In Silico Pharmacological Analysis of a Potent Anti-Hepatoma Compound of Mushroom Origin and Emerging Role as an Adjuvant Drug Lead

Mushrooms are well-known to possess a continuum of anticancer metabolites that are vital in the development of anticancer adjuvant drug leads based on natural products. Owing to the fact that conventional cancer therapeutic methods were failed to lessen mortality caused by cancer to the estimated level with occurrence of adverse side effects, anticancer agents isolated from natural mushroom sources unarguably make an experimental research area worth mass focus today. The current study was targeted on in vitro cytotoxicity and in silico predictive pharmacological analysis of a flavonoid compound isolated from Fulvifomes fastuosus mushroom. Targeted compound was isolated from the mushroom using different chromatographic methods and identified by NMR spectrometry and mass spectrometry. Cytotoxicity -3',4-dione has a high tendency to act as a good anticancer adjuvant drug in the treatment of hepatoma.


Introduction
It is well reputable that the use of medicinal mushrooms provides advantageous effects on human well-being mainly due to its therapeutic value [1]. The utilization of mushroom products as a source of medication has been commenced more than 100 years ago as they are enormously rich in biologically active secondary metabolites. Among 14,000 of mushroom species that have been identified yet, about 650 species of mushrooms have been reported to possess promising anticancer activity [2] [3]. Bioactive compounds isolated from mushrooms with anticancer properties have been identified to contain polysaccharides, polysaccharide-protein complexes, dietary fiber, certain types of proteins, phenols, terpenoids, flavonoids and steroids etc. [3] [4]. Nowadays, a major challenge encountered by the entire world community is to uncover a feasible strategy to fight neoplastic diseases as cancer is still a chronic disease that causes severe death or long term effects throughout the life [4]. Plentiful side effects given by conventional therapies to treat cancer including surgery, chemotherapy, and radiation therapy, promote the use of additional therapy to retain and improve the immune status of patients [5]. Scientists made it feasible with introduction of the use of natural drugs with conventional therapy as an adjuvant treatment, especially those that have cytotoxic activity with minor side effects. For instance, lentinan, schizophyllan and krestin are natural compounds which were isolated from Lentinus edodes, Schizophyllum commune and Trametes versicolor mu- Food and Nutrition Sciences shrooms species. All these metabolites have passed clinical trials and approved as prescribed cancer drugs in Japan to use them in adjuvant cancer therapy [6].
It is worth to note, that clinical trials for mushroom originated drugs are sufficiently expensive and long-term. Hence, researchers have done plenty of investigations for anticancer activity of medicinal mushrooms in vitro and in vivo.
However, most of the drugs have been passed clinical trials. Initial phases of clinical trials for these drugs exhibit great anticancer activity with absence of immediate or long-term toxicity [7] [8].
In spite of the potency of the natural anticancer compounds isolated from mushrooms, they must reach the target in the human body in adequate concentration, and reside there in a bioactive form long enough for the execution of expected biologic events to be effective as a drug. Hence, drug development process of a potent molecule involves assessment of absorption, distribution, metabolism and excretion (ADME) of that targeted substance inside the body [9]. In silico methods have been fostered as valid substitutes to experimental procedures for prediction of ADME, particularly at initial steps, when investigated substances are abundant but the availability of compounds is scarce [9].
In silico toxicology is one form of toxicity assessment which uses computational resources such as methods, algorithms, software, data, etc., in order to analyze, organize, simulate, model and predict toxicity of chemical substances [10]. It is knotted with in silico pharmacology, which uses data through computational tools to analyze beneficial or harmful effects of drugs in therapeutic purposes [11]. Computational methods are targeted to complement in vitro and in vivo toxicity experiments to potentially minimize the necessity for animal testing. Moreover, it aims to lessen the cost and time required for animal experiments and safety assessment [12]. Drug discovery is a multifarious process with the aim of determining efficacious molecules where their selectivity and strength are balanced with absorption, distribution, metabolism, excretion and toxicity (ADMET) properties to find out the fitting dose and dosing interval [13]. The connection between physicochemical properties and molecular structure is well known. The link between physicochemical properties and a drug's biological behavior exhibits an indirect relationship back to structure. It facilitates the prediction of a biological property using particular molecular manipulation [14]. In the point of minimizing wasted drug discovery efforts and prevention of the synthesis of high risk compounds, computational methods are used to predict the physicochemical properties accurately.
In this study, ADMET behaviors of key physicochemical properties, such as ionization, hydrophobicity, solubility, hydrogen bonding strength will be predicted in silico using SwissADME web tool [15]. Detailed analyses of the binding characteristics lead to ranking of the targets according to the tightness of binding [16]. This study will be also targeted a in silico study of drug likeliness of the isolated compound which is useful in the identification of drug targets related to hepatoma and prediction of binding-conformation of small molecule ligands to  [16]. Swiss target prediction software is used to predict target proteins for the isolated test molecule. The BOILED-Egg construction model is used via SWISSADME to assess the gastrointestinal absorption and brain penetration of the targeted compound. Gastrointestinal absorption and brain penetration data are standardized by this model and subjected to lipophilicity (WLOGP) and polarity (tPSA) computation [16].

