3.1 Basis set selection
The present study involves the potential energy scanning of herbacetin to get the lowest energy conformer. After completing potential energy scanning, the stable conformer was subjected to a geometry optimization step at the density functional theory (DFT) utilising multiple functional/basis sets combinations (see the computational methodology part), and corresponding 13C NMR chemical shifts were determined. The analysis of all these data highlighted that by comparing each computational set of data against the experimental one (Fig. 1.), and the combination B3LYP/6-31g showed best correlation. The obtained results prompted us to perform further studies using the same basis set.
3.2 Geometrical parameters
The herbacetin molecule has mainly three possible metal chelation sites due to the presence of carbonyl and hydroxyl groups and the metal ions Cu2+, and Zn2+ are coordinated to the oxygen atoms in these sites. In this work, herbacetin metal chelates of the form 1:1 is considered. The HERN-M2+ (where N=1, 2, and 3, M2+ = Cu2+, and Zn2+) complexes were fully optimised at the B3LYP/6-31g+LANL2DZ level of theory in both gas and DMSO solvent phases. The geometrical parameters associated with herbacetin and HERN-M2+ in both gas and DMSO phase are given in Tables S1-S12 in the supplementary information. Fig. 2. shows the optimized geometry of all the possible herbacetin-metal complexes with Cu2+ and Zn2+ under B3LYP/631g+LANL2DZ basis set in gas phase.
In the case of HER1-M2+complexes, HER1-Cu2+ showed shorter coordination distances than HER1-Zn2+complex. The interaction distances of O4-M2+ and O6-M2+ are found to be 1.794 and 1.799Å for Cu2+ and 1.912 and 1.910Å for Zn2+ ions respectively. The shorter interaction distances for copper complex than Zn complex in HER1-M2+ may be because of the small ionic radius of Cu2+ compared to Zn2+ ions. It is also observed that in HER1- M2+ complex, O4- M2+ shows minimum distance than O6- M2+ whereas an opposite trend was shown by HER1-Zn2+ complex. Additionally, since the herbacetin molecule is soluble in DMSO solvent medium, the metal chelates were also optimised in DMSO solvent to analyse the structure of HER1-M2+ chelates more realistically. It's worth mentioning that the solvent impact resulted in small structural alterations. The O4-M2+ and O6-M2+ distances are found to be elongated by 0.03 and 0.032Å for Cu2+ and 0.001 and 0.015Å for Zn2+ ions respectively in DMSO medium. According to the findings, the stability of the coordination bonds in HER1-Cu2+ and HER1-Zn2+ is observed to be weakened due to the action of the solvent medium.
The coordination distances of O5-M2+, and O3-M2+ interactions are found to be 1.816 and 1.778Å for HER2-Cu2+, 1.831 and 1.798Å for HER2-Zn2+ calculated at B3LYP/631g+LANL2DZ level of theory. In all the metal ligand complexes, on comparing O5-M2+ and O3-M2+ interactions, metal ions possess weaker affinity towards O5 atom because, the stronger interaction of M2+ with O3 atom slightly destabilizes the O5-M2+interactions in HER2-M2+ system. In the DMSO medium, the corresponding coordination lengths appear to be longer. The coordination distances of HER2-M2+ complexes in DMSO medium are found to be lengthened by 0.006Å for O5-Cu2+, 0.013Å for O3-Cu2+, 0.098Å for O5-Zn2+, 0.091Å for O3-Zn2+. As seen in gas phase metal ions showed a shorter interaction distance with the O3 atom than O5 atom in DMSO medium.
Furthermore, the HER3-M2+ complexes, compared to HER1-M2+, the HER2-M2+ exhibited large interaction distances and it follows the same trend observed in the HER1-M2+, and, HER2-M2+ complexes such that, the coordination distance increases with the increase in ionic radii of the metal ions. In all the HER3-M2+ complexes, metal ions possessed shorter coordination distance with O2 than O5 atom. DMSO effect were studied for HER3-M2+ complexes indicate that the solvent had a minimal impact on structural features. In solvent medium, the interaction distance decreased by 0.021 Å for both O5-Cu2+, O2-Cu2+ and increased by 0.068 Å for O5-Zn2+ and 0.035 Å for O2-Zn2+.
