Biotechnological approaches for production of bioactive secondary metabolites in Nigella sativa:¬ an up-to-date review Biotechnological approaches for production of bioactive secondary metabolites in Nigella sativa:¬ an up-to-date review

Main Article Content

Abeer Kazmi Mubarak Ali Khan Huma Ali


There is a significant count of medicinal plants and their refined natural products which being explored and are found rich in terms of therapeutic potential. A dicotyledon of the Ranunculaceae family known as Nigella sativa which is in common use for a long time in history as preservative and spice is an extensively utilized medicinal plant around the globe. It is also an eminent component of traditional medicine systems like Unani and Tibb, Ayurveda and Siddha as it has been utilized for centuries to promote health and to fight different disease. It has been widely used as antidiarrheal, analgesic, antibacterial, liver tonic, diurectic and digestive agent. It is also useful to treat several skin disorders. Furthermore, the therapeutic properties also include antidiabetic, anticancer, antihypertensive, anti-inflammatory, hepatoprotective, spasmolytic and bronchodialator. This is all because of its miraculous healing power that it has been ranked as top ranked, evidence based herbal medicines. The literature supports that the pharmacological activities of Nigella sativa are mainly because of the essential oil and its constituents particularly thymoquinone. The current review is an attempt to present a detailed literature survey regarding chemical composition, therapeutic potential and approaches to enhance the medicinal significance of this valuable plant.

Article Details

Sending to International Journal of Secondary Metabolite


[1] Khare, C.P. (2004). Encyclopedia of Indian medicinal plants. NewYork Springes-Verlag Berlin Heidelberg.

[2] Paarakh, P. M. (2010). Nigella sativa Linn.- A comprehensive review. Indian J. Nat. Prod. 1, 409- 429.

[3] Mandal, A., Datta, A. K., Bhattacharya, A. (2011). Evaluation of pollen and productive parameters, their interrelationship and clustering of eight Corchorus spp. (Tiliaceae). Nucleus 54, DOI 10.1007/s13237-011-0044

[4] Datta, A. K., Saha, A. (2003). Cytomorphological Studies and Seed Protein Characterization of Nigella sativa L. and Nigella damascena L. Cytologia 68, 51-60.

[5] Warrier, P.K., Nambiar, V.P.K., Ramankutty. (2004). Indian medicinal plants-a compendium of 500 species. Chennai Orient Longman Pvt Ltd, 139-142.

[6] Goreja, W.G. (2003). Black seed nature’s miracle remedy. New York, NY 7 Amazing Herbs Press.

[7] Al-Jassir, M.S. (1992). Chemical composition and microflora of black cumin (Nigella sativa L.) seeds growing in Saudi Arabia. Food Chemistry, 45, 239-242.

[8] Cheikh-Rouhou, S., Besbes, S., Lognay, G., Blecker, C., Deroanne, C., Attia, H. (2008). Sterol composition of black cumin (Nigella sativa L.) and Aleppo pine (Pinus halpensis Mill.) seed oils. Journal of Food Composition and Analysis, 21(2), 162-168.

[9] Bourgou, S., Ksouri, R., Bellila, A., Skandrani, I., Falleh, H., Marzouk, B. (2008). Phenolic composition and biological activities of Tunisian Nigella sativa L. shoots and roots. Comptes Rendus Biologies, 331(1), 48-55.

[10] Nickavar, B., Mojab, F., Javidnia, K., Amoli, M.A. (2003). Chemical composition of the fixed and volatile oils of Nigella sativa L. from Iran. Z Naturforsch, 58(9-10), 629-631.

[11] Katare, D.P., Aeri, V., Bora, M. (2009). Secondary metabolites and metabolic engineering. Journal of Cell Tissue Research, 9(3), 2027–2036.

[12] Bharat, B.A., Ajaikumar, B.K. (2009). Molecular Targets And Therapeutic Uses Of Spices: Modern Uses For Ancient Medicine. Woprld Scientific Publishing Company, 259-264.

[13] Mandal, A., Datta, A.K., Bhattacharya, A. (2011). Evaluation of pollen and productive parameters, their interrelationship and clustering of eight Corchorus spp. (Tiliaceae). Nucleus, 54, DOI 10.1007/s13237-011-0044-y

[14] Sharma, P.C., Yelne, M.B., Dennis, T.J. (2005). Database on Medicinal Plants Used in Ayurveda, CCRAS, New Delhi, 6, 420-440.

