Skip to main content
BMC is moving to Springer Nature Link. Visit this journal in its new home.

Abstract

Background

Echis ocellatus is a highly venomous snake that can cause serious medical complications due to the presence of toxic proteins in its venom. These proteins, such as echicetin and phospholipase A2 (PLA2), often cause severe pathophysiology in snakebite victims. Ganoderma lucidum is recognised for its pharmacological benefits against various diseases. However, the potential of this fungus as an antivenom has not yet been reported.

Objective

This study investigated the inhibitory effects of G. lucidum on haemorrhagic and anticoagulant activities induced by E. ocellatus venom, and identified its possible bioactive inhibitor compounds using in vitro, in vivo, and in silico methods.

Methods

Ganoderma lucidum was extracted using methanol in a standard procedure, and varying doses (20 and 40 mg) of the extract were tested against the biological activities E. ocellatus venom. Thereafter, the extract of the G. lucidum was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to identify its bioactive compounds. The identified compounds were docked against the catalytic active sites of echicetin and PLA2 proteins to determine the best inhibitor compound. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties of compounds were determined using the ADMETlab 2.0 web tool.

Results

The extract caused 62.96 ± 1.03% and 59.25 ± 1.59% venom-induced haemorrhage inhibition at doses of 20 and 40 mg, respectively, while plasma clotting times were shortened to 132 and 163 s at 20 and 40 mg, respectively. The GC-MS identified 29 bioactive compounds from G. lucidum extract, out of which hesperidin had the highest docking scores of – 9.3 kcal/mol and – 9.9 kcal/mol against the catalytic sites of echicetin and PLA2 enzymes, respectively.

Conclusion

The results indicate that G. lucidum has antivenom potential against E. ocellatus venom-induced toxic activities, and identified hesperidin as a promising compound for antivenom exploration against viper envenoming.

Graphical Abstract

Peer Review reports

Introduction

Snakebite is increasingly gaining global recognition as a neglected disease of significant public health importance [1]. Globally, the number of snakebite cases is estimated to be 4.5 million annually, out of which 1.8–2.7 million result in envenoming, with a mortality rate ranging between 81,000 and 138,000 [2, 3]. In low- and middle-income tropical regions, sub-Saharan African countries account for the highest burden of snakebite envenoming [4]. One of the most dangerous and venomous snakes implicated in snakebite envenoming in Africa is Echis ocellatus, commonly known as the West African Carpet Viper. It belongs to the Viperidae family and accounts for 66% of the total number of snake envenomings in Nigeria [5]. The venom of E. ocellatus is heterogeneous. It is composed of approximately 18 different protein families, primarily including metalloproteases, phospholipase A2 (PLA2), and serine proteases [5]. These venoms are toxic, and their release during a snake bite leads to systemic and local pathological effects such as haemorrhage, tissue damage, neurotoxicity, oedema, and uncoagulated blood formation [6,7,8,9]. Snake venom exerts haemorrhagic effects in different ways. For instance, zinc-dependent snake venom metalloproteases in the Viperidae family exert their haemorrhagic properties by directly degrading blood vessel components such as collagen type IV, laminin, and nidogen, thereby leading to bleeding [9,10,11]. However, in another member of Viperidae, Echis carinatus, a heterodimeric C-type lectin, echicetin, and PLA2 cause coagulopathy and bleeding by inhibiting platelet aggregation and the activation of clotting factors [12].

Antivenoms have been effective in neutralising snake venoms. However, several factors, such as the high cost of production, limited availability, unaffordability, cold chain storage requirements, and the risk of anaphylactic and pyrogenic reactions, have limited the success of antivenom therapy and necessitated the need for alternative therapies [13, 14]. Moreover, several studies have reported the efficacy of plant extracts, their bioactive compounds, and peptides against the toxic biological activities of snake venoms [3, 15,16,17,18,19]. However, there is a paucity of data on products of fungal or mushroom origin as antivenoms. Mushrooms are repositories of bioactive compounds that exhibit pharmacological properties, including antioxidant, immune-modulating, antimicrobial, anticancer, and antidiabetic effects [20, 21]. One of the medicinal mushrooms that has been reported is Ganoderma species. Mushrooms of the genus Ganoderma, especially G. lucidum, are known to possess bioactive compounds with different pharmacological properties such as antioxidant, anti-inflammatory, antiangiogenic, antimicrobial, anti-HIV, antitumor, antihepatotoxic, hepatoprotective, immunomodulatory, and cardioprotective [22, 23]. Additionally, the platelet aggregation effect of G. lucidum has been reported [24], suggesting that bioactive compounds of Ganoderma have the potential to modulate coagulation pathways for platelet formation and prevention of haemorrhagic disorders.

Computational-aided drug discovery processes are valuable in the rapid identification of natural compounds with antivenom properties and inhibitors of snake venom proteins [25, 26]. By studying the molecular interaction between compounds (ligands) and venom proteins (receptors) via molecular docking, it is possible to understand the mechanism of action of natural compounds or obtain potential lead compounds for in vitro studies [27,28,29]. Hence, the application of in vitro and in silico methods provides both experimental and mechanistic insights for validating compounds with potential inhibitory properties against venom proteins. This study aims to evaluate the anti-haemorrhagic and plasma clotting effects of G. lucidum extract on E. ocellatus venom in vivo and in vitro, respectively, and to identify the major inhibitory bioactive compound in the extract using computational approaches.

Materials and methods

Ethics statement

The ethical approval for the animal use in this study was obtained from the Osun State Health Research Ethical Committee (OSHREC), Osogbo, Osun State, Nigeria, with the authorised number: OSHREC/PRS/569T/1247. All experimental protocols in animal handling followed the rules and guidelines established for the care and use of laboratory animals in compliance with the revised ARRIVE guidelines 2.0.

Mushroom collection and identification

Mushroom basidiome exhibiting typical morphological features of G. lucidum was collected from a decomposing hardwood tree within the premises of Osun State University, Osogbo, Nigeria (7.7717° N, 4.5569° E). The mushroom displayed characteristic traits, including a reddish-brown cap with white-striped edges, a varnished surface, a kidney-shaped form, and a white to dull brown underside with fine pores (Plate 1a and b). Preliminary field identification was performed using the mobile application "Picture Mushroom", which tentatively identified the specimen as Ganoderma by matching uploaded images to an online database of fungal images. Further identification was carried out by comparing the morphological features of the specimen with descriptions and illustrations from standard mycological references [30,31,32,33]. The mushroom was confirmed and authenticated as G. lucidum by Dr. Olatunji Olusanya of the Department of Plant Biology, Osun State University, Osogbo, Nigeria.

Plate 1

The morphological appearance of the collected G. lucidum (a) The ventral part of G. lucidum showing the lacquered or shiny brown surface with whitish edges. (b) The whitish dorsal portion of G. lucidum with a brown stalk

Methanolic extraction of G. lucidum

Methanolic extraction of G. lucidum was performed using the Soxhlet method [34]. The Soxhlet extractor was filled with a thimble containing a 10 g powdered sample. The methanol (Sigma-Aldrich, USA; ≥99.9% purity; CAS number: 67-56-1)-water mixture (ratio 1:1) was put into a 500 mL round-bottom flask fitted with a condenser and connected to the Soxhlet extractor. The extractor was placed on a heating mantle to heat the solvent, which then began to evaporate as it passed through the Soxhlet extractor into the condenser. Upon reaching the siphon, the solvent containing the extract was recycled back into the round-bottom flask. The extraction solution was allowed to cool to room temperature after the process was completed. The extract was then concentrated using a rotary evaporator.

In vivo and in vitro studies

Venom and anti-venom sources

Lyophilised E. ocellatus venom was obtained from the serpentarium of the Department of Zoology, University of Ibadan, Nigeria and the standard drug was EchiTAb-Plus ICP polyvalent antivenom produced by Instituto Clodomiro Picado, University of Costa Rica, Costa Rica.

