Characterization and Phospholipase A₂ Inhibitory Potential of Guiera senegalensis Leaves Extracts via Experimental and Computational Approaches

1. Introduction

Plants comprise a huge number of phytoconstituents that synergistically act on different target elements of the complex cellular pathway (Kumar et al. 2013). Plant extracts have great potency and can be used for a variety of purposes. Approximately 80% of the world’s population relies on traditional medicine for health care, and most therapies use plant extracts and their active compounds (Winston, 1999), suggesting that two-thirds of all plant species have medicinal value ( Krishnaiah et al., 2011).

Snake envenoming is a major public health issue in the rural tropics with large numbers of envenoming and deaths (Bawaskar, 2004). The global incidence of snake bites is estimated to be around 5 million (Ameen et al., 2015). The course of snake envenomation is different across several snake species. Some snakes sting their prey without inserting the poison (dry bite) while others inject highly toxic venom through modified salivary glands resulting in death of prey. Dry bite snakes are also considered harmful as their dribble may possess Clostridium tetany which may result in death, if left untreated (Rita et al., 2011). The substitution of herbal drugs with non-pharmacological substances presents a challenge as they may lack therapeutic properties and because products derived from plants are typically variable and influenced by numerous factors, maintaining consistent product quality is crucial for the industry's survival and success (Bauer, 1998). Regulatory agencies and healthcare institutions in Africa have comparatively poor evaluations of herbal medicines compared to orthodox medicine, as opined by Falodun (2010). Thus, the evaluation of the efficacy and safety profile of medicinal plants is crucial when incorporating natural indigenous medicine into the healthcare systems of a particular country (WHO, 1996). Snakebite envenomation remains a common health problem in almost all rural areas of the tropics (Ibrahim et al., 2020). Unfortunately, in most rural areas, modern health facilities are either very poor or completely absent (Sani et al., 2020). Due to lack of hospitals in rural areas people tend to choose herbal medications from Traditional Healers (Gomez et al., 2010). Antisnake venom development is time consuming, expensive and requires ideal storage conditions (Meenatchisundaram et al., 2008). GC-MS analysis will lead to identification of the chemical composition of G. senegalensis leaves (Celestine et al., 2017). Molecular docking studies predict the probable protein–ligand interactions by minimizing the energy of the ligands and calculating their binding energies; docking algorithms utilize inhibitory and activator properties of the ligand with receptor protein and form a relationship between the drug’s structure and cytotoxicity (Divya et al., 2023). Guiera senegalensis (Family: Combretaceae) is a shrub of the savannah region of West and Central Africa (Anka et al., 2020). The plant generally occurs as a shrub that can grow to a height of 3 to 5 m according to habitat. Its stem presents numerous knots that send out branches. The grey-green leaves, darker on their upper surface, display black spots on their lower surface and are slightly downy on both sides. These features lend the plant an overall silver green color that is conspicuous in bush land (Silva et al., 2008). In a study by Sombié (2012) G. senegalensis was shown to have therapeutic effect on diarrhea, cough, bacterial infections, human herpes, malaria; inflammations, snakebite, cancers, arterial hypertension and diabetes. Its leaves extract is being used against dysentery, diarrhea, gastrointestinal pain and disorder, rheumatism and fever (Sule and Muhammed, 2006). Several studies have indicated the presence of alkaloids, flavonoids, quercetin, catechins, saponin, tannins, amino acids, ascorbic acid, anthraquinones and a bitter principle, elastine, in the roots and leaves of G. senegalensis with potential anticancer and other forms of biological activities (Jigam et al., 2011). Their functions and mechanism of actions may include the following among others: antioxidant activity, hormonal action, stimulation of enzymes, interference with DNA replication and antibacterial properties (Sule et al., 2009).

The aim of the study is to conduct phytochemical profiling and evaluate the phospholipase A₂ enzyme inhibitory potential of G. senegalensis leaves extracts via experimental and computational approaches.

