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

Medicinal and aromatic plants and their refined natural products have gained global attraction for their therapeutic potential against many human diseases. Nigella sativa is a medicinally important plant, commonly known as Black cumin or Black seed is a dicotyledon plant of the Ranunculaceae family. It is in common use for a longer time in history as preservative and spice and has also been extensively utilized by different communities around the globe. Black cumin has been an eminent component of traditional medicine systems like Unani and Tibb, Ayurveda and Siddha. Its biological activities include antidiarrheal, analgesic, antibacterial, liver tonic, diurectic, digestive agent and 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, among 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, phytochemistry, therapeutic potential and biotechnological approaches to enhance the medicinal potential of this valuable plant. ARTICLE HISTORY Received: February 10, 2019 Revised: May 15, 2019 Accepted: June 08, 2019


INTRODUCTION
N. sativa Linn, a highly potent medicinal plant of Ranunculaceae family, is an annual flowering herb which usually grows 20-90 cm tall.N. sativa locally known as Black cumin or Black seed is natively found in the regions of Southern Europe, North Africa and Southwest Asia.Currently, it is cultivated in many countries across the globe [1,2].N. sativa is a rabi crop and seeds of this crop are sown in the month of November and harvested in March or April.Sandy and loamy soil with pH 6.85, having 0.78% organic carbon is ideal for its cultivation [3,4].The morphology of N. sativa flowers comprises 5 to 10 petals with color ranging from white, yellow, pink, pale blue to pale purple (Figure 1).The fruit appears like a big, inflated capsule having 3-7 united seeds containing follicles.Seeds are small, dicotyledonous and black in color with aromatic odor and bitter taste [5].N. sativa is known with diverse names in different part of the world, such as in English: fennel flower, nutmeg flower, Roman coriander, blackseed or black caraway, black sesame; India: Assamese -kaljeera or kolajeera, Hindi/Urdu -kalaunji/ mangrail; Arabic: habbat al-barakah; French: nigelle de Crète, toute épice; Germany: Schwarzkümmel.
Due to availability of unique phytochemicals there are numerous therapeutic potentials of N. sativa such as anti-inflammatory, anti-analgesic, anti-stress, anticancer, antioxidant, antibacterial, antifungal, antiparasitic and antiasthmatic [1,[5][6][7][8].Plants have secondary metabolites for their defense mechanism whereas humans utilize these secondary metabolites for multiple purposes such as medicines, flavorings, and recreational drugs [9][10][11].Due to phytochemicals and vast therapeutic potential, seeds of N. sativa got a great economic value in local and international market such as Rs.275-500/kg in local market (Pakistan; Mingora, Dir, Peshawar, Pindi, Lahore, Gilgit).In Indian market Rs.250-300/kg, whereas it is put up for sale in international market for Rs.850-1000/gm [12].Therefore it can be one of the ideal plants for farmers to cultivate and get a good income out of it [13,14].Seeds of N. sativa can be stored for a year in airtight bags or jars to maintain its aroma.It should be kept away from other species (condiments) as it can affect the aroma and flavor of other species [13].

MEDICINAL and PHARMACOLOGICAL APPLICATIONS
To prevent and cure variety of diseases all over the world, seeds of N. sativa are used in herbal medicines.Prophet Mohammad (Peace Be Upon Him) said: "Use this Black Seed; it has a cure for every disease except death" (Sahih Bukhari).There are several ailments like skin disorders, respiratory disorders including asthma, bronchitis, disorders of joints like rheumatism and disorders of gastrointestinal track i.e. diarrhea and also hepatic one which are cured by the seeds of N. sativa.It gives strength to immune system and increase milk production in females [16].Worm treatment is also reported by using the seeds and which are also helpful in the treatment of nausea.Oil of N. sativa has the ability to work as an antiseptic and a local anesthetic [17].
Different studies have proved that N. sativa and its active secondary metabolites can be effective in different pharmacological activities such as diuretic, antihypertensive, bronchodilator, gastroprotective, hepatoprotective, antidiabetic, anticancer and immunomodulatory, analgesic, antimicrobial, analgesics and anti-inflammatory, spasmolytic, renal protective and antioxidant properties, summarized in Table 1.Antiviral, antioxidant activity enhanced RBC and platelet counts patient with hepatitis C virus (HCV) [53] Ethanolic extract Anti-parasite activity children infected with cestode worms [54] Methanolic extract of seeds Antimalarial effect, antioxidant effect hepatoprotactent Mice [55] Aqueous suspensions of seeds Antiparasite effect, anticoccidial effects coccidiosis in rabbits [39] Oil emulsions of seeds

