chennai - 600073, Tamil Nadu, India.

Department of Biochemistry ², Sri Sankara Arts and Science College, Kanchipuram - 631561, Tamil Nadu, India.

Department of Biochemistry ³, Balaji Dental College and Hospital, Chennai - 600100, Tamil Nadu, India.

ABSTRACT: In the modern lifestyle, people are prone to disease. The epidemiology study shows that the lung and colon cancer leading position in both the gender around the world at any group. As based on the traditional systems of medicine, the plants can be used for the discovery of new medicine. The aqueous and ethanol extract of Nigella sativa (N. sativa) seeds were evaluated for their genotoxicity using Ames test and cytotoxicity by MTT assay. Ames assay was conducted using strains of Salmonella typhimurium TA-100, whereas MTT assay was performed on human alveolar basal epithelial cells (A549) and human colon cancer cell line (HCT15). There is no mutagenic activity was obtained when treated with aqueous and ethanol extract of Nigella sativa seeds. But both the extracts were found to be cytotoxic against the tested cancer cell lines.  On the basis of the results, it was concluded that aqueous and ethanol extracts of Nigella sativa seeds possess cytotoxic potential but not mutagenic. It is suggested to explore the seeds further to evaluate its anticancer activity in the in-vivo model.

Nigella sativa seeds, Mutagenic, Cytotoxicity, MTT, Ames test

INTRODUCTION: Medicinal plants have been used for many centuries for treating various diseases in indigenous systems of medicine as well as folk medicines. Moreover, medicinal plants are considered to be safe as compared to allopathic medicines for disease preparation. Many researchers are focusing on medicinal plants for investing their medicinal properties, potential and mechanism of action, safety evaluation, and toxicological studies. Nigella sativa Linn is an annual herb has focused as an important medicinal plant due to their beneficial effects over the health.

The seeds of this plant are used as constringent, stimulant, diuretic, anthelmintic, dyspepsia, paralysis, piles, and skin disease in the medicinal field. Nigella sativa Linn, commonly known as Black caraway, Black seed, Black cumin, fennel flower, nigella, nutmeg flower, Roman coriander, and Kalonji, belongs to Ranunculaceae family native to south and southwest Asia.

It is used as traditional medicine for various diseases from an ancient period as a natural restorer ¹. N. sativa seed essential oils also contain an essential compound that has antioxidant and anti-inflammatory effects ^{2, 3}. Nigella sativa seeds are regarded as the stimulants for energy metabolism, and is also involved in recovery from tiredness and depression; as Islamic prophet stated, the black seed could heal all diseases except death. Due to the modern lifestyle, the mortality rate of cancer is increased. As the focus of this, many efforts have been implemented for the search of natural bioactive compound (or) chemo-preventive agent, which would reduce (or) cease the progression of cancer ⁴. Many studies explain the secondary metabolites of the plants has the ability to reduce the risks of cancer ⁵. Black seeds are also used in food like flavoring additive in the bread and pickles because it has a very low level of toxicity. In this study, we aimed to investigate in-vitro mutagenic activity and in-vitro cytotoxicity effect of N. sativa seeds extracts by Ames and MTT assay, which has been so far merely studied.

MATERIALS AND METHODS:

Collection of Plant Materials: The plant material of Nigella sativa seeds used in this study was collected from a local market, Tambaram. Authentication of plant material is carried out by prof. P. Jayaraman, Plant Anatomy Research Centre, Tambaram, Chennai 45. Following identi-fication of Voucher specimen, the plants were deposited in the herbarium. Authentication number: PARC/2017/3364. Fresh seeds of the plant were collected, cleaned, washed, shade dried and used for further studies

Bacterial Strain Collection: The mutagenic activity of plant extracts was assessed using bacterial strains based on Ames bacterial reverse mutation test ⁶. Salmonella tester strains were received from Krishgen, Bombay.

Sources of Fine Chemical: All chemicals including solvents used were of the highest purity and analytical grade purchased from Sigma Aldrich, USA and Karthick Enterprises Pvt. Ltd., Chennai – 45.

Preparation of the Extract:

Extraction: The dried seeds of Nigella sativa were pulverized to a fine powder and stored in airtight containers separately.

Preparation of Ethanol Extract: 30 g of dried seed powder were extracted with 250 ml of ethanol for 18 cycles using Soxhlet apparatus. After the running time the extract was concentrated in a rotary vacuum evaporator with temperature ranging from 30-40 ºC.

