Evaluation of Antioxidant and Antibacterial Potentials of Nigella sativa L. Suspension Cultures under Elicitation

Nigella sativa L. (family Ranunculaceae) is an annual herb of immense medicinal properties because of its major active components (i.e., thymoquinone (TQ), thymohydroquinone (THQ), and thymol (THY)). Plant tissue culture techniques like elicitation, Agrobacterium mediated transformation, hairy root culture, and so on, are applied for substantial metabolite production. This study enumerates the antibacterial and antioxidant potentials of N. sativa epicotyl suspension cultures under biotic and abiotic elicitation along with concentration optimization of the elicitors for enhanced TQ and THY production. Cultures under different concentrations of pectin and manganese chloride (MnCl2) elicitation (i.e., 5 mg/L, 10 mg/L, and 15 mg/L) showed that the control, MnCl2 10 mg/L, and pectin 15 mg/L suspension extracts greatly inhibited the growth of E. coli, S. typhimurium, and S. aureus (MIC against E. coli, i.e., 2.35 ± 0.8, 2.4 ± 0.2, and 2.46 ± 0.5, resp.). Elicitation decreased SOD enzyme activity whereas CAT enzyme activity increased remarkably under MnCl2 elicitation. MnCl2 10 mg/L and pectin 15 mg/L elicitation enhanced the DPPH radical inhibition ability, but ferric scavenging activity was comparable to the control. TQ and THY were quantified by LC-MS/MS in the cultures with high bioactive properties revealing maximum content under MnCl2 10 mg/L elicitation. Therefore, MnCl2 elicitation can be undertaken on large scale for sustainable metabolite production.


Introduction
The use of spices as food additives, flavouring agents, and also digestive stimulant tonic has been practiced widely since ancient times. These attributes, largely empirical, nevertheless efficacious, have earned them pharmacological applications in the indigenous systems of medicine. Among various medicinal spices Nigella sativa L. (family Ranunculaceae) has emerged as a miraculous herb with a wide spectrum of pharmacological activities. N. sativa seeds are most extensively studied, both phytochemically and pharmacologically. Both seeds and oils are known to possess various health properties like antitumour activity [1], antioxidant activity [2], anti-inflammatory activity [3], antibacterial activity [4], and a stimulatory effect on the immune system [5] because of which they are often used as nutritional supplement. Also it is reported that different crude extracts of N. sativa have shown effectiveness against multiantibiotic resistance bacterial isolates [6]. Black seed extracts have also proved to be potent antimicrobial agents against certain pathogenic Gram positive and Gram negative bacteria [7]. Further, it is known that its oil and fraction act as potent in vitro free radical scavengers which are correlated well with its total content of polyunsaturated fatty acids, unsaponifiables, phospholipids, and phytoconstituents as well as the initial peroxide values of crude oil [8]. These strong antimicrobial and antioxidant properties of different extracts and oil of N. sativa are due to the rich presence of chemically varied bioactive compounds.
Techniques of plant tissue culture have proved to be the best alternatives in supplementing traditional agriculture for the industrial production of medicinal plant derived metabolites [9]. Among the various techniques, elicitation has proved to be an effective strategy for the enhanced production of commercially and medicinally important bioactive compounds. Elicitors are stress agents that induce the accumulation of secondary metabolites along with phytoalexins in plants, as well as in plant cultures, and the phenomenon is known as elicitation [10]. Elicitors can be both biotic (pectin, chitin, cellulose, glycerol, jasmonic acid, etc.) and abiotic (heavy metals, salts, herbicides, pH, temperature, etc.) depending upon their nature and source. Plant cells recognize the elicitors by interacting with them via specific receptors present on the plasma membranes. The elicitor-receptor interactions generate many downstream signals which then activate nuclear plant defense genes such as phytoalexins. The action of local, systemic signal molecules and putative plasma membrane receptors is solely responsible for the initiation of the pathway. Many heavy metals (Ag, V, Cr, Ni, Pb, Hg, Cu, and Mn) and their salts are reported to have enhanced the metabolite production like copper sulphate facilitating the accumulation of cardiac glycosides up to 26 times in cell cultures of Digitalis lanata [11]. Furthermore, biotic elicitation, for example, pectin treatment, has also enhanced the induction of oleanolic acid in cell suspension cultures of Calendula officinalis [12]. The accumulation of secondary products is regarded as a part of the defense system of the intact plant.
The present study was conducted to demonstrate the antioxidant and antibacterial efficacy against different Gram positive and Gram negative bacteria of the N. sativa epicotyl suspension cultures under the effect of different concentrations of biotic (pectin) and abiotic elicitor (MnCl 2 ). Further, the effect of elicitation on cell growth, thymoquinone (TQ) and thymol (THY) production from the cell suspension cultures of N. sativa was examined.