2. Materials and Methods

2.1. Sample Collection and Identification

The leaves of G. senegalensis were collected in November 2022 from its natural habitat in Wurno Local government area of Sokoto state (13.2839 ⁰N, 5.4202 ⁰E). The fresh leaves were identified and authenticated by Musa Magaji of the Department of Pharmacognosy and Ethnopharmacy, Faculty of Pharmaceutical sciences, Usmanu Danfodiyo University, Sokoto. Where a Voucher number, PCG/UDUS/Comb/0002 was obtained.

2.2. Preparation of Plant Material

The fresh leaves of G. senegalensis collected were washed and dried in an electro thermal oven at 37 ^o C. The dried leaves were coarsely powdered with an electric blender. The sample was stored in air tight container for further use.

2.3. Extraction

The powdered sample of G. senegalensis (120 g) was defatted with hexane (500 cm³) using sohxlet apparatus at 63 -69 ^oC for 8h after which it was extracted exhaustively with methanol (500 cm³) at 60 – 65 ^oC for 8 h; the extracts were freed from solvents using rotary evaporator at 40 ^oC to obtain Hexane (HE) and Methanol (ME) leaf extracts, respectively. Some parts of ME (18 g) were suspended in distilled water and filtered and the filtrate was successively fractionated with hexane, chloroform, ethyl acetate and butanol using liquid – liquid fractionation to obtain the different fractions coded as HE (hexane), CF (chloroform), EA (ethyl acetate), BU (butanol) and AQ (aqueous), respectively.

2.4. The Gas Chromatography-Mass Spectrometry Analysis (GC-MS)

Analysis was performed on the five fractions( HE, CF, EA, BF and AQ) obtained from methanol leaf extract of G. senegalensis using Agilent Intuvo 9000 GC system coupled with detector system 5977B MSD with split/splitless injector. A DB - 5 MS (5% phenyldimethylsiloxane) fused silica capillary column 30 m, 320 µm i. d., 0.25 µm of film thickness was used with Helium gas (99.99% purity) as carrier gas at flow rates of 1.2ml min^-1 . Inlet temperature was set at 300 ^oC, MS Source at 230 ^oC and MS Quad at 150 ^oC. The oven temperature was programmed as follows: 50 ^oC for 2 minutes, increased to 250 ^oC @ 20 ^oC and hold for 2 minutes and then increased to 300 ^oC at 20 ^oC min^{-1 a} and hold for 3 minutes. Data were acquired by GCMSD/Enhanced Mass Hunter Software and processed GCMSD Data analysis software incorporated with 2017 Version of NIST Library. 1 µL of the sample extract, standard were injected in splitless mode into the GC system using Agilent Automated Liquid Sampler (ALS) G4513A (Vijisaral et al., 2014).

Elucidation of GC-MS chromatogram was done using the databases of NIST- National Institute Standard and Technology and the library of WILEY. The chromatogram of the unknown compounds was compared with the spectrum of already known compounds that are in the NIST and WILEY library. The name of the compound, peak area percentage, molecular formula, molecular weight and structure of the components of the sample were determined.

2.5. Snake Venom

The venom of an adult Naja nigricollis with an LD99 value of 5.75 mg/kg was obtained from Dr. Amina Yusuf Jega, Department of Pharmaceutical and Medicinal Chemistry, Usmanu Danfodiyo University, Sokoto. The venom was collected by the milking method of Markfalane (1967) as previously reported by Abubakar et al. (2003) and Yusuf et al. (2020) and it was lyophilized and stored at 4 ^oC until required.

2.6. Antisnake Venom (ASV)

Standard lyophilized polyvalent snake venom antiserum (African) was used as a positive control for the study. The SVA was manufactured by VINS BIOPRODUCTS LIMITED Survey No. 117, Thimmapur (V) – 509325, Kothur (Mandal), Mahaboobnagar (Dist.) Telangana, India. MFG Date: Jan 2022; Exp. Date: 31^st December, 2024

2.7. Software Used

Chimera 1.14., PyRx Virtual Screening Tool, BIOVIA Discovery studio visualizer 2020, Protein Data Bank (PDB), PUBCHEM, swissADME and ProTox-II online servers.