3.3 Interaction energies
Interaction energy has been computed at B3LYP/631g +LANL2DZ and level of theory to acquire a better understanding of the stability of the interaction of HERN-M2+ chelates, and the results for gas and DMSO solvent phases are shown in Fig. 3. The fact that all complexes have negative interaction energy values indicates that the reactions are spontaneous. The interaction energies of HER1-M2+ complexes are found to be -1445.229 and -1714.523 hartree for Cu2+ and Zn2+ ions respectively in gas phase. Compared to HER1-M2+ complexes, HER2-M2+ are found to be destabilized by 0.28 and 0.29 hartree for Cu2+ and Zn2+ respectively. Similary while comparing with HER1-M2+, the HER3-M2+ complex is found to be destabilised by 0.20 and 0.31 hartree for Cu2+ and Zn2+. HER3-Cu2+ is stabilised by a factor 0.08 hartree and HER3-Zn2+ destabilised by 0.02 hartree with respect to HER3-Cu2+ and HER3-Zn2+ respectively. It is also important to note that, solvent DMSO destabilized all the complexes. Compared to gas phase data, HER1-Cu2+ and HER1-Zn2+ are destabilised by 0.76 and 0.86 hartree in DMSO medium. The interaction energies of HER2-Cu2+, HER2-Zn2+, HER3-Cu2+, and HER3-Zn2+ are also reduced by a factor, 0.53, 0.62, 0.63 and 0.60 hartree respectively due to the solvation effect.
3.4 Molecular orbital analysis
One of the most useful molecular orbital models for assessing and analysing a molecule's chemical reactivity is the frontier molecular orbital approach [38]. This method is based on the idea that the creation of bonds between atoms is caused by the movement of electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). Thus, the HOMO orbital defines electron donating capacity, while the LUMO orbital characterises electron receiving ability [39]. The energy gap between these two orbitals causes the molecule to be more polarizable, which is often linked with high chemical reactivity and, as a result, low kinetic stability [40]. Figs. 4a) and b). lists the HOMO-LUMO orbital energies of all the complexes in gas and DMSO medium respectively. From the results, it is clear that the band gap of herbacetin is higher than that of all the studied herbacetin metal chelates in both gas phase (3.46eV) as well as DMSO solvent (3.44eV) medium. The interaction of metal ions with herbacetin, on the other hand, reduces the HOMO-LUMO energy gap, resulting in increased chemical reactivity and, as a result, a destabilisation of the kinetic stability in comparison to the isolated herbacetin. Among the chelated complexes, HER3-Zn2+ shows the lowest band gap of 0.94eV infer its high reactivity whereas HER2-Cu2+ showed highest band gap of 2.72eV in gas phase. The narrower energy gap for HER3-Zn2+complex suggests that charge transfer interactions dominate over electrostatic interactions between ligand atoms and metal ions. Additionally, including the DMSO solvent effect results in a wide separation of the HOMO-LUMO orbitals, resulting in a reduced charge transfer between them for all the HERN-M2+ chelates, which agrees with the charge transfer study.