Experimental animal

Thirty albino Wistar rats weighing between 100 and 150 g were purchased from the Animal House located at the College of Nursing Sciences, Osun State University Teaching Hospital, Osogbo, Osun State, Nigeria. The rats were transferred to the Department of Microbiology Laboratory and acclimatised for two weeks under normal conditions in a well-ventilated plastic cage at room temperature. Rats were fed with pellets and water ad libitum. Experiments were performed on the rats following standard ethical procedures.

Anti-haemorrhagic effects of G. lucidum extract

Ganoderma lucidum methanolic extract was dissolved in saline to obtain a concentration of 20 mg/mL and 40 mg/mL. The median lethal dose (LD50), 0.22 mg/kg of E. ocellatus venom [15] was reconstituted using 2 mL of normal saline. Exactly 0.2 mg/mL of each concentration of the extract and antivenom was preincubated with 0.1 mL of the reconstituted venom separately in triplicate for 30 min at 37 °C. Thereafter, 0.1 mL of the mixture was injected intradermally at the back (dorsal) of the rats in each respective group. The group injected with venom only served as the venom control, while the control group was injected with only saline. The percentage inhibition of haemorrhagic activity was calculated by measuring the size of the haemorrhagic foci using a caliper after three hours [15].

Plasma clotting effect of G. lucidum extract

Plasma clotting assay was performed as described by Adeyi et al. [35]. The LD50 of E. ocellatus venom was reconstituted with saline, and 0.2 mL was mixed with 0.2 mL of antivenom, 20 mg, and 40 mg of Ganoderma extract in separate test tubes in triplicate. Thereafter, 0.2 mL of citrated bovine plasma was added to all the mixtures and incubated at 37 °C for 30 min in a water bath before being combined with 0.2 mL of calcium chloride (2 mM). The clotting time was then recorded. For the control setups, plasma was incubated with normal saline alone.

GC-MS analysis of G. lucidum methanolic extract

GC-MS analysis was carried out using a Varian 3800/4000 gas chromatograph mass spectrometer equipped with an Agilent capillary column. Electron ionization in the GC-MS system was conducted at 70 eV with an ion source temperature of 250 °C. High-purity helium gas (99.9%) was used as the carrier gas, and an HP-5 capillary column (30 m ×ばつ 0.25 mm ×ばつ 0.32 μm) served as the stationary phase. The oven temperature was initially set at 60 °C and held for 0.5 min, then ramped to 140 °C at a rate of 4 °C/min and held for 1 min. Subsequently, the temperature was increased to 280 °C at a rate of 8 °C/min and held for 5 min. A 1 μL sample was automatically injected. Mass spectral data were interpreted by comparing the obtained spectra with reference spectra from the system’s internal library and the National Institute of Standards and Technology (NIST) database, which contains over 62,000 compound patterns.

The relative percentage amount of each component was calculated by comparing its average peak area to the total area. Measurement of peak areas and data processing were carried out by ChemStation software. This was subjected to chromatographic analysis using a Varian 3800/4000 gas chromatograph mass All the peaks were identified based on mass spectral matching (≥ 90%) from both the NIST and Wiley libraries. Only compounds with 90% or greater spectral matching accuracy are reported. No response factors were calculated.

In silico studies

Protein target (echicetin and phospholipase A2)

The in silico molecular interaction was used to determine the inhibitory potential of the compounds detected in G. lucidum methanolic extract by GC-MS against echicetin and PLA2. The 3D crystal structures of E. ocellatus echicetin and PLA2 are not available in the protein data bank (https://www.rcsb.org/ ). Hence, the 3D crystal structures of echicetin and PLA2 from Indian saw-scaled viper (Echis carinatus) deposited in the protein data bank (PDB) with PDB ID: 1OZ7 and PDB ID: 1OZ6 [6, 7], respectively, were retrieved and used as the target proteins for the molecular docking. The E. carinatus echicetin is similar to two 14–16 kDa C-type lectin-like domains of E. ocellatus involved in binding with glycoprotein Ibα, which is a molecule that interacts with von Willebrand factor (vWF) for platelet formation [8]. Phospholipase A2 protein from E. carinatus belongs to group II phospholipases and exhibits strong platelet-aggregation inhibition and induces oedema [36].

Amino acid residues in the binding and catalytic sites of the protein targets

The amino acid residues in the binding and catalytic site of the protein targets (Table 1) were obtained from previously published articles [6, 7]. Echicetin is made up of two subunits: the α-subunit, which forms the upper part of the protein binding site and the β-subunit, which forms the middle and lower part of the protein subunit [7]. The important amino acid residues required for the activity of echicetin and interaction with glycoprotein Ibα are presented in Table 1. Amino acid residues important in the catalytic activity of PLA2 are located in the active site region, hydrophobic channel, calcium-binding loop on the protein, and C-terminal region, which is the anti-platelet site (Table 1). Amino acid residues His48 and Asp49 interact via a water molecule that is required for the catalytic activity of PLA2 [6]. A unique hydrophobic channel aids the binding of PLA2 and catalytic hydrolysis of water-insoluble phospholipids. In the calcium-binding loop, the calcium ion binds as a cofactor necessary for PLA2 catalytic activity. Meanwhile, the anti-platelet site residues play an important role in the inhibition of the platelet aggregation properties of PLA2 [6].

Table 1 The important amino acid residues in the binding and active sites of Echicetin and PLA2 that are required for haemorrhagic and anti-coagulant activities, respectively

Ligands (compounds from G. lucidum)

The 2D structures of 29 constituents identified in the methanolic extract of G. lucidum via GC-MS (Table 2) were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/ ), and their interactions with echicetin and PLA2 were studied via molecular docking.

Table 2 The anti-haemorrhagic effects of G. lucidum methanolic extract against E. ocellatus venom

Molecular docking

Molecular docking was performed using Autodock Vina Wizard on the PyRx virtual screening tool [37]. Protein was prepared in the PyRx tool. Ligands were loaded on the PyRx and minimised using Open Babel. Both proteins and ligands were converted to PDBQT format before docking. The docking grid size for echicetin was x = 36.03 Å, y = 54.11 Å, and z = 65.49 Å, and for PLA2, the dimensions of the docking grid were x = 26.83 Å, y = 42.08 Å, and z = 39.35 Å. The docking results were evaluated based on the binding affinity energy (docking score), and BIOVIA Discovery Studio Visualizer 2020 was used to view the protein-ligand binding poses. The docking scores for the two proteins were validated by examining the interaction of the ligands with the key binding site residues of the protein due to the absence of the co-crystallized structure of the echicetin and PLA2 in the protein databank. Additionally, varespladib, a known inhibitor of PLA2 was included as a control, and its binding affinity was set as a cut-off to select compounds with inhibitory potential against PLA2. However, no report on echicetin inhibitor that can serve as a control.

Absorption, distribution, metabolism, excretion, and toxicity (ADMET)

The ADMET properties and drug-likeness of ligands were predicted with the ADMETlab 2.0 web tool [38], using ligands’ canonical SMILES structures. Drug-likeness of ligands was based on Lipinski’s rule of 5 (Ro5) as described by Benet et al. [39].

Statistical analysis

Statistical analysis was performed using the statistical software GraphPad Prism v. 7.00 (GraphPad Software Ltd., USA, 2003). Data were expressed as mean ± standard deviation (n = 3). Statistical significance was determined using one-way analysis of variance (ANOVA), and for the multiple comparison post hoc test, the Tukey test (p < 0.05) was used.

Results

In vivo and in vitro experiments

Anti-haemorrhagic effects of G. lucidum methanolic extract

Rats injected intradermally with 20 mg and 40 mg of G. lucidum methanolic extract had percentage haemorrhagic lesion inhibition of 62.96 ± 1.03% and 59.25 ± 1.59%, respectively, while the antivenom recorded 88.03 ± 1.61% inhibition. (Table 2).

Ganoderma lucidum methanolic extract plasma clotting activity

The methanolic extract of G. lucidum reduced the plasma clotting time following mixture with E. ocellatus venom. The 20 mg and 40 mg extract doses significantly (p < 0.05) shortened the clot time, recording 132 s and 163 s, respectively, compared to the venom control that clotted at 221 s (Fig. 2).

