Ameliorations in the Biomarker Indices of Dyslipidemia and Atherosclerotic Plaque by the Inhibition of HMG – CoA Reductase and Antioxidant Potential of Phytoconstituents of an Aqueous Seed Extract of Acacia Senegal (L.) Willd in Rabbits


 Background: The HMG-CoA inhibitor are used to control adverse cardiovascular event caused by Hypercholesterolemia and dyslipidaemia. The current study was aimed to evaluate the ability of phytoconstituents of an aqueous seed extract of Acacia senegal (L.) Willd to inhibit HMG-CoA reductase and regress the formation of atherosclerotic plaque. Methods: The chemical fingerprinting of the test extract was assessed by LC-MS. Consequently, the assessments of in-vitro, in-vivo, and in-silico were performed by following the standard methods.Results: The in-vitro assessment of the test extract revealed 74.1 % inhibition potential of HMG-CoA reductase. In-vivo evaluations of the test extract indicated that treated hypercholesterolemic rabbits exhibited a significant (𝑃 ≤ 0.001) ameliorations in the biomarker indices of the dyslipidaemia, such as the atherogenic index, Castelli risk index (I&II), atherogenic coefficient along with lipid profile. Concomitantly, significant reductions were observed in the atherosclerotic plaque area and antioxidants. The in-silico study of molecular docking shown interactions capabilities of key phytoconstituents of the test extract with target protein of HMG-CoA reductase which further validated by the molecular dynamics through potentail energy, NPT, NVT, RSMD and others. Subsequently, the ADMET analysis shown ideal druggability. Conclusion: The results indicate that phytoconstituents of an aqueous seed extract of Acacia senegal (L.) Willd. could inhibit HMG-CoA reductase and improve the levels of antioxidants activity that may reduce symptoms associated with hypercholesterolemia.


Introduction
Cardiovascular disease is one of the major reasons of mortality across the developed and developing countries. Dyslipidaemia is the process that leads to increased lipid deposition on arterial wall that promote atherosclerosis [1]. The existing therapeutics of dyslipidemia involve cholesterol lowering drugs speci cally statin and brates. The mechanism of statins involves inhibition HMG-CoA enzyme [2]. Although, there are several adverse effects associated with these synthetic drugs. In view of this, the present study explores natural plant product to have HMG-CoA reductase and antioxidant potential.
Plant products are not only used in traditional medicine but are also in demand globally as potential sources for the development of new drugs [3]. The Indigenous traditional herbal remedies contain unique formulations of local herbs and herbal extracts that have been developed based on conventional knowledge and local wisdom [4][5][6]. The ability of several traditional medicines to treat and resolve cardiovascular problems and linked metabolic disorders have been well documented [7]. In this regard, polyherbal formulation of ve local herbs (Panchkuta), such as Prosopis cineraria pod (Sangari), seed of Acacia senegal (L.) Willd. (Kumbat or Kumatiya), fruit of Capparis decidua (Ker), pulp of Casio melo (Kachar), and pulp of Mangifera indica (Amchoor) that are endemic to the Western Rajasthan region (Thar desert) of India, have been historically used to treat cardiovascular problems in rural communities [8,9]. Acacia senegal (L.) Willd. seed is one of the key ingredients in this herbal medicine (panchkuta) of which several medicinal properties have been demonstrated in our previous studies [10][11][12]. Exudates of Acacia senegal (L.) Willd., which is commonly known as gum Arabic, have also been reported to exhibit hypocholesterolemic activity in animals as well as Sudanese human subjects [13,14]. Extracts of seeds of Acacia senegal (L) also have the ability to inhibit serine proteinase activity [15]. Several reports have provided on information on the ethnopharmacological applications of foods and herbal medicines indigenous to the arid regions of African countries and the Indian subcontinent [15][16][17]. Acacia senegal (L.) Willd. is typically known by its common name, white gum tree, and is a member of the Leguminosae-Mimosoideae [18], while seed extracts of Acacia senegal (L.) Willd. is locally known as kumbat or kumatiya in Rajasthan [19,20]. The present study also identi ed the major phytoconstituents present in the seed extracts of Acacia senegal and assess its anti-atherosclerotic properties in hypercholesterolemic rabbits using a combination of in-vitro, in-silico, and in-vivo methodology.

Plant material and extraction
Dried seeds of Acacia senegal (L.) Willd. were purchased from a local store in Jodhpur (Rajasthan), India. Taxonomic con rmation 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.
Identi cation of the phytoconstituents Identi cation 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 speci c for the identi ed phytocompound. The metabolite pro le was con rmed 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 LD 50 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 IC 50 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. 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 con rmed by weekly biochemical assessments of the blood lipid pro le 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.
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 modi ed 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 xed in 10% formalin. The aortic tissues were subsequently dehydrated in a graded ethanol series and eventually embedded in para n wax. The para n-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 identi ed 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 identi ed 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 eld [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 pro le of the test compounds considered for drug development [45,46]. The test compounds were curtained by two lters: 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 coe cient ≤ 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.