2.8. Phospholipase A₂ (PLA₂) Enzyme Assay

The N. nigricollis venom PLA₂ activity was determined acidimetrically using the method described by Tan and Tan (1988) with slight modification. In this method, equal volumes of the substrate comprising calcium chloride (18 mM), sodium deoxycholate (8.1mM), and egg yolk were mixed and stirred for 10 minutes to produce a homogenous egg yolk suspension. The pH of the suspension was adjusted to 8.0 by adding sodium hydroxide (1M). 0.1 mg/cm^3. N. nigricollis was added to the above mixture to initiate the process of hydrolysis and saline was used as a control. The initial decrease in the pH of the suspension was measured after two minutes with the help of a pH meter. A decrease of 1.0 pH unit corresponded to 133 μmole of fatty acid released in the egg yolk mixture. The enzymatic activity of PLA₂ was expressed in μmole of fatty acid released per minute (Tan and Tan, 1988). For the antisnake venom activity of G. senegalensis leaf fractions, N. nigricollis venom (0.1 mg/cm³) was pre-incubated with fractions at concentrations of 1, 0.5, 0.25 and 0.125 mg/cm³ respectively for 10 minutes and 30 minutes (modification method) at 37 ^oC to neutralize the hydrolytic action of PLA₂. The inhibitory activity by the fractions against the phospholipase A₂ was calculated and expressed in terms of percentage using the following relationships.

$$Enzyme activity={\displaystyle \frac{\mu mole of fatty acid released}{Time taken in minutes}} \mathbf{E}\mathbf{q}\mathbf{u}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n} \mathbf{1}$$

$$\mathrm{\%} Enzyme activity={\displaystyle \frac{Enzyme activity of the test sample}{Enzyme activity of the control}}\times 100 \mathbf{E}\mathbf{q}\mathbf{u}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n} \mathbf{2}$$

$$\mathrm{\%} Enzyme inhibition=100-Enzyme activity\hspace{1em}\hspace{1em}\hspace{1em}\mathbf{E}\mathbf{q}\mathbf{u}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n} \mathbf{3}$$

2.9. In Silico Molecular Docking Analysis

2.9.1. Ligands Preparation

Molecular docking study was performed on the fifty (50) compounds that were characterized from G. senegalensis. PubChem (https://pubchem.ncbi.nlm.gov/) provided the SDF files for the compounds. PyRx was used to produce the ligands for docking simulations. After starting the program, each compound was imported separately using the "File" menu. The software was launched, and each compound was imported individually via the "File" menu by selecting "Load Molecules" and navigating to the directory containing its structure file. After import, the "Energy Minimization" tool was used to optimize each ligand (the 50 compounds) utilizing force fields such as Gasteiger or UFF in order to generate low-energy conformations. The final optimized structures were transformed into a PDBQT format that could be used for docking in order to guarantee accuracy. Choosing the desired ligand, going to the "AutoDock" menu, and selecting "Make Ligand" were the steps entailed in this conversion. The correctness of the PDBQT files that were generated was then verified. At last, the PDBQT format was utilized to store each ligand by simply right-clicking on their respective entries in the molecular list and choosing "Save as PDBQT." Subsequent docking simulations using PyRx were conducted with these produced ligands after receptor setup and parameter setting (Johnson et al., 2020; Yusuf et al., 2021).

2.9.2. Protein preparation

The N. nigricollis PLA₂ enzyme (Figure 1) as previously modelled by Yusuf et al. (2021) was retrieved and used for this study. Gasteiger charges were added and non-polar hydrogens were merged using AutoDock 4.2. The 3D structure of the protein was prepared using Chimera 1.14 by removing all the water molecules and non-standard residues to alleviate errors and as a clean-up of a PDB file retaining only ATOM and TER records and was modified by adding hydrogens and minimized (Johnson et al., 2020).