Fig. 1

Plasma clotting activity of the G. lucidum methanolic extract

GC-MS analysis of G. lucidum methanolic extract

GC-MS analysis of the methanolic extract of G. lucidum revealed the presence of 29 compounds (Fig. 3; Table 3). Among the identified compounds, 1,2-propanediol diformate was the most abundant (12.24%), followed by 1,2-benzenedicarboxylic acid (11.98%), 9-octadecenoic acid (Z)-, methyl ester (11.17%), dodecane, 1,12-dibromo- (9.58%), 9-tetradecenal (Z)- (9.38%), caryophyllene (8.52%), and oleic acid (7.45%). The identified compounds with documented pharmacological activities belong to various chemical classes, including phenolics, terpenes, triterpenes, fatty acids and their ester derivatives, phytosterols, flavonoids, dihydropyranones, imidazoles, aldehydes, carboxylic acids, and pyrazoles (Table 4).

Fig. 2

GC-MS chromatogram of the G. lucidum methanolic extract

Table 3 The GC-MS analysis of the methanolic extract of G. lucidum
Table 4 Pharmacological properties of compounds identified in the G. lucidum methanolic extract

In silico studies

Molecular docking

The binding affinity energy or docking score ranged between − 9.3 and − 3.3 kcal/mol for echicetin and − 9.9 kcal/mol and − 2.8 kcal/mol for PLA2 (Table 5). Hesperidin had the best docking score (echicetin = -9.3 kcal/mol and PLA2 = -9.9 kcal/mol). A strong to moderate covalent hydrogen bond was formed between four compounds and important amino acid residues of echicetin. Hesperidin formed hydrogen bonds with residues Ser98 (bond length = 2.37 Å) and His95 (bond length = 3.06 Å) located in the upper (α-subunit) and middle (β-subunit) portion of the echicetin binding site, respectively (Fig. 4a). It also interacted with Arg108 residues present in the middle portion (β-subunit) of the echicetin binding site via π-cation hydrophobic bond. 9-Octadecenoic acid (Z)-, methyl ester and tetradecanal interacted with a residue Arg108 via hydrogen bond of length 2.68 Å and 2.71 Å, respectively (Fig. 4b and c). Also, 9-tetradecenal, Z bond with Ser98 via a strong hydrogen bond of length 2.19 Å (Fig. 4d). 1,2-benzenedicarboxylic acid (phthalic acid), squalene, oleic acid, and acetic acid formed weak carbon-hydrogen bonds with the Trp72 residue located in the middle portion of echicetin (Table 6). Most of the compounds formed hydrophobic bonds with important amino acid residues of the echicetin binding site, and these include Trp72 and His95 (Table 6).

Table 5 The binding affinity energy (docking score) of compounds with potential inhibitory properties against Echicetin and PLA2
Fig. 3

The binding pose showing the interaction between amino acid residues of echicetin and (a). hesperidin (b). 9-octadecenoic acid (Z)-, methyl ester (c). tetradecanal (d). 9-tetradecenal, Z. Arrow indicates the hydrogen bond length (Å) between amino acid residues and ligands

Table 6 The bond interaction between G. lucidum methanolic extract compounds and Echicetin

All the compounds except dodecane, 1, 12-dibromo- interacted with at least one of the amino acid residues in the active site of PLA2 either with hydrogen bonds, hydrophobic bonds or both (Table 7). Hesperidin and γ-Sitosterol are the only compounds that had higher docking scores compared to the varespladib control (-8.0 kcal/mol). Varespladib interacted with the active site residues of PLA2 (Fig. 5a), however, only hesperidin interacted with two of the amino acid residues (Trp 10 and Leu17) that are important for anti-platelet activity of PLA2 via π-alkyl and alkyl hydrophobic bonding (Fig. 5b).

Table 7 The bond interaction between G. lucidum methanolic extract compounds and phospholipase A2
Fig. 4

The binding pose showing the interaction between amino acid residues of PLA2 and (a). varespladib (control) (b). hesperidin Arrow indicates the hydrogen bond length (Å) between amino acid residues and ligands

Absorption, distribution, metabolism, excretion, and toxicity (ADMET)

Hesperidin was identified as the best compound in the methanolic extract of G. lucidum with inhibitory potential against echicetin and PLA2 based on the binding affinity energy and interaction with proteins’ binding and active site residues. The ADMET result revealed that hesperidin failed the drug-likeness properties predicted according to Lipinski’s rule of 5 (Table 8). Nevertheless, it can be synthesised easily. Hesperidin is not orally toxic, haemotoxic, or neurotoxic. Hesperidin does not cross the blood-brain barrier, and it is mildly absorbed in the human intestine.

Table 8 The ADMET properties of hesperidin

Discussion

Haemorrhagic and anti-platelet effects are part of the pathophysiological manifestations of E. ocellatus (West African Carpet Viper) envenomation [59]. The haemorrhagic activity is largely due to the presence of snake venom metalloproteinase, which degrades extracellular matrix components and damages blood vessels, resulting in local and systemic haemorrhage [60]. Snake venom proteins, such as echicetin and PLA2, contribute to the haemorrhagic and anticoagulant activities of viper venom. Following E. ocellatus envenoming, echicetin and PLA2 can damage blood vessels, causing haemorrhage, inhibit platelet aggregation, and distort haemostasis, leading to coagulopathy [6, 7, 61]. Methanolic extract of the G. lucidum used in this study significantly showed anti-haemorrhagic and plasma-clotting effects against the venom of E. ocellatus. However, the Ganoderma extract exhibited better anti-haemorrhagic and plasma clotting effects at 20 mg concentration compared to 40 mg concentration. While the effectiveness of the extract at the lower dosage compared to the higher dosage was not tested further, a study has reported no dose-dependent antitumor activity of G. lucidum aqueous extract in mice [62]. Several factors, including the type of compound present in a fungus, compound interactions, and saturation of target active sites, may influence the effective dosage of the fungal extract. Therefore, it is important to determine the optimal dosage of the G. lucidum methanolic extract for anti-haemorrhagic and plasma clotting activities. Clinical trials and animal model studies have reported G. lucidum as a safe therapeutic agent with no adverse effects, good tolerability, and minimal toxicity, even when administered at a high dosage and for long periods [63, 64]. However, its long term safety in children, pregnant and lactating women, and individuals with cases of autoimmune diseases is important [64].

The public health importance of E. ocellatus and the drawbacks of conventional antivenom underscore the need to explore alternative sources for snake antivenoms. In this study, compounds identified in the methanolic extract of G. lucidum, including polyphenols, terpenes, polysaccharides, triterpenes, flavonoids, fatty acids, and steroids, are of pharmacological importance, playing important roles in the medicinal properties of the genus Ganoderma [62, 65, 66]. Phenolic compounds, including gallic acid, ferulic acid, and caffeic acid, as well as steroids and terpenoids can bind to proteins in snake venom, such as PLA2, and inhibit their activity [16, 67, 68]. Additionally, these compounds can exhibit antioxidant properties that can prevent PLA2-induced oxidative damage. Triterpenoids and sterols are known for their influence on the coagulation pathway and anti-haemorrhagic activities [69,70,71,72]. Hence, they may also contribute to the anti-snake venom property of the G. lucidum extract observed in this study. Specifically, sitosterol has been reported to possess antimyotoxic and antihaemorrhagic effects against viper and cobra venoms [66, 71, 73]. Unsaturated fatty acids identified in this study (oleic acid and 11-octadecenoic acid, (Z)-) have been reported to directly inhibit snake venom proteins like PLA2 and their neurotoxicity or prevent venom spread by maintaining cell integrity [73]. Hence, antiophidic compounds identified in this study support the potential of G. lucidum extract in the treatment of snake envenoming.

