## 3 Results and Discussion  
### Fluorescence Quenching and Enhancement  
The binary- and the ternary-system spectra were obtained at an excitation wavelength of 280 nm, while the emission wavelength ranged between 300-500 nm (Figure 2A-D). The binary-system spectra showed a decline in the fluorescence intensity with the addition of ETB to BSA. The fluorescence intensity declined further with the increase in the ETB concentration (Figure 2A). The decline in fluorescence intensity in the ternary system, including the flavonoids RUT, NAR, and SIN, decreased further than the binary system (Figure 2B-D). The spectra for the BSA-ETB system showed a redshift of 7 nm in the emission, suggesting increased polarity and reduced hydrophobicity of the fluorophore amino acids [29; 30]. The mechanism of quenching behavior of the BSA-ETB system was obtained from the Stern Volmer plot using the Stern Volmer equation [31]:  
\[\frac{F_{0}}{F}=1+K_{sv}[Q]=1+k_{q}\tau_{0}[Q]\]  
\[k_{q}=K_{sv}/\tau_{0}\]  
The fluorescence intensity of BSA is \(F_{0}\), and the fluorescence intensity of the protein-ligand (BSA-ETB) is \(F\). \(K_{sv}\) is the Stern Volmer constant, and \([Q]\) is the quencher concentration. The biomolecular quenching constant is \(kq\), and the fluorophore lifetime \(\tau_{0}\) for BSA in quencher absence is 6 ns.  
The Stern Volmer plot of the BSA-ETB system showed declined quenching constant \(K_{sv}\) values with the elevated temperature (Table 1). The Stern Volmer plots at the three studied temperatures were linear (Figure 3A). In static or dynamic quenching, \(K_{sv}\) decreases or increases, respectively, with a temperature rise. Since the BSA-ETB system showed reduced \(K_{sv}\) values with increased temperature, the BSA-ETB system followed a static-quenching mechanism [31; 32]. Further, the static quenching also suggests the formation of a stable complex between the two of them. The biomolecular quenching constant values provide a tool to establish the quenching mechanism. The static-quenching behavior of the BSA-ETB complex for the binary system is predicted based on the biomolecular quenching constant values that were higher than 2 \(\times\) 10\({}^{10}\) L mol\({}^{-1}\) s\({}^{-1}\) (Table 1). It is already well-known that \(k_{q}\) values more than the 2 \(\times\) 10\({}^{10}\) L mol\({}^{-1}\) s\({}^{-1}\) suggest a static quenching, whereas the values below indicate a dynamic quenching between binary systems [31; 32].  
Figure 3: _Cont._  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \(T\) (K) & **R** & \(K_{\alpha\
u}\times 10^{4}\pm\) SD (M\({}^{-1}\)) & \(k_{q}\times 10^{12}\) (M\({}^{-1}\) S\({}^{-1}\)) \\ \hline
298 & 0.9890 & 1.88 & 3.14 \\
303 & 0.9888 & 1.78 & 2.97 \\
307 & 0.9966 & 1.74 & 2.90 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Stern Volmer \(K_{\alpha\
u}\) and bimolecular quenching constant \(k_{q}\).  
Figure 2: The fluorescence spectra of the (**A**): BSA-ETB binary system and BSA-ETB ternary system in presence of flavonoids, (**B**): RUT; (**C**): NAR; and (**D**): SIN). BSA (1.5 uM) with a: ETB (0.00–27.5 uM); and (RUT, NAR, and SIN) = 5.5 uM at (\(\lambda_{\rm ex}\) = 280 nm and \(\lambda_{\rm em}\) = 300–500 nm).  
The \(K_{\mathit{SP}}\) and \(k_{q}\) were also evaluated for the ternary system for the BSA-ETB in the presence of RUT, NAR, and SIN (Table 2). The effect of the ternary system was determined by the Stern Volmer constant and biomolecular quenching constant values. These values were later compared to the binary BSA-ETB system.