Data collection and preparation
A library of 800 characterized phytochemicals from 17 reported medicinal plants were created [13,14,15,16,17,18,19,20,21,22,23]. The phytochemicals were obtained from literature and downloaded in the Structure data format (Sdf format) from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). Open babel was used to convert phytochemicals from their sdf formats to the protein data bank (pdb) formats. The ligprep command line was used to convert phytochemicals from the pdb formats to the protein data bank charged format (pdqt formats). The structure of COX-2 in complex with a benzopyran, (2R)-6,8-dichloro-7-(2-methylpropoxy)-2-(trifluoromethyl)-2H-chromene-3-carboxylic acid (3NTG with a crystallographic resolution of 2.19 A° was adopted for virtual screening and docking because of its high resolution and a few numbers of co-crystallized compounds when compared with several other COX-2 protein in the PDB) [26] was downloaded from the protein data bank (http://www.rcsb.org). PyMOL plugin in Autodock/Vina was used to extract the receptor. The SWISS-PdbViewer software was used to rebuild missing residues and atoms of the human COX-2 [27]. The experimental animals were randomized based on their body weight and allocated into six groups of five rats each.
Structural modeling of human COX-1 and validation
The human amino acids fasta sequences of COX-1 with accession number AAH29840.1 was obtained from the PubMed repository; this was used to carry out the homology model of COX-1 using the Swiss model server (swissmodel.expasy.org). The model was built on 2OYE [28] template with an identity of 92.8 and X-ray crystallography resolution of 2.8 Å and grid coordinates set as obtained in the co-crystallized compound, x = 251.28, y = 108.48, z = − 39.57. The modeled protein structure was validated using the Procheck server (http://servicesn.mbi.ucla.edu/PROCHECK) [29]. The active site of the modeled protein was predicted with CASTP (sts.bioe.ulc.edu).
Molecular docking and virtual high throughput screening
Virtual high throughput screening (vHTS), a computational technique used to screen a pool of compounds library against the target receptor, was used to screen phytochemicals against the binding pocket of COX-2 [23]. The grid coordinates were set exactly like those of the co-crystallized compound, x = 26.73, y = 21.49, z = 17.16.
Validation of docking results
The co-crystallized ligand was re-docked into the catalytic site of COX-2 to validate the accuracy of the docking protocols [30, 31]. Root means square deviation (RMSD), a measure of the deviation of the co-crystallized from its initial geometry when re-docked, was used to access the deviation. When the value of the RMSD is less than 2.0 Å for the re-docked pose and the co-crystallized pose, the docking protocol is accurate and fit to be used for the docking of other ligands to the receptor [32].
Identification and preparation of plant materials
The Sorghum bicolor grains were purchased from Osiele market, Abeokuta, Ogun State, Nigeria. The plant was taxonomically identified and authenticated at the Department of Botany, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The Sorghum bicolor grains were air-dried in the dark and grounded into powder form prior to extraction and isolation.
Knowledge-based extraction of flavanones from Sorghum bicolor
The vHTS technique employed revealed eriodictyol, a flavanone, as the lead. Hence, flavanones were extracted from the grains of sorghum bicolor using the method described by Bazzocchi et al. [33]. The finely grounded powdery Sorghum bicolor grains (900 g) were extracted with ethanol (5 L) in a soxhlet apparatus for a period of 24 h at around 80 °C. The solvent obtained from soxhlet extraction was evaporated under reduced pressure in a rotary evaporator at ≤ 50 °C. The crude extract obtained was dissolved in 2.5 L of dichloromethane and allowed to stand for 48 h. It was filtered; the filtrate was slowly poured into a separating funnel (2 L) placed onto a metallic stand. The filtrate was allowed to settle into immiscible phases, the organic phase (bottom layer), and the aqueous phase (top layer). The top layer was gently decanted off. The organic phase (bottom layer) was recovered and exposed in the dark for the volatile dichloromethane to escape, leaving the flavanone extract. The extract was weighed and recorded.
Calculations,
$$ {\displaystyle \begin{array}{c}\%\mathrm{Yield}=\frac{\mathrm{weight}\ \mathrm{obtained}}{\mathrm{Total}\ \mathrm{weight}}\times 100\%\\ {}\%\mathrm{Yield}=\frac{24.9\ g}{900\ g}\times 100\%\\ {}=2.77\%\end{array}} $$
Experimental animal
A sample size of 30 male Wistar rats was determined by using the G*Power 3 software for Mac OS [34], with the power set at 0.80, alpha probability at 0.05, and the effect size at 0.25. The Wistar rats weighing between 150 and 250 g were purchased from the Institute for Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan, Nigeria. They were acclimatized for 2 weeks before the onset of the experiment. The animals were housed in a wooden cage with good aeration. The animals were subjected to 12 h of light and 12 h of darkness. The animals were given standard diets and water ad libitum. The study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments in animals. The experimental protocols were also reviewed and approved by the Institutional Animal Ethics Committee (IAEC).
Experimental protocol
The experimental animals were randomized based on their body weight and allocated into six groups of five rats each:
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Group A (basal control): animals received normal chow and water ad libitum.
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Group B (negative control): Animals received 1 ml distilled water p.o for 8 days, followed by a single oral administration of 0.15 M HCl/60% ethanol (ratio 1:1) [35] on the eight day and were sacrificed 5 h after.
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Group C (positive control): Animals received 50 mg/kg body weight Diclofenac p.o for 8 days, followed by a single oral administration of 0.15 M HCl/ 60% ethanol (ratio 1:1) on the eight day and were sacrificed 5 h after.
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Group D (test group): animals received 50 mg/kg body weight Sorghum bicolor extract p.o for 8 days, a single oral administration of 0.15 M HCl/60% ethanol (ratio 1:1) on the eight day, and were sacrificed 5 h after.