2.9.3. In Silico ADMET and Drug-Likeness Prediction

The SwissADME provides an easy way to analyze results in a computer-aided drug designing platform. The ADME (absorption, distribution, metabolism and excretion) were analysed. In addition drug-likeness predictions were performed. The web-based tool provides pharmacokinetics data, physiochemical properties, lipophilicity, water solubility, and drug-likeness, and illustrates the compounds showing whether the ligand can cross the blood–brain barrier and gastrointestinal tract. Similarly protox-II was used to perform toxicity prediction of the compounds (Daina et al., 2016).

3. Results

The extraction and fractionation yield of 120 g of G. senegalensis leaf afforded the following percentages as represented in Table 1

3.1. GC-MS Analysis

GC-MS analysis of G. senegalensis leaves fractions revealed the presence of several compounds. The components were identified based on peak area (%). From the results 14, 4, 14 13 and 13 major compounds were identified for aqueous, hexane, butanol, chloroform and ethyl acetate fractions respectively. The compounds are presented in Table 2, Table 3, Table 4, Table 5 and Table 6.

3.2. PLA₂ Enzyme Assay

The antisnake venom activity of G. senegalensis leaf fractions (hexane, chloroform, ethyl acetate, butanol and aqueous) against PLA₂ enzyme are presented in Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15 and Table 16. The fractions were pre-incubated for 10 and 30 minutes, respectively, after which the antivenom efficacy was assessed. When compared to antisnake venom, there was a considerable (p<0.05) suppression of the enzymes, particularly on the aqueous fraction. After 30 minutes of incubation, a modest reduction in the effects was noticed. The findings showed that the compounds considerably reduced the deadly effects of N. nigricollis in a concentration-dependent manner; after 10 minutes of incubation, the aqueous fraction (74.67%) demonstrated the highest PLA2 enzyme inhibition, while the hexane fraction (35.54%) was the least active.

3.3. In Silico Studies

Four compounds were found to have higher binding affinity than the standards; Kaempferol and Apigenin. The docking scores of the compounds against the protein revealed binding energies ranging from -2.7 to -8.7 kcal/mol (Table 17). The docking scores of the fifty compounds (ligands) from G. senegalensis against Phospholipase A₂ enzyme are presented in the table below:

3.4. Interaction of the Compounds with PLA₂ from G. senegalensis

In order to improve the information extracted from molecular docking results, docked molecules were analyzed regarding molecular interactions as indicated in Table 18. The compounds demonstrated interactions with crucial amino acid residues, including Gly29, Phe5, Asp48, Lys6, Ile9, Tyr62, Arg30, Cys28, Leu2, Trp18, His47, Ala22, Phe21, Phe99 and Tye3. The possible mode of action for the inhibitors against the PLA₂ was observed via these molecular interactions.

2D and 3D-View of the Interactions

Figure 2. (a) 2D view of the molecular interaction of Bis (m-phenoxyphenyl) ether with PLA2 enzyme. 3D View of the molecular interaction of Bis (m-phenoxyphenyl) ether with PLA2 enzyme showing (b) aromatic Interactions (c) hydrogen bond interactions.

Figure 2. (a) 2D view of the molecular interaction of Bis (m-phenoxyphenyl) ether with PLA2 enzyme. 3D View of the molecular interaction of Bis (m-phenoxyphenyl) ether with PLA2 enzyme showing (b) aromatic Interactions (c) hydrogen bond interactions.

Figure 3. (a) 2D View of Molecular Interaction of 4, 4’-methylenebis (2, 6-dimethyl) Phenol with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) H-Bond Interactions.

Figure 3. (a) 2D View of Molecular Interaction of 4, 4’-methylenebis (2, 6-dimethyl) Phenol with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) H-Bond Interactions.

Figure 4. (a) 2D View of Molecular Interaction of Bis (p-phenoxyphenyl) ether with PLA2. 3D View of the molecular interaction of Bis (p-phenoxyphenyl) ether with PLA2 enzyme showing (b) aromatic Interactions (c) hydrogen bond interactions.

