1. INTRODUCTION:

Nile blue (NB) is an attractive molecule (Structure 1) in the class of cationic oxazine dye.Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumours due to its absorption of light in the red region of spectrum. NB efficiently react with tumour cells than normal tissues and retard the tumour growth^1,2. NB and its derivatives are favorable materials for optical and photonic devices^{3 ̵ 5}. NB employed as a reagent to assess the antioxidant activity of commercial wine and fruit juice samples⁶. NB have strong binding affinity with DNA and show extensive biological applications^7,8.

Structure 1: Structure of Nile blue

Catechols and catecholamines are potentive antioxidants and scavenge reactive oxygen species⁹. The antioxidant activity found to have direct relationship with their biological actions^10-16. The antioxidant effect depends on the chemical composition, solubility in lipids, ability to scavenge free radicals, donation of hydrogen atoms and free radicals related with chain reactions. It has been observed that presence of two hydroxyl groups in catechol molecule is responsible for the antioxidant activity. The protective effects of antioxidants vary depending on the structure of molecules¹⁷. The presence of catechol compounds in olive oil is liable for higher antioxidant activity in biological environment¹⁸. The neurological disorder, Parkinson disease arises due to the non-production of dopamine treated by administering Levodopa¹⁹.

Our prime intention is to investigate the interaction of antioxidant molecules with appropriate biological targets and in this perspective study on the fluorescence quenching of NB with catechol and phenol molecules is of utmost important in pharmaceutical point of view. We are interested in understanding the release of H^· from antioxidant molecules as it may possibly solve biological related problems^20-22. Besides, presence of substituent in catechol molecule influences the release of H^·from aromatic hydroxyl groups (-OH). Fluorescence quenching of 9-aminoacridine andacriflavine by various antioxidants such as estrogens, flavonoids, phenol and its derivatives, catechols and uracils were thoroughly investigated and found to undergo pronounced charge transfer process^23-28. Recently, NB employed as a fluorescent probe to evaluate the antioxidants of uracil molecules by abstracting the release of H^· from antioxidants via ground state complex formation²⁹. Although reports on the antioxidant activity of catechol was available, but comparison on the antioxidant activity of quencher molecules with different substitution based on steady state measurements and density functional studies are not reported so far^30-32. Thus, the present work focus on the photo induced interaction of NB with various quencher molecules using steady state and lifetime measurements. Bond dissociation enthalpy calculations reveal the efficiency of antioxidants with respect to position of the substituent.

2. EXPERIMENTAL:

2.1. Materials:

Nile blue, catechol, pyrogallol, 4-t-butyl catechol, dopamine, levodopa and 4-aminophenol were purchased from Sigma-Aldrich and used without further purification. All the stock and aliquot solutions were prepared using double distilled water.

2.2. Methods:

UV-Visible absorption and fluorescence spectra were recorded by UV-Visible absorption spectrophotometer (JASCO V-630) and Fluorescence spectrophotometer (JASCO FP-6500) respectively. The emission spectra were measured by exciting the NB at 636 nm and emission maximum of NB is observed at 669 nm in phosphate buffered at pH 7.4. In order to avoid the quenching by singlet oxygen, samples were degassed with pure nitrogen gas for 15min. Fluorescence lifetime measurements were carried out in a time correlated single photon counting (TCSPC) spectrometer. The data were analysed through software provided by IBH (DAS – 6). The kinetic trace examined by non-linear square fitting of mono exponential method.

2.3. Cyclic voltammetry measurements:

The reduction potential and oxidation potential of NB and quencher moleculeswere measured with potassium chloride (0.1 M) as the supporting electrolyte. The reduction potential for NB observed at -3.88 V versus SCE²⁹. The experimental setup consist a platinum working electrode, a glassy carbon-counter electrode and a silver reference electrode. Irreversible peak potential of quencher molecules measured at different scan rates (0.05 V/s). All samples bubbled in presence of nitrogen gas for 5 min at room temperature for deaeration.

