Plant material and extraction
Dried seeds of Acacia senegal (L.) Willd. were purchased from a local store in Jodhpur (Rajasthan), India. Taxonomic confirmation of the seeds was based on a comparison with an herbarium accession by a botanical expert in the Department of Botany, Jai Narain Vyas University, Jodhpur. Seed extract was obtained using a standard Soxhlet procedure.
Identification of the phytoconstituents
Identification of predominant phytoconstituents present in the seed extracts was based on LC-MS (Liquid chromatography and Mass spectroscopy) [21,22]. The LC-MS data were subsequently analysed using Masshunter software developed by Agilent. Peaks generated in both positive and negative modes of ionization, with ≥3500 ionization counts, were considered using a peak spacing tolerance of 0.0090m/z for reasonable resolution of the chromatogram. Chromatogram peaks were assigned masses based upon MS-MS fragmentation patterns specific for the identified phytocompound. The metabolite profile was confirmed using mass Bank workstation software along with public database information. The samples (SAIF 436) were analysed by the SAIF (Sophisticated Analytic Instrumental Facility), CDRI, Lucknow, UP, India.
Doses of standard statin drug and seed extract dosage
A supply of 20 mg tablets of Atorlip (atorvastatin) was obtained from a local pharmacy in Jodhpur and administered doses were calculated based on body weight of the test rabbits. The seed extract was administered orally at a dose of 400 mg/kg body weight per day for 45 days based on an LD50 assessment and previously published studies [23,24].
In-vitro inhibition of HMG -CoA reductase activity
The HMG-CoA reductase inhibition assay was performed in-vitro using a kit (Sigma Aldrich) according to the manufacturer’s instructions and previous reports in the literature [25,26]. The inhibitory activity of increasing concentrations (0.32mg/ml 0.62 mg/ml 1.25 mg/ml, and 5mg/ml) of the seed and a standard statin drug (Pravastatin) provided with the kit were determined by measuring absorbance at 340 nm. The IC50 was calculated based on the obtained inhibition curve for HMGR of the seed extract and the standard drug. The assay is based on the decrease in absorbance resulting from the tested compound and measures the oxidation of NADPH by the catalytic subunit of HMGR in the presence of the substrate HMG-CoA.
Groups of experimental animals
New Zealand white male adult rabbits weighing approximately 1.5±0.1 kg were used in the experiments. Four groups (two control groups and two treated groups) of rabbits were established with six rabbits in each group. Animals were acclimatized for 10 days prior to the onset of the experiment and were maintained in cages in a controlled environment (26 ± 3°C and 12 h of light and dark cycles). The animals were fed a balanced diet supplemented with micronutrients and vitamins. The experimental protocol for use of the animals was recommended by the Institutional Animal Ethics Committee (IAEC) based on the standard norms of the CPCSEA (Reg. No.1646/GO/a/12/CPCSEA valid up to 27.03.23).
Experimental groups were assigned as follows:
Group I: Vehicle control
Group II: Hypercholesterolemic control
Group III: Group administered seed extracts of Acacia senegal (L.) Willd.
Group IV: Group administered standard statin drug (Atorvastatin).
The duration of the experiment was 60 days inclusive of the time needed to induce hypercholesterolemia (15days) and administer the treatments (45days)
Induction of hypercholesterolemia
Hypercholesterolemia was induced in the test rabbits by feeding them a high fat diet and a cholesterol powder supplement for 15 days. The cholesterol powder supplement was formulated at 500mg cholesterol powder/kg body weight per day mixed with 5ml coconut oil [27,28]. The induction of hypercholesterolemia was confirmed by weekly biochemical assessments of the blood lipid profile and calculation of the atherogenic index using standard methods.
Collection of serum samples for biochemical analysis and histopathology
Twenty-four-hour fasted animals were autopsied under mild anaesthesia at the completion of the experiment and blood samples were obtained from direct cardiac and hepatic vein puncture. The collected blood was kept in EDTA-coated vials and serum was separated by centrifugation for 15 min at 3000 rpm.
Serum lipid profile and atherogenic index
Total cholesterol[29], HDL-cholesterol[30], and triglyceride (TG)[31] were determined using standard methods and the lipid profile was constructed following Friedewald’s formula (Kumar, 2014). The following indices were calculated using the indicated formulas:
LDL-cholesterol = Total cholesterol - HDL-cholesterol - VLDL-cholesterol
Where VLDL= triglyceride/5
The Castelli risk index – I (Total cholesterol/HDL), Castelli risk index – II (LDL/HDL)[33] and the Atherogenic index = Log (Triglyceride / HDL-cholesterol) [34].