3.5 Natural population analysis
The net atomic charge of metal ions in complexes provides useful information concerning charge transfer between metal ions and donor ligand atoms (oxygen atoms in herbacetin). The difference between the charges of the metal ions in the complex and the isolated state can be used to calculate how much charge the metal ions gain from the donor ligand atoms during complexation. Natural population analysis (NPA) has been performed in both gas and DMSO solvent phases at the B3LYP/631g+(LANL2DZ) level of theory to determine the individual atomic charges of the donor ligand atoms and metal ions in the complexes. The amount of charge on donor ligand atoms, O2, O3, O4, O5, and O6 of herbacetin before complexation were determined to be -0.70280, -0.68907, -0.72364, -0.65573, and 0.67597e respectively. Natural population analysis of HER1-M2+ complexes in both gas and DMSO medium is shown in Fig. 5. Natural population analysis of HER2-M2+ and HER3-M2+ complexes in both gas and DMSO medium is shown in Figs. S1 and S2, respectively. After complexation, it was noted that the net charge on O4 atom was found to be increased by 0.01, and 0.02e for HER1- Cu2+, and HER1- Zn2+ respectively. It was also observed that the charge on the O6 atom was increased by 0.056, and 0.084e for HER1- Cu2+, and HER1- Zn2+ complexes respectively. Similarly, the calculated net charges on metal ions for HER1-M2+ complexes are found to be 1.141e for Cu2+, and 1.027e for Zn2+ complexes. The quantity of positive charges neutralised in the gas phase calculations for Cu2+, and, Zn2+ complexes is 0.859, and 0.973e, respectively. The order in which metal ions gain charge is Zn2+ > Cu2+, which is perfectly correlated with the interaction energy order.
Similarly, the calculated net charges on metal ions for HER2-M2+ complexes are found to be 1.200e for Cu2+, and 1.452e for Zn2+ complexes in gas phase. The quantity of positive charges neutralised in the gas phase calculations for Cu2+, and Zn2+ complexes is 0.800, and 0.548e, respectively. For HER3-M2+ complexes, 0.897 amount of positive charge for Cu2+, and 0.734e for Zn2+ complexes have been neutralised. The order in which metal ions gain charge is Cu2+ > Zn2+ in both HER2-M2+, HER3-M2+, which is not correlated with the interaction energy order. This could be because, in addition to ligand-to-metal charge transfer interactions, metal-to-ligand charge transfer interactions are also detected, as shown by the data. The metal to ligand charge transfer contact is observed to be stronger in the HER1- Zn2+, complex than in the HER1- Cu2+ complex, resulting in the Zn2+ ion competitively retaining its positive charge. The total amount of the charge on metal ions of studied complexes in DMSO medium are found to be 1.009, and 1.608e for HER1- Cu2+, and HER1- Zn2+ respectively. The amount of positive charge neutralised during complexation are found to be 0.571 and 0.548e for Cu2+, 0.28e and 0.29 for Zn2+ ions in HER2-M2+and HER3-M2+complexes respectively. Generally, it is hypothesised that using DMSO as a solvent reduces the quantity of charge transfer between metal ions and donor ligand atoms. All the complexes in DMSO medium showed metal-to-ligand charge transfer interactions together with ligand-to-metal charge transfer interactions.
3.6 Natural bond orbital (NBO) analysis
NBO analysis was performed to explain the charge transfer [41, 42] between herbacetin and metal ions in HER1-M2+ complex and the related data is given in Table 1. The stability scale for donor-accepter interaction in complex is denoted by E(2) value. Higher the E(2) more stable will be the complex. The study revealed that the most favourable donor-acceptor interaction is due to the charge from lone pair of oxygen atoms of herbacetin to the anti-bonding orbital of metal ions, Cu2+ and Zn2+. The higher E(2) value of HER1- Zn2+ indicate the strong interaction of herbacetin molecule with Zn2+ than Cu2+ ion.
Table 1
The computed E(2) value (in kcal/mol) of HER1-M2+ complexes from NBO analysis
Complex
|
Charge transfer
|
E(2)
|
Gas
|
DMSO
|
HER1- Cu2+
|
LP(O4) LP*Cu31
|
27.30
|
22.07
|
|
LP(O6) LP*Cu31
|
26.27
|
21.09
|
HER1- Zn2+
|
LP(O4) LP*Cu31
|
49.15
|
46.38
|
|
LP(O6) LP*Cu31
|
43.96
|
42.86
|
3.7 Molecular electrostatic potential map (MESP)
The molecular electrostatic potential (MESP) maps are extremely effective for identifying and predicting a molecule's reactive locations [43]. The ESP maps of herbacetin and HER1-M2+ in gas and DMSO phase are shown in Fig. 6. The blue areas reflect nucleophilic sites, while the red areas show relative electron abundance electrophilic site [44]. According to MESP the reactive nucleophilic centres for coordination with metal ions are oxygen atoms.