Figure 4. (a) 2D View of Molecular Interaction of Bis (p-phenoxyphenyl) ether with PLA2. 3D View of the molecular interaction of Bis (p-phenoxyphenyl) ether with PLA2 enzyme showing (b) aromatic Interactions (c) hydrogen bond interactions.

Figure 5. (a) 2D View of Molecular Interaction of 6-(4-ethyl phenyl)-2-phenyimidazo [1, 2-b] [1, 2, 4] triazine with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) hydrogen bond.

Figure 5. (a) 2D View of Molecular Interaction of 6-(4-ethyl phenyl)-2-phenyimidazo [1, 2-b] [1, 2, 4] triazine with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) hydrogen bond.

Figure 6. (a) 2D View of Molecular Interaction of 2-methyl -7-phenylindole with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) hydrogen bond.

Figure 6. (a) 2D View of Molecular Interaction of 2-methyl -7-phenylindole with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) hydrogen bond.

Figure 7. (a) 2D View of Molecular Interaction of 3, 4-nitrophenylamino Indole with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) hydrogen bond.

Figure 7. (a) 2D View of Molecular Interaction of 3, 4-nitrophenylamino Indole with PLA2 enzyme. 3D View of the molecular interaction showing (b) aromatic Interactions (c) hydrogen bond.

4.5. In Silico ADME and Drug-Likeness

ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) plays a pivotal role in drug discovery and development (Norinder and Bergstrom, 2006). Understanding the ADMET properties of a compound is crucial for predicting its behavior in the body, identifying potential drug candidates, and reducing the risk of clinical failures; the studies provide vital information on how a drug is absorbed, distributed, metabolized, and excreted, as well as its potential toxicity, allowing drug developers to assess the safety and efficacy of a drug candidate. The ADME and Drug-likeness of the compounds are presented in Table 19.

3.5. Toxicity Profile

The toxicity predictions of the compounds are presented in Table 20; none of the compounds showed indication of hepatotoxicity, carcinogenicity, Immunotoxicity, cytotoxicity and mutagenicity. Similarly only 6-(4-ethylphenyl)-2 phenylimidazo [1, 2-b] [1, 2, 4] triazine has the potential to be neurotoxic.

4. Discussion

The methanol extract at 60-65 ⁰C has greater yield (19.04 %) than extraction with hexane (5.35 %) at 63-69 ⁰C (Table 1). The efficiency of methanol as a solvent used for extraction of the plant might be due to it being an organic polar solvent. The methanol polarity makes it able to have strong interactions with polar substances (Lokeswari et al., 2011). The color of extract observed were brown in methanol and yellowish green in hexane extract. The color of fractions observed were yellowish green in hexane, light green in chloroform, reddish brown in ethyl acetate, brown in butanol and light brown in aqueous fraction (Table 1).

The compounds present in the fractions of G. senegalensis were identified by GC-MS analysis. Fifty compounds which were the major components were identified. The active principles with their molecular formula and concentration (% Area) in the methanol fractions of G. senegalensis leaves are presented in Table 2, Table 3, Table 4, Table 5 and Table 6.

The result revealed that Dodecanoic acid, methyl ester (5.31%), 4-Cyclopropylmethylbenzonitrile (5.48%), Methyl tetradecanoate (3.09%) in aqueous fraction are the major compounds. On the other hand Hexadecanoic acid, methyl ester (13.03%), n-hexadecanoic acid (14.14%), 10-Octadecenoic acid, methyl ester (5.07%), Methyl stearate (3.27%), Tricosanoic acid (3.42%) were prevalent in hexane fraction. In addition 2-methyl-1-methylmannopyranoside (1.99%), 1H-Indole-3-carboximidamide (1.94%), 1, 6-anhydro .beta.-D-Glucopyranose (7.03%) were the major compounds in Butanol Fraction. Hexachloro-ethane (3.09) in chloroform fraction. 2-Methoxy-5-methyl thiophene (2.09%), 4, 4’ –methylenebis (2, 6-dimethyl) Phenol (2.84%), Methyl.beta.-D-glucopyranose(2.13%), 2-(1,3-benzodioxol-5-yl)methoxy-5-Benzofuranpropanol (13.98%), 6-(4-ethylphenyl)-phenyl Imidazo([1, 2-b] 1,2,4-triazine (13.17%) and 2-Methyl-7-phenylindole(6.68%) in ethyl acetate fraction were found as the major compounds.

