Profiling of phytochemical and antioxidant activity of wild mushrooms: Evidence from the in vitro study and phytoconstituent's binding affinity to the human erythrocyte catalase and human glutathione reductase

Abstract This study was undertaken to evaluate the appearance of phytochemicals and antioxidant activity of seven wild mushrooms of the University of Chittagong campus. Phytochemical screening was performed using standard methods, whereas DPPH radical scavenging assay was used to elucidate the antioxidant effect. Besides, in silico studies were implemented using the targets of human erythrocyte catalase 3‐amino‐1,2,4‐triazole, human glutathione reductase, and selected compounds. Again, the absorption, distribution, metabolism, elimination and toxicity (ADME/T) analysis has been determined by using online tools. Both Ganoderma lucidum (Curtis) Karst. and Ganoderma applanatum (Pers.) Pat. showed a significant (p < .001) increase in the percentage of scavenging activity at 400 μg/ml concentration when compared with ascorbic acid. The methanol extract of G. lucidum, G. applanatum, and Rhodofomes cajanderi (P. Karst.) B. K. Cui, M. L. Han & Y. C. Dai showed strong antioxidant activity with an IC50 value. In addition, molecular docking studies of the previously isolated compounds from three selective mushrooms revealed that the targeted compounds along with positive controls were able to interact strongly (range: −3.498 to −8.655) with the enzymes. The study concludes that the G . lucidum, G. applanatum, and R. cajanderi mushrooms can be a strong source in the management of oxidative stress‐induced diseases.


| INTRODUC TI ON
Oxidation is a vital process in humans that enables the transformation of nutrients such as carbohydrates, protein, and fat into energy (Sánchez, 2016). During this normal metabolic process, reactive oxygen species (ROS) are generated as a by-product. Highly reactive, unstable, and partially reduced oxygen derivatives such as superoxide radicals (O 2 •-), hydroxyl radicals (•OH), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ) are known as ROS (Chio & Tuveson, 2017). At low-level, ROS are crucial for various physiological processes and act as secondary messengers (Rajendran et al., 2014). ROS at high concentrations may exert harmful effects on cellular components such as DNA mutations, lipids peroxidation of membrane lipids, and membrane protein damage (Karim et al., 2020). The variation between the creation of ROS and the antioxidant defense capacity of the body is known as oxidative stress (Liguori et al., 2018). Oxidative stress is responsible for causing several diseases such as cancer, atherosclerosis, cardiovascular disease, diabetes, and metabolic disorders (Pizzino et al., 2017). Oxidative stress is regarded as an important characteristic for the pathogenesis and development of type 2 diabetes mellitus, but whether it is a simple combination of inflammatory responses or of a clinical entity that is based on different physiological variables is still disputed (Rehman & Akash, 2017). Antioxidants donate electrons to stabilize ROS to prevent cell and tissue damage (Khatua et al., 2013). Catalase is a vital enzyme of the antioxidant system of the human body. It helps to keep the redox stability of the immune system by reducing hydrogen peroxide (Wang et al., 2013).
Another essential antioxidant enzyme is glutathione reductase (GR).
GR in reduced form controls the ROS at the cellular level (Carlberg & Mannervik, 1985). Endogenous antioxidants and exogenous antioxidants are two known types of antioxidants. The human body makes endogenous antioxidants, which play an important role at low concentrations by scavenging the free radicals to keep maximum cellular functions. However, in case of oxidative stress, these endogenous antioxidants are found to be insufficient to protect the body from the harmful effect of ROS. Diet or dietary supplements may be required as exogenous antioxidants to maintain optimal cellular function (Kurutas, 2016). Nowadays, the industry that is responsible for producing food uses several synthetic antioxidants that have shown carcinogenicity. As a result, there is an urgency to search for antioxidants from natural sources (Abdullah et al., 2012). Recently, consumption of edible mushrooms has increased greatly because they are high in carbohydrates, protein, fiber, essential amino acids, and vitamins while low in fat, cholesterol, sodium, and calories (Rashidi & Yang, 2016).
In many cultures, edible mushrooms have been used traditionally as a source of home remedy from long ago due to the presence of biologically active compounds to protect the body from various oxidative stress-induced diseases (Chen et al., 2012). Several scientific reports have reported the medicinal properties of mushrooms including free radical scavenging, antioxidant (Sánchez, 2017), immunomodulating (Shamtsyan et al., 2004), antitumor (Singdevsachan et al., 2016), antidiabetic (Stojkovic et al., 2019), antihypercholesterolemia (Wasser, 2017), antibacterial, and antiviral effects (Roncero-Ramos et al., 2017). Hence, the aim of this study was the evaluation of phytochemicals and the antioxidant potential of the wild mushrooms found on the campus of the University of Chittagong.

