Anti-inflammatory effect of seeds and callus of Nigella sativa L. extracts on mix glial cells with regard to their thymoquinone content

Abstract

Anti-inflammatory effect of the alcoholic extracts of N. sativa seeds and its callus on mix glial cells of rat with regard to their thymoquinone (TQ) content was investigated. Callus induction was achieved for explants of young leaf, stem, petiole, and root of N. sativa on solid Murashige and Skoog (MS) medium containing 2,4-D (1 mg/l) and kinetin (2.15 mg/l). TQ content of the alcoholic extracts was measured by HPLC. Total phenols were determined using Folin-Ciocalteu method and antioxidant power was estimated using FRAP tests. The mix glial cells, inflamed by lipopolysaccharide, were subjected to anti-inflammatory studies in the presence of various amounts of TQ and the alcoholic extracts. Viability of the cells and nitric oxide production were measured by MTT and Griess reagent, respectively. The leaf callus obtained the highest growth rate (115.4 mg/day) on MS medium containing 2,4-D (0.22 mg/l) and kinetin (2.15 mg/l). Analyses confirmed that TQ content of the callus of leaf was 12 times higher than that measured in the seeds extract. However, it decreased as the calli aged. Decrease in the TQ content of the callus was accompanied with an increase in its phenolic content and antioxidant ability. Studies on the inflamed rat mix glial cells revealed significant reduction in the nitric oxide production in the presence of 0.2 to 1.6 mg/ml of callus extract and 1.25 to 20 μl/ml of the seed extracts. However, the extent of the effects is modified assumingly due to the presence of the other existing substances in the extracts.

Fig. 1

Proposed pathway for TQ biosynthesis (44)

Fig. 2

Primary mix glial cell culture after 14 days observed by an inverted microscope a 32×, and b 40×

Fig. 3

a N. sativa seeds, b sprouted seeds, and c plantlet growing on hormnon-free 1/2MS medium at 25°C applying a photoperiod of 16:8 h (light–dark cycle). d Excised explants from N. sativa young plantlet on MS medium supplemented with 2,4-D (1 mg/l) and kinetin (2.15 mg/l). e, f Induced calli to grow on MS medium supplemented with 2,4-D (0.22 mg/l) and kinetin (2.15 mg/l). Calli grown g in the absence of light and h in the presence of light at 25°C

Fig. 4

Weights of the wet calli at the end of first subculture (dark bars), second subculture (dotted bars), and third subculture (gray bars). Calli were subcultured every 3 weeks on solid MS medium supplemented with 2,4-D (0.22 mg/l) and kinetin (2.15 mg/l). See the “MATERIALS AND METHODS” section for details

Fig. 5

HPLC chromatograms of the extracts of a the seeds, b bright parts of the leaf callus at the second week and c fourth week of the second subculture. See the “MATERIALS AND METHODS” section for the experimental details

Fig. 6

Nitrite concentration in the supernatant of the mix glial cell cultures after 48 h of treatment with different concentrations a of NSC (0.2, 0.4, 0.8, 1.6, and 3.2 mg/ml) designated as NSC1 to NSC5, b of NSO (0.5, 1.25, 2.5, 5, 10, and 20 μl/ml) designated as NSO1 to NSO6. Results of MTT test for the mix glial cells treated with c five different concentrations of NSC, and d six different concentrations of NSO