Based on the docking score and molecular interactions with key amino acid residues, the in silico analysis revealed hesperidin as the best compound that can bind to echicetin and PLA2, exerting potential anti-haemorrhagic effects and clot formation, respectively. Computational methods are useful in identifying compounds that can bind to the active sites of venom proteins. Studies have reported strong inhibitory activities of bioactive compounds such as kaempferol [3], methanol, 6, 8, 9-trimethyl-4-(2-phenylethyl)-3-oxabicyclo [3.3.1] non-6-en-1-yl)- and paroxypropione [73], 2-Hydrazino-8-hydroxy-4-phenylquinoline [74,75,76] against active sites of snake venom enzymes using computational approaches. In protein-ligand interactions, the formation of a hydrogen bond between a ligand and the target protein is important for a molecule’s function [77]. Also, according to Jeffrey and Jeffrey (2003) [78], hydrogen bond with distances ranging between 2.2 and 2.5 Å is classified as strong covalent hydrogen bonds, while moderate and weak electrostatic hydrogen bonds have distances ranging between 2.5 and 3.2 Å and 3.2–4.0 Å, respectively. Hence, the presence of strong to moderate covalent hydrogen bond interactions between hesperidin and key binding and active site residues of the venom proteins (echicetin and PLA2) encourages the formation of stronger and robust ligand-protein complexes [79]. Furthermore, some electrostatic bonds, including π-sigma, amide π-stacked, alkyl, π-alkyl, and π-cation, may also contribute to the stabilisation of the echicetin-hesperidin and PLA2-hesperidin complexes.

Hesperidin is a biflavonoid abundantly present in citrus fruits [80]. It is a biologically active compound with antioxidant, anti-allergy, antimicrobial, antiviral, anticancer, anti-inflammatory, cardioprotective, and neuroprotective effects [52, 80,81,82,83,84]. It is also important in the management of cutaneous damage and wound healing [84]. The antioxidant and anti-inflammatory properties of hesperidin [85, 86] may be significant in ameliorating the oxidative stress, inflammation and tissue damage caused by PLA2. Abdel-Aty et al. [87] reported for the first time the abundance of hesperidin in mango extract (3000 ± 112 mg/100 g seed and 55.6% of total polyphenols) and the therapeutic effect of the hesperidin-rich mango seed kernel extract against viper venoms, inhibiting PLA2 and neutralising haemorrhage. The result from the in silico analysis in this study showed the inhibitory potential of hesperidin against viper echicetin and PLA2. However, further experimental studies using pure hesperidin compound are important to confirm its anti-snake venom properties.

The Graph Attention-based assessment of Synthetic Accessibility (GASA), a graph neural network architecture that evaluates the ease of compound synthesis [88], predicted hesperidin as an easy-to-synthesise compound. According to Lipinski (2000) [89], the potency of a compound depends on its oral absorption, which is determined by the level of permeability or solubility. Compounds which comply with Lipinski’s rule of five (Ro5), which include molecular weight < 500 g/mol, hydrogen bond donor < 5, hydrogen bond acceptor < 10, and Log P (octanol–water partition coefficient) < 5 are said to be drug-like and orally bioavailable [39]. Hesperidin violated the Lipinski Ro5 based on the results from the ADMETlab 2.0 (supplementary file S1), hence suggesting its poor oral bioavailability. Nevertheless, with chemical modification, intravenous administration, encapsulation, and application of an absorption enhancer, the bioavailability of hesperidin can be improved [90, 91]. The molecular weight of compounds can interfere with enzyme activity [92]. For instance, high molecular weight compounds may not fit into the binding site of the enzyme, while low molecular weight compounds may not be able to bond with the key amino acid residues of the enzyme. Hence, the ability of a compound to fit into an enzyme binding site and interact with critical amino acid residues can affect an enzyme’s activity. In this study, hesperidin has high molecular weight of 610.57 g/mol (Table 8), when compared with the Lipinski Ro5 criteria, however, it interacted with the key amino acid residues of echicetin and PLA2 suggesting its enzyme inhibitory potential.

Hesperidin metabolism produces hesperetin, a better drug-like compound and a potent venom protein inhibitor [93, 94]. Hesperidin does not cross the blood-brain barrier, and it is moderately absorbed in the human intestine. The toxicity result predicted that hesperidin is not orally toxic, haemotoxic or neurotoxic. This agrees with previous in silico and in vivo ADMET reports on hesperidin [86, 94]. ADMETlab 2.0 software predicted the potential AMES toxic property of hesperidin with a probability of 0.569 (supplementary file S1). However, this prediction contradicts the report of Aja et al. [95], which supported the non-mutagenic and non-carcinogenic properties of hesperidin. The docking results and ADMET features of hesperidin make it a suitable drug candidate for the development of antivenom.

Conclusion

The study is the first report on the antivenom properties of G. lucidum extract. The experimental work reported the anti-haemorrhagic and plasma clotting activities of the methanolic extract of G. lucidum against E. ocellatus venom. With the limited information on the antivenom properties of G. lucidum, this baseline study has demonstrated the potential of G. lucidum as an anti-snake venom. However, future studies, including proteomic, toxin-specific neutralisation, enzyme inhibition mechanism, and toxicity assays, will further support the application of G. lucidum antivenom properties. From the in silico analysis, hesperidin binds to the key amino acid residues of echicetin and PLA2, which are important proteins in viper snake venoms, involved in haemorrhage and plasma clotting inhibition, respectively. This showed the potential of hesperidin to inhibit the activities of echicetin and PLA2. Further experiments, which involve testing different concentrations of G. lucidum extract to determine the best antiophidic concentration, are suggested. With the promising potential of hesperidin, further experiments are needed to better understand its antivenom properties.

Data availability

Data generated and materials during this study are included in this published article. Any needed additional data will be made available on request.

Abbreviations

PLA2:

Phospholipase A2

GC-MS:

Gas Chromatography-Mass Spectrometry

ADMET:

Absorption, Distribution, Metabolism, Excretion, and Toxicity

LD50 :

Median lethal dose

NIST:

National Institute of Standards and Technology

PDB:

Protein Data Bank

GASA:

Graph Attention-based assessment of Synthetic Accessibility

References

  1. Warrell DA, Williams DJ. Clinical aspects of snakebite envenoming and its treatment in low-resource settings. Lancet. 2023;401:1382–98. https://doi.org/10.1016/S0140-6736(23)00002-8.

    Article CAS PubMed Google Scholar

  2. Alcoba G, Chabloz M, Eyong J, Wanda F, Ochoa C, Comte E, Nkwescheu A, Chappuis F. Snakebite epidemiology and health-seeking behaviour in Akonolinga health district, cameroon: Cross-sectional study. PLoS Negl Trop Dis. 2020;14:e0008334. https://doi.org/10.1371/journal.pntd.0008334.

    Article CAS PubMed PubMed Central Google Scholar

  3. Ajisebiola BS, Oladele JO, Adeyi AO. Kaempferol from Moringa oleifera demonstrated potent antivenom activities via Inhibition of metalloproteinase and Attenuation of Bitis arietans venom–induced toxicities. Toxicon. 2023;233:107242. https://doi.org/10.1016/j.toxicon.2023.107242

    Article CAS PubMed Google Scholar

  4. Nduagubam OC, Chime OH, Ndu IK, Bisi-Onyemaechi A, Eke CB, Amadi OF, Igbokwe OO. Snakebite in children in nigeria: a comparison of the first aid treatment measures with the world health organization’s guidelines for management of snakebite in Africa. Ann Afr Med. 2020;19:182–7. https://doi.org/10.4103/aam.aam_38_19

    Article PubMed PubMed Central Google Scholar

  5. Dingwoke EJ, Adamude FA, Salihu A, Abubakar MS, Sallau AB. Toxicological analyses of the venoms of Nigerian Vipers Echis ocellatus and Bitis arietans. Trop Med Health. 2024;52:15. https://doi.org/10.1186/s41182-024-00581-9

    Article PubMed PubMed Central Google Scholar

  6. Jasti J, Paramasivam M, Srinivasan A, Singh TP. Structure of an acidic phospholipase A2 from Indian saw-scaled Viper (Echis carinatus) at 2.6 Å resolution reveals a novel intermolecular interaction. Acta Cryst. 2004;60:66–72. https://doi.org/10.1107/S090744490302208X.

