Multispectroscopic and molecular docking studies on the interaction of diltiazem hydrochloride with bovine serum albumin and its application to the quantitative determination of diltiazem hydrochloride

2.1 Reagents and chemicals

Procurements of BSA (fraction V, about 96%) and DTZ (>99%) were done from HiMedia Laboratories (India) and Sigma Aldrich (U.S.A.), respectively. Warfarin (>98%; TCI, Japan), ibuprofen (>98%; Sigma Aldrich) and indomethacin (98.5–100.5%; Sigma Aldrich) were used for site marker studies. NaH₂PO₄·H₂O (99%) and Na₂HPO₄·2H₂O (99%) were obtained from Central Drug House (P) Ltd, India to prepare a buffer solution (pH 7.4). The stock solutions of BSA (10 μΜ) and DTZ (1 mg/mL) were made in phosphate buffer solution (0.1 M; pH 7.4) and distilled water, respectively.

2.2 Fluorescence measurement

All fluorescence analyses were performed using a Fluorescence Spectrophotometer (Model: 2700, Hitachi High-Tech Corporation, Tokyo, Japan) fitted with quartz cells of 1 cm and a xenon lamp. Fluorescence spectra of solutions containing 5 µM BSA and varied concentration of DTZ (5–35 µg/mL) were scanned in the emission wavelength range from 300 to 540 nm using 280 nm as excitation wavelength at 283, 298 and 308 K. The measured fluorescence intensities were corrected using Eq. (1):

2.3 UV-absorption spectra

The absorption spectra of BSA (5 µM), DTZ (5–20 µg/mL) and DTZ-BSA were scanned from 200 to 350 nm using Perkin-Elmer Lamda-45 spectrometer. In DTZ-BSA system, varying concentration of DTZ was mixed with BSA (5 µM) and measured at 298 K.

2.4 Fluorescence resonance energy transfer (FRET)

The absorption spectrum of DTZ (5 μM) was measured in the wavelength range 300–550 nm whereas the fluorescence spectrum of BSA (5 μM; λ_ex = 280 nm) was also obtained in the same wavelength region. The overlapping of absorption spectrum of DTZ with fluorescence spectrum of BSA was utilized to compute the energy transfer.

2.5 Circular dichroism (CD)

CD experiments were conducted using Jasco J-815 circular dichroism spectrometer fitted with temperature-controlled Peltier device, 150 W Xenon arc lamp and 2.0 mm quartz cuvette. Instrument specifications were response time 1 s, scanning rate 100 nm min⁻¹ and wavelength range 190–300 nm. The instrument was continuously purged with N₂ gas to remove the moisture content before and during the experiment.

2.6 Fourier transform infrared (FTIR) measurement

FTIR spectra of BSA and DTZ-BSA were scanned from 4000 to 400 cm⁻¹ using FTIR spectrometer (PerkinElmer).

2.7 Molecular docking

BSA crystal structure was obtained from the protein data bank (PDB ID: 3 V03). Diltiazem structure was drawn in chemdraw 12.0 with energy minimization of MM-2 method implemented in Chem.3D Pro 12.0 and structure was saved in pdb format. Auto Dock Vina program was used for docking. All water molecules were pulled out and all polar hydrogens were added for smooth docking without hindrance. Grid parameters were set as 76 × 76 × 76 Å with grid center x = 38.078, y = 22.708, z = 40.771 to envelope all active site residues. Kollman and Gasteiger charges were added to drug and protein with Auto Dock Tools (ADT), respectively and the file was saved in pdbqt format. BSA was held as rigid whereas DTZ was flexible. Accelrys Discovery Studio 4.5 software was used to examine the energy minimized output files of the auto dock vina.

2.8 Analytical application

3.1 Fluorescence quenching mechanism

BSA exhibits a fluorescence emission peak at 340 nm when excited at 280 nm while DTZ showed no intrinsic fluorescence. The fluorescence intensity of BSA decreases successively and the maximum emission wavelength moved from 340 nm to 351 nm with increasing concentration of DTZ at 298 K (Fig. 1(a)). The decrease in BSA fluorescence indicated the complex formation between DTZ and BSA. Additionally, the blue shift in maximum wavelength emission described the alteration in conformation of BSA. The blue shift also pointed towards decrease in polarity around the tryptophan residues and increase in hydrophobicity. The experiments were also conducted under the same conditions at 283 K and 308 K and it was found that the quenching decreases with increasing temperature. The fluorescence quenching caused by DTZ was analyzed by Stern-Volmer equation which is expressed as:

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Fig. 1

(a) Fluorescence quenching spectra of BSA (b) Stern-Volmer plots for BSA-DTZ system (c) Lineweaver-Burk plots for BSA-DTZ system and (d) binding plots for BSA-DTZ system; with varying concentration of DTZ [(1) without DTZ (2) 5 µM (3) 7 µM (4) 10 µM (5) 15 µM (6) 20 µM (7) 25 µM (8) 30 µM and (9) 35 µM at pH = 7.4.