LCMS analysis of the seed extract
The monoisotopic mass obtained for phytoconstituents was calculated as M + H or M-H ions in QTOF mass hunter software and veri ed by MS/MS and identi ed using the data METLINE software and published literature. Results indicated that the seed extract contained nine major phytoconstituents (Table 1and Fig. 1).

In-vitro inhibition of HMG-CoA reductase activity
The seed extract and the standard statin drug, pravastatin, exhibited a maximum 74.1 % and 91.4% inhibition of HMG-CoA reductase activity, respectively. Increasing gradient of concentrations of the seed extract were assessed. Enzyme activity was calculated based on the product rate per minute. The IC 50 of the seed extract, calculated from the inhibition curve, was 0.064µg/ml ( Fig. 2A &2B).

Effect On Peroxidation And Antioxidants Levels
The levels of peroxidation and antioxidants (SOD, CAT and GSH) were abnormal in hypercholesterolemic rabbits. In contrast, however, administration of the seed extract or atorvastatin resulted in signi cant reduction (P ≤ 0.001) in MDA in hypercholesterolemic rabbits, relative to the untreated, hypercholesterolemic rabbits. Moreover, the levels of catalase, SOD and GSH were signi cantly elevated in hypercholesterolemic rabbits treated with the seed extract. Increased levels of total antioxidants were observed in the rabbits treated with the seed extract, as determined by using a FRAP assay (Fig. 5).

Histology And Morphometric (planimetric) Analysis Of The Aorta
The aortal wall of the vehicle control group (non-hypercholesterolemic) of rabbits was composed of three distinct layers (intima, media and adventitia) and exhibited a compact wall area and enlarged lumen (Fig. 6A). In contrast, the aortal wall of hypercholesterolemic rabbits exhibited an abnormal wall area with the presence of bulging structures of atherogenic plaque and a reduced lumen volume (Fig. 6B). Treatment of the hypercholesterolemic rabbits with the seed extract resulted in a signi cant (P ≤ 0.001) reduction in the aortal total wall area and plaque along with an enlargement in lumen volume relative to the untreated, hypercholesterolemic rabbits. The effect was even greater than the reduction exhibited in response to treatment with the standard statin drug (Fig. 6C and 6D; Fig. 7).

Molecular Docking
HMG-CoA has a catalytic groove comprising amino acid residues from 426 to 888. The catalytic portion is composed of Cys688, Thr689, Asp690 and Lys691. The side chain of Lys691 is positioned in the middle of the active site. The ap, primarily composed of Glu559 and Asp767, is in the front of the active site. Among the identi ed phytoconstituents, eicosanoic acid, linoleic acid, digallic acid, and avan-3-ol displayed polar interactions with the catalytic residues of the receptor protein ( Table 2, Fig. 8A-8E). In contrast, gallocatechin, taxifolin, and myricetin did not exhibit any interaction with the HMG-CoA molecule.  Admet Analysis Of Pharmacokinetics ADMET studies of the identi ed phytoconstituents indicated that, among the identi ed phytoconstituents in the seed extract, only the avonoid, avan-3-ol, conforms to the Lipinski rule of ve along with the potential to cross the BBB. Although eicosanoic acid and linoleic acid both displayed a molecular interaction with HMG-CoA in the docking analysis, they did not conform with the Lipinski rule of ve for an ideal drug molecule. Fisetinidol and taxifolin exhibited ideal drug pro les but lack the ability to cross the BBB and did not interact with the target protein in the docking analysis (Table 3).

