Comparative evaluation of two analytical methods for thymoquinone determination and their correlation with phenolic and flavonoid contents in Nigella sativa biomass.

The present study re-examines the efficacy of prevalent methods of thymoquinone (TQ) estimation in the biomass of Nigella sativa L. using RP-HPLC. The differential efficacy in terms of magnitude, accuracy and cognate correlation with metabolically affiliated groups of biomolecules, phenolics, and antioxidants opens new vistas. The study was conducted to develop a more accurate and sensitive method for quantitative assessment and correlation among TQ content, total phenolics, and flavonoids in six different plant parts of N. sativa, which are used in culinary applications, therapeutic formulations, nutraceuticals, and biowaste. The re-examination was based on the extraction of TQ-rich fractions with solvents of differential polarity (methanol and benzene), followed by quantitative assessment by RP-HPLC using methanol: water (80:20) as the mobile phase. RP-HPLC data revealed that TQ in methanolic extracts was highest in root (0.00959±0.00032mg/g FW), followed by flower (0.00882±0.00013mg/g FW), and lowest in fruit (0.00488±0.00014mg/g FW). Benzene extracts yielded the same pattern but with substantially higher magnitude, particularly in flower (0.0634±0.0272mg/g FW) and fruit (0.0616±0.0416mg/g FW), making the system more resolvable. With the objective to understand the correlation with compounds of antioxidant nature, total phenolic content (TPC) and total flavonoid content (TFC) were determined and correlated with TQ content estimated using two different solvents. TPC was highest in leaf (0.208±0.010mg GAE/g) and lowest in fruit. TFC was highest in bud (0.344±0.004mg QE/g) and lowest in root. Pearson correlations using triplicate values showed very weak associations between methanolic TQ and TPC (r=0.008; P<0.05) and between methanolic TQ and TFC (r=-0.330; P<0.05). Benzene-extracted TQ also showed a negligible positive correlation with TPC (r=0.110; P<0.01) and a very poor negative correlation with TFC (r=-0.016; P<0.01). TPC and TFC remained moderately positively correlated (r=0.450; P=0.05). The results indicate that TQ and phenols accumulate largely independently in different plant parts of N. sativa. The TQ estimation using benzene extracts appears far superior to the method relying on methanolic extracts, given the large-scale and magnitude differences, making it more resolvable during screening.