Plant Material.
Seeds of N. sativa were procured from a local grocery store in Lucknow, India, and authenticated at the National Botanical Research Institute, Lucknow. Seeds were surface-sterilized by washing thoroughly under tap water containing few drops of Tween-20 and then rinsed with 70% ethanol for 30 seconds followed by washing with sterile water twice. They were then immersed in 0.2% mercuric chloride solution for 5 min after which they were rinsed with sterile water twice, were allowed to grow in glass petri plates having threefold of damp blotting paper in distilled water at 25 ± 2 ∘ C under aseptic condition in dark for three days till sprouting, and, thereafter, were exposed to light (photoperiod of 14/10 h, 100 mol m −2 s −1 ), where complete germination with leaf, epicotyl, hypocotyl, and root took eleven days [13]. (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50) and segments (0.5 cm) of epicotyl, hypocotyl, and root from the 11th day seedling were surface-sterilized and cultured in triplicate on solidified Murashige and Skoog (MS) medium supplemented with kinetin (Kn) 2 mg/L + naphthalene acetic acid (NAA) 1 mg/L [14]. Cultures were kept under a photoperiod of 16/8 h white fluorescent light at 25 + 2 ∘ C for a month. Cell suspension cultures were derived from friable epicotyl callus in Erlenmeyer flasks (250 mL) containing 100 mL of liquid MS medium supplemented with Kn 2 mg/L + NAA 1 mg/L in triplicate. Cultures were maintained for a month at 110 rpm (Remi Orbital Shaker Incubator, Model number IHC-2410) at 25 ± 2 ∘ C, 3000 lux, and 16/8 h photoperiod and growth was determined by loss of weight by dissimilation [14].

Effect of Biotic and Abiotic Stress on Growth, Biomass
Accumulation, and Metabolite Production. A cell biomass of 50-75 mg from the stock culture was transferred into Erlenmeyer flasks of separate groups, containing 100 mL medium with Kn 2 mg/L + NAA 1 mg/L in triplicate under three different concentrations of pectin and MnCl 2 , that is, 5 mg/L, 10 mg/L, and 15 mg/L (as they have given the best results in enhancing metabolite production in the epicotyl callus of N. sativa in our previous study) [15]. pH of the medium was adjusted to 5.8 before autoclaving. Cultures were maintained at 110 rpm, 16/8 h, and 25 ± 2 ∘ C for a month and growth was determined by loss of weight by dissimilation, TQ and THY production was estimated by TLC and LC-MS/MS, and growth index was calculated by the following equation:

Estimation of Thymol and Thymoquinone by TLC
2.5.1. Extraction Method. Cells from different suspension cultures were ground in 100% of methanol using pestle and mortar at room temperature with periodical mixing on shaker overnight, filtrated using Whatman Grade number 1 filter paper, medium particle retention (pore size 11 m), and concentrated at 45 ∘ C up to 5 mL. Extracts were stored at −20 ∘ C for further use. concentrations of crude extract and 0.1 mM methanolic DPPH solution were added to 3 mL of reaction mixture, incubated for 30 minutes. Absorbance was taken at 517 nm lower absorbance of the reaction mixture indicating higher free radical inhibition activity. All extracts were analyzed in triplicate. Ascorbic acid was taken as a standard: where 1 is the absorbance of the sample and 0 is the absorbance of the control, respectively.  [20] with some modifications. Samples were finely ground; 10 mL of 100 mM phosphate buffer (KH 2 PO 4 /K 2 HPO 4 ) pH 7.0, containing 0.1 mM Na 2 EDTA and 0.1 g of polyvinylpyrrolidone (PVP), was added to them. The homogenate was filtered through Whatman filter and centrifuged at 15000 ×g for 10 min at 4 ∘ C; supernatant was recentrifuged at 18000 ×g for 10 min; the supernatant was stored at 4 ∘ C for enzyme assay.

