Materials and Methods

Drugs, chemicals and kits

Drugs: Scopolamine was purchased from Cooper (Copper, France) and donepezil from Biwole Abondo Pharmacy (Yaounde, Cameroon).

Chemicals: All the chemicals used were of analytical grade and purchased from Sigma Co., Louis, MO, USA.

Plant material, harvesting and processing

Terminalia macroptera (T. macroptera) barks were collected in Baligui in January 2021 (Centre, Cameroon) and identified at the National Herbarium of Cameroon, Yaoundé (in comparison with specimen N° 43688/HNC). The barks were washed, dried, and ground to obtain a powder, which was stored in opaque vials. This powder was used to prepare the TT-TeMac™.

Preparation of the TT-TeMac™

It was carried out by optimizing the protocol of Adey et al. (2021). 1000 g of bark powder was extracted with 3.5 L of methanol. The extract was concentrated using a rotary evaporator, yielding a blue-black solid mass of the methanol bark extract. A mass of 184.2 g of extract with a yield of 18.42% was obtained. The T. macroptera bark extract (100 g) was reconstituted in 300 mL of distilled water and subjected to liquid-liquid partition. The reconstituted extract was placed in a separating funnel, and 300 mL of hexane was added sequentially as a 1:1 (v/v) solution and stirred. The sample was allowed to stand for 60 min in the separating funnel until a thin separation line appeared, clearly indicating the supernatant above the sediment before desorption. This process was repeated until exhaustion, and the hexane fraction was collected and concentrated using a rotary evaporator. The same procedure was repeated sequentially with ethyl acetate and n-butanol to obtain the ethyl acetate fraction, n-butanol fraction and residual water fraction, respectively. The concentrated ethyl acetate fraction constituted the TT-TeMac™ and was used for further work.

Determination of total terpenoid content

Terpenoids were measured using the method of Ghorai et al. (2012). Two hundred microliters (200 µL) of TT-TeMac™ (4 mg/mL) were added to 1.5 mL of chloroform. The mixture was homogenized using the vortex. Then 100 µL of sulfuric acid 6 N was added to each tube (white, trial and standards). The mixture was cooled using an ice pack for 10 min. Absorbance was measured at 538 nm after 2 h of incubation at room temperature in the dark. Calibration was performed using oleanolic acid (0–1000 μg/mL).

Determination of total tannins content

The method of Medini et al. (2014) was used to estimate the tannin of TT-TeMac™. 1 mL of extract (4 mg/mL) was mixed with 5 mL of the working solution (50 g of vanillin plus 4 mL of 1 N HCl in 100 mL of distilled water) and the mixture was incubated at 30°C for 20 min. Absorbance was read at 500 nm against blank. Tannic acid (0–1000 μg/mL) was used as standard.

Identification of terpenoids and Tannins TT-TeMac™

Terpenoids and tannins were identified by liquid chromatography-mass spectrometry (LC-MS) as previously described by Teixeira et al. (2016). Raw LC-MS spectra data were converted with MSconvert, processed and analyzed with MZmine 2.53 software for Windows.

Computational Study

Network pharmacology analysis

Identification of potential targets of active compounds

Structural on active compounds of TT-TeMac™ were drawn from chemdraw Professional 15.0 (version 15.0, PerkinElmer Informatics, USA). Potential human target genes of TT-TeMac™ compounds were identified using the SwissTarget Prediction (http://swisstargetprediction.ch/) and PharmMapper (http://www.lilab-ecust.cn/pharmmapper/) databases. Duplicate genes were removed using Microsoft Excel to obtain the final list of target genes.

Identification of potential targets of cholinergic dysfunction

Potential human target genes associated with cholinergic dysfunction were harvested from online databases, including GeneCards (http://www.genecards.org/), DisGeNET (https://www.disgenet.org/).

The keyword “Cholinergic Dysfunction” was used to perform the search, and duplicate genes were removed to obtain a comprehensive list of genes associated with human cholinergic dysfunction.

