## 3 Results and Discussion
### Comparison of Binary and Ternary-System Interactions  
The binding constants were compared to each other for both the ternary and binary systems. A decrease in the binding constants was observed with a temperature rise in the binary system compared to the ternary system. It was observed that the addition of the flavonoids in the BSA-ETB system interfered in the binding and quenching constants of the BSA-ETB system (Table 2). The interaction with flavonoids (RUT, NAR, and SIN) and BSA was previously reported to decrease albumin fluorescence, and the binding constants for the three flavonoids RUT, NAR, and SIN were of the magnitude of >106, >104, and >105, respectively [34; 35; 36; 37]. The presence of RUT, NAR, and SIN increased the quenching constants in the ternary system. In the binary system, ETB quenched the fluorescence of the serum albumin by 27%, while as in the ternary system, RUT, NAR, and SIN in the ETB quenched the fluorescence of serum albumin by 29%, 33%, and 37%, respectively (Figure 4). Therefore, the influence of the studied flavonoids varied, with RUT affecting the least followed by NAR and the highest with SIN. The elevated quenching constants for the ternary system suggest that RUT, NAR, and SIN might have increased the accessibility of the ETB to the BSA, improving its quenching efficiency or the flavonoids had a synergistic effect. Hence, the quenching increased (Table 2) [38]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**T (K)** & \(\mathbf{K_{b}}\)\(\pm\)**SD** & \(n\) & \(\mathbf{\Delta G^{\circ}\pm SD}\) & \(\mathbf{\Delta H^{\circ}\pm SD}\) & \(\mathbf{\Delta S^{\circ}\pm SD}\) \\  & & **(kJ mol\({}^{-1}\))** & **(kJ mol\({}^{-1}\))** & **(J mol\({}^{-1}\).K\({}^{-1}\))** \\ \hline
298 & \(2.02\times 10^{4}\) & \(1.0073\) & \(-24.59\) & & \\
303 & \(7.91\times 10^{3}\) & \(0.9871\) & \(-22.57\) & \(-144.69\) & \(-403.02\) \\
307 & \(2.12\times 10^{3}\) & \(0.9833\) & \(-20.96\) & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Binding parameters and thermodynamic parameters for BSA-ETB system. The results from the present study corroborate earlier studies where a moderate binding between BSA and ETB was established [32], and the temperature rise led to a further decrease in the binding constants. In addition, interaction studies of BSA with the RUT, NAR, and SIN have established that RUT and SIN bind very firmly to BSA with binding constants greater than \(10^{5}\) M\({}^{-1}\), whereas NAR is moderately bound to BSA [34; 35; 36; 37]. Hence, the binding constants for the BSA-ETB system were calculated for the ternary system separately for RUT, NAR, and SIN since the studied flavonoids either strongly or moderately interact with BSA, which may, in turn, influence the ETB binding to BSA as it is moderately bound to BSA. As is apparent from (Table 2), the binding constant values of the ternary system were less than that of the binary system. Further, the binding cooperativity obtained from the Hill equation is less than one, which suggests negative cooperativity. Hence, the presence of RUT, NAR, and SIN in the BSA-ETB system lowers the affinity of the ETB towards BSA. In addition, it suggests that ternary-complex formation is less favorable than the ligands binding independently. Thus, the effect of RUT, NAR, and SIN in the systemic circulation might increase the free ETB fraction. The high plasma-ETB concentration has been associated with some severe adverse events in patients on ETB therapeutic regimens. Furthermore, adherence to the ETB treatment regimen has been affected by the severe adverse effects in NSCLC patients, which may be related to therapeutic plasma-ETB concentrations [39]. Some of the common adverse events associated with ETB use are skin-related, including acne, rash, pruritus, xeroderma, and paronychia. These adverse events sometimes become severe, which necessitate disruption or complete termination of the treatment with ETB [40].