# Exploring the Multifaceted Phytochemical Profile of Nigella sativa and the Therapeutic Potential of Thymoquinone

## TL;DR

This study explores the chemical makeup of Nigella sativa seeds and finds that thymoquinone has strong antibacterial, antioxidant, and anti-diabetic properties.

## Contribution

The study isolates and purifies thymoquinone from Nigella sativa and evaluates its multi-target therapeutic potential in vitro.

## Key findings

- Thymoquinone inhibited Gram-positive and Gram-negative bacteria with MICs ranging from 62.5 to 500 µg/mL.
- Thymoquinone showed strong antioxidant activity with DPPH and ABTS IC50 values of 52.3 and 41.6 µg/mL, respectively.
- Thymoquinone inhibited α-glucosidase (92%) more effectively than α-amylase (84%) at 128 µg/mL.

## Abstract

Background: Nigella sativa (black cumin) seeds are renowned for their ethnomedicinal significance and are rich in bioactive phytochemicals, which contribute to food preservation and the prevention of various diseases through their antimicrobial and antioxidant properties. Accordingly, this study aimed to characterize the phytochemical composition of N. sativa seed extracts, isolate thymoquinone, and assess their antibacterial, antibiofilm, antioxidant, anti-inflammatory and antidiabetic activities. Methods: Nigella sativa seed extracts were prepared using solvents of increasing polarity and analyzed for phytochemical content. Metabolite profiling was performed using UHPLC/QTOF-MS. Thymoquinone, the major constituent, was isolated via thin-layer chromatography (TLC), further purified using semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC), and evaluated in vitro for antibacterial, antibiofilm, antioxidant, anti-inflammatory, and antidiabetic activities. Results: Extraction yields ranged from 5.5% to 8.4% (w/w), with methanol yielding the highest phenol (6.34 ± 0.31 mg GAE/mL) and flavonoid (5.12 ± 0.26 mg QE/mL) contents. UHPLC/QTOF-MS revealed a chemically diverse profile dominated by thymoquinone (58% relative abundance), alongside p-cymene, carvacrol, longifolene, and nigellidine. Thymoquinone (Rf = 0.56) was initially isolated from the methanolic extract with a yield of 270 mg/g and further purified from preparative TLC fractions using semi-preparative RP-HPLC, affording 82 mg of >95% pure compound with a 68.3% recovery, suitable for subsequent biological assays. It inhibited Gram-positive and Gram-negative bacteria, with MICs of 62.5 µg/mL against Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes; 125–250 µg/mL against Escherichia coli and Salmonella typhimurium; and 500 µg/mL against Pseudomonas aeruginosa. Thymoquinone reduced biofilm formation (>80% at 25–50 µg/mL; MBIC50 ≈ 5.4–11.6 µg/mL), exhibited antioxidant activity (DPPH IC50 = 52.3 ± 2. 1 µg/mL; ABTS IC50 = 41.6 ± 1.9 µg/mL), stabilized erythrocyte membranes (IC50 ≈ 14.8 µg/mL), and inhibited carbohydrate-hydrolyzing enzymes, with stronger inhibition of α-glucosidase (~92%) than α-amylase (~84%) at 128 µg/mL. Conclusions: Thymoquinone is a major bioactive constituent of N. sativa seeds, exhibiting consistent multi-target in vitro activity. These findings highlight its functional relevance and in vivo investigations to establish therapeutic potential.