Construction and analysis of the protein-protein interaction (PPI) network

Common target genes were obtained using Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). Common target genes were submitted to the STRING database to construct a PPI network. To ensure data reliability, a confidence score ≥ 0.4 was set for target acquisition with “Homo sapiens”. PPI results were imported into Cytoscape software for network generation and analysis. Significant genes were identified using the CytoHubba plugin, based on three parameters: maximum clique centrality (MCC), maximum neighbourhood component (MNC), and degree. The ten most significant genes were identified based on their scores. The data was imported into a Venn and an intersection was constructed to identify likely hub genes in the network.

Molecular Docking

MEO (version 2014.0901, Chemical Computing Group Inc., Montreal, QC, Canada) software was used to analyse the interactions between TT-TeMac™ compounds and the identified hub targets. The three-dimensional structures of the target proteins; were obtained from the RCSB Protein Data Bank in PDB format based on human origin and their high resolutions.

The docking study was completed carried out as such; (1) water molecules were removed from the protein structure; (2) hydrogen atoms with standard geometry were added to the structure, broken bonds were reconnected, the potential was fixed; (3) large sites in the enzyme structure were identified using the MEO Alpha Site Finder, and dummy atoms were generated from the resulting alpha spheres; (4) the interaction of the ligand with the active sites of amino acids was analysed. The ligand binding affinity was evaluated using the scoring function; dock function (S, Kcal/mol) created by the MOE 2014 software (Al-Karmalawy et al., 2021). Active ligands with the highest docking score have the most negative values.

Experimental animals

To validate the in silico results, an experimental study was carried out on male Wistar rats treated with scopolamine

Ethical approval

Animals were treated following the guidelines of the ethical committee of the University of Yaoundé 1 (BTC-JIRB2024–101) and the Guide of the Care and Use of Laboratory Animals (8th edition). The number of animals has been reduced to the bare minimum and the protocols have been optimized to limit animal suffering.

Experimental design

Twenty (20) male Wistar rats, weighing between 230 and 240 g, were obtained from the animal house of the Laboratory of Nutrition and Nutritional Biochemistry, Department of Biochemistry, Faculty of Science, University of Yaoundé 1. The animals were housed under controlled conditions with a constant temperature of 25 ± 2 °C, relative humidity of 50 ± 5%, and a 12/12-hour light/dark cycle. They had free access to food and water.

Animals were divided into 4 experimental groups of 5 rats each and treated as follows:−(1) Normal Control group received distilled water (5 mL/kg bw/day; po)−(2) SCO + H2O group received scopolamine (1 mg/kg bw/day, ip) and distilled water (5 mg/kg bw/day; po)−**(**3) SCO + TT-TeMac™ group received scopolamine (1 mg/kg bw/day; ip) and TT-TeMac™ (100 mg/kg BW/day; po)−(4) SCO + Done group received scopolamine (1 mg/kg/bw/day; ip) and donepezil (5 mg/kg bw/day; po).

Scopolamine was administered daily by intraperitoneal injection (1 mg/kg b.w) for 7 days to induce cognitive decline. The TT-TeMac™ and Donepezil were administrated by oral intubation through an esophageal tube 60 min after scopolamine injection. TT-TeMac™, donepezil, and distilled water were administered at a dose of 5 mL/kg b.w. The novel object recognition test was assessed on days 1, 2 and 3; and Morris Water Maze test on days 4, 5, 6, 7 and 8.

Animal Sacrifice

At the end of the study, after a 12-hour fast, the rats were deeply anesthetized with ether, blood was collected by cardiac puncture, until the animals were euthanized by exsanguination, and their brains were carefully removed after cervical incision. Their brains were weighed and part was used to prepare homogenates and the other part to perform histopathological examinations.

