In silico results
Prediction of drug-likeness
Physicochemical descriptors and properties such as molecular weight, predicted central nervous system (CNS) activity, QPlogo/w, QPlogHERG, QPlogBBB, QPlogCaco, QPlogS, and % human oral absorption were estimated using ‘QikProp' tool in Maestro. DHZ demonstrated a predicted percentage of human oral absorption of 89.89%. Predicted apparent QPPCaco for DHZ was found to be 949.609 nm/sec. This indicates better intestinal absorption property of the DHZ molecule. All the pharmacokinetic properties for DHZ showed optimum values for QPlogS, QPlogo/w and QPlogHERG, predicting the drug-likeness of DHZ. The results are presented in Table 1.
Molecular docking analysis
The molecular docking predicts binding orientation and structural stability of the ligand–protein complex. The docking process involves the search algorithm in the generation of different poses and scoring function that is involved in the evaluation of the binding interaction of different poses and protein .
The validation of the generated grid was done by redocking the inbound ligand and calculating the RMSD. RMS of 0.5235 Ǻ was observed after the superimposition of redocked ligand and protein inbound ligand, which validates the generated grid, as seen in Fig. 1. XP docking study was performed which suggested that the docking score of DHZ was better than the standard mooclobemide molecule, as listed in Table 2.
The inhibitor binding site for MAO-A consists of the cavity with a flavin molecule extending from residues 210–216 . The natural ligands inhibiting MAO-A has been reported to show π–π interactions with TYR444, PHE208, TYR407 and PHE352 as well as H-bond interactions with ASN 181, TYR197, and TYR444 . DHZ with XP score of -7.431 showed hydrophobic interactions with TYR69, PHE352, TYR197, TYR407, TYR444, ILE180, ILE207, PHE208, ILE325, CYS323, ILE335, and LEU337 and polar interactions with ASN181, GLN215, and THR336. A similar interaction was observed in moclobemide with XP score of − 7.147 and π–π stack interaction with PHE208.
Molecular dynamics (MD) simulation
Molecular docking can only predict the interaction of the static protein and ligand. To understand the interaction pattern of the ligands with the dynamic protein in an environment similar to physiological conditions, DHZ was subjected to the MD simulation study for 30 ns. A stable RMSD of 3.6 Å was observed for DHZ for 3–12 ns duration. The formation of the H-bond and water bridge interactions between the hydroxyl group of DHZ and residue ASN181 was predominant during the 3–12 ns duration. After 12 ns, the H-bond and water bridge type of interactions of ASN181 were lost. Stable interactions, mainly hydrophobic and water bridge, were observed with residue PHE208 of MAO-A and DHZ after 15 ns of the simulation, which remained stable during 30 ns of MD simulation. These interactions have led to a decrease in RMSD of ligand to 1.8 Å. During the XP docking study, there was no H-bond interaction with ILE180, ASN181, CYS323, TYR407 and TYR444 between DHZ and MAO-A, whereas the formation of H-bond interaction was observed during the MD simulation study. For the entire simulation study, residues TYR 407 (mainly H-bond and hydrophobic interaction), TYR444 (mainly H-bond), ILE 180 (mainly H-bond and hydrophobic interaction) and ILE 335 (hydrophobic interaction) were involved in the interaction with the ligand DHZ. The protein molecule was stable throughout the simulation study. RMSD fluctuation from 1.0 to 3.5 Å was observed during the entire simulation study. RMSF plot showing the fluctuation of 4.8 Å in the protein structure was mainly due to the tail region residue from 450 to 500. However, RMSF fluctuation was comparatively less in the case of ligand (1–2 Å), DHZ. MD simulated interactions are represented in Fig. 2.
In vivo study
Characterization of dehydrozingerone
The FTIR spectra of DHZ showed absorption bands at 3311.78 cm−1 due to the OH stretching (-C6H5OH), 2999.31 cm−1 (aromatic CH stretch), 2848.86 cm−1 (aliphatic CH stretch -OCH3), 1635 carbonyl stretching (–C=C–), 1585.49 cm−1 (alkene stretch –C=C–) and 1516 cm−1 (aromatic –C=C– stretch) (Fig. 3). The ESI mass spectra in the positive ionization mode illustrated the base peak at 193 m/z, corresponding to the molecular weight of DHZ. (Fig. 4).