Patents
There are five different FDA (Food and Drug Administration) patents in the U.S.A. of Nigella sativa for the treatment of following diseases [56]

Cultivation Requirements and Challenges
N. sativa is a highly medicinal plant and its demand especially for its magical oil is increasing day by day.The Nigella crop takes around 140-160 days to reach to its harvesting period as it is a rabi (cool season) crop so it grows during the winter season in India.The requirements for sowing the seeds are warm weather with a temperature range of 20-25°C and cold weather is required for the early growth period.The seed formation also requires the warm sunny weather thus it is the main requirement for N.sativa from seed sowing to seed formation [57,58].Root rot is one of the common infections of N. sativa which is instigated by Rhizoctonia and Fusarium.In this disease, first the leaf color turn from green to yellow and early drying of plant occurs, which significantly decreases the crop yield.No unspoiled control procedures are available for this disease.Aphids (small sap-sucking insects), larvae of armyworm Spodoptera litura and Cercospora nigellae are also involved in damaging the crop [58].All these issues with Nigella in wild grown conditions are dragging us towards an alternate method to meet the demands and requirements of this modern era.
Therefore, in vitro cultures are attractive technique that can play a vital role in fulfilling these demands by providing metabolites within reasonable time and quantity.Plant tissue culture is a technique through which one can grow and multiply cells, tissues and organs of plants on defined solid or liquid media under contamination free and ideal conditions.Different important metabolic compounds such as alkaloids, phenols, terpenoids, vitamins and other highly medicinal compounds can easily be isolated from this technology [59].

BIOTECHNOLOGICAL ASPECTS 4.1. Cell Culture Technology
Plant cell culture technology is an essential tool in basic and applied research for the production of seedlings and plants and it is very important on a commercial scale.To succeed in tissue culture, medium composition is very important [60].Hormones such as auxins, cytokinins and gibberellic acid are used to control cell growth and division can be supplemented to the growth medium at the right time which poses an important role in the formation of callus, regeneration of other plant parts or organogenesis.These hormones can also lead to increase the amount of phenolics, flavonoids and terpenoids in different cultures such as, thidiazuron (TDZ)-induced stimulated production of phenolics and flavonoids in callus and cell suspension cultures of A. absinthium [61,62].Likewise, callus cultures of A. absinthium displayed the maximum levels of phenolic and flavonoid content in response to combining thidiazuron (TDZ) and naphthaleneacetic acid (NAA) [63].Artemisinin concentration was found highest in A. absinthium callus cultures when treated with benzyl adenine (BA; 2.0 mg/L) [64].Some parts of cultivated plants need auxin to produce callus while some require only cytokinin however most cultures need both.Optimal formula of medium varies depending on the species, type of genotype within the species, origin and the age of tissue culture.In most experiments in this field, culture has been conducted in base medium of Morashige and Skoog [65] at different ratios of plant hormones.
The most important feature of callus is that, this cellular mass has the necessary potential for organogenesis, embryogenesis and complete plant production.Beside PGRs callus formation also depends on the type of the explants used.A variety of explants are used for the callus and suspension cultures, organogenesis and embryogenesis which are summarized in Figure 4.