    Article CAS Google Scholar

  7. Jasti J, Paramasivam M, Srinivasan A, Singh TP. Crystal structure of Echicetin from Echis carinatus (Indian saw-scaled viper) at 2.4 Å resolution. J Mol Biol. 2004;335:167–76. https://doi.org/10.1016/j.jmb.2003年10月04日8.

    Article CAS PubMed Google Scholar

  8. Wagstaff SC, Sanz L, Juarez P, Harrison RA, Calvete JJ. Combined snake venomics and venom gland transcriptomic analysis of the ocellated carpet viper, Echis ocellatus. J Proteom. 2009;71:609–23. https://doi.org/10.1016/j.jprot.200810003.

    Article CAS Google Scholar

  9. Abd El-Aziz TM, Shoulkamy MI, Hegazy AM, Stockand JD, Mahmoud A, Mashaly AM. Comparative study of the in vivo toxicity and pathophysiology of envenomation by three medically important Egyptian snake venoms. Arch Toxicol. 2020;335 – 44. https://doi.org/10.1007/s00204-019-02619-y

    Article PubMed Google Scholar

  10. Gutiérrez JM, Rucavado A. Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage. Biochim. 2000. https://doi.org/10.1016/S0300-9084(00)01163-9. 82:841 – 50.

    Article Google Scholar

  11. Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming. Nat Reviews Disease Primers. 2017;3:1–21. https://doi.org/10.1038/nrdp.2017.63.

    Article Google Scholar

  12. Clemetson KJ, Lu Q, Clemetson JM. Snake venom proteins affecting platelets and their applications to anti-thrombotic research. Curr Pharm Des. 2007;13:2887–92. https://doi.org/10.2174/138161207782023702.

    Article CAS PubMed Google Scholar

  13. Alangode A, Rajan K, Nair BG. Snake antivenom: challenges and alternate approaches. Biochem Pharmacol. 2020;181:114135. https://doi.org/10.1016/j.bcp.2020.114135.

    Article CAS PubMed Google Scholar

  14. Habib AG, Musa BM, Iliyasu G, Hamza M, Kuznik A, Chippaux JP. Challenges and prospects of snake antivenom supply in sub-Saharan Africa. PLoS Negl Trop Dis. 2020;14:e0008374. https://doi.org/10.1371/journal.pntd.0008374.

    Article CAS PubMed PubMed Central Google Scholar

  15. Adeyi AO, Adeyemi SO, Effiong EO, Ajisebiola BS, Adeyi OE, James AS. Moringa oleifera extract extenuates Echis ocellatus venom-induced toxicities, histopathological impairments and inflammation via enhancement of Nrf2 expression in rats. Pathophysiology. 2021;28:98–115. https://doi.org/10.3390/pathophysiology28010009.

    Article PubMed PubMed Central Google Scholar

  16. Omara T, Kagoya S, Openy A, Omute T, Ssebulime S, Kiplagat KM, Bongomin O. Antivenin plants used for treatment of snakebites in uganda: ethnobotanical reports and pharmacological evidences. Trop Med Health. 2020;48:1–6. https://doi.org/10.1186/s41182-019-0187-0

    Article Google Scholar

  17. Bala AA, Mohammed M, Umar S, Ungogo MA, Hassan MA, Abdussalam US, Ahmad MH, et al. Preclinical efficacy of African medicinal plants used in the treatment of snakebite envenoming: a systematic review protocol. Ther Adv Infect Dis. 2022;20499361211072644. https://doi.org/10.1177/20499361211072644

  18. Adrião AA, Dos Santos AO, de Lima EJ, Maciel JB, Paz WH, da Silva FM, Pucca MB, Moura-da-Silva AM, Monteiro WM, Sartim MA, Koolen HH. Plant-derived toxin inhibitors as potential candidates to complement antivenom treatment in snakebite envenomations. Front Immunol. 2022;13:842576. https://doi.org/10.3389/fimmu.2022.842576.

    Article CAS PubMed PubMed Central Google Scholar

  19. Mouane A, Telli A, Tedjani A, Achab D, Djehiche R, Gahtar A, Kadri M, Abid A, Alayat MS, Mekhadmi NE, Aouadi A. Exploring ethnobotanical remedies: medicinal plants for snakebite envenoming treatments in the Oued Righ region (Northern Algerian Sahara). Toxicon. 2025;255:108259. https://doi.org/10.1016/j.toxicon.2025.108259.

    Article CAS PubMed Google Scholar

  20. Yadav D, Negi PS. Bioactive components of mushrooms: processing effects and health benefits. Food Res Int. 2021;148:110599. https://doi.org/10.1016/j.foodres.2021.110599.

    Article CAS PubMed Google Scholar

  21. Bolesławska I, Górna I, Sobota M, Bolesławska-Król N, Przysławski J, Szymański M. Wild mushrooms as a source of bioactive compounds and their antioxidant properties—preliminary studies. Foods. 2024;13:2612. https://doi.org/10.3390/foods13162612

    Article CAS PubMed PubMed Central Google Scholar

  22. Boh B, Berovic M, Zhang J, Zhi-Bin L. Ganoderma lucidum and its pharmaceutically active compounds. Biotechnol Annu Rev. 2007;13:265–301. https://doi.org/10.1016/S1387-2656(07)13010-6.

    Article CAS PubMed Google Scholar

  23. Baby S, Johnson AJ, Govindan B. Secondary metabolites from Ganoderma. Phytochemistry. 2015;114:66–101. https://doi.org/10.1016/j.phytochem.2015年03月01日0.

    Article CAS PubMed Google Scholar

  24. Swallah MS, Bondzie-Quaye P, Wu Y, Acheampong A, Sossah FL, Elsherbiny SM, Huang Q. Therapeutic potential and nutritional significance of Ganoderma lucidum–a comprehensive review from 2010 to 2022. Food Funct. 2023;14:1812–38. https://doi.org/10.1039/D2FO01683D.

    Article CAS PubMed Google Scholar

  25. Yusuf AJ, Ibrahim N, Abdullahi MI, Adeboyega AE, Salihu M. Exploring the antisnake venom potential of Zingiber officinale and its bioactive compounds against Naja nigricollis venom through computational approaches and experimental validation. Chem Biodivers. 2025;3:e202402449. https://doi.org/10.1002/cbdv.202402449.

    Article CAS Google Scholar

  26. Poole DA III, Albulescu LO, Kool J, Casewell NR, Geerke DP. Computational strategies for broad spectrum venom phospholipase A2 inhibitors. J Chem Inf Model. 2025;65:4593–601. https://doi.org/10.1021/acs.jcim.5c00045.

    Article CAS PubMed PubMed Central Google Scholar

  27. Oyedara OO, Adeyemi FM, Adetunji CO, Elufisan TO. Repositioning antiviral drugs as a rapid and cost-effective approach to discover treatment against SARS-CoV-2 infection. In: Medical biotechnology, biopharmaceutics, forensic science and bioinformatics. CRC Press; 2022. p. 155–66. https://doi.org/10.1201/9781003178903-10

  28. Oyewole KA, Oyedara OO, Awojide SH, Olawade MO, Abioye OE, Adeyemi FM, Juárez-Saldivar A, Adetunji CO, Elufisan TO. Antibacterial and in silico evaluation of β-lactamase inhibitory potential of Pinus sylvestris L. (Scots pine) essential oil. Proc Natl Acad Sci India Sect B Biol Sci. 2023;4:967–77. https://doi.org/10.1007/s40011-023-01490-3

    Article CAS Google Scholar

  29. Oyedara OO, Fadare OA, Franco-Frías E, Heredia N, García S. Computational assessment of phytochemicals of medicinal plants from Mexico as potential inhibitors of Salmonella enterica efflux pump AcrB protein. J Biomol Struct Dyn. 2023;41:1776–89. https://doi.org/10.1080/07391102.2021.2024261.