Antioxidants and peroxidation assays of serum
Serum antioxidant levels were determined for catalase[35], superoxide dismutase (SOD)[36], GSH (reduced glutathione)[37], and FRAP (Ferric reducing antioxidant potential) [38] using standard protocols based on redox reaction end products measured as absorbance at an appropriate wavelength. The degree of lipid peroxidation (LPO) in serum was determined by assessing thiobarbituric acid reactive substances (TBARS) and is represented as malondialdehyde (MDA) content, following the modified method of Ohkawa [39].
Histology and planimetric (morphometry) study of aorta
A 2-3 cm length of the ascending aorta of autopsied animals was removed and fixed in 10% formalin. The aortic tissues were subsequently dehydrated in a graded ethanol series and eventually embedded in paraffin wax. The paraffin-embedded samples of aorta were sectioned at a thickness of 5 microns and processed for staining and histopathological analysis [10,40]. The morphometric measurements and planimetric assessments of the sectioned samples of aorta were performed using a Camera Lucida [27,40].
Molecular Docking
Molecular interactions of identified compounds with HMG-CoA reductase was analyzed using Autodock 4.2[41,42]. The catalytic portion of human HMG-CoA reductase (1HW8) was downloaded from a protein data bank and processed using PyMol to extract the co-crystallised ligand inhibitor atorvastatin, remove unwanted water molecules, and correct for chain integration. Three-dimensional structures of the compounds identified in the seed extract and the known inhibitors (pravastatin, atorvastatin) were downloaded from Pubchem Database. Ligand processing was performed using PyMol and hydrogen was added to the structures. The developed docking protocol was validated by performing re-docking with prepared co-crystalized ligand and prepared receptor protein and maps were generated. Post-validation of the docking protocol of the test compounds was performed by independently docking them with target receptor proteins. The parameters of molecular interactions were obtained through ligand conformations, binding energies, and linked assessments.
Molecular dynamics
Molecular dynamics (MD) simulation studies were performed using GROMACS to understand conformational dynamics of docked complexes (Atorvastatin, Eicosonoid, Flavan-3-ol, Linoleic acid and Pravastatin) with 1HW8. All atoms simulation method was used to gain the insight by solving newton’s equation of motion. MD simulations of Atorvastatin_1HW8(HMG-CoA reductase), Eicosonoid_1HW8, Flavan-3-ol_1HW8, Linoleic acid_1HW8 and Pravastatin_1HW8 complexes were performed with the GROMACS 2020.2 package using CHARMM36 force field[43]. The topology of 1HW8 was generated using pdb2gmx modules of GROMACS In addition, PRODRG 2.5 an automated server was used to generate the topology of ligand separately[44]. For solvation of protein, dodecahedron box was used, and protein was placed at least 1.0 nm from the edge of the box. Energy minimization was performed after adding required charges to the system. In one phase potential energy was minimized at maximum force of 1000.0 KJ/mol/nm using 50,000 energy minimization steps cut-off. The temperature coupling was performed by considering the protein structure and ligand as one at a temperature of 310K for 100ps and coordinates of the complex was saved after every 10ps. Pressure equilibrium was also attained using Parrinello-Rahman pressure coupling. The LINCS algorithm was used for constraining all the bonds. Finally, the systems were submitted to molecular dynamics simulation for 1ns to observe stability of Atorvastatin_1HW8, Eicosonoid_1HW8, Flavan-3-ol_1HW8, Linoleic acid_1HW8 and Pravastatin_1HW8 complexes. Structural analysis (RMSD, RMSF and Radius of Gyration) was performed using rmsd, rmsf and gyrate modules of GROMACS and their graphs were generated with xm grace (Graphing, Advanced Computation and Exploration program).
Pharmacokinetic Analysis
ADMET analysis was performed using Drulito software with the standard protocol used to determine the ideal pharmacokinetic profile of the test compounds considered for drug development [45,46]. The test compounds were curtained by two filters: the Lipinski rule and the blood brain barrier (BBB) requirement. The Lipinski rule indicates that an ideal drug molecule should weigh below 500g/mol, the number of hydrogen bond donors should be less than or equal to 5 and the number of hydrogen bond acceptor should be ≤ 10, with a partition coefficient ≤ 5. The test compound should pass the BBB if the number of hydrogen bonds present is approximately 8-10 and no acidic groups should be present in the molecule. TPSA (total polar surface area) represents the bioavailability of the drug molecule according to Veber’s rule which indicates that a TPSA less than or identical to 140Å will have good oral bioavailability.
Statistical Analysis
The data on the biochemical parameters are expressed as a mean ± SEM (standard error of the mean). A one-way analysis of variance (ANOVA) was conducted followed by Tukey’s multiple comparison tests using GraphPad Prism 7.0 software. Graphical representations of the data were constructed using MS Excel 2018.