3.8 Quantum theory of atoms in molecules (QTAIM)
QTAIM analysis at the bond critical points (BCPs) in gas and DMSO medium was done to characterize the nature of interactions between herbacetin and metal ions [45]. The AIM results of HER1-M2+ is provided in Table 2. A stronger interaction corresponds to an increase in electron density ( ) at the BCP. The high value of M-O electron density in the HER1-Zn2+ complex indicating that Zn2+ and herbacetin interact more. The estimated (r) values of the complexes suggest that herbacetin-metal interaction through the O4 is greater than O6 in the HER1-Cu2+ complex, but the HER1-Zn2+ complex shows a reverse tendency in both gas and DMSO medium. Covalent bonds are represented by negative values of the Laplacian electron density ( 2 ), whereas non-covalent bonds are represented by positive values [46]. From the table, the positive 2 of M-O bonds in both the complexes indicate their non-covalent nature. Furthermore, the hypothesised ratio of kinetic energy density (G(r)) to potential energy density (V(r)) at the BCP is employed to characterise the type of interaction. The ratio of -G(r)/V(r) for M-O bond is close to one in all complexes indicate the electrostatic interaction [30]. Covalent interaction is characterised by higher ELF and LOL values, while electrostatic interaction is characterised by lower ELF and LOL values [34]. The small values of ELF and LOL again confirm the electrostatic interaction of M-O bonds in both the complexes and the topological view of ELF and LOL are shown in Fig. 7.
Table 2
Calculated topological parameters (in a.u) at the BCPs of M-O bonds in gas phase
Complex
|
Bond
|
Solvent
|
|
2
|
-G
|
ELF
|
LOL
|
|
|
|
|
|
|
|
|
HER1-Cu2+
|
Cu-O4
|
Gas
|
0.1292
|
0.6404
|
0.8299
|
0.1818
|
0.3202
|
DMSO
|
0.1188
|
0.5978
|
0.8397
|
0.1668
|
0.3086
|
Cu-O6
|
Gas
|
0.1277
|
0.6331
|
0.8307
|
0.1797
|
0.3187
|
DMSO
|
0.1168
|
0.5883
|
0.8404
|
0.1638
|
0.3062
|
HER1-Zn2+
|
Zn-O4
|
Gas
|
0.9637
|
0.4041
|
0.7891
|
0.1510
|
0.2967
|
DMSO
|
0.9643
|
0.4156
|
0.7933
|
0.1465
|
0.2930
|
Zn-O6
|
Gas
|
0.9703
|
0.4054
|
0.7880
|
0.1525
|
0.2978
|
DMSO
|
0.9382
|
0.4019
|
0.7942
|
0.1440
|
0.2909
|
3.9 Effect on metal ions on the anti-oxidant activity of herbacetin
Herbacetin is an effective free radical scavenger, and this free radical scavenging ability is thought to be responsible for its antioxidant activity. Even though, the metal ions are complexed at the hydroxyl groups of herbacetin and the other phenolic OH groups remain intact, it is claimed that the complex, in addition to parent herbacetin, can participate in free-radical scavenging activities. In order to understand the effect of metal ion on the anti-oxidant activity of herbacetin molecule, we have calculated all the numerical parameters (bond dissociation enthalpy (BDE), ionisation potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA), electron transfer enthalpy (ETE) ) associated with the possible anti-oxidant mechanisms, hydrogen atom transfer (HAT), single-electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET) [47, 48].