2.4. BDE calculation:

All the organic quencher molecules were optimized by B3LYP^33-36 method with 6-31G**^37-39 basis set. Harmonic vibrational frequency calculations were carried out on these optimized geometries to confirm that they are saddle point with all positive frequencies. RB3LYP method applied for quencher molecules and UB3LYP method applied for radicals. All these computational methods have been used as employed in the Gaussian 16 software package⁴⁰. The bond dissociation energy (BDE) of O–H bond was calculated by

E_BDE= E_RO• + E_H• - E_RO–H

where, E_RO•, E_H• and E_RO–H represent the energies of RO^•, H^• and RO–H, respectively.

3. RESULT AND DISCUSSION:

3.1 UV-Visible absorption spectra:

The absorption of NB is characterized by a strong band at 636 nm. UV Visible spectral studies have been performed to reveal the presence of ground state interaction between NB and quencher molecules. All the quencher molecules show no absorption bands in the range of 600 -700 nm. Interestingly, addition of quencher molecules decreases the absorbance of NB followed with an observable red shift (longer wavelength). This shows the existence of ground state complex formation between NB and quencher molecules⁴¹. Figure 1 indicates UV-Visible absorption study of NB with increasing concentration of dopamine in phosphate buffered media at pH 7.4. It is worthy to note that similar behavior noticed for other quencher molecules.

Figure 1: Absorption spectra of NB (5X10^-6M) in the presence of various concentrations of dopamine (2,4,6,10 x 10^-4M) in phosphate buffered media at pH 7.4. (The arrows indicate decrease in absorbance followed with red shift)

Figure 2: Emission spectra ofNB (5x10^-6M)in the presence of Catechol (0 - 8 X 10^-5M)in phosphate buffered media at pH 7.4. (The down arrow indicates decrease in emission intensity of NB).

3.2 Effect of quenchers in emission spectra of NB:

The emission spectra of NB were measured in absence and presence of quencher molecules by exciting at 636 nm. It has been observed that on increasing the concentration of quencher molecules, the emission intensity of NB decreases. Figure 2 depicts the fluorescence quenching of NB in absence and presence of catechol. The Stern –Volmer rate constant (K_sv) calculated from the following Stern – Volmer equation as follows, I₀/I = 1 + K_sv[Q] = 1 + k_q.t₀

The Stern - Volmer (S-V) plot has been obtained from the plot of I₀/I versus quencher concentration. It yields a straight line as shown in figure 3. The bimolecular quenching rate constant (k_q) was calculated and compiled in table 1. The extent of quenching efficiency are found in the order as follows.

Dopamine > L-DOPA > pyrogallol > 4-aminophenol > 4-t-butyl Catechol > Catechol

Table 1: Fluorescence quenching rate constant and electrochemical data of NB with quencher molecules

S. No	Quencher	k_q ( x 10¹¹M^-1s^-1)^a			E_oxvs. SCE (V)^b	∆G_et (eV)^c
S. No	Quencher	15 °C	25 °C	35 °C	E_oxvs. SCE (V)^b	∆G_et (eV)^c
1	Dopamine	11.32	9.83	8.05	0.67	4.55
2	levodopa	9.65	7.73	5.41	0.58	4.46
3	Pyrogallol	8.72	6.45	4.54	0.78	4.66
4	4-aminophenol	7.72	5.72	4.11	0.39	4.27
5	4-t-butyl catechol	6.68	5.35	3.85	0.6	4.48
6	Catechol	5.34	4.16	2.89	0.8	4.68

^adetermined by steady state fluorescence quenching in phosphate buffer media (τo=1.74ns)

^bOxidation potential of quencher molecules inV vs SCE

^cCalculated byRehm-Weller equation DG_et= E_ox (D) – E_red (A) – E* + C, the reduction potential of NB is−3.88

V vs. SCE, E*=1.93eV. Error ± 3%

Figure 3: Comparison of Stern - Volmer Plot of NB (5x10^-6M) a) in the presence of quencher molecules at various concentrations (0 - 8 X 10^-5M).