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The Lineweaver equation (Abdelhameed et al., 2015) was also applied to analyze the type of quenching which is expressed as:

The plot of ${(F_0-F)}^{-1}$ vs $C_Q^{-1}$ yields a straight line (Fig. 1(c)) which indicates static quenching. Moreover, the K_LB values (Table 1) decreased as the temperature rises, pointing towards static quenching.

3.2 Binding constant and binding site

The binding constant and number of binding sites for a system obeying static quenching can be obtained from Eq.4:

Here, $k_a$ and n describe the binding constant and the number of binding sites, respectively. The $k_a$ and n values were computed from log $\left(F_0-F\right)$ /F vs $log\left[Q\right]$ plots at 283, 298 and 308 K (Fig. 1d). The results are listed in Table 1. The $k_a$ value increased as a function of temperature which suggested more stability of DTZ-BSA system at higher temperature. The $k_a$ values were higher at all the temperatures studied which illustrated a strong interaction between BSA and DTZ. This demonstrated that DTZ could be transported by BSA via blood circulation to other organs in vivo. Also, the n value is almost 1 which suggested the presence of one binding site for DTZ in BSA molecule.

3.3 Thermodynamic study

The thermodynamic parameters such as ΔG, ΔH and ΔS were assessed at 283, 298 and 308 K using Eqs. (5) and (6):

The values of binding constants at 283, 298 and 308 K were obtained from Eq. (4). R is a gas constant (8.314 J mol⁻¹ K⁻¹). The ΔH and ΔS values were computed from Van’t Hoff plot (Fig. S2). The values of ΔG were −31.57, −40.91 and −47.14 kJ mol⁻¹ at 283, 298 and 308 K, respectively which demonstrated the spontaneous nature of interaction between DTZ and BSA. The ΔH and ΔS values were computed as 144.74 kJ mol⁻¹ and 0.62 kJ mol⁻¹ K⁻¹, respectively. The value of ΔS indicated that the water molecules which are present in an ordered manner around the drug and protein molecules get more randomness due to hydrophobic interactions. Therefore, positive values of ΔH and ΔS illustrated the role of hydrophobic interactions in the binding of DTZ to BSA (Ross and Subramanian, 1981).

3.4 UV–Visible spectroscopy

UV–visible absorption spectra of BSA, DTZ and BSA-DTZ complex are depicted in Fig. S3. The absorption spectrum of BSA showed two bands centered at 210 nm and 280 nm. The peak at 280 nm represents the π-π* transitions arising from phenyl rings of aromatic amino acids. The peak at 210 nm characterizes the α-helical structure of BSA (Rudra et al., 2018). The addition of DTZ to BSA caused a gradual increase in absorption intensity of peak at around 210 nm with a red shift in maximum wavelength of absorption. The results confirmed the formation of complex between DTZ and BSA which also illustrated the alteration in the conformation of BSA.

3.5 Conformational studies.

3.5.1

3.5.1 FTIR spectroscopy

The FTIR spectrum of diltiazem hydrochloride (Fig. 2) shows a band at 1637 cm⁻¹ which refers to C⚌O stretching. FTIR spectrum of BSA indicates the amide I band peaking at 1667 cm⁻¹ which is mainly arising from C⚌O stretching of amide group. The band centered at 1528 cm⁻¹ characterizes the amide II band which presents C—N stretching together with N—H bending. The amide I and amide II bands are considered as most valuable vibrational modes of protein backbone and are related to secondary structure of protein. The FTIR spectrum of DTZ-BSA complex revealed that the amide I band moved from 1667 cm⁻¹ to 1643 cm⁻¹ and amide II band disappeared which indicated that DTZ has reacted with BSA and also pointed towards the change in conformation of BSA structure.

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Fig. 2

FTIR Spectra of (a) BSA (b) BSA-DTZ (c) DTZ.