Superoxide Dismutase
Activity. SOD activity was measured in a reaction solution (3 mL) according to the method of Giannopolitis and Ries [21]. SOD activity was determined by measurement of inhibition of photochemical reduction of nitro blue tetrazolium (NBT) at 560 nm, where one unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of photochemical reduction of NBT.

Catalase Activity.
CAT activity was assayed by the method of Aebi [22] in a reaction solution (3 mL [26,27]. Media plates (11 cm in diameter) were prepared with nutrient agar. A total of four wells (7 mm in diameter) per agar plate were cut. For test, three doses of extract (25,50, and 75 L/well) were used. Standard antibiotics, streptomycin (30 g), ciprofloxacin (10 g), doxycycline (30 g), ampicillin (10 g), and ofloxacin (5 g), were used as positive control and dimethyl sulfoxide (75 L/well) was used as negative control. A 100 L (10 5 cfu) of diluted microbial suspension was swabbed on nutrient agar plates. Extracts and positive control were added separately to each well and allowed to diffuse at room temperature for 15-20 min. Plates were incubated at 37 ∘ C for 24 h after which they were examined for zones of growth inhibition and the diameter of these zones was measured. The assay was repeated three times for each extract. The antimicrobial effects were recorded as the mean diameter of the resulting inhibition zones of growth in millimeter.

Estimation of Thymol and Thymoquinone by LC-MS/MS.
Extraction from different suspension cultures was done in 100% of HPLC grade methanol using mortar and pestle with periodical mixing on shaker overnight and centrifuged at 5000 ×g for 10 min, and then the supernatant (5 mL) was collected for analysis. Analysis was performed using Triple Quadrupole LC-MS/MS Mass Spectrometer (MS-Manufacturer-AB Sciex Instruments, Model 1034067 V, Serial number V210201201) turbo spray interface. The separation was carried out in Acquity UPLC BEH apparatus with an autosampler equipped with C18 column dimension-2.1 × 50 mm. Column temperature was kept ambient, mobile phase methanol and water in ratio of 90 : 10, source temperature 200.0 ∘ C at set point, injection volume 10.0 L, and run time 2 min. All acquisitions were performed under positive ionization mode with a capillary voltage of 3500 V. Data acquisition and processing were done using the software version Analyst 1.6. Q 1 MS total chromatogram was run at positive mode and the nebulizer gas (GS1, 10.00), heater gas (GS2, 10.00), temperature (TEM, 200.00), declustering potential (DP), entrance potential (EP, 10.00), collision energy (CE), and collision exit potential (CXP, 9.00) of the compounds were optimized. CXP gave the daughter ion of the parent ion. Multiple Reaction Monitoring (MRM) of the compound's daughter ion was developed. The chromatogram developed gave separate peaks of different area for two compounds which was used to quantify the two compounds in the samples against the standard thymol and thymoquinone in terms of g/gm. Best peak of thymoquinone was developed at 151.100/109.100 (DP-start 52.00; stop 52, CE-start 17.00; stop 17.00) and best peak of thymol was developed 165.100/137.100 (DP-start 70.00; stop 70.00, CE-start 35.00; stop 35.00).

Statistical
Analysis. Data were statistically analyzed and the results were expressed as means (±SD) of average of three replicates ( = 3). values (≤0.05) were considered as significant compared to the respective controls.