Effect of DHZ treatment in OFT
Number of Line Crossings The number of line crossings in the normal control group was found to be 6.75 ± 1.32 on day 1 and 4.25 ± 0.48 on day 7. The treatment with escitalopram showed a significant increase in the number of line crossings on day 1 (F4,12 = 14.37, p = 0.0023) and day 7 (F4,13 = 17.11, p = 0.0004) as compared to normal control. DHZ treatment did not show any significant change in the number of line crossings on day 1 after 1 h (F4,12 = 14.37, p = 0.9187), 3 h (F4,12 = 14.37, p = 0.8140) and 6 h (F4,12 = 14.37, p = 0.1320) of DHZ dosing compared to normal control. Statistical values 1st day Line crossing was: FDFn,DFd, where DFn is degrees of freedom numerator, and DFd is degrees of freedom denominator and p value:: F4,12 = 14.37 and p = 0.0002.
Similarly, no significant change in line crossing was seen on day 7 after 1 h (F4,13 = 17.11, p = 0.898), 3 h (F4,13 = 17.11, p = 0.8613) and 6 h (F4,13 = 17.11, p = 0.2929) of DHZ dosing as compared to normal control animals. Statistical values 1st day line crossing were: FDFn,DFd and p value:: F4,13 = 17.11 and p < 0.0001. (Fig. 5).
Number of Centre square entry The number of centre square entry in the normal control group was found to be 1.80 ± 0.20 on day 1 and 2.50 ± 0.29 on day 7. Treatment with escitalopram showed a significant increase in the number of centre square entry on day 1 (F4,18 = 16.57, p < 0.0001) and on day 7 (F4, 12 = 16.41, p = 0.0027) as compared to normal control animals. DHZ treatment also showed a significant increase in the number of centre square entry on day 1 after 1 h (F4,18 = 16.57, p = 0.0005) and 3 h (F4,18 = 16.57, p = 0.0141) dosing compared to normal control animals. However, no significant change was observed in this parameter after 6 h of DHZ treatment (F4,18 = 16.57, p = 0.2098) compared to normal control animal on day 1. On day 7, DHZ treatment did not show any significant change in the number of centre square entry after 1 h (F4, 12 = 16.41, p = 0.3723), 3 h (F4, 12 = 16.41, p = 0.3723) and 6 h (F4, 12 = 16.41, p = 0.1675) of dosing as compared to normal control animal. Statistical values were: FDFn,DFd and p value:: F4,18 = 16.57 and p < 0.0001 on 1st day centre square entry, and F4, 12 = 16.41 and p < 0.0001 for 7th day centre square entry. (Fig. 5).
Effect of DHZ treatment on immobility time of mice in FST and TST
FST The immobility time in the normal control group was 4.89 ± 0.22 s and 2.92 ± 0.52 s on 1st day and 7th day respectively. The immobility time of mice was significantly reduced on both 1st (F4,24 = 19.53, p = 0.0008) and 7th day (F4,18 = 76.82, p = 0004) after 1 h of treatment with DHZ (100 mg/kg po) and escitalopram (F4,24 = 19.53 with p < 0.0001 and 1.44 ± 0.05 s with p = 0.0002 respectively) as compared to normal control. However, there was no significant reduction in the immobility time in the mice after 3 h (4.77 ± 0.29 s and 2.27 ± 0.07 s) and 6 h of DHZ (5.00 ± 0.27 s and 5.28 ± 0.08 s) treatment on both day 1 and day 7 respectively. Statistical values were as follows: FDFn,DFd and p value:: F4,24 = 19.53 and p < 0.0001 for FST at day 1; F4,18 = 76.82 and p < 0.0001 for FST at day 7. (Fig. 6).