Among the identified phytochemicals n- Hexadecanoic acid have the property of antioxidant activity as reported by earlier studies (Lalitharani et al., 2009; Maruthupandian and Mohan, 2011). All the compounds have previously been reported from a number of other plants species.

It is well known that GC-MS can reveal the composition and concentration distribution of compounds that are identifiable when analyzing unidentified components of plant origin (Liang et al., 2010).

Beta-D-Glucopyranose, 1, 6-anhydro- shows antibacterial, and antioxidant activity (Igwe et al., 2013).

Furthermore, n-Hexadecanoic acid was reported to have antioxidant, hypocholesterolemic, nematicidal, pesticidal, hemolytic, 5-alpha reductase inhibitor, anti-androgenic, hemolytic (Kumar et al., 2010), and anti-inflammatory activity (Aparna et al., 2013).

Hexadecanoic acid, methyl ester possess some biological activity such as antioxidants, hypocholesterolemic, nematicide and pesticide (Sheela et al., 2013).

Febri et al. (2016) reported the antibacterial activity of Dodecanoic acid against Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium and Escherichia coli at a concentration of 5%. This is consistent with a similar study conducted by Moigradean et al. (2013) which indicated the presence of Hexadecanoic acid which is also called as Palmitic acid which is also present in walnut and coconut oil, and has anti-bacterial property (Yff et al., 2002).

Bis (p-phenoxy phenyl) ether was found to possess anti-androgenic activity (Abeer et al., 2017). Studies have shown that 2-(1, 3-benzodioxol-5-yl) methoxy-5-benzofuranpropanol have good antioxidant activity (Rhinde et al., 2010).

6-(4-ethylphenyl)-2-phenylimidazo [1, 2-b] [1, 2, 4] triazine possess antituberculosis (Gong et al., 2015), antioxidant, antiviral, antitumor (Hamama et al., 2016), antifungal (Lafleur et al., 2011) and antiparasitic (Thompson et al., 2020) activities. The prevailing compounds found in the leaf of G. senegalensis fractions are lipid components. Fatty acids and derivatives in plant extracts have been widely reported to inhibit cancer cell proliferation by induction of apoptosis (Alasmari et al., 2015. Leyva-Peralta et al.,2015).A clear example of this was the extract from Euphorbia kansui, which contained a high amount of octadecenoic acid, methyl ester as the major compound that triggered apoptosis in human gastric cancer cell line (Ismail et al., 2019).

All the major compounds from the different fractions are biologically active molecules. Considering these findings, the traditional uses of the leaves appeared to be justified.

The study of G. senegalensis fractions' antisnake venom effectiveness demonstrated the plant's ability to suppress PLA₂ enzyme activity. After 10 minutes and 30 minutes of incubation, respectively, N. nigricollis venom released 122.36 μmol/FA and 119.70 μmol/FA, respectively, with 100% enzyme activity. In contrast, standard antisnake venom produced 14.63 μmol/FA with good inhibition (82.3%) of PLA₂ enzyme activity after 10 minutes of incubation.

After 10 minutes, Table 4.19 showed that the aqueous fraction at the lowest concentration (0.125 mg/cm³) produced the most percentage inhibition (74.67%), while the highest concentration (1 mg/cm³) produced the lowest percentage inhibition (66.18%).

Furthermore, the lowest concentration (0.125 mg/cm³) of Butanol fraction produced the highest percentage of inhibition (69.93%) while an inhibition of (60.78%) was recorded by 1 mg/cm³ concentration (1 mg/cm³) after 10 minutes (Table 4.17).

