Nigella sativa callus treated with sodium azide exhibit augmented antioxidant activity and DNA damage inhibition

Abstract

Nigella sativa L. (NS) is an herbaceous plant, possessing phytochemicals of therapeutic importance. Thymoquinone is one of the active phytochemicals of NS that confers noteworthy antioxidant properties. Sodium azide, an agent of abiotic stress, can modulates antioxidant system in plants. In the present investigation, sodium azide (0, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM and 200 µM) doses administered to the in vitro NS callus cultures for production/modification of secondary metabolites with augmented activity. 200 µM sodium azide treated NS callus exhibited maximum peroxidase activity (1.286 ± 0.101 nanokatal mg^-1 protein) and polyphenol oxidase activity (1.590 ± 0.110 nanokatal mg^-1 protein), while 100 µM sodium azide treated NS callus for optimum catalase activity (1.250 ± 0.105 nanokatal mg^-1 protein). Further, 200 µM sodium azide treated NS callus obtained significantly the highest phenolics (3.666 ± 0.475 mg g^-1 callus fresh weight), 20 µM sodium azide treated NS callus, the highest flavonoids (1.308 ± 0.082 mg g^-1 callus fresh weight) and 100 µM sodium azide treated NS callus, the highest carotenes (1.273 ± 0.066 mg g^-1 callus fresh weight). However, NS callus exhibited a decrease in thymoquinone yield/content vis-à-vis possible emergence of its analog with 5.3 min retention time and an increase in antioxidant property. Treatment with 200 µM sodium azide registered significantly the lowest percent yield of callus extract (4.6 ± 0.36 mg g^-1 callus fresh weight) and thymoquinone yield (16.65 ± 2.52 µg g^-1 callus fresh weight) and content (0.36 ± 0.07 mg g^-1 callus dry weight) and the highest antioxidant activity (3.873 ± 0.402%), signifying a negative correlation of the former with the latter. DNA damage inhibition (24.3 ± 1.7%) was recorded significantly maximum at 200 µM sodium azide treatment. Sodium azide treated callus also recorded emergence of a new peak at 5.3 min retention time (possibly an analog of thymoquinone with augmented antioxidant activity) whose area exhibits significantly negative correlation with callus extract yield and thymoquinone yield/content and positive correlation with antioxidant activity and in vitro DNA damage inhibition. Thus, sodium azide treatment to NS callus confers possible production of secondary metabolites or thymoquinone analog (s) responsible for elevated antioxidant property and inhibition to DNA damage. The formation of potent antioxidants through sodium azide treatment to NS could be worthy for nutraceutical and pharmaceutical industries.

Figure 1

NS seed germination on MS medium and callus formation using plant tissue culture technique. (A) NS seeds on MS media; (B) NS seedlings; (C) Callus of NS in MS media supplemented 2,4-D; (D) Sodium azide treatment on NS Callus (n = 3).

Figure 2

Graded doses of sodium azide significantly (p < 0.001) affect callus extract yield (mg g⁻¹ callus FW) and thymoquinone yield (µg g⁻¹ callus FW)/content (mg g⁻¹ callus extract) in Nigella sativa, FW = Fresh weight. Vertical bars refer to ± standard deviation and histograms bearing the different alphabets in a series of data are significantly different from each other (Tukey’s HSD test).

Figure 3

Graded doses of sodium azide significantly (p < 0.001) affect activities of POX, PPO and CAT in callus extract of Nigella sativa. Vertical bars refer to ± standard deviation and histograms bearing the different alphabets in a series of data are significantly different from each other (Tukey’s HSD test).

Figure 4

Graded doses of sodium azide significantly (p < 0.001) affect contents of phenolics, flavonoids and carotene in callus extract of Nigella sativa. Vertical bars refer to ± standard deviation and histograms bearing the different alphabets in a series of data are significantly different from each other (Tukey’s HSD test).

Figure 5

Graded doses of sodium azide significantly (p < 0.001) affect antioxidant activity of callus extract of Nigella sativa that also significantly (p < 0.001) corresponds to in vitro calf DNA damage inhibition (%). Vertical bars refer to ± standard deviation and histograms bearing the different alphabets in a series of data are significantly different from each other (Tukey’s HSD test).

Figure 6

DNA damage inhibition on NSE after treatment with various concentrations of sodium azide. L1 Standard calf thymus DNA; L2, L3, L4, L5, L6, L7, L8 representing calf thymus DNA and NSEs after 0 (Control), 5, 10, 20, 50, 100, 200 µM treatment of sodium azide, respectively; L9 showing calf thymus DNA with 1 mM thymoquinone.

Figure 7

RP-HPLC histograms depict sodium azide dose-dependent new peak with retention time of 5.3 min along with thymoquinone peak (RT 7.3 min). (A) Control (0 µM sodium azide), (B) 5 µM sodium azide, (C) 10 µM sodium azide, (D) 20 µM sodium azide, (E) 50 µM sodium azide, (F) 100 µM sodium azide and (G) 200 µM sodium azide.

Figure 8

Significant negative correlation (p < 0.01) between peak area of RT 5.3 (New peak) and RT 7.3 (Thymoquinone).

Figure 9

Sum of both peak areas (RT 5.3 and RT 7.3) across graded doses of sodium azide. Values on histograms denote comparison (%) with Control (100) and are not significantly different from each other.

Figure 10

Peak area at 5.3 min retention time correlates negatively with callus yield (p < 0.05), thymoquinone yield and content (p < 0.01) and positively with antioxidant activity (p < 0.01) and DNA damage inhibition (p < 0.01).