[15] Mehta, B.K., Mehta, P., Gupta, M. (2009). A new naturally acetylated saponin from Nigella sativa. Carbohydrate, 344, 149-151.

[16] Al-Ali, A., Alkhawajah, A.A., Randhawa, M.A., Shaikh, N.A. (2008). Oral and intraperitoneal LD50 of thymoquinone, an active principle of Nigella sativa, in mice and rats. Journal of Ayub Medical College Abbottabad, 20(2), 25-27.

[17] Morsi, N.M. (2000). Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-resistant bacteria. Acta Microbiol, 49, 63–74.

[18] Umar, S., Zargan, J., Umar, K., Ahmad, S., Katiyar, C.K., Khan, H.A. (2012). Modulation of the oxidative stress and inflammatory cytokine response by thymoquinone in the collagen induced arthritis in Wistar rats. Chemico Biological Interaction, 197(1), 40-46.

[19] Evirgen, O., Gokçe, A., Ozturk, O.H., Nacar, E., Onlen, Y., Ozer, B. (2011). Effect of thymoquinone on oxidative stress in Escherichia coli-Induced Pyelonephritis in Rats. Current Therapeutic Research, Clinical and Experimental, 72, 204–215.

[20] Nemmar, A., Al-Salam, S., Zia, S., Marzouqi, F., Al-Dhaheri, A., Subramaniyan, D. (2011). Contrasting actions of diesel exhaust particles on the pulmonary and cardiovascular systems and the effects of thymoquinone. British Journal of Pharmacology, 164(7), 1871-1882.

[21] El-Abhar, H.S., Abdallah, D.M., Saleh, S. (2003). Gastroprotective activity of Nigella sativa oil and its constituent, thymoquinone, against gastric mucosal injury induced by ischaemia/reperfusion in rats. Journal of Ethnopharmacology, 84(2-3), 251-8.

[22] Mohideen, S., Ilavarasan, R., Sasikala, E.R., Thirumalai, K.R. (2003). Hepatoprotective Activity of Nigella sativa Linn. Indian journal of pharmaceutical sciences, 65(5), 550-551.

[23] Keshri, G., Singh, M.M., Lakshmi, V., Kamboj, V.P. (1995). Post-coital contraceptive efficacy of the seeds of Nigella sativa in rats. Indian Journal of Physiology and Pharmacology, 39(1), 59-62.

[24] Agarwal, C., Narula, A., Vyas, D.K., Jacob, D. (1990). Effect of seeds of kalaunji on fertility and sialic acid content of the reproductive organs of male rat. Geo Bios, 17, 269-272.

[25] Aqel, M., Shaheen, R. (1996). Effects of the volatile oil of Nigella sativa seeds on the uterine smooth muscle of rat and guinea pig. Journal of Ethnopharmacology, 52(1), 23-26.

[26] Zaoui, A., Cherrah, Y., Mahassini, N., Alaoui, K., Amarouch, H., Hassar, M. (2002). Acute and chronic toxicity of Nigella sativa fixed oil. Phytomedicine, 9(1), 69-74.

[27] Mbarek, A., Elabbadi, N., Bensalah, M., Gamouh, A., Aboufatima, Benharref. (2007). Anti-tumor properties of blackseed (Nigella sativa L.) extracts. Brazilian Journal of Medical and Biological Research, 40, 839-847.

[28] Bakathir, H.A., Abbas, N.A. (2011). Detection of the antibacterial effect of Nigella sativa ground seeds with water. African Journal of Traditional Complementry and Alternative Medicines, 8, 159–164.

[29] Monika, T., Sasikala, P., Vijaya, Bhaskara, Reddy, M. (2013). A investigational of antibacterial activities of Nigella sativa on mastaitis in dairy crossbred cows. International Journal of Advanced Scientific and technical Research, 3, 263–272.

[30] Hannan, A., Saleem, S., Chaudhary, S., Barkaat, M., Arshad, M.U. (2008). Anti-bacterial activity of Nigella sativa against clinical isolates of methicillin resistant Staphylococcus aureus. Journal of Ayub Medical College Abbottabad, 20, 72–74.