Preparation of Aqueous extract: 20 g of dried seed powder were defatted by soaking the seed with petroleum ether for one day & 250 ml of distilled water were added for extraction & kept for 18 h. After that it’s filtered with cheesecloth, the extract were poured in Petri plates and lyophilized to make it as powder then they are stored in airtight container.

Mutagenicity Assay (Ames test): The assays were performed using tester strain TA100 in both the presence and absence of S9 metabolic activator, positive controls were also evaluated. All test and controls items doses were evaluated using plates. Tester strains were exposed to the samples via the plate incorporation methodology described by Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, the tester strain, samples, are combined in molten top agar, which is then overlaid onto a minimal bottom agar plate.

Tester Strain Density and Samples Preparation: The optical density of the overnight cultures were determined at 650 nm in a spectrometer and found to be within the range of 0.04 to 0.06, which demonstrates the cultures were in late exponential or early stationary phase with ≥ 10⁹ cells / ml. The sample of the extracts was dissolved in DMSO. Dose formulations were prepared on the day of use. Dose formulation of varies concentrations of samples were prepared by serial dilution for the study.

Plating Procedures: These procedures were used in the dose range-finding and mutagenicity assays.  Each plate was labeled with the samples, test phase, tester strain, activation condition and dose. Treatments in both the presence and absence of S9 were performed by adding 100 µl tester strain and 100 µl test or control are added to 2.5 ml molten diluted top agar (maintained at 45 ± 2 ºC).  The mixtures were vortexes and overlaid onto the surface of bottom agar dishes.

After the overlay solidifies; the plates were inverted and incubated for 72 h at 37 ± 2 ºC.  After incubation the plates were evaluated for the condition of the background lawn for the evidence of cytotoxicity and samples precipitate in comparison with the control and the plates were evaluated for the number of revertant colonies.

In-vitro Cytotoxicity Assay:

Cell Lines Maintenance: Cancer cell lines A549 and HCT15 were procured from Tissue Culture section of Virology Department at King Institute of Preventive Medicine. These cell lines were cultured in culture dish containing minimum essential medium 10% - Eagles MEM (Growth Medium), 2 % - Eagles MEM (Maintenance Medium) respectively. Both the cell lines were also maintained in Trypsin, phosphate buffer saline, Versene, Glucose, solution (T.P.V.G reagent) for subculturing. The culture dishes were incubated at 37 ºC in a carbon dioxide incubator ⁷ till the experiments starts. Cytotoxicity assay implies inhibition of cell growth. The cell undergoes morphological alteration in the cell layer or cell shape.

Treatment of Cancer Cell Line with Plant extract: The cell lines were treated with crude extracts, 1 ml of MEM with Sodium pyruvate 10% FCS was added to the dissolved Nigella sativa aqueous seed extract, Nigella sativa ethanol seed extract were filtered through Whatman syringe filter (pore size-0.2 µm).

The filtered crude extracts were made up into 1 ml stock. 100 µl of diluted Nigella sativa aqueous extract, Nigella sativa ethanol extract was added in the 96 well plates. The 96 well plates was kept in 5% CO₂ incubator at 36 ºC and observed after 24 h, 48 h and 72 h.

Cytotoxicity Test for Plant Extracts: The determination of cell cytotoxicity of Nigella sativa seeds extracts on both the cell lines was carried out using colorimetric assay that measures the reduction of yellow-3-(4, 5Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium            Bromide) by mitochondrial succinate dehydrogenase enzyme described by Mosmann in 1983. The number of surviving cells and % of inhibition is directly proportional to the level of the formazan product created. The color can then be quantified using a simple colorimetric assay. The results can be read on a multi-well scanning spectrophotometer (ELISA reader) ⁸.

RESULTS AND DISCUSSION:

Mutagenic Potential: The number of revertant colonies when exposed to TA-100 strains  and treated with both  the seed extracts for assessing the mutagenic action  were present in Table 3,  in both the presence and absence of (-S9) metabolic activation.

TABLE 3: MUTAGENIC ACTIVITY OF AQUEOUS EXTRACT AND ETHANOL EXTRACT OF PLANT USING STRAIN TA 100.  MEAN COLONY COUNT - STRAIN TA 100 - SPONTANEOUS MUTATION

PC: positive control (Na azide), NC: Normal control (DMSO), Aq. Ex: Aqueous extract, Et. Ex: Ethanol extract