Callusing Response and Establishment of Suspension Cultures.
Callusing results showed that, among the different explants (leaf, epicotyl, hypocotyl, and root), epicotyl segments (0.5 cm long) gave the best and fast callusing response with creamish white friable callus. The stock suspension cultures were initiated by these epicotyl calluses which were subcultured every week. A sigmoidal growth curve was obtained with all five growth phases, that is, lag, exponential, linear, stationary, and progressive decline. Maximum growth and longest stationary phase were achieved in suspension culture supplemented with Kn (2 mg/L) + NAA (1 mg/L) combination.
Plant tissue culture techniques are of immense importance for the production of myriad of useful secondary metabolites as compared to the whole plant or microbial system [28,29]. In the present study, an epicotyl suspension culture was established and maintained under suitable conditions of aeration, agitation, light, temperature, and other physical parameters [30].  (Table 1). In contrast to this control cultures gave maximum growth index of 3.413 followed by 5 mg/L and 10 mg/L of pectin and MnCl 2 elicitation where it was 2.850, 2.786 and 2.906, 2.500, respectively. The abiotic elicitation thus proved to be more deleterious than the biotic elicitation towards cell division, fresh weight accumulation, and growth index and, therefore, showed a dose-dependent growth inhibition. Pectin elicitation used in the present study clearly supports the facts of the previous findings that elicitation facilitates growth and enhances metabolite production. Pectin elicitation showed a dose-dependent effect on the cell growth and biomass accumulation of the N. sativa epicotyl suspension cultures as seen in the cultures of Mucuna pruriens in a study conducted by Raghavendra et al. [31]. Further, results of our study regarding the effect of abiotic elicitor (MnCl 2 ) on culture growth are in accordance with the results of Ghorpade et al. [32] conducted on Boswellia serrata callus cultures which showed a deleterious effect of increasing concentration of the abiotic elicitors on the biomass accumulation.   the control culture and also reported the presence of other two spots recorded in 5 mg/L but of more intensity. Elicitation of MnCl 2 15 mg/L totally inhibited the occurrence of TQ as no spot of TQ was reported, thus reporting serious harmful effect of elevated concentrations of abiotic elicitors on TQ and THY production.

TLC Analysis for TQ and THY under Different
Elicitors are known to cease the in vitro culture growth temporarily or permanently. This cessation may also lead to a defense response by switching from primary metabolism to secondary one [33]. TLC results and high concentrations quantified in LC-MS/MS results for both the metabolites in 15 mg/L of pectin elicitation are justified by the above statement where higher concentration leads to the conversion of the primary metabolites to secondary ones in order to prevent cell death. Earlier reports have attempted to correlate changes of HMGR enzyme activity with the induced synthesis of particular isoprenoids as this is a key enzyme in the terpenoid biosynthesis. Suzuki et al. [34] correlated a transient induction of HMGR activity with the accumulation of ipomeamarone, a furanosesquiterpenoid, in sweet potato tissue infected with Ceratocystis fimbriata. Stermer and Bostock [35] have shown a transient induction of HMGR activity in potato discs stimulated to produce sesquiterpenoids by application of arachidonic acid. Therefore, MnCl 2 elicitation somehow triggered the TQ and THY production by inducing the activity of HMGR though no such evidence is still available. Further, the study of Moses and Mukundan [36] on THY enhancement by lower concentration MnCl 2 did not exhibit noticeable increase in THY accumulation; in contrast to this our results showed that even lower dose of MnCl 2 , that is, 10 mg/L, elicited the metabolite production.

DPPH Free Radical Inhibition
Activity. DPPH radical inhibition activity of various tissue culture generated extract is depicted in Figure 1. The inhibition activity recorded for the cultures under pectin elicitation increased with the increasing elicitor concentration and pectin 15 mg/L elicitation recorded maximum inhibition comparable to the cultures without elicitation (control suspension), epicotyl explant extract, and to some extent the standard, that is, ascorbic acid over the 18.62 IC 50 value (Table 3). In the case of MnCl 2 elicitation 5 mg/L and 10 mg/L elicitation inhibited DPPH radical at higher level than the pectin elicited cultures, but maximum inhibition was shown by cultures under 10 mg/L elicitation which was higher than the epicotyl explant extract and cultures without elicitation (control cultures) also comparable to the standard ascorbic acid used over a value of 16.44 IC 50 ( Table 3). The inhibition caused by cultures under biotic and abiotic elicitation was far less than the pure TQ used but very near to THY. Standards and all the extracts showed a dose-dependent inhibition on the DPPH radicals.
In the present work, epicotyl suspension cultures under less favourable conditions (biotic and abiotic elicitation) showed increased levels of antioxidant metabolites, that is, TQ and THY, which contributed to reducing the stress generated in these circumstances. The observed high levels of DPPH radical scavenging capacity in the MnCl 2 elicited cultures were comparable to THY and ascorbic acid could reflect the expression of antioxidant biosynthetic pathways resulting in prevention or minimization of the cytotoxic impact of ROS formed during stress conditions, as described against chilling [37] and salt stress [38]. The previous studies done on the antioxidant activity of N. sativa seeds were in the range of IC 50 2.26-28.8 mg/mL [2,39]. The values of both IC 50 and EC 50 obtained in this study were lower than those of previous studies, thereby indicating a higher antioxidant activity in the suspension samples. The results of this study are also in accordance with the previous study that the higher enzyme activity and antioxidant properties are attributed to increased TQ and THY production in them under elicitation which are known antioxidants.