[31] Hanafy, M.S., Hatem, M.E. (1991). Studies on the antimicrobial activity of Nigella sativa seed (black cumin) Journal of Ethnopharmacology, 34, 275–278.

[32] Nair, M.K.M,, Vasudevan, P., Venkitanarayanan, K. (2005). Antibacterial effect of black seed oil on Listeria monocytogenes. Food Control, 16, 395–398.

[33] Salem, E.M., Yar, T., Bamosa, A.O., Al-Quorain, A., Yasawy, M.I., Alsulaiman, R.M. (2010). Comparative study of Nigella sativa and triple therapy in eradication of Helicobacter Pylori in patients with non-ulcer dyspepsia. Saudi Journal of Gastroenterology, 16(3), 207-214.

[34] Chaieb, K., Kouidhi, B., Jrah, H., Mahdouani, K., Bakhrouf, A. (2011). Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial biofilm formation. BMC Complementry and Alternative Medicines, 11, 1–6.

[35] Harzallah, H.J., Kouidhi, B., Flamini, G., Bakhrouf, A., Mahjoub, T. (2011). Chemical composition, antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone. Food Chemistry, 129, 1469–1474.

[36] Hasan, N.A., Nawahwi, M.Z., Malek, H.A. (2013). Anti microbial activity of Nigella sativa seed extract. Sains Malaysiana, 42, 143–147.

[37] Deepak, S.S., Sikender, M., Garg, V., Samim, M. (2011). Entrapment of seed extract of Nigella sativa into thermosensitive (NIPAAm–Co–VP) co-polymeric micelles and its antibacterial activity. International Journal of Pharmaceutical Science and Drug Research, 3, 246–252.

[38] Hosseinzadeh, H., Fazly-Bazzaz, B.S., Motevaly-Haghi, M. (2007). Antibacterial activity of total extracts and essential oil of Nigella sativa L. seeds in mice. Pharmacology online, 2, 429–435.

[39] Baghdadi, H.B., Al-Mathal, E.M. (2011). Anti-coccidial activity of Nigella sativa L. Journal of Food and Agricultural Enviroment, 9, 10–17.

[40] Aljabre, S.H., Randhawa, M.A., Akhtar, N., Alakloby, O.M., Alqurashi, A.M., Aldossary, A. (2005). Antidermatophyte activity of ether extract of Nigella sativa and its active principle, thymoquinone. Journal of Ethnopharmacology, 101, 116-119.

[41] Rogozhin, E.A., Oshchepkova, Y.I., Odintsova, T.I., Khadeeva, N.V., Veshkurova, O.N., Egorov, T.A. (2011). Novel antifungal defensins from Nigella sativa L. seeds. Plant Physiology and Biochemistry, 49(2), 131-137.

[42] El-Nagerabia, S.A., Al-Bahryb, S.N., Elshafieb, A.E., AlHilalib, S. (2012). Effect of Hibiscus sabdariffa extract and Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and Aspergillus parasiticus strains. Food Control, 25, 59–63.

[43] Sunita, M., Meenakshi, S.H. (2013). Chemical composition and antidermatophytic activity of Nigella sativaessential oil. African Journal of Pharmacy and Pharmacology, 7, 1286–1292.

[44] Bita, A., Rosu, A.F., Calina, D., Rosu, L., Zlatian, O., Dindere, C. (2012). An alternative treatment for Candida infections with Nigella sativa extracts. Eurpean Journal of Hospital Pharmacy, 19, 162.

[45] Fierro, I.M., Barja-Fidalgo, C., Cunha, F.Q., Ferreira, S.H. (1996). The involvement of nitric oxide in the anti-Candida albicans activity of rat neutrophils. Immunology, 89, 295–300.

[46] Khan, M.A., Ashfaq, M.K., Zuberi, H.S., Mahmood, M.S., Gilani, A.H. (2003). The in vivo antifungal activity of the aqueous extract from Nigella sativa seeds. Phytotherapy Research, 17, 183–186.

[47] Abdel-Azeiz, A.Z., Saad, A.H., Darweesh, M.F. (2013). Efficacy of thymoquinone against vaginal candidiasis in prednisolone-induced immunosuppressed mice. Journal of American Science, 9, 155–159.

[48] Mahmoud, M.R., El-Abhar, H.S., Saleh, S. (2002). The effect of Nigella sativa oil against the liver damage induced by Schistosoma mansoni infection in mice. Journal of Ethnopharmacology, 79, 1–11.