    Article CAS PubMed Google Scholar

  30. Wang XC, Xi RJ, Li Y, Wang DM, Yao YJ. The species identity of the widely cultivated Ganoderma,‘G. Lucidum’ (Ling-zhi), in China. PLoS One. 2012;7:e40857. https://doi.org/10.1371/journal.pone.0040857

    Article CAS PubMed PubMed Central Google Scholar

  31. Richter C, Wittstein K, Kirk PM, Stadler M. An assessment of the taxonomy and chemotaxonomy of Ganoderma. Fungal Divers. 2015;1–5. https://doi.org/10.1007/s13225-014-0313-6

  32. Luangharn T, Karunarathna SC, Dutta AK, Paloi S, Promputtha I, Hyde KD, Xu J, Mortimer PE. Ganoderma (Ganodermataceae, Basidiomycota) species from the greater Mekong subregion. J Fungi. 2021;7:819. https://doi.org/10.3390/jof7100819.

    Article CAS Google Scholar

  33. Liu Y, Long Y, Liu H, Lan Y, Long T, Kuang R, Wang Y, Zhao J. Polysaccharide prediction in Ganoderma lucidum fruiting body by hyperspectral imaging. Food Chem X. 2022;13:100199. https://doi.org/10.1016/j.fochx.2021.100199.

    Article CAS PubMed Google Scholar

  34. Mokaizh AA, Nour AH, Alazaiza MY, Mustafa SE, Omer MS, Nassani DE. Extraction and characterization of biological phytoconstituents of Commiphora gileadensis leaves using Soxhlet method. Processes. 2024;12:1567. https://doi.org/10.3390/pr12081567.

    Article CAS Google Scholar

  35. Adeyi AO, Ajisebiola SB, Adeyi EO, Alimba CG, Okorie UG. Antivenom activity of Moringa oleifera leave against pathophysiological alterations, somatic mutation and biological activities of Naja nigricollis venom. Sci Afr. 2020;8:e00356. https://doi.org/10.1016/j.sciaf.2020.e00356

    Article CAS Google Scholar

  36. Kemparaju K, Krishnakanth TP, Gowda TV. Purification and characterization of a platelet aggregation inhibitor acidic phospholipase A2 from Indian saw-scaled Viper (Echis carinatus) venom. Toxicon. 1999;37:1659–71. https://doi.org/10.1016/S0041-0101(99)00104-X.

    Article CAS PubMed Google Scholar

  37. Dallakyan S, Olson AJ. Small-molecule library screening by docking with PyRx. In: Chemical biology: methods and protocols. New York, NY: Springer New York; 2014. p. 243–250. https://doi.org/10.1007/978-1-4939-2269-7_19

  38. Xiong G, Wu Z, Yi J, Fu L, Yang Z, Hsieh C, Yin M, Zeng X, Wu C, Lu A, Chen X. ADMETlab 2.0: an integrated online platform for accurate and comprehensive predictions of ADMET properties. Nucleic Acids Res. 2021;49:W5–14. https://doi.org/10.1093/nar/gkab255.

    Article CAS PubMed PubMed Central Google Scholar

  39. Benet LZ, Hosey CM, Ursu O, Oprea TI. BDDCS, the rule of 5 and drugability. Adv Drug Deliv Rev. 2016;101:89–98. https://doi.org/10.1016/j.addr.201605007.

    Article CAS PubMed PubMed Central Google Scholar

  40. Yang C, Wang B, Wang J, Xia S, Wu Y. Effect of pyrogallic acid (1, 2, 3-benzenetriol) polyphenol-protein covalent conjugation reaction degree on structure and antioxidant properties of pumpkin (Cucurbita sp.) seed protein isolate. Lwt. 2019;109:443–9. https://doi.org/10.1016/j.lwt.2019年04月03日4.

    Article CAS Google Scholar

  41. Ferdosh S. The extraction of bioactive agents from Calophyllum inophyllum L., and their Pharmacological properties. Sci Pharm. 2024;92:6. https://doi.org/10.3390/scipharm92010006

    Article CAS Google Scholar

  42. Chen F, Zhang X, Wang J, Wang F, Mao J. P-coumaric acid: advances in pharmacological research based on oxidative stress. Curr Top Med Chem. 2024;24:416–36. https://doi.org/10.2174/0115680266276823231230183519

    Article CAS PubMed Google Scholar

  43. Kaur J, Gulati M, Singh SK, Kuppusamy G, Kapoor B, Mishra V, Gupta S, Arshad MF, Porwal O, Jha NK, Chaitanya MV. Discovering multifaceted role of vanillic acid beyond flavours: nutraceutical and therapeutic potential. Trends Food Sci Technol. 2022;122:187–200. https://doi.org/10.1016/j.tifs.2022年02月02日3.

    Article CAS Google Scholar

  44. Hadidi M, Liñán-Atero R, Tarahi M, Christodoulou MC, Aghababaei F. The potential health benefits of gallic acid: therapeutic and food applications. Antioxidants. 2024;13:1001. https://doi.org/10.3390/antiox13081001

    Article CAS PubMed PubMed Central Google Scholar

  45. Espíndola KM, Ferreira RG, Narvaez LE, Silva Rosario AC, Da Silva AH, Silva AG, Vieira AP, Monteiro MC. Chemical and pharmacological aspects of caffeic acid and its activity in hepatocarcinoma. Front Oncol. 2019;9:541. https://doi.org/10.3389/fonc.2019.00541

    Article PubMed PubMed Central Google Scholar

  46. Zhai Y, Wang T, Fu Y, Yu T, Ding Y, Nie H. Ferulic acid: a review of pharmacology, toxicology, and therapeutic effects on pulmonary diseases. Int J Mol Sci. 2023;24:8011. https://doi.org/10.3390/ijms24098011

    Article CAS PubMed PubMed Central Google Scholar

  47. Dickson K, Scott C, White H, Zhou J, Kelly M, Lehmann C. Antibacterial and analgesic properties of beta-caryophyllene in a murine urinary tract infection model. Molecules. 2023;28(4144). https://doi.org/10.3390/molecules28104144

  48. Cheng L, Ji T, Zhang M, Fang B. Recent advances in squalene: biological activities, sources, extraction, and delivery systems. Trends Food Sci Technol. 2024;104392. https://doi.org/10.1016/j.tifs.2024

  49. Santa-María C, López-Enríquez S, Montserrat-de la Paz S, Geniz I, Reyes-Quiroz ME, Moreno M, Palomares F, Sobrino F, Alba G. Update on anti-inflammatory molecular mechanisms induced by oleic acid. Nutrients. 2023;15:224. https://doi.org/10.3390/nu15010224.

    Article CAS PubMed PubMed Central Google Scholar

  50. Semwal P, Painuli S, Badoni H, Bacheti RK. Screening of phytoconstituents and antibacterial activity of leaves and bark of Quercus leucotrichophora A. Camus from Uttarakhand himalaya. Clin Phytosci. 2018;4:1–6. https://doi.org/10.1186/s40816-018-0090-y.

    Article CAS Google Scholar

  51. Mostofa MG, Reza AA, Khan Z, Munira MS, Khatoon MM, Kabir SR, et al. Apoptosis-inducing anti-proliferative and quantitative phytochemical profiling with in silico study of antioxidant-rich Leea aequata L. leaves. Heliyon 202;10. https://doi.org/10.1016/j.heliyon.2023.e23400

  52. Balamurugan R, Duraipandiyan V, Ignacimuthu S. Antidiabetic activity of γ-sitosterol isolated from Lippia nodiflora L. in streptozotocin induced diabetic rats. Eur J Pharmacol. 2011;667:410–8. https://doi.org/10.1016/j.ejphar.2011年05月02日5

    Article CAS PubMed Google Scholar

  53. Pereira V, Figueira O, Castilho PC. Hesperidin: a flavanone with multifaceted applications in the food, animal feed, and environmental fields. Phytochem Rev. 2024;1–5. https://doi.org/10.1007/s11101-024-10008-2

  54. Yu X, Zhao M, Liu F, Zeng S, Hu J. Identification of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H-pyran-4-one as a strong antioxidant in glucose–histidine Maillard reaction products. Food Res Int. 2013;51:397–403. https://doi.org/10.1016/j.foodres.2012年12月04日4

    Article CAS Google Scholar

  55. Siwach A, Verma PK. Synthesis and therapeutic potential of imidazole containing compounds. BMC Chem. 2021;15:1–69. https://doi.org/10.1186/s13065-020-00730-1.

