Pod procurement, authentication, and extraction method
Dried pods of Prosopis cineraria (L.) Druce were obtained from a local provisional store and their taxonomic identity was confirmed by a botanical expert using an old herbarium voucher. The dried pods were ground in a mixer, macerated after boiling in hot water, and then vacuum dried. The resulting sticky extract was stored under desiccated conditions. Atorvastatin, a type of statin, was purchased from a local medical store and used as a positive control. All chemicals and reagents were purchased from local suppliers, Jodhpur (Rajasthan), India.
Phytochemical analysis of the extract
A phytochemical analysis and identification of the small molecular weight compounds in the extract was conducted by LCMS, GCMS, and FTIR analysis. The small molecular weight phytoconstituents were identified using METLIN software which identified the masses of the obtained peaks and identified them based on the monoisotopic mass of standard compounds using M and M+H ions in QTOF mass hunter software. The default series for mass identification used a value greater than 100m/z ratio.
LC/MS chemical analysis
A metabolomic analysis based on LC-MS was conducted to characterize the chemical fingerprint of the plant extract. Several mobile phase sequences were analysed in the analysis to obtain a comprehensive characterization of chromatographic peaks .
Gas chromatography with tandem mass spectrometry (GC-MS/MS) analysis
GC-MS analysis of an ethanol pod extract of P. cineraria was conducted using a standard protocol. The sample was injected into a gas chromatograph interfaced with a mass spectrometer (GC-MS) . The LC-MS/MS and GC-MS/MS was used to identify the phytoconstituents in the extract based on the separation and isolation of small molecular weight compounds based on their molecular weight, retention time, as well as other chromatographic techniques.
An FTIR Spectrophotometer (Bruker Co., Germany) equipped with a standard detector and a germanium beam splitter, interfaced with a computer and analytical software, was utilized for the analysis. The KBr pellet technique was used to obtain a spectrum in the mid IR region of 400-4000 cm 1. The spectrum was characterized using the attenuated Total Reflectance (ATR) technique .
HMG -CoA reductase activity
In-vitro inhibition of HMG CoA reductase activity by the plant extract was performed using an HMG-CoA reductase assay kit (Sigma-Aldrich), based on the measurement of absorbance in a spectrophotometer. The assays utilized increasing concentrations of the pod extract (1.56μg/ml, 3.13 μg/ml, 6.25μg/ml, 12.50μg/ml and 25μg/ml) and pravastatin as a positive control. The concentration of the HMG-CoA reductase enzyme solutions ranged between 0.50-0.70mg/ml. The different concentrations of the aqueous extract were mixed with a reaction mixture containing NADPH, HMG-CoA substrate, and HMGR. Pravastatin (Sigma Aldrich co.) was used as a positive control and distilled water served as a negative control [20,23].
The in-vitro antioxidant potential of the pod extract was evaluated using both ABTS and FRAP assays . The FRAP reagent was prepared freshly at a ratio of 10:1:1 by mixing 300mM acetate buffer (pH 3.6), 10mM of 2,4,6-tripyridyl-s-triazine (TPTZ), and 20mM of FeCl3⋅6H2O. The assays were carried out in triplicate in a 96-well plate by mixing 20μl of plant extract at different concentrations with 180μl of FRAP reagent. The reaction was incubated at 37°C in the dark for 30 minutes and absorbance was measured at 593nm. The antioxidant capacity was calculated as ferrous equivalents using a standard curve generated with FeSO4.
The ABTS reagent was prepared by mixing a 7mM ABTS solution with 2.4mM potassium persulfate. The reagent was stored in the dark for 16-24 hours to stabilize it prior to use. A working solution was prepared by diluting the reagent with ethanol to obtain an absorbance of 0.70 at 734nm at 37°C. Subsequently, 10μl of the plant extract was mixed with 190μl of diluted ABTS reagent in a 96-well plate. Trolox was used as a positive standard to measure the relative percentage scavenging activity of the aqueous pod extract of P. cineraria.