Dopamine show higher k_q value than other quencher molecules. Dopamine consists of two hydroxyl groups at adjacent position with ethylamine as a side chain. The presence of electron releasing group proliferate the electron density inside the ring and improves the antioxidant activity⁴². Thus, it leads to superior release of H^·. L-DOPA shows lesser k_qthan dopamine. The observed behaviour attributed to the effect of electron withdrawing nature of COOH group. The presence of electron withdrawing group diminishes the effect of electron releasing group (-NH₂) in the quencher molecule. Pyrogallol shows less k_qvalue than dopamine and L-DOPA. Pyrogallol consists of three hydroxyl groups at adjacent positions but the absence of electron releasing species in the molecule might be plausible reason for lower k_qvalue. 4-Aminophenol shows higher k_qvalue than 4-t-butylcatechol. The presence of electron releasing amine group enhances the electron density and favour the possibility of releasing the H^·from the molecule. Unsubstituted catechol shows very less k_qvalue among the quencher molecules due to the absence of electron releasing substituent in the molecule. The presence of electron releasing species in the antioxidants greatly influences the fluorescence quenching of NB. Similar type of observations documented in the literature^27-29.

The fluorescence quenching experimentswas executed at different temperatures and observed the presence of significant change in the excited state of NB in existence and non-existence of quencher molecules. The quenching titrations were conceded at various temperatures ranging from 15 to 35^oC. The bimolecular quenching rate constant (k_q) falls with increasing the range of temperatures (shown in Table 1). The observed resultsspecify the existence of static quenching between NB and quencher molecules.

3.3 Lifetime measurements:

The fluorescence quenching of NB with quencher molecules were carried for understanding the decay mechanism through excited state lifetime measurements. The fluorescence quenching shall be either dynamic or static⁴³. The lifetime of NB was recorded with different concentrations of quencher molecules. The decay curve properly fit well with single exponential decay. The excited state lifetime of NB was observed and foundto be 1.74 ns⁴⁴. The lifetime of NB in presence and absence of quencher molecules was noted. Interestingly, the lifetime of the NB molecule remains unaffected. The decay process was plotted and looks like single decay curve. The excited state lifetime measurement of NB in absence and presence of catechol was shown in figure 4 and indicate the existence of static quenching between NB and quencher molecules. The presence of static quenching embraces the possibility of ground state complex formation. Similar behaviour observed for NB in presence of other quencher molecules. Hence, the quenching pursues static mechanism.

Figure 4: Fluorescence lifetime decay curve of NB (5x10^-6M) in absenceand presence ofpyrogallol (8 X 10^-5 M) in phosphate buffered media at pH 7.4.

3.4 Mechanism of fluorescence quenching:

The fluorescence quenching of NB with quencher molecules can be rationalised by various mechanisms. The possibility of energy transfer mechanism can be eliminated as the absorption spectrum of catechol and phenol molecules unsuccessfully overlay with the fluorescence spectrum of NB. The prospect of either electron transfer or proton transfer mechanism, were evaluated by employing Rehm – Weller expression, shown as follows,

DG_et= E_ox (D) – E_red (A) – E* + C

The DG_etvalues were positive and imply the probability of proton transfer mechanism⁴⁵. The obtained values are displayed in table 1.

The forces acting between NB and quencher molecules are favored with weak interactions forces such as hydrogen bond formation, electrostatic interaction, hydrophobic interaction and Vander Waals forces⁴⁶. The binding mode is authenticatedby using thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) of binding reaction. As based on thermodynamic point of view, ΔH > 0and ΔS > 0 indicate a hydrophobic interaction; ΔH<0 and ΔS<0 implies the Vander Waals forces or hydrogen bond formation and ΔH~ 0 and ΔS>0 suggest an electrostatic force exist between fluorophore and quencher molecules⁴⁷.

The thermodynamic parameters were calculated using the following equation and the values are displayed in Table 2.

ΔG = ̶ RT ln K

ln K = ̶ ΔH/RT + ΔS/R

The ΔG value is negative and signifies the interaction process is spontaneous. The ΔH and ΔS value point out the non-bonded (Vander Waals) interactions and hydrogen bond formation⁴⁷. Thus, quencher molecules are destined to NB due to Vander Waals interaction and hydrogen bond formation. The ∆H and ∆S values predict the possibility of charge transfer and hydrogen bonding interaction. NB possesses high reduction potential and quencher molecules own oxidation potential.The charge transfer occurs between NB and quencher molecules. The charge transfer process might be one of the promising evidence for the quenching mechanism of the non-radiative processes.