The average revertant colonies per plate were treated with the control in both the presence and absence of metabolic activation system, which was found to be within the acceptable limits of the spontaneous revertant control values of the range 5-0.62 mg/ml of respective Salmonella strains TA 100. As there where a lack of papers regarding the mutagenic activity of N. sativa of aqueous and ethanol extracts, our study provide as evidence for the non-mutagenic effect of extracts. Among the several other works, ⁹ suggested that the anti-genotoxic effect of N. sativa seed extracts were investigated for a chromosomal mutation in bone marrow cells and spleen of male albino mice infected with schistosomiasis. The same result was found even in the in-vitro investigation when exposed to Thymoquinone or extracts. The paper reported by ¹⁰ & ¹¹ that aqueous seed extracts of N. sativa have the ability to reduce the chromosome aberration in spermatocytes of the mouse when it was treated with CCl₄. The protective effect of methanol extract of roots& shoots of Nigella sativa plant is used in assessing the anti-mutagenic potential on the bacterial strains of TA 1535 along with induced mutagen such as sodium azide ¹² & also by bleomycin-induced cell damage in human lymphocytes has shown the result in decreasing the chromosome changes ¹³. In other study using Nigella sativa seed, oil & TQ reduced the tumor effect in female rats induced by DMBA treatment ^{14, 15}. Our study result clearly exhibits that the number of revertant colonies of TA100 strains is not induced by both the extract of Nigella sativa seeds with or without the presence of metabolic activation of S9 mix as compared to the positive control. The result was expressed in mean value shows that no significant difference among the two extracts, hence the crude extracts of Nigella sativa seed of the plant are assessed to be as the non-mutagenic effect could to be due to the presence of the anti-mutagenic property.

Subsequently, the extracts were also analyzed for cytotoxicity effect over the cancer cell lines such as A549 and HCT15 at 48 h. In the previous study of Nigella sativa seeds has shown better cytotoxicity effect against many cancer cell lines such as Hela cells, cos31, MCF7, BG-1 cells 16 using the methanolic extract. The study reveals that the methanolic extract has the ability to down-regulate the expression of the anti-apoptotic gene and upregulate the expression of apoptotic genes, which suggest that the action is due to the presence of the active compounds. The in-vitro cytotoxicity studies of various species of Nigella sativa seed extract were assessed by neutral red uptake test; the investigation reveals that the extract has the ability to induce the anticancer properties by decreasing the cell proliferation, reducing the percentage of abnormalities over the chromosome ¹⁷ indicates that irrespective of any kind of Nigella species have anti-proliferative activity. A study reported that active component of the plant seed exerted as an antitumor effect by negative regulation of signal cascade mechanism in colorectal cancer ¹⁸.

As the previous study has revealed about the cytotoxic effect of Nigella sativa in several different types of cancer, where our in-vitro experimental results show that the low concentration of ethanol extract itself has good ability to triggers the anti-proliferative mechanism by stimulating the apoptosis process which brings the cell death (or) inhibit the cell growth as compared to aqueous extract on both lung cancer cell line and colon cancer cell line. The in-vitro Cytotoxicity Potential of crude extract of Nigella seed was calculated for IC₅₀ value at 48 h & depicted in the graph.

GRAPH 1: ANTI-PROLIFERATIVE ACTIVITY OF NIGELLA SATIVA IN A549 ADENOCARCINOMA HUMAN ALVEOLAR BASAL EPITHELIAL CELL LINE

GRAPH 2: ANTI-PROLIFERATIVE ACTIVITY OF NIGELLA SATIVA IN HCT 15 COLON ADENOCARCINOMA CANCER CELL LINE

Graph 1 and 2 shows the dose-dependent changes in the viability of Cancer cell lines such as A549 and HCT15. The results clearly confirmed that the efficacy of the extracts does not only depend on the concentration of the dosage would also be based on the presence of bioactive compounds.

CONCLUSION: Hence our study rendered that the crude extracts of Nigella sativa seed has no mutagenic effect and also has the ability to inhibit the proliferation of cancer cells at dosage depended manner. Therefore, further investigation is required for the isolation and characterizations of bioactive compounds from crud seed extract.

ACKNOWLEDGEMENT: This research work is supported by the Department of Biochemistry, Prince Shri Venkateshwara Arts & Science College, Gowrivakkam, Chennai, Tamil Nadu, India.

CONFLICTS OF INTEREST: There are no conflicts of interest among all the authors with the publication of the manuscript.