At the time of the study, the use of ether had been approved (BTC-JIRB2024–101) by the Institutional Ethics Committee of the Faculty of Sciences at the University of Yaoundé 1. This ethics committee follows the guidelines of the National Committee for the Protection and Use of Animals regarding animal euthanasia. However, the researchers acknowledge that the use of ether is no longer recommended and that safer and more humane methods will be used in future studies**.**

Behavioural analysis

Novel Object Recognition (NOR)

The novel object recognition test is used to determine learning, memory, recognition. In this study, the NOR test was performed in an Open Field device following the protocol adapted by Djiogue et al. (2018). The test consisted of 3 phases: habituation or pre-training, training, and trial or testing. During habituation phase, took place on the first day, each animal was placed in the field to explore the empty device box for 5 min and thereafter returned in the initial cage. During the training phase, animals were placed in the field for 5 min to explore two identical objects. The test was performed on the second day, 3 h after the training phase, to assess short-term memory, and on the third day, 24 h after training, to assess long-term memory. Exploration was defined as when the rat moved its snout toward or touched an object. The time spent exploring familiar and novel objects was recorded using a video recording system consisting of a camera positioned above the workspace and connected to a computer. After each trial, the box and objects were cleaned with a 70% ethanol solution to prevent any disturbance. The videos were analysed with the Any-Maze 7.1 software. The recognition index (RI) at short, and long-term memory was obtained. RI= (time spent exploring the new object) / (time spent exploring the new object +time spent exploring the familiar object)

Morris Water Maze (MWM)

The MWM test provides an accurate and reproducible measure used to evaluate spatial learning and memory; as well as hippocampal damage (Morris, 1984). In the present study, the MWM test was performed in a black circular tank (diameter 120 cm × height 50 cm) half filled with water and divided into four equal quadrants. A black drainage platform was placed in a fixed position (south quadrant of the apparatus), submerged 1.0 cm below the surface of water. The test included a 4-day acquisition phase and a retention phase on day 5. The acquisition phase consisted on three trials per day with 15-minute break between trials. At the end of each trial, the animals were properly cleaned and returned to their home cages. During the acquisition trial, animals were left to explore the pool searching for the hidden platform. Once the animal located the platform, it was left there for 10 s. However, if an animal failed to reach the platform within 60 s it was guided there and kept for 10 s. During the retention phase, the platform was removed, and each rat was placed into the pool at one of the fixed targets facing the target quadrant and had 60 s to swim. All the parameters were recorded using a video recording system with a camera placed above the pool and connected to a computer. The videos were analysed with the Any-maze 7.1 software. The latency time (s) to reach the exact position of the platform and the number of entries in the target quadrant of the platform were expressed.

Histopathological examination

One of the hemispheres of brain of each rat was fixed in 10% formaldehyde, embedded in kerosene, cut (5 µm sections in the coronal plane), and processed for hematoxylin-eosin and Cresyl violet staining using standard procedures (Garman, 2011). Brain tissue damage was assessed in the hippocampal formation using a microscope connected to a camera (Axioskop 40, Zeiss, Hallbermoos, Germany). In addition, neurons in Cornus Ammonis (CA) regions 1, 2, 3 (CA1, CA2, CA3), and Dentate gyrus (DG) were counted using Image J software (version 1.52a).

Preparation of brain homogenate

The brain tissues were homogenized in 10% (w/v) ice-cold phosphate buffer saline, and centrifuged at 1500_g_ for 10 min at 4°C. The brain homogenate (the supernatant) was immediately collected and stored at - 80°C for the brain cholinergic transmission evaluation.

Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) activities assays

Brain AChE and BuChE enzyme activities were determined using Ellman’s method. Acetylthiocholine iodide/S-butyrylthiocholine chloride was used as a substrate for AChE/BuChE. Thiocholine, produced forms a yellow complex with 5,5-dithiobis (2-nitrobenzoic acid). The absorbance was measured at 412 nm. The results are expressed in µmol/min/mg of protein (Ellman et al., 1961).

Total proteins determination

Protein estimation was carried out by the method described by Lowry et al. (1951).

Statistical analysis

GraphPad Prism software version 10.2.2 was used for statistical analysis. Normality test was confirmed by Shapiro-Wilk test, one-way analysis of variance (ANOVA) with Tukey test was performed for comparison between groups. Results are expressed as mean value ± standard deviation (n = 5 for in vivo tests). P values < 0.05 were considered significant.