[49] Aboul-Ela, E.I. (2002). Cytogenetic studies on Nigella sativa seeds extract and thymoquinone on mouse cells infected with schistosomiasis using karyotyping. Mutation Research, 516, 11–17.

[50] Mohamed, A.M., Metwally, N.M., Mahmoud, S.S. (2005) Sativa seeds against Schistosoma mansoni different stages. Memórias do Instituto Oswaldo Cruz, 100, 05–211.

[51] Shenawy, E.l., Nahla, S., Soliman, M.F., Reyad, S.I. (2008). The effect of antioxidant properties of aqueous garlic extract and Nigella sativa as anti- schistosomiasis agents in mice. Revista do Instituto de Medicina Tropical de São Paulo, 50, 29–36.

[52] Salem, M.L., Hossain, M.S. (2000). Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection. International Journal of Immunopharmacology, 22, 729–740.

[53] Barakat, E.M.E.l., Wakeel, L.M., Hagag, R.S. (2013). Effects of Nigella sativa on outcome of hepatitis C in Egypt. World Journal of Gastroenterology, 19, 2529–2536.

[54] Akhtar, M.S., Riffat, S. (1991). Field trial of Saussurea lappa roots against nematodes and Nigella sativa seeds against cestodes in children. Journal of Pakistan Medical Association, 41, 185–187.

[55] Okeola, V.O., Adaramoye, O.A., Nneji, C.M., Falade, C.O., Farombi, E.O., Ademowo, O.G. (2011). Antimalarial and antioxidant activities of methanolic extract of Nigella sativa seeds (black cumin) in mice infected with Plasmodium yoelli nigeriensis. Parasitology Research, 108, 1507–1512.

[56] Datta, A.K., Saha, A., Bhattacharya, A., Mandal, A., Paul, R., Sengupta, S. (2012). Black cumin (Nigella sativa L.) – a review. Journal of Plant Development Sciences, 4 (1), 1-43. 2012

[57] Pruthi, J.S. (2001). Minor Spices and Condiments. ICAR, New Delhi, 1–782.

[58] Malhotra, S.K. (2002). Nigella cultivation practices (in Hindi). NRCSS, Ajmer. Extension Folder No. 7, 1–4.

[59] Shengwei, Z., Jingsam, S. (2000). Rapid plant regeneration from cotton Gossypium hirsutum L. Chinese Science Bulletin, 45(19), 1772-1773.

[60] Thorpe, T. A. (1990). The current status of plant tissue culture. Plant Tissue Culture, Applications and Limitations (Bhojwani, S. S., ed.), Elsevier, Amsterdam, 1–33.

[61] Ali, M., Abbasi, B.H., Ihsan-ul-Haq. (2013). Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L. Industrial Crops Production, 49, 400–406.

[62] Ali, M., Abbasi, B.H. (2014). Thidiazuron-induced changes in biomass parameters, total phenolic content, and antioxidant activity in callus cultures of Artemisia absinthium L. Applied Biochemistry and Biotechnology, 172, 2363–2376.

[63] Tariq, U., Ali, M., Abbasi, B.H. (2014). Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L. Journal of Photochemistry and Photobiology B, 130, 264–271.

[64] Zia, M., Mannan, A., Chaudhary, M.F. (2007). Effect of growth regulators and amino acids on artemisinin production in the callus of Artemisia absinthium. Pakistan Journal of Botany, 39, 799–805.

[65] Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15, 473-497.

[66] Grech-Baran, M., Pietrosiuk, A. (2012). Artemisia species in vitro cultures for production of biologically active secondary metabolites. BioTechnologia, 93, 371–380.

[67] Bourgaud, F., Gravot, A., Milesi, S. (2001). Production of plant secondary metabolites: a historical perspective. Plant Science, 161, 839–851.

[68] Zhao, J., Davis, L.C., Verpoorte, R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23, 283–333.

[69] Zhong, J.J., Bai, Y., Wang, S.J. (1996). Effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of Panax quinquefolium. Journal of Biotechnology, 45, 227–234.

[70] Okumuş, V., Pirinc, V., Onay, A. (2011). In vitro propagation of Diyarbakır watermelons and comparison of direct-seeded and transplanted watermelon. Turkish Journal of Biology, 35, 601–610.