The numerical values of herbacetin, HER1-Cu2+ and HER1-Zn2+ complexes associated with anti-oxidant mechanisms are given in Table 3. The findings demonstrate that the parameters governing the three mechanisms are of vastly different magnitudes, and this evidence can be used to determine which anti-oxidant mechanism is preferred over the others. For describing the HAT mechanism, the BDE is the most trustworthy thermodynamic parameter and this approach includes the transfer of a H atom from an antioxidant's hydroxyl group to a free radical [49]. The lowest BDE is associated with higher radical scavenging activity[50]. The lowest BDE value of 15-OH of herbacetin (in both gas and DMSO medium) indicates its strong anti-oxidant effect compared to the other phenolic OH groups. In HER1-Cu2+ and HER1-Zn2+ complexes, 12-OH showed low BDE value hence high anti-oxidant activity. BDE value of 12-OH, 14-OH, and 22-OH of HER1-Zn2+ were lower than the corresponding values of isolated herbacetin indicating the enhanced antioxidant activity in both gas and DMSO medium. The BDE values of HER1-Cu2+ were also comparable with herbacetin molecule. Except 15-OH, the BDE values of all other phenolic OH groups of herbacetin in gas phase were found to be lower than that of corresponding values in DMSO medium. It was also noted that, the hydrogen atom transfer was enhanced in DMSO for HER1-Zn2+complex where as an opposite trend was observed for HER1-Cu2+ complex. Among the numerical parameters, BDE of herbacetin and HER1-M2+ complexes have the lowest value in both gas and DMSO medium, implying the preferred mechanism is HAT.
In SET-PT, one electron is transferred from the antioxidant to the free radical, resulting in the creation of a radical cation, which is then deprotonated. The most essential parameters in determining this mechanism's feasibility are IP and PDE. The higher electron donating capacity is associated with low IP value [51]. In the present study, the IP values of HER1-M2+ in both the medium were found to be lower than herbacetin molecule indicating that the chelated complexes are more active in radical scavenging activity than the isolated one. The energy required to complete the entire SET-PT reaction (IP+PDE) are significantly higher than those required by the HAT mechanism, implying that this is not the preferable method. PA and ETE are the numerical parameters associated with SPLET mechanism. From Table 2 it is clear that the energy required for the first step (PA) is much higher than the second step (ETE) in this mechanism. As in the case of SET-PT mechanism, the overall energies of both steps appear to rule out the possibility of the described process occurring in the gas phase and DMSO medium.
Table 3
Numerical parameters associated with anti-oxidant mechanism of herbacetin, HER1-Cu2+ and HER1-Zn2+ complexes
Molecule
|
Hydroxyl group
|
BDE
(kcal/mol)
|
IP
(kcal/mol)
|
PDE
(kcal/mol)
|
PA
(kcal/mol)
|
ETE
(kcal/mol)
|
gas
|
dmso
|
gas
|
dmso
|
gas
|
dmso
|
gas
|
dmso
|
gas
|
dmso
|
Herbacetin
|
12-OH
|
78.9
|
74.9
|
164.6
|
129.5
|
228.8
|
259.9
|
335.1
|
294.4
|
58.3
|
95.0
|
|
14-OH
|
90.2
|
84.5
|
164.6
|
129.5
|
240.2
|
269.6
|
345.5
|
300.3
|
59.2
|
98.9
|
|
15-OH
|
67.9
|
69.0
|
164.6
|
129.5
|
217.8
|
254.2
|
329.8
|
290.5
|
52.6
|
93.2
|
|
16-OH
|
83.8
|
79.6
|
164.6
|
129.5
|
233.7
|
264.7
|
333.2
|
291.1
|
65.1
|
103.1
|
|
22-OH
|
78.6
|
78.4
|
164.6
|
129.5
|
228.6
|
263.6
|
327.2
|
293.0
|
65.9
|
100.1
|
HER1-Cu2+
|
12-OH
|
78.9
|
85.6
|
156.5
|
129.2
|
237.0
|
271.1
|
324.4
|
297.4
|
69.0
|
102.9
|
|
14-OH
|
83.8
|
88.7
|
156.5
|
129.2
|
241.9
|
274.2
|
332.0
|
305.6
|
66.4
|
97.9
|
|
22-OH
|
88.0
|
95.4
|
156.5
|
129.2
|
246.0
|
280.9
|
322.8
|
294.2
|
79.8
|
115.9
|
HER1-Zn2+
|
12-OH
|
74.7
|
70.7
|
154.7
|
106.4
|
234.6
|
279.0
|
327.0
|
299.1
|
62.2
|
86.3
|
|
14-OH
|
81.6
|
75.9
|
154.7
|
106.4
|
241.4
|
284.1
|
336.1
|