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3.5.2

3.5.2 Circular dichroism spectra

CD spectra of BSA with and without DTZ at 298 K were recorded (Fig. 3). The CD spectrum of BSA exhibited two negative bands peaking at 208 nm and 222 nm which characterize the π-π* and n-π* transitions of α-helix peptide bonds, respectively. The addition of DTZ decreased the CD signal of BSA and no significant shift in band position was observed which illustrated the loss of α-helix content of BSA and alteration in protein secondary structure. These observations suggested the formation of DTZ-BSA complex. Further, α-helix content of BSA can be assessed after addition of DTZ. CD results are usually represented as mean residual ellipticity (MRE). The MRE (deg cm² dmol⁻¹) and α-helix content were quantified by Eqs. (7) and (8), respectively.

(7)

$$\mathit{MRE}=\frac{\mathit{observedCD}(mdeg)}{C_p\times n\times l\times 10}$$ where C_p is BSA concentration (mol/L). n and l are number of amino acid residues (583), and path length (1 mm), respectively. The α-helix content of BSA and combined BSA can be computed using Eq. (8):

(8)

$$\alpha -helix=\frac{{\mathit{MRE}}_{208}-4000}{33000-4000}$$

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Fig. 3

CD spectra of BSA-DTZ system.

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Herein, ${\mathit{MRE}}_{208}$ is the experimental MRE obtained at 208 nm. The α-helix content of BSA (free) at 208 nm was 65.64%. The addition of 4.434 × 10^-5 M DTZ reduces the α-helix content of BSA from 65.64% to 51.99%. Further addition of DTZ (8.87 × 10^-5 M) reduced the α-helix content to 37.24%. These results suggested that the change has occurred in BSA secondary structure on interaction with DTZ.

3.5.3

3.5.3 Synchronous fluorescence spectra

When the Δλ value ( ${\lambda }_{\mathit{em}}-{\lambda }_{\mathit{ex}}$ ) is held at 15 and 60 nm, the synchronous fluorescence imparts characteristic properties of tyrosine and tryptophan residues of BSA, respectively. The effect of varying concentrations of DTZ on the synchronous fluorescence of BSA is depicted in Fig. 4. It is evident that the maximum emission wavelength ( ${\mathrm{\lambda }}_{\mathit{em}})$ shifted towards longer wavelength (6.5 nm) when Δλ was set at 15 nm while a red shift of 9.5 nm in ${\mathrm{\lambda }}_{\mathit{em}}$ was noticed on setting Δλ at 60 nm upon addition of DTZ to BSA (Fig. 4b). Additionally, the synchronous fluorescence intensity of tyrosine and tryptophan residues decreased upon addition of DTZ. The results indicated the increase in polarity around tyrosine and tryptophan residues and decrease in hydrophobicity which suggested the involvement of tyrosine and tryptophan residues in binding with DTZ.

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Fig. 4

Synchronous fluorescence spectra (A) at Δλ = 15 nm (B) Δλ = 60 nm for BSA-DTZ system with varying concentration of DTZ [(1) without DTZ (2) 5 µM (3) 7 µM (4) 10 µM (5) 15 µM (6) 20 µM (7) 25 µM (8) 30 µM and (9) 35 µM] at 298 K and pH = 7.4.

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3.5.4

3.5.4 Three-dimensional (3D) fluorescence spectroscopy

3D fluorescence spectra and the corresponding contour plots of BSA and DTZ-BSA are displayed in Figs. 5(a) and (b) respectively, and the characteristic parameters are presented in Table 2. Peak a represents the second order scattering peak (λ_em = 2 λ_ex) while peak b is related to Raleigh scattering peak (λ_em = λ_ex). The 3-D fluorescence spectrum also exhibited peak 1 and peak 2. Peak 1 demonstrated the fluorescence spectral nature of tryptophan and tyrosine residues. Peak 2 illustrated the fluorescence characteristics of polypeptide backbone of BSA. The fluorescence intensity of peaks 1 and 2 decreases markedly with the addition of DTZ, indicating that the protein molecule's microenvironment is changed because of complex formation.

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Fig. 5a

3D fluorescence spectra of (a) BSA (5 μM), (b) BSA + 5 µg/ml DTZ, (c) BSA + 10 µg/ml DTZ.

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Fig. 5b

Contour plot of (a) BSA (5 μM), (b) BSA + 5 µg/ml DTZ, (c) BSA + 10 µg/ml DTZ.

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Table 2 Three-dimensional fluorescence parameter.