| Reagents
1,1-Diphenyl-2-picrylhydrazyl (DPPH) was obtained from Sigma Chemical Co., USA. Ascorbic acid was purchased from SD Fine Chemicals Ltd., Biosar, India. Other chemicals of analytical grade were supplied by the Department of Pharmacy, University of Chittagong.

| Preparation of extract
After shade drying, the mushrooms were milled for efficient extraction. Exactly 100 g of milled mushroom powder was soaked in 500 ml methanol in a clean, sterilized, and flat-bottomed glass container for 7 days accompanying occasional stirring and agitation at room temperature. It was then filtered using filter papers (Whatman size no. 1). The filtrate was allowed to evaporate the solvent by using a rotary evaporator. These extracts were kept in tightly closed glass containers and stored in the refrigerator for further use.

| Antioxidant activity
Antioxidant activity of different mushroom extracts was carried out using the method of Alam et al. S. Alam, Rashid, et al., 2021). Two milliliters of each mushroom extract with different concentrations (12.5, 25, 50, 100, 200, and 400 μg/ml) was mixed with 3 ml of a 0.004% w/v methanol solution of DPPH.
Then, the tubes containing the mixture were kept at room temperature for 30 min in a dark place to complete the reaction. The absorbance was taken at 517 nm against using an ultraviolet-visible spectrophotometer (Halo SB-10 single-beam spectrophotometer, Dynamica Scientific Ltd., UK). Ascorbic acid was used as a positive control. The capability to scavenge the DPPH radical was calculated from [(A 0 -A 1 )/A 0 ] × 100, where A 0 is the absorbance of the control reaction (DPPH + Methanol) and A 1 is the absorbance of the sample.

| Protein preparation
For the current experiment, we have selected two enzymes of the cellular antioxidant mechanism, catalase and GR, respectively, for the demonstration of the inhibitory potential of the targeted chemical constituents of three mushrooms, that is, G. lucidum, G. applanatum, and R. cajanderi. Three-dimensional (3D) structures of human erythrocyte catalase (PDB ID: 1DGH) and human GR (PDB ID: 1XAN) were downloaded from the Protein Data Bank (www.rcsb.org/pdb) in PDB format. The structures were prepared and refined using the Protein Preparation Wizard of Schrödinger-Maestro v10.1. Charges and bond orders were assigned, hydrogens were added to the heavy atoms, selenomethionines were converted to methionines, and all waters were deleted. Using force field OPLS_2005, minimization was carried out, setting maximum heavy atom root-mean-square deviation to 0.30 Å.
In addition, we have used dihydro-nicotinamide adenine dinu-

| Receptor grid generation
Receptor grids were calculated for the prepared proteins for the observation of poses by various ligands, which bind within the active predicted site during the docking procedure. In Glide, grids were generated, keeping the default parameters of van der Waals scaling factor 1.00 and charge cutoff 0.25 subjected to the OPLS_2005 force field. A cubic box of specific dimensions centered on the centroid of the active site residues was obtained for the receptor. The bounding box was set to 14 × 14 × 14 Å for docking experiments.
2.6.4 | Glide standard precision ligand docking Standard precision flexible ligand docking was carried out in Glide of Schrödinger-Maestro v10.1 Friesner et al., 2004Friesner et al., , 2006 within which penalties were applied to non-cis/trans amide  where G = EMM + GSGB + GNP.

| Ligand-based Absorption, Distribution, Metabolism, Elimination, Toxicity analysis
The pharmacokinetic properties of all the selected bioactive compounds were evaluated and screened for drug candidacy using Lipinski's rule of five (RO5) and Veber's rule (C. A. Lipinski et al., 2001;Veber et al., 2002). According to Lipinski's RO5, a compound may show optimal drug-likeness if it fulfills at least four of the five criteria, namely, molecular weight (not more than 500 g/mol), hydrogen bond donors (≤5), hydrogen bond acceptors (≤10), lipophilicity (<5), and molar refractivity (between 40 and 130). The other filter we considered is Veber's rule, according to which the number of rotatable bonds (≤10) and topological polar surface area (TPSA; ≤140 Å²) in a compound must be within the specified limit to show drug-like behavior. The web tool SwissADME, which is indicated as a very useful tool in drug discovery, was used to analyze the abovementioned properties of the selected compounds (Emon, Alam, Rudra, et al., 2021;Veber et al., 2002). Compounds passing both Lipinski's filter and Veber's filter can be considered as suitable candidates for new drug development.