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Group E (test group): received 100 mg/kg body weight Sorghum bicolor extract p.o for 8 days, a single oral administration of 0.15 M HCl/60% ethanol (ratio 1:1) on the eight day, and were sacrificed 5 h after.
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Group F (test group): received 100 mg/kg body weight Sorghum bicolor extracts p.o for 8 days and were sacrificed 5 h after.
Note: animals’ chow and water were withdrawn 24 h prior to sacrifice.
Sacrifice, blood collection, and organs excision
The experimental animals were sacrificed through cervical dislocation. The blood samples were collected via abdominal vein and centrifuged at 2500 rpm for 10 min. The supernatants were collected into clean Eppendorf tubes and stored for further biochemical assays. The Livers and the stomachs were also harvested, cleansed of superficial connective tissue in ice-cold normal saline, and later blotted with tissue paper and weighed. The tissues were homogenized for biochemical assays.
Preparation of tissue homogenates
The livers tissues were rinsed in cold sodium phosphate buffer (1:3, w/v), and subsequently homogenized with phosphate buffer, pH 7.4. The homogenate was centrifuged at 4000 rpm for 10 min, after which the clear supernatants were used for the various biochemical assays.
Biochemical assays
Determination of serum alanine aminotransferase activity
The alanine aminotransferase (ALT) activity was determined according to the method described in Lab Kit, Canovelles, Barcelona. One tablet of the substrate was dissolved in 15 ml of buffer, 30 μl of the sample was added to 300 μl of the working reagent, and the absorbance was taken every minute for 3 min at 340 nm [36].
Determination of serum aspartate aminotransferase activity
Aspartate aminotransferase (AST) activity was determined according to the method described in Lab Kit, Canovelles, Barcelona. One tablet of the substrate was dissolved in 15 ml of the buffer and 30 μl of the sample was added to 300 μl of the working reagent and the absorbance was taken every minute for 3 min at 340 nm [36].
Determination of liver superoxide dismutase activity
Superoxide dismutase activity was determined by the method of Zou et al. [37]. The medium for the estimation was prepared as shown in the table below and the reaction was allowed to run for 60 s each time for 3 min before the absorbance was read against the reagent blank at 340 nm.
| Test (ml) | Blank (ml) |
Buffer | 0.10 | 0.15 |
Distilled water | 0.83 | 0.83 |
Sample | 0.05 | -------- |
Incubate at 25°C for 10 min. | | |
Pyrogallol | 0.02 | 0.02 |
Determination of lipid peroxidation
Lipid peroxidation was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the method of Buege and Aust [38].
This method is based on the reaction between 2-thiobarbituric acid (TBA) and malondialdehyde.
Determination of liver reduced glutathione level
The method of Beutler et al. [39] was followed in estimating the level of reduced glutathione (GSH). Then, 0.2 ml of sample was added to 1.8 ml of distilled water and 3 ml of precipitating solution was mixed with sample. The mixture was allowed to stand for approximately 5 min and then filtered. At the end of the fifth minute, 1 ml of filtrate was added to 4 ml of 0.1 M phosphate buffer. Finally, 0.5 ml of Ellman’s reagent was added and the optical density was measured at 412 nm. A blank was prepared with 4 ml of the 0.1 M phosphate buffer, 1 ml of diluted precipitating solution, and 0.5 ml of the Ellman’s reagent.
Reverse transcription-polymerase chain reaction
The total RNA of the liver tissues was isolated using TRIzol reagent (Gibco). The RNA was dissolved with the RNA-free DNase (Roche, Switzerland) for a period of 15 min at a temperature of 37 °C. The RNeasy kit (Qiagen, Germany) was used to purify the RNA. The cDNA was synthesized by incubating 40 μg of the total RNA at 37 °C for a period of 1 h with the reverse transcriptase (GE Healthcare, UK) and also with random hexanucleotides, following the manufacturer’s instructions. The primers were designed using Snap gene software and ordered from Sigma-Aldrich, USA. The primers used to specifically amplify the genes of interest were,
Target gene | Forward 5′-3′ | Reverse 5′-3′ |
COX-2 | CTCAGCCATGCAGCAAATCC | GGGTGGGCTTCAGCAGTAAT |
COX-1 | TTGGAACTTCGAAGCCAT | CTGACAAGAAACAAGAACAAG |
Cyclophilin | TGGAGAGCACCAAGACAGACA | TGCCGGAGTCGACAATGAT |
Cyclophilin is the internal control gene. The genes were amplified for 50 cycles, 2 h and 20 min using the thermocycler. The amplified PCR products were run on 1.0% agarose gels and visualized with the aid of ethidium bromide (EtBr) staining [40].
Histopathology studies
The gastric mucosal tissues from the stomach of the experimental animals, groups A–F, were fixed in 10% buffered formalin (100 ml 37–40% formaldehyde, 4 g sodium phosphate monobasic, and 65 g of sodium phosphate dibasic in 900 ml of distilled water, pH 7.0) for 24 h [41]. The fixative was removed by washing with running water overnight. After dehydration, the tissues were cleaned in methyl-benzoate and embedded in paraffin wax. The section was cut into 3–5 μm thickness and later stained with hematoxylin and eosin. The sections were mounted and observed under light microscope.
Statistical analysis
The results obtained were expressed as the means ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by Turkey’s multiple range tests and one-tailed test statistics were used to analyze the results. Conformation to the assumptions of the ANOVA test was ascertained. P < 0.05 were regarded as being significant, using the IBM-SPSS version 21.0 and GraphPad Prism version 7.0. The ImageJ software was used to quantify the bands from gene expression profiling (Fig. 1).