Table 2: Thermodynamic parameters of NB with quencher molecules

S. No	Quencher	∆G (kcal mol^-1)	∆H (kcal mol^-1)	∆S (cal K^-1mol^-1)
S. No	Quencher	∆G (kcal mol^-1)	∆H (kcal mol^-1)	∆S (cal K^-1mol^-1)
1	Dopamine	-16.31	-13.17	-42.11
2	Levodopa	-16.93	-21.13	-52.43
3	Pyrogallol	-17.4	-22.14	-59.86
4	4-aminophenol	-17.7	-22.89	-60.57
5	4-t-butyl catechol	-18.25	-24.52	-65.99
6	Catechol	-19.35	-25.67	-68.42

Figure 5 Comparison of bimolecular quenching rate constant with BDE value

3.5 Bond Dissociation Enthalpy calculation:

Density functional theory (DFT) calculations were carried out to understand the radical scavenging performance of chosen phenolic quencher molecules, Bond dissociation enthalpy (BDE) found to be as a common descriptor for radical scavenging activity of quencher molecules. The radical scavenging of quencher molecules was defined as

Q–H + ROO^• Q^•+ ROOH

where Q–H and ROO^•correspond to the quencher molecule and peroxyl radical, respectively.

The BDE of O-H bond in the quencher molecule may act as parameters to envisage the pathway of scavenging free radicals by quencher molecule. The lowest BDE, indicates the most preferred mechanism of scavenging the ROO^• radical. The lower the BDE value, weaker the O–H bond strength and greater the free radical scavenging ability of organic quencher molecules. The weakest O–H bond are recognized for the quencher molecules and compared with corresponding k_qvalues (shown in figure 5). The calculated BDE values are shown in figure 6. The quencher molecules with smallest BDE value, exhibit highest k_qvalues and correlate well between them. Intriguingly, analogous trend observed in the steady state measurements.

Among the quenchers, dopamine shows lower BDE (70.63 kcal/mol) value and found that outmost high antioxidant activity. This is owing to the electron rich olefin, electron releasing (-NH₂) group and especially, intramolecular hydrogen bond between two -OH group in dopamine⁴⁸. On comparing L-DOPA and pyrogallol, the former show less BDE value due to presence of electron releasing -NH₂ functional group and intramolecular hydrogen bond. Pyrogallol consists of three hydroxyl groups and one of the hydroxyl group in pyrogallol molecule show less BDE value and it account for high antioxidant activity than 4-aminophenol (70.36 kcal/mol). The trend indicates the presence of electron releasing species at C-4^th position has great impact in radical scavenging potential of quencher molecules. Thus 4-t-butyl catechol show less BDE than catechol molecule. The observed behaviour is owing to the presence of electron releasing methyl group at the 4^th position and intramolecular hydrogen bond. The existence of substituent at C-4^th position plays an important role in assessing the BDE of quencher molecules. The interpretations disclose the prominence of H in determining the antioxidant activity. The present research reveals the activity of radical scavenging confides on the position and electron releasing property of substituent in quencher molecules.

Figure 6: Structure of quencher molecules with BDEs at B3LYP/6-31G**/LANL2DZ level of theory (energies are given in kcal/mol)

4 CONCLUSION:

The fluorescence quenching of NB in presence of antioxidant molecules investigated by using steady state, lifetime measurements and BDE calculations. The formation of ground state complexes between NB and quencher molecules were confirmed using UV-Visible spectroscopy and lifetime measurements. The calculated bimolecular quenching rate constant (k_q) depend on the substituent in quencher molecules. The effect on fluorescence spectra of NB in existence of quencher molecules were carried out at diverse temperatures on the way to estimate the thermodynamic parameters. The BDE value wasdeliberated to examine the discharge of H*in the quencher molecules. Based on the fluorescence quenching experiments and BDE calculations, it suggests that the hydrogen atom transfer between the NB and quencher molecules as one of the possible quenching mechanism. The present study demonstrates a new way in designing novel molecules with great antioxidant activity.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. ACKNOWLEDGMENT:

One of the authors, N. Vedichi thanks Vinayaka Mission’s Research Foundation (Deemed to be University) for providing financial support in the form of seedmoney (Research Grant No: VMRF/SeedMoney-Phase2/2020-10/VMKVASC/Salem/2).

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Received on 15.07.2022 Modified on 06.10.2022

Research J. Pharm. and Tech 2023; 16(9):4350-4356.

DOI: 10.52711/0974-360X.2023.00712