*2.2. Steady-State Fluorescence*

The inhibitory effect of caffeic and coumaric acid on α-amylase was studied by exploiting the fluorescence ability of the protein. Quenching studies were carried out against the protein in the presence of both the natural compounds, using a spectrofluorometer (Jasco FP-750, JASCO Corporation, Tokyo, Japan). The protein (5 µM α-amylase) was excited at 295 nm, and the emission spectra were recorded at higher wavelengths between 300–400nm [32–35]. Caffeic acid (0–40 µM) and coumaric acid (0–30 µM) were titrated with the α-amylase at three different temperatures (25, 30 and 35 ◦C). All these binding experiments were conducted at physiological pH of 7.4. The obtained quenching data were put into Stern–Volmer (Equation (1)) and modified Stern–Volmer equation (Equation (2)) as per earlier published literature [20,21,36] to find various binding parameters. The binding experiment was performed at physiological pH of 7.4.

*F*<sup>0</sup> corresponds to the maximum fluorescence intensity of free protein, \**F* shows the fluorescence intensity of complex, \**K* corresponds to the binding site, *n* depicts the binding sites and \**C* refers to the concentration of ligand.

$$F\_0/F = 1 + \text{Ksv} \text{ [C]} \tag{1}$$

$$\log\left[ (F\_0 - F)/F \right] = \log K + n \log \left[ C \right] \tag{2}$$

Using the van't Hoff equation, (Equation (3)) [37,38], thermodynamic parameters for ligand–protein interaction such as Gibbs free energy change (∆*G* **0** ), enthalpy change (∆*H***<sup>0</sup>** ) and entropy change (∆*S* **0** ) can be calculated.

$$
\Delta G^{\mathbf{0}} = -\mathbf{R} \mathbf{T} \mathbf{L} \mathbf{n} \mathbf{K} = \Delta H^{\mathbf{0}} - \mathbf{T} \Delta S^{\mathbf{0}} \tag{3}
$$

The mode of quenching was further confirmed from the value of the bimolecular quenching rate constant, *K*q, which was calculated as per Equation (4)

$$\mathbf{Kq} = \mathbf{Ksv} / \pi\_0 \tag{4}$$

τ<sup>o</sup> refers to the average integral fluorescence lifetime of tryptophan and is reported to be 10−<sup>8</sup> .