| Bioactivity prediction of the selected
The selected compounds were subjected to bioactivity calculations using an online validation tool, Molinspiration cheminformatics server (www.molin spira tion.com; M. M. Alam, Rashid, et al., 2021;Emon, Alam, et al., 2020;Rakib et al., 2020). Molinspiration calculates the molecular property associated with the drug-likeness and predicts the bioactivity including G protein-coupled receptor (GPCR) ligand, ion channel inhibitor, nuclear receptor ligand, kinase inhibitor, protease inhibitor, and enzyme inhibitors. The calculated bioactivity score for each of the selected compounds was compared with the specific activity of each compound, and the results were compared with the standards. In case of organic molecules, the probability is if the bioactivity score is more than 0, then it is active; if it is between −5.0 and 0.0, then moderately active; and if the score is <−5.0, then it is inactive.

| Statistical analysis
All experiments were carried out in triplicate. The data are pre-   (Bribi, 2018). Except for C. lactineus and G. lucidum, alkaloids were present in all mushrooms of this experiment. Glycosides are organic compounds formed of a

| Phytochemical screening
sugar group (glycon) and a non-sugar group (aglycon) linked together by a glycosidic bond. Glycosides are used as an analgesic, antirheumatic, antibiotic, cardiotonic, demulcent, and purgative agent (Kren & Martinkova, 2001). This study showed that glycosides were absent in only L. squarrosulus, among all mushrooms. The presence of steroids was determined in every mushroom. Steroids are widely used for the treatment of inflammation and several autoimmune diseases.
Anesthesia can be induced by using steroids (Shaikh et al., 2012).
Carbohydrate-based therapeutics are widely used in the treat-

| Antioxidant activity
At 400 μg/ml concentration, both G. lucidum and G. applanatum showed significant (p < .001) increase in the percentage of scavenging activity when compared with ascorbic acid. An increase in the scavenging activity of DPPH radical was found with the increasing concentration of the mushroom extracts ( Figure 1).  Table 3. Different pharmacological activities have shown phenolic compounds among which antioxidant and antimicrobial effects are more prominent (Bahri et al., 2014).
Several reports have also suggested the utilization of flavonoids and many other phenolic compounds as free radical scavenging, anticancer, anti-inflammatory, cardioprotective, and immune system promoting agents (Tungmunnithum et al., 2018). Flavonoids were present in every mushroom, whereas tannins were absent in D.   (Thakur et al., 2018). So the investigated mushrooms with antioxidants can be used to prevent diseases.

| In silico molecular docking analysis
Molecular docking analysis allowed us to identify the binding mode of the targeted ligand molecules with the selective receptor(s). The  Phe78, and Gly439 residues were shown interacting through hydrophobic interaction (Figure 6b). The findings of the docking experiment with GR revealed that 5 showed the highest interaction with the enzyme (Table 5) (Putnam et al., 2000). In addition, like NADPH, both 3 and 6 interacted with Tyr358 residue with hydrophobic interaction, and Tyr358 residue is responsible for the reactivity of electron donation towards the iron-heme group of catalase (Putnam et al., 2000). Moreover, both compounds along with NADPH were interacted with Gly147 residue, which is an alignment with a previous study, showing that aminotriazole interacted with Gly147 residue (Sahoo et al., 2015). Importantly, Val74 and Phe153 are among the residues that are crucial for allowing small molecules to reach the heme molecule (Putnam et al., 2000).

1-5 along with NADPH yielded hydrophobic interaction towards
Arg354, and this amino acid residue might change the metal site by removing charges from Tyr358 residue. Additionally, Arg354 is not only responsible for reducing the charge repulsion, but also the multiple protonations of this amino acid residue are crucial for stabilizing oxidation produced electrostatic fields (Putnam et al., 2000). Further, we also have done molecular docking analysis for the targeted compounds with GR receptor (PDB ID: 1XAN), using FAD as the positive control, which possessed the best docking score of −7.27 kcal/mol. The key interaction includes three hydrogen bond interactions with Lys67, Asn71, and Glu442 residues, and hydrophobic interaction with Tyr106, Val68, Thr72, His75, Val74, Trp70, Phe78, and Gly439 residues. But 6 interacted with four hydrogen bonding with Ser470, Tyr407, Met406, and His82 residues, though the docking score of 6 was only −4.507 kcal/ mol. Also, 5 yielded a hydrogen bond interaction with Met406 and Tyr407 residues. It has been reported that a hydrogen bond with Tyr407 residue is crucial for maintaining the human GR complex, and His82 is pivotal for the disulfide bond with Cys90. In addition, His82 along with His75, Phe78, Met79, and Asp81 contains the active site disulfide (Savvides & Karplus, 1996). Moreover, 1, 7, 8,   9, and 10 along with FAD interacted with His75 residue, 5, 6, 7, 9, and 10 as well as FAD interacted with Phe78 residue, 1, 2, 4, and 8 interacted with Met79, and 5, 6, 7, 9, and 10 interacted with His82

