## 3 Results and Discussion
### Total Flavonoids Content (TFC)  
The total flavonoids content in extracts from the bark of walnut (_Iuglans regia_ L.) ranged from 28.0 to 88.3 mg(QE)/100 g (DM), varying according to the experimental conditions. Flavonoids contents as a function of the extraction temperature, raw material fraction size and process time are presented in Figure 3.  
Multivariable analysis of variance (ANOVA) performed based on analytical data shows that the flavonoids content in subcritical water extracts strictly depends on the temperature, raw material fraction size and process time. The relationship between the temperature and flavonoids content was described by a second-order equation. The content of flavonoids increases with an increasing temperature value up to 131.6 \({}^{\circ}\)C and then decreases. The impact of raw material fraction size and process time changes is linear. Details of ANOVA analysis are presented in Table 6.  
Figure 3: Flavonoids content for different subcritical extraction parameters (**a**) Flavonoids content as a function of extraction temperature; (**b**) Flavonoids content as a function of raw material fraction size; (**c**) Flavonoids content as a function of process time.  
An F-value of 162.42 indicates that the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise. _p_-values less than 0.05 indicate the significance of the model terms. The significant model terms are A, B, C, AC, A\({}^{2}\). The lack of fit F-value of 0.2831 implies the lack of fit is not significant relative to the pure error. The predicted R\({}^{2}\) (0.9744) and adjusted R\({}^{2}\) (0.9829) are in reasonable agreement. The signal-to-noise ratio is referred as Adeq Precision. A ratio of 40.916 indicates an adequate signal. The obtained model is statistically significant and can be used to navigate the analyzed space.  
The total flavonoids content can be determined using Equation (6).  
\[TFC=-0.02067~{}T^{2}+5.4995~{}T-0.01016~{}T\cdot t-10.0747~{}Fs+1.0769~{}t-262.9 \tag{6}\]  
where: TFC--total flavonoids content [mg(QE)/100 g (dry mass)]; T--temperature [\({}^{\circ}\)C]; Fs--fraction size of raw material [mm]; t--process time [min].  
There are no studies available on the flavonoids content in the subcritical water extracts of _Iuglans regia_ L. bark. Taking into account the main goal of this study, that is to investigate the effect of process parameters on the chemical composition and antioxidant activity of obtained extracts, changes in flavonoids and polyphenols contents in relation to process parameters were compared. Flavonoids content varies with a temperature change in a similar manner to polyphenols. In the temperature range from 110 to 131.6 \({}^{\circ}\)C, both flavonoids and polyphenols contents increase; with a further temperature increase, both flavonoids and polyphenols contents decrease. Similar trends in both flavonoids and polyphenols contents were observed in relation to the raw material fraction size and process duration. An increase in the raw material size and process duration negatively affects both the polyphenols and flavonoid contents. This phenomenon can be caused by the worst contact of the solvent with desired compounds in the case of a larger raw material size, whereas a longer process duration at elevated temperatures may be the reason for the occurrence of thermal degradation of the extract.