    Article CAS Google Scholar

  56. Fan Q, Ning M, Zeng X, He X, Bai Z, Gu S, Yuan Y, Yue T. Anti-Vibrio parahaemolyticus mechanism of hexanal and its inhibitory effect on biofilm formation. Foods. 2025;14:703. https://doi.org/10.3390/foods14040703.

    Article CAS PubMed PubMed Central Google Scholar

  57. Ryssel H, Kloeters O, Germann G, Schäfer T, Wiedemann G, Oehlbauer M. The antimicrobial effect of acetic acid—an alternative to common local antiseptics? Burns. 2009;35:695–700. https://doi.org/10.1016/j.burns.200811009.

    Article CAS PubMed Google Scholar

  58. Karrouchi K, Radi S, Ramli Y, Taoufik J, Mabkhot YN, Al-Aizari FA, Ansar MH. Synthesis and pharmacological activities of pyrazole derivatives: a review. Molecules. 2018;23:134. https://doi.org/10.3390/molecules23010134

    Article CAS PubMed PubMed Central Google Scholar

  59. Howes JM, Kamiguti AS, Theakston RD, Wilkinson MC, Laing GD. Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus on blood coagulation and platelets. Biochim biophys Acta Gen Subj. 2005;1724:194–202. https://doi.org/10.1016/j.bbagen.2005年03月01日1

  60. Gutiérrez JM, Escalante T, Rucavado A, Herrera C. Hemorrhage caused by snake venom metalloproteinases: a journey of discovery and Understanding. Toxins. 2016;8:93. https://doi.org/10.3390/toxins8040093.

    Article CAS PubMed PubMed Central Google Scholar

  61. Spolaore B, Fernández J, Lomonte B, Massimino ML, Tonello F. Enzymatic labelling of snake venom phospholipase A2 toxins. Toxicon. 2019;170:99–107. https://doi.org/10.1016/j.toxicon.2019.09

    Article CAS PubMed Google Scholar

  62. Furusawa E, Chou SC, Furusawa S, Hirazumi A, Dang Y. Antitumour activity of Ganoderma lucidum, an edible mushroom, on intraperitoneally implanted Lewis lung carcinoma in synergenic mice. Phytother Res. 1992;6:300–4. https://doi.org/10.1002/ptr.2650060604

    Article Google Scholar

  63. Klupp NL, Kiat H, Bensoussan A, Steiner GZ, Chang DH. A double-blind, randomised, placebo-controlled trial of Ganoderma lucidum for the treatment of cardiovascular risk factors of metabolic syndrome. Sci Rep. 2016;6:29540. https://doi.org/10.1038/srep29540.

    Article CAS PubMed PubMed Central Google Scholar

  64. Plosca MP, Chiș MS, Fărcaș AC, Păucean A. Ganoderma lucidum—from ancient remedies to modern applications: chemistry, benefits, and safety. Antioxidants. 2025;14:513. https://doi.org/10.3390/antiox14050513

    Article CAS PubMed PubMed Central Google Scholar

  65. Cör Andrejč D, Knez Ž, Knez Marevci M. Antioxidant, antibacterial, antitumor, antifungal, antiviral, anti-inflammatory, and nevro-protective activity of Ganoderma lucidum: an overview. Front Pharmacol. 2022;13:934982. https://doi.org/10.3389/fphar.2022.934982.

    Article CAS PubMed PubMed Central Google Scholar

  66. Oke MA, Afolabi FJ, Oyeleke OO, Kilani TA, Adeosun AR, Olanbiwoninu AA, Adebayo EA. Ganoderma lucidum: unutilized natural medicine and promising future solution to emerging diseases in Africa. Front Pharmacol. 2022;13:952027. https://doi.org/10.3389/fphar.2022.952027.

    Article CAS PubMed PubMed Central Google Scholar

  67. Ahmad MF, Alsayegh AA, Ahmad FA, Akhtar MS, Alavudeen SS, Bantun F, et al. Ganoderma lucidum: insight into antimicrobial and antioxidant properties with development of secondary metabolites. Heliyon. 2024;10(3). https://doi.org/10.1016/j.heliyon.2024.e25607

  68. Carvalho BM, Santos JD, Xavier BM, Almeida JR, Resende LM, Martins W, Marcussi S, Marangoni S, Stábeli RG, Calderon LA, Soares AM. Snake venom PLA2s inhibitors isolated from Brazilian plants: synthetic and natural molecules. Biomed Res Int. 2013;1:153045. https://doi.org/10.1155/2013/153045.

    Article CAS Google Scholar

  69. Kankara IA, Abdullahi I, Paulina GA. Ethnomedicinal plants: a source of phytochemical compounds against snake venom PLA2s activity. J Pharm Phytochem. 2020;9:1270–5. https://doi.org/10.13140/RG.2.2.36280.37124

    Article CAS Google Scholar

  70. Poniedziałek B, Siwulski M, Wiater A, Komaniecka I, Komosa A, Gąsecka M, Magdziak Z, Mleczek M, Niedzielski P, Proch J, Ropacka-Lesiak M. The effect of mushroom extracts on human platelet and blood coagulation: in vitro screening of eight edible species. Nutrients. 2019;11:3040. https://doi.org/10.3390/nu11123040.

    Article CAS PubMed PubMed Central Google Scholar

  71. Galappaththi MC, Patabendige NM, Premarathne BM, Hapuarachchi KK, Tibpromma S, Dai DQ, Suwannarach N, Rapior S, Karunarathna SC. A review of Ganoderma triterpenoids and their bioactivities. Biomolecules. 2022;13:24. https://doi.org/10.3390/biom13010024.

    Article CAS PubMed PubMed Central Google Scholar

  72. Mors WB, do Nascimento MC, Pereira BM, Pereira NA. Plant natural products active against snake bite—the molecular approach. Phytochem. 2000. https://doi.org/10.1016/S0031-9422(00)00229-6. 55:627 – 42.

    Article Google Scholar

  73. Gomes A, Saha A, Chatterjee I, Chakravarty AK. Viper and Cobra venom neutralization by β-sitosterol and stigmasterol isolated from the root extract of Pluchea indica Less. (Asteraceae). Phytomedicine. 2007;14:637–43. https://doi.org/10.1016/j.phymed.2006年12月02日0

    Article CAS PubMed Google Scholar

  74. Gopi K, Renu K, Jayaraman G. Inhibition of Naja naja venom enzymes by the methanolic extract of Leucas aspera and its chemical profile by GC–MS. Toxicol Rep. 2014;1:667 – 73. https://doi.org/10.1016/j.toxrep.2014年08月01日2

  75. Adeyi AO, Mustapha KK, Ajisebiola BS, Adeyi OE, Metibemu DS, Okonji RE. Inhibition of Echis ocellatus venom metalloprotease by flavonoid-rich ethyl acetate sub-fraction of Moringa oleifera (Lam.) leaves: in vitro and in silico approaches. Toxin Rev. 2022;41:476–86. https://doi.org/10.1080/15569543.2021.1893334

    Article CAS Google Scholar

  76. Adeyi AO, Jimoh AO, Ajisebiola BS, Adeyi OE, Metibemu DS, Okonji RE. Inhibition of phospholipase A2 from Naja haje and Naja nigricollis venoms by active fraction of Moringa oleifera leaves: in vitro and in silico methods. Toxin Rev. 2023;42:629 – 39. https://doi.org/10.1080/15569543.2023.2205538

  77. Wu MY, Dai DQ, Yan H. PRL-dock: Protein‐ligand Docking based on hydrogen bond matching and probabilistic relaxation labeling. Proteins Struct Funct Bioinform. 2012;80:2137–53. https://doi.org/10.1002/prot.24104.

    Article CAS Google Scholar

  78. Jeffrey GA, Jeffrey GA. An introduction to hydrogen bonding. Volume 12. New York: Oxford University Press; 1997. p. 228.