residue.
For the verification of the docking experiments, we performed prime/MM-GBSA analysis, which is a groundbreaking quantum mechanics/molecular mechanics (QM/MM) attribute, to calculate the relative binding affinities utilizing best poses from a ligand-receptor interaction. We observed that most of the compounds yielded the highest binding score. The results of the ADME/T analysis were shown in Table 6. In the current study, ADME analysis was done along with molecular docking simulation. The 10 selected compounds were further checked by online-based prediction server SwissADME to explore their drug candidacy, pharmacokinetic parameters, and physicochemical properties. The pharmacokinetic properties of a drug candidate usually rely on the chemical descriptors of the molecules (Shahinozzaman et al., 2018). According to Lipinski's rules of 5 (RO5), compounds violating any of the rules may have problems with permeability, absorption, and bioavailability as it was reported that compounds with lower molecular weight, hydrogen bond capacity, and lipophilicity may exhibit higher permeability (Duffy et al., 2015), better absorption, and bioavailability (Daina et al., 2014;Christopher A. Lipinski et al., 1997). Importantly, six compounds including 1, 2, 3, 4, 7, and 8 exhibited orally active optimal drug-likeness characteristics, according to Lipinski's RO5. In addition, the parameters involved in Veber's rule suggested that the number of rotatable bonds elicits molecular flexibility, which can be a good descriptor for potential drug candidates. On the contrary, passive molecular transport of drugs through membranes is expressed by the values of the TPSA (Shahinozzaman et al., 2018). Interestingly, almost all compounds except 2, 5, and 6 met the specified requirements under Veber's rule.

| ADME/T analysis
After analyzing the results, we have found five compounds, namely 1, 3, 4, 7, and 8, that satisfied the descriptors of both Lipinski's RO5 and Veber's rule, indicating that these compounds can be deliberated as potential drug molecules with receptor-based optimization.
The bioactivity score provides useful information on the binding cascade of a drug, which can describe the beneficial effects of the drug molecules inside the living body. A drug molecule is supposed to bind with a biological target, also referred to as drug target, and these drug targets are common proteins and can include enzymes, ion channels, and receptors. Molinspiration cheminformatics was utilized to predict bioactivity score for important drug targets like binding to GPCR ligand and nuclear receptor ligand, ion channel inhibition, kinase inhibition, protease inhibition, and enzyme activity inhibition. Importantly, the findings from the biological activity of our targeted compounds depicted no bio-inactivity and moderate to high bioactivity towards all the parameters. Interestingly, the compounds passing both Lipinski's RO5 and Veber's filter were predicted to be compatible with the biological targets inside the living body in comparison with the controls.

| Bioactivity scores of the compounds
Molinspiration cheminformatics server was used to predict the bioactivity score for the selected compounds. NADP and FAD were taken as standard compounds. Selected compounds except 2, 3, 5, and 6 were found to be highly bioactive towards GPCR ligands (>0), which indicates that they could bind more effectively with GPCR and were very close to the standard nicotinamide adenine dinucleotide phosphate (NADP) and better than FAD. The ion channel inhibiting property of 8 and 10 delineated more than the positive controls. Kinase inhibitor activities were observed to be moderate among the selected compounds, whereas binding to nuclear receptor ligand of all the compounds possessed greater bioactivity score in comparison with the controls. Besides, 1, 4, 8, and 10 exhibited higher (>0) bioactivity values for protease inhibition and were close to the standard NADP and better than FAD. Interestingly, the enzyme inhibition score was high (>0) for all the compounds and close to the standard compounds as well. The findings of the biological activities were shown in Table 7.

| CON CLUS ION
The results of the present study exhibited that G. lucidum, G. applanatum, and R. cajanderi mushrooms exert strong antioxidant effects. This could be due to the presence of phenolic compounds like flavonoids and tannins in these mushrooms. Moreover, in silico studies also unleashed that the selective compounds from the mushrooms were biologically active and also interacted with the enzymes from the human antioxidant system. Furthermore, the selected compounds also possessed drug-likeness properties. From the results stated in the preceding sections, we conclude that ROS and oxidative stress play a significant role in many consequences, whereas mushroom originated antioxidant therapy is one of the greatest ways to improve the adverse effects of oxidative stress.
After that, further research on the phytoconstituents of the chosen mushrooms is recommended to understand the precise mechanism by which the mushrooms exhibit their antioxidant capabilities.

ACK N OWLED G EM ENTS
We are thankful to the Research and Publication Cell, University of Chittagong for providing methodological support and the Department of Pharmacy, University of Chittagong for technical and laboratory support.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any competing interests.

E TH I C A L A PPROVA L
As this research works based on in vitro and in silico studies, ethical approval is not required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the analyzed data are available in the manuscript.