Fungal Material
The specimen of F. fastuosus was collected from the dry zone forest reserves of Voucher specimens were deposited at the same Institute (UOC:DAMIA:D27b).

Large Scale Extraction of Fruiting Bodies of F. fastuosus
Mature fruiting bodies of F. fastuosus were cleaned, dried in the oven at 40˚C to a constant mass and pulverized. Shredded and ground mushroom specimen of fruiting bodies from F. fastuosus (1 kg) was subjected to sonication extraction with methanol (4 L), for 5 -6 hours at 37˚C. Methanol extract was filtered twice through Whatman No. 1 filter paper and same extraction procedure was repeated for three times. Filtrates were pooled and evaporated to dryness at 40˚C under reduced pressure using rotary evaporator. The resulting dried methanol extract was dissolved in distilled water (500 mL) with maximum solubility and partitioned into hexane by liquid-liquid extraction. Remainder in water was partitioned into dichloromethane. Dichloromethane layer was dispensed and aqueous layer was extracted into ethyl acetate using same extraction procedure. Ethyl acetate fraction was evaporated in a rotary evaporator to yield dried extract.

Isolation of Test Compound by Normal Phase Column Chromatography
The ethyl acetate extract was fractionated by normal phase column chromatography using the following protocol. The dried ethyl acetate extract was dis- nm UV lamp. Further purifications were performed using reverse phase preparative HPLC to obtain the purified product of the targeted compound.

Purification by High Performance Liquid Chromatography (HPLC)
Dried compound was further purified by a recycling preparative-HPLC (Model LC-908, JAI Co., Japan) eluting with a linear gradient from 9:1 to 3:7 water:methanol acidified with 0.04% trifluoroacetic acid at a flow rate of 5 mL/min. Prime sphere C 18 HC (10 g) 50 × 250 mm with a 50 × 30 mm guard column was used in the preparative HPLC. In this manner, purified HPLC fraction comprising the compound 2-

Identification and Characterization of the Compound by Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker avance AV-400 or 500 MHz spectrometer in deuterated acetone: methanol (1:1) mixture. One and two-dimensional NMR experiments, including 1 H-NMR (proton NMR), 13 C-NMR, DEPT 13  Overhauser Effect Spectroscopy (NOESY) provided molecular structure information. In addition, HREIMS (High Resolution Electrospray Ionization Mass Spectrometry) was performed to obtain the high resolution mass spectrum.

Cell Lines and Cell Culture
Hep-G2 and CC-1 cell lines (ATCC) were cultured in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), 3% glutamine, sodium bicarbonate and antibiotic (penicillin/ streptomycin). The cells were incubated at 37˚C in a humidified CO 2 incubator.

Morphological Determination and DNA Fragmentation of Apoptosed Cells
The

SwissADME Web Tool
Assessment of in silico pharmacokinetic parameters, physicochemical parameters, drug likeliness, lipophilicity and medicinal chemistry friendliness properties for the molecule were performed using SwissADME web tool.

BOILED-Egg Construction Model
The BOILED-Egg construction model was obtained via SWISSADME to assess the gastrointestinal absorption and brain penetration of the targeted compound.
This model standardizes gastrointestinal absorption and brain penetration datasets and converts into SMILES notation which in turn subjected to lipophilicity (WLOGP) and polarity (tPSA) computation.

Prediction of Target Proteins of the Compound
The target proteins for the isolated molecule have been predicted using the Swiss target prediction software. Interactions were selected based on following criteria; I) involve human proteins (II) annotate as direct binding with an activity (K i , K d , IC 50 or EC 50 ) ≤ 10 µM (III) involve molecules consisting of 80 heavy atoms and (IV) involve targets that are single proteins or protein complexes.

Calculations and Statistics
All experiments were carried out in triplicate and set values are representative for at least three independent experiments. All the results of the experiments were presented as the mean ± standard deviation (Mean ± SD). The results were statistically analyzed by SPSS statistic software package. A p-value less than 0.05 was considered as statistically significant.

Structure Elucidation of the Flavonoid Compound Isolated from Fulviformes fastuosus
The spectroscopic methods including 1D and 2D NMR experiments were used to identify the chemical structure of the isolated compound. It was confirmed as    [19]. Apart from this, this compound can be biosynthesized via rearrangement of interfungins A, which is resulted from the condensation of hispidin and hispolon moieties (Figure 2).

