Evaluation of phenolic profile, enzyme inhibitory and antimicrobial activities of Nigella sativa L. seed extracts

Abstract

Black cumin (Nigella sativa L. [N.sativa]) seed extracts demonstrated numerous beneficial biological effects including, among others, antidiabetic, anticancer, immunomodulatory, antimicrobial, anti-inflammatory, antihypertensive, and antioxidant activity. To better understand the phytochemical composition of N. sativa seeds, methanol seed extracts were analyzed for phenolic acid and flavonoid content. Furthermore, we tested N. sativa methanol, n-hexane, and aqueous seed extracts for their inhibitory activity against butyrylcholinesterase (BChE) and catalase (CAT) as well as for antimicrobial activity against several bacterial and a yeast strains. The phenolic content of N. sativa was analyzed using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The inhibition of BChE was assessed by modified Ellman's method, and the inhibition of CAT was determined by monitoring hydrogen peroxide consumption. The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Salmonella enterica, and Escherichia coli using the agar diffusion method. The UHPLC-MS/MS method allowed the identification and quantification of 23 phenolic compounds within 15 minutes. The major components found in N. sativa seed extract were sinapinic acid (7.22 ± 0.73 µg/mg) as a phenolic acid and kaempferol (11.74 ± 0.92 µg/mg) as a flavonoid. All extracts showed inhibitory activity against BChE, with methanol seed extract demonstrating the highest inhibitory activity (inhibitory concentration 50% [IC50] 79.11 ± 6.06 µg/ml). The methanol seed extract also showed strong inhibitory activity against CAT with an IC50 value of 6.61 ± 0.27 µg/ml. Finally, the methanol extract exhibited considerable inhibitory activity against the tested microbial strains. Overall, this is the first study to investigate the ability of black cumin seed extracts to inhibit CAT. Our results indicate that N. sativa seed can be considered as an effective inhibitor of CAT activity.

FIGURE 1

Preparation of Nigella sativa seed extracts for ultra-high performance liquid chromatography tandem mass spectrometry method. Using this isolation method, it is possible to separate plant phenolics into four classes, i.e., free phenolics (NS-P1), esters (NS-P2), phenolic glycosides (NS-P3), and phenolics non-soluble in 80% MeOH (NS-P4), which was used as the extraction solvent. All phenolic compounds are present in free form, which is the most convenient form for their reversed-phase chromatographic separation, due to the solubility in the mobile phase and faster separation through the column.

FIGURE 2

Structure of major phenolic compounds in Nigella sativa (N. sativa) seed extracts. All four fractions contained ferulic acid, sinapinic acid was detected in three fractions (NS-P2, NS-P3, and NS-P4), and salicylic acid was also found in three fractions (NS-P1, NS-P2, and NS-P4). The majority of phenolic acids in the seeds of N. sativa are actually ester-bound phenolics (bound to the cell walls), followed by the corresponding esters and glycosides.