## 3 Results and Discussion
### TQ Content Estimation
#### 3.2.2 Changes in TQ Content During Thermal Treatment of BCSO  
The analysis of the TQ amount demonstrated that its content in BCSO (9.5 mg/mL and 6.65 mg/mL for the UV and HPLC assay, respectively) was in accordance with previous research [5,38]. As can be seen in Figure 4, during storage at 60 \({}^{\circ}\)C, its content was changed depending on the storage conditions. For \"open\" samples (Figure 4a), after more than three weeks of heating, the intensity of the bands discussed above were clearly decreased, indicating a reduction in TQ concentration in the oil samples at about 45%, 65%, and 75% of the initial value obtained for FTIR, UV, and HPLC dependence, respectively. In BCSO, during heating in open bottles, TQ, as the volatile compound, decreases under thermal storage, but, as reported in other research, simultaneously remained more stable compared to most of the other terpen [17]. The differences in the quantity amounts of TQ obtained by the spectrophotometric and HPLC methods could be due to the presence of other oil compounds, containing chromophores and functional groups that absorb at the same TQ wavelength range [4,35]. However, the results obtained from each of the methods used showed the same trend during the entire heating process. The calculated Pearson correlation coefficients between the HPLC and spectroscopic results (\(r\) = 0.7416 \(\div\) 0.9949) confirmed a statistically significant relationship between the used methods. This study demonstrated that spectroscopic methods are a good alternative to the quantitate determination of TQ by an HPLC assay during the accelerate storage test. Interestingly, in the first days of heating, the TQ content in \"open\" probes has clearly grown (at about 10%) compared to the no treatment oil, which is evidently seen for all three dependences presented at Figure 4a. In addition, for \"closed\" samples (Figure 4b), the TQ content after 3 days of heating increased at about 25%, keeping that higher value by almost all over days. A statistically significant decrease occurred only at the end of heating. Moreover, after 24 days of heating, the amount of TQ was still higher compared to BCSO before starting storage at 60 \({}^{\circ}\)C. In order to check in which period of time this rise in TQ  
Figure 4: Change in thymoquinone (TQ) content in BCSO: (**a**) “open” and (**b**) “closed” samples expressed as FTIR intensity band at 1659 cm\({}^{-1}\) (black line—right scale) and quantity amount of TQ calculated spectrophotometrically and from HPLC assay (bars—left scale). Error bars show the variations in three determinations in terms of standard deviation. content occurs, an additional experiment was performed, where, initially, the oil was heated in a closed vial (first part for 3 days, and second part for 6 days), and then the vial was opened. Also, in that test, an increase of about 25% in the TQ concentration was observed after 3 days, which was kept during the time when the dish was closed and a systematic decrease in its content was started after opening the vial (Figure S2). The obtained results indicate the growth in TQ content, presumably as a result of the conversion of some other compounds present in the oil to TQ which occurred at the initial period of heating. It is known that, in plants, the biosynthesis of TQ primarily occurs through the terpene biosynthetic pathway, where thymol or carvacrol serve as the intermediate products [8; 9; 11]. In our study, the increasing TQ content in BCSO probably ensued from the conversion of one or both of the above-mentioned or other monoterpenes, which may occur at a higher temperature. Such a hypothesized mechanism of the transition between quinones by the oxidation of thymol under controlled heat finally leading to the formation of TQ was indicated in [39]. However, it cannot be ruled out that the presence of other oil compounds is necessary for the process we observed to take place. As previous results indicate, the volatiles' accumulation, including TQ, in _N.