Significance of Plant in Vitro Cultures
There are numbers of drugs and medicines available in market for treatment of different diseases but we are still in search of novel chemical compounds which can help us in the decreasing the number of diseases and outbreaks.Therefore, we have to look towards natural resources where our synthetic drugs fail to cure us.Plants are rich with these novel chemical compounds known as phytochemicals or plant secondary metabolites, these phtochemicals are used by humans against different diseases [66].Due to less number of availability of plant and high demand of phytochemicals, different approaches like in vitro cultures, are utilized to improve the quality and quantity of these metabolites [67].One of the main reasons for utilization of in vitro cultures is the presence of trace amounts of these secondary plant compounds in the plants.The most likely reason for this is that different genes coordinate at different developmental stages indicating that production of useful important metabolites is growth dependent.There is also a general perception that during in vitro growth of the cells, the chemicals produced are mainly those which support the growth of the plant and production of secondary metabolites might be unnecessary or even toxic thus leading to deceased secondary metabolites production in vitro.Therefore, there is a need to optimize the in vitro growth conditions increased secondary metabolites production that would be a key to overcome this hurdle.

Strategies to Enhance Biomass and Secondary Plant Compounds
There are different strategies mentioned in Figure 5, which can enhance the phytochemicals in in vitro cultures.Studies have revealed that elicitation is the most effective

Effects of PGRs
Plant growth regulators are signaling compounds which help the plant growth and development and production of secondary metabolites [68].Plant growth regulators (PGRs) have a significant effect on explant in plant tissue culture; they have a great impact of cell growth, differentiation, regeneration, and metabolite formation [69][70][71].Different PGRs have been studied on different plants including N. sativa and each PGR has its own role in initiating in vitro growthand biomass formation.Datta et al. reported that 2,4-D and kinetin help in callus formation from hypocotyl segment [76].In another study, MS media added with 2,4-D, NAA and IAA produced a significant amount of callus from leaf of Nigella sativa [75].PGRs not only responsible for culture initiation and biomass formation, but also enhance the metabolites accumulation.Chaudhry et al. and Hoseinpanahi et al. concluded that combination of Kn + NAA and BAP + IAA enhanced terpenoid and thymol production in suspension culture of N. sativa [72,73].Al-Ani also reported that 2,4-D and Kn initiated callus formation from leaf explant of Nigella sativa with enhanced thymol concentration [74].By enhancing metabolic content the antioxidant activity of culture is also increased, Further, TDZ + NAA enhanced biomass and antioxidant activity of callus of Nigella sativa [77].

Role of Elicitors
Elicitation is one of the most effective approachs for the enhancement and biotechnological production of secondary metabolites [134,135].An "elicitor" is a substance which initiates or stimulates the production of particular metabolites when applied at optimal concentrations.These compounds stimulate plant defense by promoting secondary metabolism for the protection of plant cell, to cope with the stress created by the them, as a result plant through a series of reactions such as activation of NADPH oxidase, production of reactive oxygen and nitrogen species, expression of defensive genes and secondary metabolites production [136][137][138][139]. Elicitors may be abiotic such as metal ions and inorganic compounds, or biotic from fungi, bacteria, viruses or herbivores, plant cell wall components [68].Jasmonic acid (JA) is naturally synthesized inside plant and is responsible for different functions along with activation of production of secondary metabolites, therefore different mediators can be used to activate JA pathway [78,79].In several studies PGRs stimulated the production of callus, organogenesis, phytochemicals which were further enhanced by the elicitors in many medicinal plants.GA3 enhanced artemisinin accumulation in hairy root cultures and shoot culture of A. annua [80,81,[83][84][85][86] and A. dubia [82].Similarly, Salicyclic acid (SA), AgNPs, MeJ, SPD and Chitosan enhanced stevioside biosynthesis in various cultures of Stevia [140][141][142][143].A variety of elicitors have been used in different studies to initiate cultures of N. sativa with higher production of secondary metabolites.Casein hydrolysate promoted callus formation and embryogenesis with enhance biomass of N. sativa [87,107].In another study, SA enhanced the physiological parameters and also produced healthy biomass of Callus in N. sativa [88].Increase in levels of monodesmosidic triterpene saponins α-hederin and kalopanaxsaponin I (KsI) in the leaves of Nigella sativa were observed when treated with methyl jasmonate (MeJA) [110], GA3 enhanced germination rate of N. sativa [89], AgNO3 and SA increased secondary metabolites (fatty acids and essential oil) in callus culture [90], Nano-silver and yeast extract increased total phenol and flavonoids in N. sativa [91] (Table 2).