Induction of hypercholesterolemia and experimental groups
A rabbit (New Zealand white males) animal models were selected for use in the study having an approximate weight of 1.5 ± 0.5 kg, 6-9 months in age and were deemed healthy after an inspection by a veterinarian. The animals were kept under standard environmental conditions with a 12-hour light/dark cycle. The rabbits were used for experimentation after ten days of acclimatization to the laboratory conditions.
Hypercholesterolemia was induced by oral administration of 500 g of cholesterol powder mixed with 5 ml of coconut oil for 15 days, along with a high fat diet(21 % fat) .
Animals were divided into four groups, with each group containing five rabbits. The experiment ran for 60 days. The experimental groups were as follows:
Group 1: Vehicle Control, treated with only distilled water for 60 days.
Group 2: Hypercholesterolemic control, diseased animal model.
Group 3: Treatment with aqueous pod extract of Prosopis cineraria (500mg/kg for 45 days, oral).
Group 4: Treatment with atorvastatin (0.25mg/kg).
Blood was collected from animals in each of the treatment groups and serum was separated from blood by centrifugation using a standard protocol and stored at -20oC. After thawing, assessments were made of total cholesterol , triglyceride , HDL – cholesterol , glucose , and total protein. All measurements were conducted by following the standard methods. The lipid profile and atherogenic indices were calculated using Friedewald’s formula[31,32].
Serum antioxidant assay
The antioxidant capacity of the serum was evaluated by measuring catalase, SOD (superoxide dismutase), GSH, lipid peroxidation (LPO), and total antioxidant activity. Thiobarbituric acid reactive substances (TBARS) were examined as an index of lipid peroxidation.
Histopathology and planimetric analysis
The aortas of the four different experimental groups were obtained from autopsied animals and processed for histopathological examination. The aortas were fixed in 10% formalin, processed for embedding and sectioning. Tissue sections were mounted on glass slides, stained, and then covered with a cover slip for evaluation under a microscope. Planimetric studies of the aorta wall, lumen volume, and atherosclerotic plaque were conducted using a Camera Lucida .
A set of 16 molecules (Table 1) were docked onto human HMG-COA reductase receptors (PDB ID:1HWK). The molecules in structure data format (sdf) were downloaded from Pubchem using their structure CIDs. The geometry of the molecules was optimized using OPLS2005 force field and low energy conformers generated in the LigPrep module of Schrodinger. The generated conformers were then used in the molecular docking studies.
The three-dimensional crystal structure of human HMG-COA reductase was obtained from a protein data bank and resolved using X-ray diffraction method (PDB ID: 1HWK). The protein structure was prepared in the “PrepWiz” module of Schrodinger. Hydrogens were added, bond orders were assigned, and proper ionization states were assigned during pre-processing. The structure was optimized using restrained minimization by OPLS2005 force field. The prepared structure was used in the docking studies.
Grid generation and docking
Co-crystallized ligand was used as a reference to define the receptor binding sites and a receptor grid was generated around the centroid of the co-crystallized ligand (Atorvastatin). The binding pocket residues at MET657, SER661, VAL683, ARG590, SER684, CYS688, ASN686, ASP690, LYS691, and LYS692 were used for grid generation. The docking was done using the Glide module of Schrodinger in a standard precision (SP) mode. The molecules were ranked after docking on the basis of their glide g-score which utilizes different parameters such as lipophilic terms, hydrogen bond terms, metal ligand interaction, Vander Waals interaction, solvation, π–π, and cation–π interactions to calculate the glide score.
ADMET and BBB studies
Analysis of absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the identified compounds utilizing by Drulito online software. The violation of ideal drug properties such as molecular weight (MW), partition coefficient (logP), octanol-water partition coefficient (AlogP), H-bond donor (HBD), H-bond acceptor (HBA), total polar surface area (TPSA), nHB (number of Hydrogen Bonds), and the number of acidic groups present were evaluated for violation of the Lipinski rule of five and for the ability of the identified compounds to pass through blood brain barrier (BBB) filters.
Results of the biochemical assessments, organ weights, and planimetric studies are expressed as a mean ± standard error of the mean (SEM). Significant statistical differences were determined by one-way ANOVA and student ‘t‘test.