    Google Scholar

  79. Majewski M, Ruiz-Carmona S, Barril X. An investigation of structural stability in protein-ligand complexes reveals the balance between order and disorder. Commun Chem. 2019;2:110. https://doi.org/10.1038/s42004-019-0205-5.

    Article Google Scholar

  80. Pyrzynska K, Hesperidin. A review on extraction methods, stability and biological activities. Nutrients. 2022;14:2387. https://doi.org/10.3390/nu14122387.

    Article CAS PubMed PubMed Central Google Scholar

  81. Lee NK, Choi SH, Park SH, Park EK, Kim DH. Antiallergic activity of hesperidin is activated by intestinal microflora. Pharmacology. 2004;71:174–80. https://doi.org/10.1159/000078083.

    Article CAS PubMed Google Scholar

  82. Devi KP, Rajavel T, Nabavi SF, Setzer WN, Ahmadi A, Mansouri K, Nabavi SM, Hesperidin. A promising anticancer agent from nature. Ind Crops Prod. 2015;76:582–9. https://doi.org/10.1016/j.indcrop.2015年07月05日1.

    Article CAS Google Scholar

  83. Hajialyani M, Hosein Farzaei M, Echeverría J, Nabavi SM, Uriarte E, Sobarzo-Sánchez E. Hesperidin as a neuroprotective agent: a review of animal and clinical evidence. Molecules. 2019;24:648. https://doi.org/10.3390/molecules24030648.

    Article CAS PubMed PubMed Central Google Scholar

  84. Ma R, You H, Liu H, Bao J, Zhang M. Hesperidin: a citrus plant component, plays a role in the central nervous system. Heliyon. 2024;10(21). https://doi.org/10.1016/j.heliyon.2024.e38937

  85. Man MQ, Yang B, Elias PM. Benefits of hesperidin for cutaneous functions. Evid Based Complement Alternat Med. 2019;1:2676307. https://doi.org/10.1155/2019/2676307.

    Article Google Scholar

  86. Garg A, Garg S, Zaneveld LJ, Singla A. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phyto Res. 2001;8:655 – 69. https://doi.org/10.1002/ptr.1074

  87. Abdel-Aty AM, Salama WH, Hamed MB, Fahmy AS, Mohamed SA. Phenolic-antioxidant capacity of mango seed kernels: therapeutic effect against viper venoms. Rev Bras Farmacogn. 2018;28:594–601. https://doi.org/10.1016/j.bjp.201806008

    Article CAS Google Scholar

  88. Yu J, Wang J, Zhao H, Gao J, Kang Y, Cao D, Wang Z, Hou T. Organic compound synthetic accessibility prediction based on the graph attention mechanism. J Chem Inf Model. 2022;62:2973–86. https://doi.org/10.1021/acs.jcim.2c00038.

    Article CAS PubMed Google Scholar

  89. Lipinski CA. Drug-like properties and the causes of poor solubility and poor permeability. J Pharmacol Toxicol Methods. 2000. https://doi.org/10.1016/S1056-8719(00)00107-6. 44:235 – 49.

    Article PubMed Google Scholar

  90. Oyedara OO, Agbedahunsi JM, Adeyemi FM, Juárez-Saldivar A, Fadare OA, Adetunji CO, Rivera G. Computational screening of phytochemicals from three medicinal plants as inhibitors of transmembrane protease serine 2 implicated in SARS-CoV-2 infection. Phytomedicine Plus. 2021;1:100135. https://doi.org/10.1016/j.phyplu.2021.100135

    Article PubMed PubMed Central Google Scholar

  91. Molaakbari E, Aallae MR, Golestanifar F, Garakani-Nejad Z, Khosravi A, Rezapour M, et al. In silico assessment of hesperidin on SARS-CoV-2 main protease and RNA polymerase: molecular docking and dynamics simulation approach. Biochem Biophys Rep. 2024;39:101804. https://doi.org/10.1016/j.bbrep.2024.101804

  92. Ciulli A, Abell C. Fragment-based approaches to enzyme Inhibition. Curr Opin Biotechnol. 2007;18:489–96. https://doi.org/10.1016/j.copbio.200709003.

    Article CAS PubMed PubMed Central Google Scholar

  93. Yamamoto M, Jokura H, Hashizume K, Ominami H, Shibuya Y, Suzuki A, Hase T, Shimotoyodome A. Hesperidin metabolite hesperetin-7-O-glucuronide, but not hesperetin-3′-O-glucuronide, exerts hypotensive, vasodilatory, and anti-inflammatory activities. Food Funct. 2013;4:1346–51. https://doi.org/10.1039/C3FO60030K.

    Article CAS PubMed Google Scholar

  94. Vander dos Santos R, Villalta-Romero F, Stanisic D, Borro L, Neshich G, Tasic L. Citrus bioflavonoid, hesperetin, as inhibitor of two thrombin-like snake venom serine proteases isolated from Crotalus simus. Toxicon. 2018;143:36–43. https://doi.org/10.1016/j.toxicon.201801005

    Article CAS PubMed Google Scholar

  95. Aja PM, Awoke JN, Agu PC, Adegboyega AE, Ezeh EM, Igwenyi IO, Orji OU, Ani OG, Ale BA, Ibiam UA. Hesperidin abrogates bisphenol A endocrine disruption through binding with fibroblast growth factor 21 (FGF-21), α-amylase and α-glucosidase: an in Silico molecular study. J Genet Eng Biotechnol. 2022;20:84. https://doi.org/10.1186/s43141-022-00370-z.

    Article CAS PubMed PubMed Central Google Scholar

Download references

Funding

Nil.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Osun State University, Osogbo, Nigeria

    O. O. Oyedara & O. O. Oluyide

  2. Department of Animal and Environmental Biology, Osun State University, Osogbo, Nigeria

    B. S. Ajisebiola

  3. Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria

    O. E. Abioye

  4. Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria

    O. A. Fadare

  5. Department of Plant Biology, Osun State University, Osogbo, Nigeria

    O. A. Olatunji

  6. Department of Microbiology, Osun State University, Osogbo, Nigeria

    F. M. Adeyemi, M. N. Enahoro, S. F. Popoola & Z. A. Adeyemi

  7. Department of Biotechnology, School of Science, Engineering and Environment, University of Salford, Manchester, M5 4WT, UK

    Z. A. Adeyemi

Authors
  1. O. O. Oyedara
  2. B. S. Ajisebiola
  3. O. E. Abioye
  4. O. A. Fadare
  5. O. A. Olatunji
  6. F. M. Adeyemi
  7. M. N. Enahoro
  8. S. F. Popoola
  9. O. O. Oluyide
  10. Z. A. Adeyemi

Contributions

OOOyedara and BSA: Conceptualisation and project supervision; OAO, OEA, OAF: Fungal identification, extraction, and analysis; MNE, SFP: Laboratory analysis; OOOyedara and BSA: In silico analysis; FMA, OOOluyide, ZAA: Manuscript development and proofreading. All authors read, discussed, reviewed, and approved the manuscript.

Corresponding author

Correspondence to O. O. Oyedara.

Ethics declarations

Ethical approval

The ethical approval for the animal use in this study was obtained from the Osun State Health Research Ethical Committee (OSHREC), Osogbo, Osun State, Nigeria, with the authorised number: OSHREC/PRS/569T/1247. All experimental protocols in animal handling followed the rules and guidelines established for the care and use of laboratory animals in compliance with the revised ARRIVE guidelines 2.0.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

About this article

Cite this article

Oyedara, O., Ajisebiola, B., Abioye, O. et al. Evaluation of the pharmacological potential of Ganoderma lucidum against haemorrhagic and anticoagulant activities of Echis ocellatus venom. BMC Biotechnol 25, 105 (2025). https://doi.org/10.1186/s12896-025-01044-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • DOI: https://doi.org/10.1186/s12896-025-01044-7

Keywords

BMC Biotechnology

ISSN: 1472-6750

Contact us

AltStyle によって変換されたページ (->オリジナル) /