FRAP-Ferric Ion Scavenging Activity. Results of Fe (III)
reduction demonstrated that all the tissue culture generated extracts had lower reducing ability than the radical    (Table 3). The chelating effect of all the extracts increased with an increase in their concentrations which may be due to the increase in the amount of the secondary metabolites present in the extracts. The protective effects of plant are ascribed because of their several components; correspondingly the metabolites like thymoquinone, carvacrol, thymol, cymene, t-anethole, and 4-terpineol confer major antioxidant properties to N. sativa [40,41]. Results of the study clearly have stated the positive effect of both elicitors towards TQ and THY production which provided the cultures with remarkable scavenging activity.

Antioxidant Enzymes Activity under Biotic and Abiotic Elicitation
3.5.1. Superoxide Dismutase. Results showed that both biotic and abiotic elicitors had an effect on the activity of antioxidant enzyme SOD. As the concentration of elicitors was increased the activity of the enzyme was also enhanced. MnCl 2 (10 mg/L) proved to be the best inducer (Figure 3) with respect to SOD activity as compared to pectin. On the contrary, SOD enzyme activity showed a remarkable decrease when compared to the cultures without elicitation (control). This might be due to the excessive consumption of the enzyme in combating the increased ROS level under elicitation. The change in the activity of SOD under elicitation was too prominent; therefore, it can be said that its presence in all samples suggests that this enzyme may participate in protecting suspension cells against free superoxide radicals.
There are many reports in support of the increased activities of SOD under abiotic stresses induced with tissue culture techniques in a wide range of plant species, including heavy metals, such as Al, Cd, Mn, Cr, and Cu, salt, and drought [42][43][44][45][46][47][48]. Therefore, the increased level of SOD activity under MnCl 2 elicited suspension cultures in comparison to the pectin elicitation is in justification of the previous studies.

Catalase.
In the activity of CAT enzyme a significant increase was observed in the elicited cultures when compared to the epicotyl explant extract and cultures without elicitation (control suspension culture). Pectin 10 mg/L and MnCl 2 5 mg/L reported almost similar level of enzyme activity, whereas pectin 15 mg/L, MnCl 2 10 mg/L, and MnCl 2 15 mg/L reported a higher enzyme activity (Figure 4). The considerable increase seen in the catalase enzyme activity particularly under MnCl 2 elicitation might have aroused so as to neutralize the large amount of generated H 2 O 2 in the elicited cultures as the end product of SOD catalyzed reaction when compared to control cultures.
Plant products are rich sources of phytochemicals as is the extract of this study and have been found to possess a variety of biological activities including antioxidant, cytotoxic, and hepatoprotective potentials. They are excellent reducing agents and reverse oxidation by donating electrons and/or hydrogen ions [49]. Increased CAT enzyme activities reported in the suspension cultures under abiotic elicitation in this study are supported by the facts of previous findings which have demonstrated that CAT activities are also induced under abiotic stresses in different in vitro conditions in different plants, including heavy metals, such as Al, Cd, Mn, Cr, and Cu, salt, and drought [42][43][44][45][46][47][48].