For hexane fraction, the lowest concentration (0.125 mg/cm³) produced the highest percentage inhibition (35.54%) while the concentration (1 mg/cm³) produced (5.23%) inhibition (Table 4.11) after 10 minutes. Chloroform fraction at the least concentration (0.125 mg/cm³) demonstrated highest percentage inhibition (50.49%) while the concentration (1 mg/cm³) produced the lowest percentage inhibition (23.29%) (Table4.13) after 10 minutes.

Similarly, after 10 minutes, the concentration of Ethyl acetate fraction at the lowest level (0.125 mg/cm³) provided the highest percentage inhibition (62.50%), while the concentration at the highest level (1 mg/cm³) gave the lowest percentage inhibition (46.24%) (Table 4.15).

The percentage inhibition for all the fractions after 30 minutes exhibited significant increase in enzyme activity compared to the 10 minutes incubation results, hence they have lower percentage enzyme inhibitions. Interestingly, higher concentrations of all the compounds resulted in lower inhibitions. The findings showed that the fractions, in a concentration-dependent manner, significantly reduced the deadly activities of N. nigricollis venom.

Therefore, this pattern of inhibition at low concentration may be attributed to their high potency in effectively detoxifying the potent toxic effect of PLA₂ enzyme. The findings aligned with those reported by Abubakar et al. (2000), which showed that, the extract of the leaves of G. senegalensis was found to detoxify (in vitro) venom from two common northern Nigerian snake species, E. carinatus and N. nigricollis, in separate experiments. Thus, supporting the present studies. It’s important to note that the effectiveness of the compounds might vary depending on the snake venom composition.

To understand how the compounds interact with the snake venom enzyme PLA₂, computer simulations (molecular docking) was performed and the findings are shown in Table 17.

Bis (m-phenoxyphenyl) ether (-8.7 kcal/mol) and 4, 4-methylenbis, 2, 6-dimethylphenol (-8.6 kcal/mol) exhibited higher binding affinity compared to Kaempferol (-8.4 kcal/mol) and Apigenin (-8.4 kcal/mol) that were used as standard ligands. Thus, the compounds showed a favourable binding affinity to the PLA₂ enzyme as outlined by the docking scores in Table 17.

The binding energy for the best pose against phospholipase A₂ enzyme ranged from -8.7 to -8.1 kcal/mol. This suggests they can interact with key amino acids such Trp18, Tyr62, Gly29, Phe99, Cys44, Asp48, Arg30 (Table 18) crucial for enzyme function (Murakami & Kudo, 2002). Anti-snake venom activity of nitrogen containing compounds have been reported (Cesar et al., 2014), which might explain the good inhibition of PLA₂ enzyme of the compounds. The compounds from G. senegalensis exhibited molecular interactions such as conventional hydrogen bond, Van der Waals, pi-sigma, pi-alkyl, pi-pi T-shaped, carbon hydrogen bond and amide pi stacked (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7). These interactions contribute significantly to the higher docking scores observed for the compounds compared to the standard ligands Kaempferol and Apigenin. The strength and type of intermolecular interactions determine the binding affinity and selectivity of the ligand to the protein in molecular docking (Fan et al., 2019).

Despite the PLA₂ inhibitory potential exhibited by the G. senegalensis compounds, their ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties are crucial factors that will determine their therapeutic effectiveness in treating snake envenomation. In Silico ADMET prediction offers a rapid and cost-effective approach to evaluate whether compounds will be readily absorbed, efficiently distributed to their target site of action, favourably metabolized, and easily eliminated from the body without causing toxic side effects (Ntie-Kang, 2013). The properties of the compounds can be seen from Table 19 revealing their solubility, lipophilicty, physicochemical and pharmacokinetics.