[71] Verma, S.K., Yücesan, B.B., Gürel, S. (2011). Indirect somatic embryogenesis and shoot organogenesis from cotyledonary leaf segments of Digitalis lamarckii Ivan, an endemic medicinal species. Turkish Journal of Biology, 35, 743–750.

[72] Chaudhry, H., Fatima, N., Ahmad, I.Z. (2014). Establishment of callus and cell suspension cultures of nigella sativa l. For thymol production. International Journal of Pharmacy and Pharmaceutical Sciences, 6, 0975-1491.

[73] Hoseinpanahi, S., Majdi, M., Mirzaghaderi, G. (2016). Effects of growth regulators on in vitro callogenesis and regeneration of black cumin (Nigella sativa). Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 24(2), 242.

[74] Al-Ani, N.K. (2008). Thymol Production from Callus Culture of Nigella sativa L. Plant Tissue Culture & Biotechnology, 18(2), 181-185.

[75] Chand, S., Roy, S. C. (1978). Effects of different auxins on callus tissues of Nigella sativa. Cell Chro, News Lett, 1, 10.

[76] Datta, A.K., Biswas, A.K., Ghosh, P. D. (1983). Chromosomal variations in callus tissues of two species of Nigella. Nucleus, 26, 173-177.

[77] Bibi, A., Khan, M.A., Adil, M., Mashwani, Z.U.R. (2018). Production of callus biomass and antioxidant secondary metabolites in black cumin. The Journal of Animal and Plant Sciences, 28(5).

[78] Gundlach, H., Müller, M.J., Kutchan, T.M. (1992). Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proceedings of the National Academy of Sciences USA, 89, 2389–2393.

[79] Mueller, M.J., Brodschelm, W., Spannagl, E. (1993). Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proceedings of the National Academy of Sciences USA, 90, 7490–7494.

[80] Cai, G., Li, G., Ye, H. (1995). Hairy root culture of Artemisia annua L. by Ri plasmid transfdormation and biosynthesis of artemisinin. Chinese Journal of Biotechnology, 11, 227–235.

[81] Liu, C.Z., Guo, C., Wang, Y. (2003). Factors influencing artemisinin production from shoot cultures of Artemisia annua L. World Journal of Microbiol Biotechnology, 19, 535–538.

[82] Ali, M., Kiani, B., Mannan, A. (2012). Enhanced production of artemisinin by hairy root cultures of Artemisia dubia. Journal of Medicinal Plant Research, 6, 1619–1622.

[83] Woerdenbag, H.J., Lüers, J.F.J., van-Uden, W. (1993). Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L. Plant Cell, Tissue and Organ Culture, 32, 247–257.

[84] Smith, T.C., Weathers, P.J., Cheetham, R.D. (1997). Effects of gibberellic acid on hairy root cultures of Artemisia annua: growth and artemisinin production. In Vitro Cellular and Developmental Biology- Plant, 33, 75–79.

[85] Paniego, N.B., Giulietti, A.M. (1996). Artemisinin production by Artemisia annua L.-transformed organ cultures. Enzyme and Microbial Technology, 18, 526–530.

[86] Wang, H., Ye, H., Li, G. (1999). Effects of fungal elicitors on cell growth and artemisinin accumulation in hairy root cultures of Artemisia annua. Acta Botanica Sinica, 42, 905–909.

[87] Youssef, A.A., Rady, M.R., Ghanem, S.A. (1998). Growth and some primary products in callus cultures of Nigella sativa as influenced by various cultural conditions and salt stress. Fitoterapia, LXIX, 4, 329-336.

[88] Khabir, E., Moradi, P.A. (2016). Study on impact of auxin and elicitors on tissue culture and proliferation of Nigella sativa L. 11(10), 1990-6145.

[89] Boselah, N.A.E. (1995). Seed germination of Nigella sativa L.

[90] Ibrahim, M.M., Arafa, M.N., Matter, M.A. (2015). Effect of some elicitors on chemicals composition for Nigella sativa callus cultures. World Journal of Pharmaceutical Sciences, 2015, 2321-3086.

[91] Ali, S.A., Solouki, M., Bahman, F.B. (2017). Optimization of Callus Induction and Effects of Biological and Nonbiological Elicitors on Content of Phenol/ Flavonoid Compounds in Nigella sativa under In-Vitro Conditions. Journal of Cell & Tissue (JCT), 8(2), 165-184.