Light Effect
Light is a basic requirement for majority of plants for their growth and development and production of primary, secondary metabolites [92,93].Light in Plant tissue culture can play a very major part, it is a type of physical elicitation, fluctuation of intensity and color of light can produce some sort of stress in plant which may lead to initiate culture and also stimulate the production of phytochemicals.Light has stimulated the production of protopine in suspension cultures of Fumaria, [94] phenolics and flavonoids in callus cultures of Stevia rebaudiana, [95] caffeic acid derivatives in hairy root cultures of Echinacea purpurea, [93] phenolic acids in in vitro cultured Ruta graveolens and Ruta graveolens divaricata [96].Several reports are available on light stimulated cultures of N. sativa.Complete dark helped in callus initiation from seeds [97], leaf explants [98], and stem of N. sativa [99], Somatic embryo formation was promoted when explants of N. sativa were kept in dark [98].In another study, controlled dark conditions stimulated Melanin production in suspension culture of N. sativa [115] (Table 2).

Plant Cell Cultures Strategies for Phytochemical Production
For evaluation of biomass kinetics, optimize conditions for production of highly medicinal and important secondary metabolites, Cell suspension cultures play a very central role [100].The factors making cell suspension cultures suitable for the analysis of complex physiological processes include the homogeneous nature of cell population, the availability of material in bulk, accelerated growth of cells and conditions reproducibility [101].Some recent examples of medicinal secondary metabolites in suspension cultures include zerumbone production in Zingiber zerumbet, [102] ursolic acid production in Eriobotrya japonica, [103] lutein and tocopherol in carrot, [104] rosmarinic acid in Satureja khuzistanica [105] and taxane in Taxus chinensis [106], enhanced terpenoid and thymol production in N. sativa [72].Al-Ani reported that from callus culture of N. sativa higher thymol concentration was extracted [74].Enhanced biomass and antioxidant activity showed by the Cotyledon derived callus culture of Nigella sativa [77].Enhanced Thymoquinone concentration was also extracted from callus culture of Nigella sativa [133].

NIGELLA SATIVA IN NANOTECHNOLOGY
Plants have majority of phytochemicals such as phenols, acids, tannins, steroids, terpenes etc which can be utilize in synthesis of nanoparticles (Green synthesis).Plants derived nanoparticles are environment friendly with low cost and can be used in majority of therapeutic and pharmacological applications such as antibacterial, antitumor, and can also be used as biosensor.Seed extract of N. sativa and AgNO3 resulted in the formation of silver nanorods, which showed antidiabetic property, in vitro [126].Silver nanorods were also prepared from the leaf extract of N. sativa [127].Gold nanoparticles AuNPs have also been prepared from seed extract of N. sativa and aqueous chloroauric acid solution [128].Plant extracts of Nigella sativa, Dioscorea alata was used to produce phytochemical capped Silver nanoparticles, thymoquinone, dioscorin and ferulic acid worked as capping agents [129].Encapsulation of TQ into nanoparticles enhances its anti-proliferative, anti-inflammatory effects and can be used in variety of biomedical applications [130] Silver nanoparticles prepared from essential oil of N. sativa, showed inhibitory activity against pathogenic Vibrio harveyi and V. parahaemolyticus [131].

CONCLUSIONS
Nigella sativa has shown substantial therapeutic effects on several biological systems.The volatile oil as well as organic and aqueous fractions of the seeds has been proven to possess beneficial effects in terms of medicinal significance.The presence of active proteins and lipid soluble elements provide the clue to the several mechanisms of actions behind therapeutic potential.Although, the Nigella sativa has become the topic of research worldwide, still there is lot of room to be explored regarding this phytotherapeutic source and no doubt clinical trials need to be done to validate the therapeutic efficacy of the plant.

Conflicts of Interest
All the authors declared that there is no conflict of interest with regards to any part of the manuscript.

Figure 4 .
Figure 4. Explants of N. sativa used in different cultures and increasing the production of secondary metabolites in in vitro cultures.

Figure 5 .
Figure 5. Strategies to enhance secondary metabolites

Table 2 .
Strategies used to enhance Secondary metabolites (SMs) in various cultures of Nigella sativa.