Solvent based optimization for extraction and stability of thymoquinone from Nigella sativa Linn. and its quantification using RP-HPLC

Abstract

The Nigella sativa pharmacological properties are mainly ascribed to its volatile oil, of which thymoquinone is an important bioactive component. Surprisingly, till date, no standard formulation or thymoquinone rich N. sativa extract is under clinical use probably due to its poor extraction and lesser stability in the already used solvents. In the present investigation solubility, extraction, percent composition and total antioxidant activity from the seeds of N. sativa was explored using five solvents. An HPLC method was standardized in an isocratic system (C-18 column, flow rate of 1.0 ml/min, mobile phase-water:methanol: 30:70, detection wavelength-254 nm, retention time-8.77 min) for quantification of thymoquinone. To further confirm the presence of thymoquinone in the respective extracts absorbance spectra analysis has been carried out and compared with pure thymoquinone. Additionally total antioxidant activity of Nigella sativa extracts has been evaluated using ascorbic acid as standard. Our results showed maximum percentage yield in aqueous extract while methanol having the least yield and the ethanol, benzene and hexane extracts exhibited moderate yields. A linear standard calibration curve of thymoquinone showed R² as 0.999 and % RSD as 7.166. The HPLC analysis revealed maximum percentage composition of thymoquinone in the benzene extract, whereas in the hexane and methanol extracts the content was less. Aqueous and ethanol extracts displayed insignificant thymoquinone content. Absorbance spectra analysis confirms the presence of thymoquinone peak in the benzene, hexane and methanol extracts while aqueous and ethanol extracts showed minimal absorbance. Maximum total antioxidant activity was observed in the aqueous extract while minimum was observed in the methanolic extract. Weak positive (+ 0.3676) correlation was established between percent composition of thymoquinone and antioxidant activity among different extracts indicating that thymoquinone may not be the only factor for antioxidant activity, but other phytochemicals might also contribute. However, we for the first time demonstrated that the benzene extract of N. sativa has better solubility and percent composition of thymoquinone as compared to other solvents. It can be concluded that the solubility, differential composition of bioactive components among these extracts may have diverse effects on the total antiradical activity. Thus, our study provides insights on optimization and standardization of bioactive rich formulation of N. sativa.

Keywords: Antioxidant; Benzene; Nigella sativa; RP-HPLC; Solvent extraction; Thymoquinone.

Fig. 1

Variation of the potassium permanganate concentration after adding 1 ml of ascorbic acid 0.01 (mmol/ml)

Fig. 2

Differential percentage yields of extracts in five different solvents

Fig. 3

UV-Visible spectrum scan of thymoquinone standard and various extracts using Shimadzu Spectrophotometer

Fig. 4

Thymoquinone standard curve (a) with mean coefficient of determination (R²) = 0.999 using concentration range (0.0312–2.0 μg/ml). Thymoquinone standard peak (b) was detected at retention time of 8.77 min. Data analyzed and acquired using Lab Solution Lite software

Fig. 5

Variable percentage composition (content) of thymoquinone in different extracts

Fig. 6

Chromatogram showing peaks of Thymoquinone in aqueous (a), benzene (b), ethanol (c), hexane (d) and methanol (e) extracts. Data was analyzed and acquired using Lab Solution Lite software

Fig. 7

Total antioxidant activity of different extracts of Nigella sativa