[92] Ali, M., Abbasi, B.H. (2014). Light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of Artemisia absinthium L. Journal of Photochemistry and Photobiology B, 140, 223–227.

[93] Abbasi, B.H., Tian, C.L., Murch, S.J. (2007). Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of Echinacea purpurea. Plant Cell Reports, 26, 1367–1372.

[94] Georgieva, L., Ivanov, I., Marchev, A. (2015). Protopine production by Fumaria cell suspension cultures: effect of light. Applied Biochemistry and Biotechnology, 176, 287–300.

[95] Ahmad, N., Rab, A., Ahmad, N. (2015). Light-induced biochemical variations in secondary metabolites production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert). Journal of Photochemistry and Photobiology B, 154, 51–56.

[96] Szopa, A., Ekiert, H., Szewczyk, A. (2012). Production of bioactive phenolic acids and furanocoumarins in in vitro cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams under different light conditions. Plant Cell, Tissue and Organ Culture, 110, 329–336.

[97] Chand, S., Roy, S. C. (1980a). Study of callus tissues from different parts of Nigella sativa (Ranunculaceae). Experientia, 36(3), 305–306. doi:10.1007/bf01952291

[98] Elhag, H., El-Olemy, M.M., Al-Said, M.S. (2004). Enhancement of somatic embryogenesis and production of developmentally arrested embryos in Nigella sativa L. Horticulture Science, 39, 321-323.

[99] Landa, P., Marsik, P., Vanek, T., Rada, V., Kokoska, L. (2006). In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species. Biologia, 61(3). doi:10.2478/s11756-006-0052-6

[100] Srivastava, P., Sisodia, V., Chaturvedi, R. (2011). Effect of culture conditions on synthesis of triterpenoids in suspension cultures of Lantana camara L. Bioprocess and Biosystem Enginering, 34, 75–80.

[101] Moscatiello, R., Baldan, B., Navazio, L. (2013). Plant cell suspension cultures. Methods in Molecular Biology, 953, 77–93.

[102] Jalil, M., Annuar, M.S.M., Tan, B.C. (2015). Effects of selected physicochemical parameters on zerumbone production of Zingiber zerumbet Smith cell suspension culture. Evidence-Based Complementary and Alternative Medicine (eCAM), 2015, 757514.

[103] Li, H.H., Yao, D.H., Xu, J. (2015). Research on ursolic acid production of Eriobotrya japonica cell suspension culture in WAVE bioreactor. Zhongguo ZhongYao ZaZhi, 40, 1693–1698.

[104] Smith, J., Rogers, R., Jeon, S. (2015). Production of uniformly labeled 13C-Lutein and 13C-α-tocopherol in vitro using carrot cell suspension culture. FASEB Journal, 29, 604.

[105] Sahraroo, A., Mirjalili, M., Corchete, P. (2016). Establishment and characterization of a Satureja khuzistanica Jamzad (Lamiaceae) cell suspension culture: a new in vitro source of rosmarinic acid. Cytotechnology, 68, 1415–1424. doi.10.1007/s10616-015-9901-x.

[106] Dong, Y., Duan, W., He, H. (2015). Enhancing taxane biosynthesis in cell suspension culture of Taxus chinensis by overexpressing the neutral/alkaline invertase gene. Process Biochemistry, 50, 651–660.

[107] Banerjee, S., Gupta, S. (1976). Embryogenesis and differentiation in Nigella sativa leaf callus in vitro. Physiologia Plantarum, 38, 115-120. doi: 10.1111/j.1399-3054.1976.tb04869.x

[108] ElNour, E.M., Mawahib, Mahmood, Z.A., Futooh, Yagoub, O., Sanaa. (2015). In Vitro Callus Induction and Antimicrobial Activities of Callus and Seeds Extracts of Nigella Sativa L. Research & Reviews: Journal of Biology, 3(3), 21-28.

[109] Haroon, A., Qamar, S., Shireen, F. (2016). In vitro regenration protocol of nigella sativa using different plant growth regulators. International Conference on Forestry and Enviroment; Challenges and Prospects, University of Agriculture, Faisalabad, Pakistan, November, 138, 21-22.