Peaks	BSA		BSA: DTZ (1:20)			BSA: DTZ (1:40)
	Peak position (λex/λem) nm/nm	Stokes shift	Peak position (λex/λem) nm/nm	Stokes shift	Relative intensity	Peak position (λex/λem) nm/nm	Stokes shift	Relative intensity
Peak1	275/340	65	285/340	55	0.78	285/340	55	0.66
Peak2	225/340	115	225/340	115	0.08	225/345	120	0.04

3.6 Forster resonance energy transfer (FRET)

The overlapping of absorption spectrum of DTZ with the emission spectrum of BSA is displayed (Fig. S4). The E (Efficiency of energy transfer) value was determined using Eq. (9) (Forster, 1996).

$\varphi$ defines quantum yield of donor molecule, N presents medium’s refractive index, $k^2$ is dipole’s spatial orientation factor, and $J$ is overlap integral. $J$ can be estimated as:

In the wavelength range λ to λ + Δλ, normalized fluorescence intensity is given by $F(\lambda )$ and the molar absorptivity of the acceptor is provided by ε(λ).

The established values of $k^2$ , N, $\varphi$ are 2/3, 1.36, 0.15, respectively (Senthilkumar et al., 2016). The computed values of R₀, r and $J$ are 0.54 nm, 1.40 nm and 2.37 × 10⁻¹⁸ cm³ M⁻¹, respectively. The average distance of <8 nm was obtained between the donor and acceptor, which is evidence for the transfer of energy from BSA to DTZ with high probability. The obtained r value illustrated that the reduction in the fluorescence intensity is due to static quenching mechanism (Suryawanshi et al., 2016).

3.7 Molecular docking

The 3D structure of BSA is heart-shaped with three analogous domains, which are further divided into two subdomains. Auto dock vina gives 9 different conformers of DTZ of varying Gibbs free energy that can bind with the BSA. The best fit model with minimum energy was chosen and the result shows that DTZ was mainly located at subdomain I-B (Yang et al., 2019). Fig. 6 gives 2D View of BSA-DTZ docking whereas Fig. S5 gives the detailed overview. The Gibbs free energy calculated from the docking result is slightly different from the experimental results because the X-ray crystal structure of BSA is different from that which is in the aqueous solution (Neelam et al., 2010). The results of molecular docking are reported in Table S1.

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Fig. 6

2D View of Docked structure of DTZ-BSA complex.

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3.8 Displacement studies

Competitive binding experiments were performed using indomethacin (site IB), warfarin (site IIA) and ibuprofen (site IIIA) as specific binding site markers to locate the DTZ binding site on BSA (Zsila, 2013). The protein and site probes were kept at a constant molar ratio (1:1) and DTZ was varied up to 35 μg/mL. The fluorescence spectra were obtained for BSA-DTZ system with and without site markers at 298 K.

The values of K_sv for BSA-DTZ in the presence of indomethacin, warfarin and ibuprofen were calculated from Stern-Volmer plots (Fig. S6) and found to be 7.30 × 10³, 1.24 × 10⁴ and 1.00 × 10⁴ L mol⁻¹, respectively. The K_sv value for BSA-DTZ system without any site marker was 1.03 × 10⁵ L mol⁻¹. On comparing the K_sv values, it was noticed that in presence of indomethacine K_sv value decreased more dramatically. These results suggested that DTZ competes with indomethacin for I-B site in BSA.

3.9 Method validation

The calibration curve (F₀/F vs concentration of DTZ) exhibited linearity in the range of 3–50 µg/mL DTZ with R² > 0.99. The detection (LOD) and quantification limits (LOQ) were evaluated using Eqs. (12) and (13) (Rahman and Khan, 2016; Lutfullah et al., 2008).

S.D and b are standard deviation of intercept and slope of regression line, respectively. $\mathit{LOD}$ and $\mathit{LOQ}$ values were 0.51 µg/mL and 1.70 µg/mL, respectively.

Within-day and between-day precision were assessed with three concentrations (10,20,30 µg/ml) by performing analysis 5 times per day for within-day whereas 5 times once a day for between-day precision (Table S2). The values of RSD (%) were found in the range 0.60–0.87% and 0.71–0.89% for with-in-day and between-day precision, respectively which revealed that the proposed method has acceptable precision.

3.10 Analytical application

The proposed and reference methods were utilized to determine the quantity of DTZ in tablets. The point and interval hypothesis tests were conducted to compare the results of proposed method with the results of reference method (Table 3). At 95% confidence level, the calculated F and t (paired) values were well below the indexed values which illustrated that no significant difference occurred between the methods compared. The interval hypothesis test (Rahman and Khan, 2019) was also conducted to compare the performance of proposed and reference methods based on the true bias which was evaluated using Eq.(14):