Morphological Changes of Apoptosed Cells
Apoptosis is an orderly process of the terminal morphological and biochemical events of programmed cell death which is represented by the specific changes in

Determination of the in Silico Toxicological Parameters of the Isolated Compound Using SwissADME
The SwissADME tool has estimated the key physicochemical, pharmacokinetic, drug-like and and medicinal chemistry friendliness properties for the molecule. This SwissADME section gives access to five different rule-based filters, with diverse ranges of properties inside of which the molecule is defined as drug-like. These filters often originate from analyses by major pharmaceutical companies aiming to improve the quality of their proprietary chemical collections. The Lipinski (Pfizer) filter is the pioneer rule-of-five implemented rules. The Ghose (Amgen), Veber (GSK), Egan (Pharmacia) and Muegge (Bayer) methods have made other 4 rules for drug likeliness. Multiple estimations allow consensus views or selection of methods best fitting the end-user's specific needs in terms of chemical space or project-related demands. Any violation of any rule described here appears explicitly in the output panel [21]. The 2-(3,4-dihydroxyphenyl)-6-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-5'-methylspiro[2H-furo[3,2-c]pyran-3,2'-furan]-3',4-dione was first described by its chemical structure and canonical SMILES together with the Bioavailability Radar. Bioavailability Radar displays rapid appraisal of drug-likeness [21]. Six physicochemical properties were taken into account: lipophilicity, size, polarity, solubility, flexibility and saturation, pharmacokinetics, drug-likeness and medicinal Chemistry (Figure 9).
SWISS-ADME has analyzed pharmacokinetics parameters including the response towards CYP and P-gp/MDR1proteins that are localized at the apical/luminal membrane of enterocytes [21]. P-gp/MDR1 and CYP3A proteins act as a major protective barrier for the bioavailability of orally administered drugs synergistically. Moreover, P-gp/MDR1 also plays a role in counteracting active transport of drug molecules back to the lumen after it is passively absorbed into the enterocytes [21]. The localization of P-gp/MDR1 and CYP3A represents that  of the MDR1 gene. It leads to therapeutic failure for many drugs that are P-gp substrates [22]. Although the targeted test compound is a non-P-gp substrate, accidental increase of P-gp will not affect the therapeutic failure of the compound.
The activity of CYP enzymes is increased or decreased due to the impact of drug via altering the rate at which the drug is metabolized and removed from the body. If the drug increases the activity of a CYP protein, it causes the drug to become ineffective, due to the rapid removal of drug from the body [22]. On contrary, if the drug inhibits a CYP protein, CYP can prevent the accumulation of drug to toxic levels, even to the level of causing an overdose. Among several families of CYP proteins, CYP1, CYP2, CYP3 and CYP4 are the most essential proteins in terms of drug biotransformation; particularly CYP3A4, act as the most prevalent CYP in the body and involves in metabolizing many drugs [23].
As this compound act as a non-inhibitor for CYP3A4, it is not responsible for increasing activity of other CYP proteins allowing better absorption and clearance of the drug. Furthermore, the resulted output was compared with the  Figure 10).
Molecules targeting oral administration, solubility are one foremost property that influences absorption to deliver an adequate quantity of active ingredient in a small volume [24]. Moreover, it greatly facilitates various drug development activities, mostly the ease of handling and formulation. Since this targeted compound is having soluble in water, it demands for drug developmental activities.

BOILED-Egg Construction Model of the Molecule
The BOILED-Egg model is a perceptive graphical classification model which predicts the propensity for a given small molecule to permit passive human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) permeation ( Figure 11).  The white region is the physicochemical space of molecules with highest probability of being absorbed by the gastrointestinal tract, and the yellow region (yolk) is the physicochemical space of molecules with highest probability to permeate to the brain. Yolk and white areas are not mutually exclusive. This model represents the tendency for 2-(3,4-dihydroxyphenyl)-6-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-5'-methylspiro[2H-furo[3,2-c]pyran-3,2'-furan]-3',4-dione in permitting passive human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) permeation.

Prediction of Target Proteins of the Compound
It appears that Glyoxalase I, Matrix metalloproteinase 9, Matrix metalloproteinase 2 and Receptor protein-tyrosine kinase erbB-2 have a higher probability to act as target proteins for 2-  Figure 12). All target proteins fit into the target classes namely enzymes, proteases, kinases, Phosphodiesterase and Lyase. Glyoxalase I is an enzyme and Matrix metalloproteinase 9, Matrix metalloproteinase 2 are proteases. Receptor protein-tyrosine kinase erbB-2; Epidermal growth factor receptor erbB1 and Vascular endothelial growth factor receptor 2 are kinases. Frequency of the target classes is represented in the pie chart given in Figure  12. 3',4-dione acts as a inhibitor for CYP2C9 protein and non-inhibitors for CYP1A2, CYP2C19, CYP2D6 and CYP3A4 proteins allowing better absorption and clearance of the targeted compound. Moreover, as test compound act as a non-substrate for P-gp/MDR1, it will not influence the therapeutic failure of the compound.
As most of the mushrooms belong to the family hymenochaetaceae, which is the family of F. fastuosus have been used as edible forms or are known to be non-toxic [25] [26] test compound has a greater potential to act safely in human body without producing any complications. Also, this fact was greatly supported by the high selectivity index shown by the 2- terfere with the anticancer activity of the test compound [27]. Apart from it, substance can be added to fruit juice or nutrient drinks comprising nutraceuticals such as dietary supplements, vitamins and minerals.