[110] Scholz, M., Lipinski, M., Leupold, M., Luftmann, H., Harig, L., Ofir, R., Müller, K. J. (2009). Methyl jasmonate induced accumulation of kalopanaxsaponin I in Nigella sativa. Phytochemistry, 70(4), 517–522. doi:10.1016/j.phytochem.2009.01.018

[111] Ghosh, A., Gadgil, V.N. (1979). Shift in ploidy level of callus tissue: A function of growth subtances. Indian Journal of Experimental. Biology, 17, 562-564.

[112] Farag, M.A., El Sayed, A.M., El Banna, A., Ruehmann, S. (2015). Metabolomics reveals distinct methylation reaction in MeJA elicited Nigella sativa callus via UPLC–MS and chemometrics. Plant Cell, Tissue and Organ Culture (PCTOC), 122(2), 453–463.

[113] Banerjee, S., Gupta, S. (1975). Suspension culture of Nigella sativa. Cellular and Molecular Life Sciences, 31, 792-795. doi: 10.1007/BF01938469

[114] Al-Salih, H.S. (2012). Evaluation of Deltamethrine Pesticide Effect in the Plant Cell Growth Using Nigella sativa L. Callus Cultures. Rafidain journal of science, 23(4A), 128-136

[115] Haseeb, A., Elhag, H. inventor; Haseeb, A., Elhag, H., assignee. (2012). Process for producing melanin using cultures of the genus Nigella. WIPO patent, WO 2012125091A1.

[116] Chand, S., Roy, S. C. (1980b). Cytological Abnormalities During Culture of Nigella sativa. Protoplasma, 104, 353-357.

[117] Chand, S., Roy, S.C. (1981). Induction of Organogenesis in Callus Cultures of Nigella sativa L. Annals of Botany, 48(1), 1–4. doi:10.1093/oxfordjournals.aob.a086087

[118] Sokmen, A., Jones, B. M., Erturk, M. (1999). Antimicrobial activity of extracts from the cell cultures of some Turkish medicinal plants. Phytotherapy Research, 13(4), 355–357. doi:10.1002/(sici)1099-1573(199906)13:4<355::aid ptr454>;2-e

[119] Jha, T.B., Roy, S.C. (1979). Rhizogenesis From Nigella sativa Protoplasts. Protoplasma, 101, 139-142.

[120] Gany, Z.S.A., Mahdi, M.F. (2008). Cytotoxic Assay of Nigella sativa Leaf Callus Extract (Thymol) on Hep-2 Cell Line Using ELISA Assay. Iraqi Journal of Pharmaceutical Sciences, 17(2).

[121] Rasheed-uz-zafar., Kausar, A. (2013). Biotransformation of limonene by freely suspended and immobilised cells of Nigella sativa. International Journal of Pharmacy and Pharmaceutical Sciences, 5, 23-26.

[122] Mohammad, A.M.S., Jumma, N.E. (2006). Partial Purification of Glutamate Dehydrogenase from the Callus of Stems of (Nigella sativa L.) in the Presence of 2,4- D or PDA. Rafidain Journal of Science, 17, 80-93.

[123] AL-Noaimy, M.M., AL-Saleh, H.S. (2010). The Role of Interaction of some Growth Regulators with Salfanilamide on Initiation and Growth of Cell Suspension Culture of Black Seed Nigella sativa L. Rafidain Journal of Science, Journal of Mesopotamia, 21, 56-72.

[124] Al-Dulaimee, H.M., Abood, S.A. (2006). Presence of Dihydrofolate Reductase in Seedlings and Callus of Nigella Sativa L. Plant. Rafidain Journal of Science, Journal of Mesopotamia, 17, 26-38.

[125] Sobhanizadeh, A., Solouki, M., Fazeli-Nasab, B. (2017). Optimization of Callus Induction and Effects of Biological and Non- biological Elicitors on Content of Phenol/ Flavonoid Compounds in Nigella sativa under In-Vitro Conditions. Journal of Cell & Tissue, 8(2), 165-184.

[126] Kumar, P.M., Vinmathi, V., Gautam, P., Wilson, A.H., Jacob, S.J.P. (2015). Green Synthesis of Silver Nanorods Using Aqueous Seed Extract of Nigella Sativa and Study of its Antidiabetic Activity. Australian Journal of Basic and Applied Sciences, 9(10), 295-298.