The Lipinski filter has proven effective in screening potential drug candidates for oral drug-likeness based on their molecular weights, hydrogen bond acceptors and donors, and lipophilicity (Belal, 2018). The ADME and drug-likeness properties of the best four compounds from G. senegalensis were assessed using swissADME, with the analysis performed based on Lipinski's rule of five (RO5) and Veber's rule (Yusuf et al., 2021). These rules evaluate whether a compound is likely to be membrane permeable and readily absorbed via passive diffusion in the human intestine. Lipinski's rule of five helps determine if a biologically active chemical or drug is likely to have chemical and physical properties conducive to oral bioavailability, based on the following criteria: no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, molecular weight less than 500 Da, and partition coefficient not greater than 5 (Lipinski, 2004; Yusuf et al., 2021).

Veber's rule states that for a drug to be a good candidate, it should have a polar surface area (TPSA) ≤140 and a rotatable bond count (RB) ≤10 (Yusuf et al., 2021). The compounds adherence to these rules indicates that they have passed the absorptivity test for oral medication. The 0.55% bioavailability score of the compounds means that the compounds has about a 55 % probability of a minimum of 10 % oral absorption in rat or human colon carcinoma cells (Johnson et al., 2022).

CYP enzymes were shown to be inhibited by 4, 4' –methylenebis [2, 6-dimethyl] Phenol (CYP2C9), Bis (m-phenoxyphenyl) ether (CYP3A4), and Bis (p-phenoxyphenyl) ether (CYP3A4). Fascinatingly, 6-(4-ethylphenyl)-2-phenylimidazo [1, 2-b][1,2,4]triazine did not exhibit CYP enzyme inhibition, suggesting a decreased possibility of medication interactions (Daina, 2017). The calculated total polar surface area (TPSA) was within the range 27.69 -43.08A². The hydrophilicity of the compounds determined by calculating the log p value indicated that the compounds have good absorption in the digestive tract (Table 19). Thus, higher log p values result in poor absorption (Yusuf et al., 2022). Aromatic interactions and hydrogen bond donors and acceptors as evident in Figures 2 - 25, provide a valuable insight into the binding mode and potential mechanism of action of the compounds; thus, it assisted in pinpointing the specific functional groups or residues crucial ligand- PLA₂ binding (Brinkley and Gupta, 2001; Hunter et al., 2001).

The toxicity predictions for the best four compounds from G. senegalensis appeared favourable (Table 20). The oral toxicity prediction for Bis (m-phenoxyphenyl) ether and, 4, 4’ –methylenebis [2, 6-dimethyl) Phenol and Bis (p-phenoxyphenyl) ether falls under toxicity class 5,while 6-(4-ethylphenyl) - [1, 2-b] 1, 2, 4-triazine-2-phenyl imidazo falls under toxicity class 3, with predicted LD₅₀ values of 3140,3430, 2460 and 3,000 mg/kg, respectively, indicating that they are slightly toxic. Neither compound showed any indication of hepatotoxicity or cytotoxicity; however, 6-(4-ethylphenyl) - [1, 2-b] 1, 2, 4-triazine-2-phenyl imidazo has the potential to be neurotoxic.

5. Conclusion

From the study, phytochemical analysis showed the presence of secondary metabolites. The identified metabolites are believed to be responsible for the plants therapeutic activity.

The G. senegalensis extract exhibited concentration dependent antisnake venom activity by inhibiting the lethal actions of N. nigricollis venom using an in vitro model. Aqueous fraction demonstrated best inhibitory activity although all the fractions were less potent than the standard antivenin. Four compounds were identified by the molecular docking scores to have better binding affinities than the standards; Kaempferol and Apigenin, which suggest that they have good binding affinity to the target. Furthermore, In Silico ADME and Drug-likeness assessments of the compounds using swissADME revealed that 4, 4’ –methylenebis [2, 6-dimethyl) Phenol and 6-(4-ethylphenyl)-2-phenylimidazo[1,2-b][1,2,4]triazine are a good drug candidates, as they did not violate any of the Lipinski's rule of five and Veber's rule. Toxicity predictions revealed that the four compounds analyzed are relatively non-toxic.