Discussion
The prevailing strategy for the management of hypercholesterolemia is the use of HMG-CoA reductase inhibitors which work by inhibiting cholesterol synthesis by HMG-CoA reductase in the liver and removal of excess cholesterol level in peripheral circulation by several mechanisms of reverse cholesterol transport [47,48]. Excess cholesterol in the circulatory system is indicated by biomarker indices of dyslipidaemia and abnormal lipoproteins ratios, which can be regulated by proper fractional esteri cation of cholesterol and reverse cholesterol transport (RCT) [49,50]. Cholesterol present in the intestine is rst absorbed in the form of chylomicron (triglyceride rich complex) and is then modi ed and packaged as high-density lipoprotein (HDL) cholesterol. Therefore, the ratio of triglyceride to HDL is indicative of the levels of peripheral cholesterol in circulation. Abnormal cholesterol esteri cation rates in apoB-lipoproteindepleted plasma (fractional esteri cation) and lipoprotein particle size result in dyslipidaemia [49,51]. In animal model, speci cally hypercholesteraemic rabbits, exhibit elevated levels of the biomarker indices of dyslipidaemia, such as the logarithm of the TG/HDL ratio, total cholesterol/ HDL (Castelli risk index -I (CRI-I)) and LDL-cholesterol/HDL-cholesterol (Castelli risk index-II (CRI-II)). In the present study, the treatment of hypercholesterolemic rabbits with an aqueous seed extract of Acacia senegal (L.) Willd. caused a signi cant reduction in the atherogenic index and CRI -I&II, indicating improved fractional esteri cation of cholesterol and reverse cholesterol transport. These results are similar to a previously reported study [33]. The lipid pro le i.e. total cholesterol, triglyceride, VLDL-cholesterol, and LDLcholesterol were signi cantly improved by treatment with the aqueous seed extract of Acacia senegal (L.) Willd. The seed extract appears to signi cantly inhibit cholesterol biosynthesis in hepatic tissues, as demonstrated in the in-vitro HMG-CoA reductase inhibition assay, as well as the in vivo studies in hypercholesterolemic rabbits. A variety of phytocompounds have been reported to have capacity to inhibit HMG-CoA reductase, a key enzyme in cholesterol biosynthesis, by inducing the activation of sterol regulatory element binding protein-2 (SERBP-2) and modi cations in LDL receptors that lead to reduced cholesterol production and other parameters of the lipid pro le [48,52].
Excessive amounts of peripheral LDL-cholesterol induce the generation of an excessive level of free radicals resulting in oxidative stress. This causes endothelial dysfunction and leads to the further progression of atherosclerotic plaque and reduced lumen volume in the aorta. Similar observations have been noted in hypercholesterolemic animals accompanied by an excess level of cholesterol in the peripheral circulatory system, as well as the progression of atherosclerosis. In the present study, hypercholesterolemic rabbits treated with the seed extract exhibited lower levels of free radicals and elevated levels of catalase, SOD and GSH, which are responsible for scavenging and degrading free radicals. In addition, treatment with the seed extract also resulted in a signi cant regression in atherosclerotic plaque which would have reversed the progress of atherosclerosis. Previous studies have indicated that hypercholesterolemia promotes atherosclerosis by generating oxidative stress which causes an imbalance between host antioxidant capability and the level of oxidative stress-inducing molecules including reactive oxygen (ROS), nitrogen (RNS), and halogen species, non-radical as well as free radical species. Oxidative stress leads to peroxidation of cellular proteins, lipids, and DNA, resulting in cell injury or cell death, which activates cell death signalling pathways that are responsible for accelerating atherogenesis [53]. In the present study, treatment of hypercholesterolemic rabbits with the seed extract elevated the levels of catalase, SOD and GSH and thus the free radical scavenging capacity of the cell. This effect reduced the atherogenic plaque area and increased the lumen volume. Natural and synthetic antioxidants have been reported to play a crucial role in the prevention and treatment of atherosclerosis through different mechanisms, including inhibition of LDL oxidation [54], decreasing the generation of ROS [55], inhibition of cytokine discharge, the regression of atherosclerotic plaque formation [56] and platelet accumulation [57], the prevention of mononuclear cell in ltration, improvement in endothelial dysfunction [53] and vasodilation, increasing nitric oxide (NO) bioavailability [58], modulating the expression of adhesion molecules, and reducing foam cell formation [57]. The phytochemical analysis of the seed extract identi ed several predominant phytoconstituents, including setinidol, linoleic acid, eicosanoic acid, lupenone, avan-3-ol, myricetin, digallic acid, taxifolin, and gallocatechin. The in silico molecular docking analysis indicated that eicosanoic acid, linoleic acid, and avan-3-ol are capable of binding to the target enzyme, HMG-CoA reductase [41]. Accordingly, the molecular dynamics simulation validates the stability of the complex system in polar solution was observed using the parameters of RMSD (root mean square deviation), RMSFs (root means square uctuations), and radius of gyration [59]. The system pressure and temperature are encouraging the structural deviations distress the density, viscosity, thermodynamic properties, and chemical kinetics within melts as indicated by respective graphs [60,61]. Compassionately, the ADMET pro le of the major phytoconstituents present in the seed extract indicated that the compounds have ideal pharmacokinetic properties conforming to the Lipinski rule, have good bioavailability, and are capable of crossing the blood brain barrier [45].

Conclusion
In This study was not conducted by speci c research grant.

Availability of data and materials
All data used in this study has been included in this article and supplementary data can be provided upon reasonable request.
Ethics approval and consent to participate.
The experimental protocol was approved by IAEC (Institutional Animal Ethical Committee) Department of Zoology, JNVU, Jodhpur which is registered under CPCSEA (Reg. No.1646/GO/a/12/CPCSEA valid up to 27.03.23). All the participants agreed to publish this work.

Consent for publication
All the participants approved the nal version for the publication and provided their consent.

Competing interests
It is declared that there are no con icts of interest for the authors participating in this study.

Funding
This study was not conducted by speci c research grant.

Con icts of interest/Competing interests
It is declared that there are no con icts of interest for the authors participating in this study.

Figure 6
Effect of an aqueous extract of Acacia. senegal (L.) Willd. seed on planimetry of aorta