[127] Amooaghaie, R., Saeri, M.R., Azizi, M. (2015). Synthesis, Characterization and Biocompatibility of silver nano-particles synthesized from Nigella sativa leaf extract in comparison with chemical silver nanoparticles. Ecotoxicology and Environmental Safety, 120, 400-408.

[128] Fragoon, A.L., Zhu, J., Zhao, J. (2012). Biosynthesis of Controllable Size and Shape Gold Nanoparticles by Black Seed (Nigella Sativa) Extract. Journal of Nanoscience and Nanotechnology, 12(3), 2337-2345.

[129] Sangeetha, J., Sandhya, J., Philip, J. (2014). Biosynthesis and Functionalization of Silver Nanoparticles Using Nigella sativa, Dioscorea alata and Ferula asafetida. Science of Advanced Materials, 6(8), 1681-1690.

[130] Ravindran, J., Nair, H.B., Sung, B., Prasad, S., Tekmal, R.R., Aggarwal, B.B. (2010). Thymoquinone Poly (lactideco-glycolide) Nanoparticles Exhibit Enhanced Antiproliferative, Anti-inflammatory and Chemosensitization Potential. Biochemical Pharmacology, 79(11), 1640-1647.

[131] Manju, S., Malaikozhundan, B., Chen, J.C., Vaseeharan, B. (2014). Essential Oil of Nigella Sativa Based Synthesis of Silver Nanoparticles and Its Effect on Pathogenic Vibrio Harveyi and Vibrio Parahaemolyticus isolated from Aquatic Environments. Journal of The Fisheries Society of Taiwan, 41(2), 123- 134.

[132] Gilani, A.U.H., Jabeen, Q., Khan, M.A.U. (2004). Pakistan Journal of Biological Sciences, 7, 441-451.

[133] Alemi, M., Sabouni, F., Sanjarian, F., Haghbeen, K., Ansari, S. (2013). Anti-inflammatory effect of seeds and callus of Nigella sativa L. extracts on mix glial cells with regard to their thymoquinone content. AAPS PharmSciTechn, 14, 160-167.

[134] Yang, L., Stöckigt, J. (2010). Trends for diverse production strategies of plant medicinal alkaloids. Natural product reports, 27(10), 1469-1479.

[135] Yue, W., Ming, Q.L., Lin, B., Rahman, K., Zheng, C.J., Han, T., Qin, L.P. (2016). Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites. Critical reviews in biotechnology, 36(2), 215-232.

[136] Ferrari, S. (2010). Biological elicitors of plant secondary metabolites: Mode of action and use in the production of nutraceutics. Bio-Farms for Nutraceuticals, 152-166.

[137] Zhang, B., Zheng, L.P., Wang, J.W. (2012). Nitric oxide elicitation for secondary metabolite production in cultured plant cells. Applied microbiology and biotechnology, 93(2), 455-466.

[138] Zhao, J., Davis, L.C., Verpoorte, R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology advances, 23(4), 283-333.

[139] Ramirez-estrada, K., Vidal-limon, H., Hidalgo, D., Moyano, E., Golenioswki, M., Cusido, R.M. (2016). Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories. Molecules, 21(2), 182.

[140] Golkar, P., Moradi, M., Garousi, G.A. (2018). Elicitation of Stevia glycosides using salicylic acid and silver nanoparticles under callus culture. Sugar Tech. 3, 1-9.

[141] Tahmasi, S., Garoosi, G., Ahmadi, J., Farjaminezhad, R. (2017). Effect of salicylic acid on stevioside and rebaudioside A production and transcription of biosynthetic genes in in vitro culture of Stevia rebaudiana. Iranian Journal of genetics and plant breeding, 6(2), 1-8.

[142] Lucho, S.R., Do-amaral, M.N., Milech, C., Ferrer, M.Á., Calderón, A.A., Bianchi, V.J., Braga, E.J.B. (2018). Elicitor-Induced Transcriptional Changes of Genes of the Steviol Glycoside Biosynthesis Pathway in Stevia rebaudiana Bertoni. Journal of Plant Growth Regulation, 37(3), 971–985.

[143] Bayraktar, M., Naziri, E., Karabey, F., Akgun, I., Bedir, E., Röck-okuyucu, B., Gürel, A. (2018). Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana. International Journal of Secondary Metabolite, 5 (4), 362-374.