TST The immobility time in normal control animals were 3.32 ± 0.07 s and 3.33 ± 0.05 s on 1st day and 7th day respectively. The immobility time of mice was significantly reduced 1 h post DHZ treatment (F4,22 = 19.02, p < 0.0001 and F4,17 = 18.68, p = 0023) and escitalopram treatment (F4,22 = 19.02 with p < 0.0001 and F4,17 = 18.68 with p = 0.0055) on day 1 and day 7 respectively as compared to normal control. Even 3 h post DHZ treatment showed a significant decrease in immobility (F4,22 = 19.02 with p = 0.0124) on 1st day as compared to normal control animals. However, no significant reduction in the immobility time was observed in the mice 3 h post DHZ treatment (3.14 ± 0.17 s) on day 7 as compared to normal control animals. 6 h post DHZ treatment on day 1 (F4,22 = 19.02, p = 0.9998) and day 7 (F4,17 = 18.68, p = 0.9529) did not show a significant decrease in immobility time as compared to normal control. Statistical values were as follows: FDFn,DFd and p value:: F4,22 = 19.02 and p < 0.0001 for TST at day 1; F4,17 = 18.68 and p < 0.0001 for TST at day 7. (Fig. 6.)
LC–MS method optimization
Optimized chromatographic conditions are as follows: Column: (HILIC Kinetix 50 × 2.1 mm, 2.6 µm), Mobile phase: 0.1% Formic acid in water (A) and 0.1% Formic acid in acetonitrile (B), Gradient programme: (0.0 min %A (80); 2.5 min to 5.0 min %A (70); 9.0 min to 14.0 min %A (80), Flow rate 0.2 mL/min, Injection volume 10 µl; Column temperature: 25 °C; autosampler temperature: 6 °C. The retention time for the analytes in the extracted ion chromatogram in the MS2 mode (Fig. 7) was found to be 5.71, 6.01, 5.51 and 5.65 min respectively for dopamine, noradrenaline, serotonin and isoprenaline (internal standard) .
Optimized mass spectrometric conditions are as follows: Ion source: Heated Electron Spray Ionization source (HESI), Analyzer: Ion Trap, Operating mode: Positive polarity with SRM (Selective Reaction Monitoring), Capillary voltage: 48.48 V, Capillary temperature: 300 °C, Source heater temperature: 400 °C, Sheath gas flow rate: 50 arb, Auxiliary gas flow rate: 12 arb, Sweep gas flow rate: 0 arb. SRM transition and collision energy (CE) were as follows: dopamine (154.12–136.86, CE 25), noradrenaline (170.00–152.30, CE 17), serotonin (177.10–159.93, CE 30), isoprenaline (212.00–151.89, CE 55).
Estimation of neurotransmitters level in the mice brain
LC–MS method was used to measure the level of serotonin, -noradrenaline and dopamine in the mice brain. Statistically significant changes were seen for interaction (F8,75 = 1027 and p < 0.0001) and neurotransmitters (F2,75 = 14,671 and p < 0.0001) within groups (F4,75 = 3514 and p < 0.0001). Normal control animals showed 5310.4 ± 31.48, 817.3 ± 6.58 and 4103.5 ± 43.43 ng/g of tissue, respectively, for dopamine, noradrenaline and serotonin levels in brain homogenate. Escitalopram treatment showed a significant increase in dopamine (p < 0.0001), noradrenaline (p = 0.0008) and serotonin (p < 0.0001) levels as compared to normal control. An increase in the level of these neurotransmitters was observed after treatment with DHZ as compared to normal control (Fig. 8). The significantly highest increase was found in the mice 1 h post-treatment for dopamine (p < 0.0001), noradrenaline (p < 0.0001) and serotonin (p < 0.0001) followed by 3 h post-treatment (p < 0.0001 for all three neurotransmitters) and 6 h post-treatment (p < 0.0001, p = 0.1214 and p < 0.0001 respectively) of DHZ s compared to normal control. The level of dopamine demonstrated as four times increase in comparison to the normal control group while serotonin levels were increased by nearly three times. Observed neurotransmitter levels are represented as bar diagrams and LC–MS chromatogram in Fig. 8