309.4
|
60.0
|
81.2
|
|
22-OH
|
78.5
|
76.0
|
154.7
|
106.4
|
238.3
|
284.3
|
324.8
|
295.4
|
68.2
|
95.3
|
3.10 Molecular docking
Docking studies were conducted with the herbacetin molecule to assess its qualified binding poses within the selected target proteins [52], AChE2, and BChE. For AChE2, and BChE, tacrine [53] was chosen as a control ligand. Herbacetin presented good binding interaction with all the selected target proteins which were comparable with the well-known standard drug tacrine. Molecular docking studies showed that herbacetin possessed a negative binding energy value -9.0 kcal/mol and -9.6 kcal/mol with AChE2 and BChE respectively which indicate the stability of the complex. Interestingly these binding affinity values were higher than that of tacrine with AChE2 (-8.6 kcal/mol) and BChE (-8.2 kcal/mol). Figs. 8. a), b), c) and d) shows the 3D image of herbacetin-AChE2 complex, interaction analysis of herbacetin-AChE2 complex, 3D images of herbacetin-BChE complex, and the interaction analysis of herbacetin-BChE complex respectively.
The interaction analysis revealed that, herbacetin shows conventional hydrogen bonds, pi-sigma, pi-pi T shaped, amide pi stacked and van der Waals interactions with neighbouring amino acid residues of AChE2 protein. In particular, residues GLU202, SER203, and HIS447 were formed three hydrogen bonds with herbacetin molecule. A pi-sigma was observed between the GLY121 and pi electron cloud of the molecule. Apart from that, two types of molecular interactions were formed via pi-pi T shaped interaction with residues TYR124, PHE338, and TYR341 and amide pi stacked interaction with GLY120. The molecule is well interacted with amino acid residues, TYR337, GLY448, TYR133, ALA127, LEU130, GLY126, TRP86, SER125, PHE297, and PHE295, through van der Waals interactions. Herbacetin was found to be involved in hydrogen bonding interaction with the residues, SER198, and GLY116 of BChE. A significant number of van der Waals interaction were observed between herbacetin and BChE. Precisely, amino acid residues, ALA328, TYR332, TRP430, GLU197, GLY115, ALA199, GLY117, PHE398, and VAL288. The amino acid residue, HIS438 possessed a pi-cation interaction with the molecule. Few other residues, TRP82, PHE329, and TRP231 participated in pi-pi T shaped interaction. A pi- alkyl interaction was found between LEU82 with herbacetin molecule. It was also noted that the molecule formed van der Waals interactions with the aminoacid residues, ALA328, TYR332, TRP430, GLU197, GLY115, ALA199, GLY117, PHE398, and VAL288 within the active site.
3.11 In-silico ADME and drug-likeness prediction
The ADME analysis is a crucial step in weeding out a few interesting chemical entities from a vast chemical dataset. The ADME profile of the herbacetin molecule was thus be predicted using SwissADME, which is publically available at http://www.swissadme.ch [54]. Molecular weight, lipophilicity, water solubility, as well as Lipinski's rule of five [55] of herbacetin molecule are given in Table S13. Orally active drugs must follow the Lipinski five rule, which states that molecular weight (MW) should not exceed 500 g/mol, hydrogen bond acceptors (HBA) should not exceed 10, hydrogen bond donors (HBD) should not exceed 5, LogP value should not exceed 5, and the number of rotatable bonds should not be less than 10, with a violation of two or more rules indicating that the molecule is not orally active. The molecular weight of herbacetin was 302.24 g/mol and it comes within the limit 500 g/mol according to Lipinski rule. The logP value of the herbacetin was less than 5, ranging from -0.56 to 2.17. The HBA number was 7 and less than 10; the number of HBD atoms was 5. Hence herbacetin follow Lipinski five rule with no violation. According to aqueous solubility methods, log S (ESOL), log S (ALI), and log S (SILICOS-IT), herbacetin is soluble in water.