In Vitro Antimicrobial Effect of Different Suspension
Extracts. The different methanol extracts of epicotyl and suspension cultures grown under biotic and abiotic elicitors were studied for their antibacterial potential against five Gram positive and Gram negative pathogenic bacterial strains, results of which indicated that different extracts showed different degrees of growth inhibition higher than the standard drugs used depending on the quantity of the metabolites quantified, type of elicitation, dose, and bacterial strains ( Table 5). Among the five tested bacterial strains, almost all the extracts showed a maximum degree of inhibition with lowest minimum inhibitory concentrations (MIC) towards E. coli and B. cereus, that is, inhibiting the growth of both Gram positive and Gram negative bacteria ( Figure 5). Among the biotic and abiotic elicitation, control cultures, and the in vitro germinated epicotyl extract, control suspension culture reported highest activity against E. coli (32 ± 0.4) with MIC value 2.35 ± 0.8 g mL −1 at lowest dose of 25 L (25 mg sample) (Table 4). Likewise, in the case of cultures grown under elicitation of pectin 15 mg/L and MnCl 2 10 mg/L, highest zone of inhibition was recorded against B. cereus (30 ± 0.7) and E. coli (30 ± 0.2) ( Table 5) with MIC values 2.55 ± 0.6 and 2.46 ± 0.2 g mL −1 (Table 4), respectively, at lowest dose of 25 L (25 mg sample). Further, MnCl 2 15 mg/L elicited culture recorded least inhibitory activity followed by epicotyl, with maximum inhibitions against E. coli 18 ± 0.2, MIC 4.15 ± 0.3 g mL −1 ; 22 ± 0.3, MIC 3.61 ± 0.5, respectively. Results of the in vitro generated extracts were far better when compared with standard antibiotics used as positive control.
N. sativa is known to have very strong antibacterial background mainly due to lethal effects of its active components, that is, TQ and THY, towards the microbial growth [50][51][52]. Antibacterial results of our study are in accordance with the results of Islam et al. [25] and Kamal and Ahmad [53], as the methanolic extracts of different elicited cultures proved to be potent natural inhibitors. The inhibitory activity of plant or tissue culture extracts depends upon the type of explant used, concentration, and microbes tested [54]. Findings of the present study are an extension of our previous work    where MnCl 2 , CoCl 2 , cellulose, and pectin elicited epicotyl callus extracts showed remarkable antibacterial properties [15]. Therefore, this may be a reason for the variation in the inhibitory activity of different extracts of N. sativa against the bacterial strains where maximum inhibition was marked by MnCl 2 10 mg/L elicited epicotyl followed by pectin 15 mg/L elicited culture extracts.

Estimation of Thymol and Thymoquinone by LC-MS/MS.
Suspension cultures which marked the prominent presence of thymoquinone and thymol in the TLC analysis and also proved to be potent inhibitors towards the pathogenic bacterial isolates and different reactive free radicals were further quantified for the presence of TQ and THY in them.
Quantitative analysis of the epicotyl explant showed a total content of TQ 1.843 ± 0.34 g/gm and THY 1.08 ± 0.27 g/gm (Figures 6(a) and 6(b)), whereas in control suspension cultures, it was TQ 2.82 ± 0.41 g/gm and THY 1.99 ± 0.21 g/gm (Figures 7(a) and 7(b)). In contrast to this MnCl 2 10 mg/L elicitation marked the highest presence of the metabolites, that is, TQ 2.90 ± 0.33 g/gm and THY 1.

Conclusion
The present study was carried out to exploit the potential of N. sativa epicotyl suspension culture for thymoquinone and thymol production and their efficacy as potent antioxidants and antibacterial agents. Therefore, this is a first report on response of growth and metabolite production in epicotyl suspension cultures of N. sativa under the effect of biotic and abiotic elicitation. Results showed that MnCl 2 elicitation enhanced the production of thymoquinone and thymol; further, these extracts demonstrated strong antioxidant and antibacterial properties against Gram positive and Gram negative bacteria due to increased phytochemical accumulation. Henceforth, it could be concluded that these elite cell lines can be used for sustainable production of thymoquinone and thymol which will be helpful in understanding the biosynthetic mechanism of these metabolites under in vitro conditions.