REFERENCES:

1. Harzalah H, Kouidhi H, Flamini B, Bakhrouf G, Mahjoub A and Touhami: Chemical composition, antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone .Food chemistry 2011; 129(4): 1469-74.
2. Gali-Muhtasib HU, Fakhoury IH, Zaki AM, El-Baba CO and Schneider-Stock R: Thymoquinone: Fifty years of success in the battle against cancer models. Drug Discovery Today 2014; 19: 18-30.
3. Woo CC, Kumar AP, Sethi G and Tan KH: Thymoquinone: Potential cure for inflammatory disorders and cancer. Biochem Pharmacol 2012; 83: 443-51.
4. Perše M and Cerar A: “The dimethyl hydrazine induced colorectal tumors in rat-experimental colorectal carcinogenesis,” Radiology and Oncology 2005; 39(1): 61–87.
5. Lipkin SM, Moens PB, Wang V, Lenzi M, Shanmugarajah, Gilgeous A, Thomas J, Cheng J, Touchman JW and Green EDD: Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat Genet 2002; 31: 385-90.
6. Maron DM and Ames BN: Revised Methods for the Salmonella Mutagenicity Test. Mutation Research/ Environmental Mutagenesis and Related Subjects 1983; 113: 173-15.
7. Razak MFA, Aidoo KE and Candlish AGG: Mutagenic and cytotoxic properties of three herbal plants from Southeast Asia Tropical Biomedicine 2007; 24(2): 49-59.
8. Thounaojam MC, Sankhari JM, Jadeja RN and Devkar RV: Anthocyanin‐rich red cabbage (Brassica oleracea ) extract attenuates cardiac and hepatic oxidative stress in rats fed an atherogenic diet journal of the sciences of food and agriculture 2012; 92 (8): 1688-93
9. Nguyen T, Talbi H and Hilali A: In-vitro toxicity, genotoxicity and antigenotoxicity of Nigella sativa extracts from different geographic locations. South African Journal of Botany 2019; 126: 132-41.
10. Abdel-Moneim AM, Essawy AE, Hamed SS, Abou-Gabal AA and Alzergy AA: Protective effect of Nigella sativa seeds against spermatocyte chromosomal aberrations and genotoxicity induced by carbon tetrachloride in mice. Enviro Science and Pollution Res 2017; 24: 11677-82.
11. El-Baba C, Mahadevan V, Fa hlbusch FB, S SM, Rau TT, Gali-Muhtasib H and Schneider-Stock R: Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer. Molecular Cancer 2014; 13: 201-05
12. Bourgou S, Ksouri R, Bellila A, Skandrani I, Falleh H and Marzouk B: Phenolic composition and biological activities of Tunisian Nigella sativa shoots and roots. Comptes Rendus Biologies 2007; 331: 48-55.
13. Galhena BP, Samarakoon SSR, Thabrew MI, Paul SFD, Perumal V and Mani C: Protective effect of a polyherbal aqueous extract comprised of Nigella sativa (seeds), Hemidesmus indicus (roots) and Smilax glabra (rhizome) on Bleomycin induced cytogenetic damage in human lymphocytes. Biomed Res International 2017; 1-7.
14. Linjawi SA, Khalil WK, Hassanane M and Ahmed ES: Evaluation of the protective effect of Nigella sativa extract and its primary active component thymoquinone against DMBA-induced breast cancer in female rats. Archives of Medical Science 2015; 11: 220-29.
15. Liu W, Liu J, Yin D and Zhao X: Influence of ecological factors on the production of active substances in the anti-cancer plant Sinopodophyllum hexandrum (Royle) TS Ying. PLOS One 2015: e0122981.
16. Talbi H, Talbi J, Charaf B and Hilali A: The in-vitro micronucleus assay for antigenotoxic effect evaluation of the Nigella sativa seeds extract on cultured human peripheral blood lymphocytes. International Research Journal of Pharmaceutical and Biosciences 2016: 3: 23-34.
17. Dilshad A, Abulkhair O, Nemenqani D and Tamimi W: Anti-proliferative Properties of Methanolic Extract of Nigella sativa against the MDA-MB-231 Cancer Cell Line, Asian Pacific Journal of Cancer Prevention 2012; 13:(11) 5839-42.
18. Jrah-Harzallah H, Ben-Hadj-Khalifa S, Almawi WY, Maaloul A, Houas Z and Mahjoub T: Effect of Thymoquinone on 1, 2-dimethyl-hydrazine-induced oxide-tive stress during initiation and promotion of colon carcinogenesis. Eur J Cancer 2013; 49: 1127-35.
    

    ---

    How to cite this article:

    Durga B, Prakash MVD and Julius A: In-vitro mutagenic and cytotoxic activity of Nigella sativa seed extract by AMES and MTT assay. Int J Pharm Sci & Res 2020; 11(11): 5760-65. doi: 10.13040/IJPSR.0975-8232.11(11).5760-65.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---