Studies on the extracts of black cumin (Nigella sativa L.) obtained by supercritical fluid carbon dioxide.

The pharmacologically valued black cumin seeds of Nigella sativa L. plant of
Ranunculaceae family are used in folk medicine, as they contain a complex of more
than hundred phytochemicals, some of which are yet to be identified, as they are
highly unstable. Of the major quinonic phenol compounds thymoquinone (TQ),
dithymoquinone (DTQ), thymohydroquinone (THQ) and thymol (THY) present in
the seed as bioactives, whereas thymoquinone is the most pharmacologically active
compound. Conventional SoxtecTM and supercritical carbon dioxide (SCCO2)
extractions were carried out for the seeds. Response surface methodology (RSM)
along with Box-Behnken experimental design was employed to maximise the extract
yield and TQ content by varying the temperature, pressure and material in SCCO2
extraction. The SCCO2 extraction at optimized conditions (120 bar pressure, 40°C
temperature and solvent to material ratio of 45kg/kg) showed higher TQ content and
lower of extract yield. SCCO2-1 extract as major volatile part (at 120 bar pressure
with 40°C) and total extract obtained as SCCO2-2 (at 280 bar pressure with 50°C)
and HD SCCO2 (Hydrodistillation of SCCO2-2) were chemically characterized to
report 47 compounds. Of these compounds, 16 have been reported for the first time
in Nigella sativa seed. The occurrence of four major quinonic phenol compounds;
TQ, DTQ, THQ and THY in SCCO2-2 extract was confirmed by 2D HSQCT NMR.
Further, the major bioactive compound TQ was isolated and characterized from the
SCCO2-2 extract. The SCCO2 extracts showed higher antibacterial activities than
SoxtecTM extracts. The isolated TQ showed lower MIC (minimum inhibitory
concentration) and MBC (minimum bactericidal concentration) values against the
major food borne bacteria used in this study and proved to be an antibacterial
principle. Antioxidant activities were evaluated by DPPH and FRAP assays with
total phenolic content (TPC), where also SCCO2 extracts indicated higher
antioxidant activity than Soxtec™ extracts. The isolated TQ from SCCO2 extract
proved with higher antioxidant properties and higher TPC. The synthesis of water
soluble thymol glycosides was carried out using amyloglucosidase (from Rhizopus
mold) and thymol with different carbohydrates in DMF (dimethyl formamide) under
SCCO2 conditions. Thymol formed glycosides with D-galactose, D-mannose, Dfructose,
D-ribose and D-arabinose. Spectral characterization studies revealed that
the reaction occurred between the phenolic OH group of thymol and 1-O/2-O groups
of D-fructose and C-1 group of D-galactose, D-mannose, D-ribose and D-arabinose
resulting in monoglycosylated/arylated derivatives. Of the glycosides synthesized,
thymol-6-O-D-galactopyranoside (α and β forms), thymol-6-O-D-mannopyranoside
(α and β forms), thymol-6-O-2-O-D-fructofuranoside and thymol-6-O-1-O-Dfructofuranoside,
thymol-6-O-D-ribofuranoside (α and β forms) and thymol-6-O-Darabinofuranoside
(α and β forms) were reported for the first time. The synthesized
thymol glycosides have showed better bioavailability and pharmacological
properties for future applications. Hence, TQ appears to be promising as a potent
natural antibacterial as well as an antioxidant principle, which could replace the
synthetic antibiotics and antioxidants in food and pharmacological applications.