separations

: Fusarium oxysporum is one of the most harmful soil-borne pathogens that cause root rot, damping-off, and wilt disease in many plant species. Management of Fusarium oxysporum diseases is often by using many harmful and expensive chemical fungicides which have many harmful effects on the environment and human health. The current study was conducted to identify the chemical constituents of black cumin seeds’ methanolic extract and investigate the ability of the major constituents to inhibit the Fusarium oxysporum trypsin-like serine protease, which play an important role in F. oxysporun pathogenicity. The HPLC-MS analysis of black cumin seeds’ methanolic extract revealed the presence of seven major compounds: amentoﬂavone, Procyanidin C2, Quercetin3-O-sophoroside-7-O-rhamnoside, 5,7-Dihydroxy-3,4-dimethoxyﬂavone, Borapetoside A, tetrahydroxy-urs-12-en-28- O-[b-D-glucopyranosyl (1-2)-b-D-glucopyranosyl] ester, and kudzusapongenol A-hexA-pen. The results of molecular docking between these compounds and the active site of Fusarium oxysporium trypsin showed that only four compounds were able to bind to the active site of F. oxysporum trypsin. Amentoﬂavone, 5,7-Dihydroxy-3,4-dimethoxyﬂavone, and Quercetin3-O-sophoroside-7-O-rhamnoside have the highest binding energy, − 6.4, − 6.5, and − 6.5 Kcal/mol, respectively. In addition, the results clarify that 5,7-Dihydroxy-3,4-dimethoxyﬂavone was the only compound to form a hydrogen bond with Asp189 (the residue responsible for substrate speciﬁcity). The results of the study strongly indicate that ﬂavonoids of black cumin seeds’ methanolic extract could be used as effective inhibitors for the F. oxysporum trypsin-like serine protease.


Introduction
Fusarium oxysporum is one of the main pathogens that cause serious damage to numerous monetarily significant crops worldwide. Phytopathogenic fungi including Fusarium oxysporum possess a number of extracellular cell-wall-degrading enzymes such as cellulases, pectic enzymes, and proteases; those enzymes cause a lot of damage to plant cell walls during pathogenesis [1,2]. Proteases are important for the pathogenicity and growth of phytopathogenic fungi. Among the proteases that are secreted by phytopathogenic fungi is the trypsin-like serine protease [2]; the serine protease takes its name from the serine residue in its catalytic site. The hydroxyl group of the serine residue is responsible for the first step in peptide bond hydrolysis through acting as a nucleophile [3]. The F. oxysporum trypsin-like serine protease consists of two domains; each domain consists of six beta strands. The active site of the F. oxysporum trypsin-like enzyme consists of a catalytic site located in the cleft between the two domains and the specificity bucket that is located near the catalytic site serine residue [4]. Until now, the main method for controlling F. oxysporum worldwide is chemical fungicides. With the increasing awareness of the damage caused by chemical fungicides to the environment, for instance, affecting non-target organisms and soil micro-organisms and contaminating water sources, in addition to the decrease in their effect on phytopathogenic fungi as a result of the development of resistant strains, the search for other means to control phytopathogenic fungi including F. oxysporum has become urgent. One of the possible methods for controlling phytopathogenic fungi is the use of natural compounds. Several studies were conducted to investigate the ability of different natural compounds and plant extracts to inhibit cell-wall-degrading enzymes including proteases as a key role in phytopathogenic fungi control [5,6]. Previous studies have reported the inhibitory effect of black cumin seeds' (N. sativa) oil and extracts on phytopathogenic fungi growth. It has been reported that black cumin seeds' oil decreased 56.67% of the severity of root rot disease caused by Rhizoctonia solani at a concentration of 5% [7], and the different extracts of black cumin seed can inhibit 76-100% of Fusarium oxysporum growth at a concentration of 50 mg/mL [8].
Nigella sativa, commonly known as black cumin (family: Ranunculaceae), is native to south Asia. Black cumin seeds contain a large variety of compounds such as fatty acids, alkaloids, saponins, flavonoids, terpenoids, and quinones [9]. Previous studies showed that black cumin seeds' oil contains high percentage of unsaturated fatty acids, and the major fatty acid was linoleic acid [10]. Moreover, previous studies reported the presence of phenolic compounds including coumaroyl acid derivative and thermoquinol glucoside, flavonoids including quercetin and apigenin, alkaloids including norargemonine and nigellimine, 2-(4-Nitrobutyryl), and saponins such as alpha-hederin, a triterpene saponin in black cumin seeds' extracts [9,11]. Black cumin seeds have been widely used in traditional medicine in the Middle East [12]. In addition, black cumin seeds have been reported to have antibacterial, antifungal, and antioxidant properties [8,13,14].
Recently, several studies have demonstrated that black cumin seeds have an inhibitory effect on different types of proteases including the phytopathogenic fungi R. solani extracellular protease. It has been reported that black cumin seeds' methanolic extract at 2000 ppm inhibited 74% of R. solani protease activity [15]. Moreover, it has been reported that black cumin seeds' oil inhibited 75.4% and 91.1% of the serine protease elastase type I and type II activity, respectively, and 92.4% of collagenase activity at a concentration of 300 µg/mL [16]. On the other hand, there is no available information about the effect of black cumin seeds on the extracellular trypsin-like protease of F. oxysporum.
Molecular docking is a tool used widely in drug discovery studies; it based on using software to predict the interaction between a known 3D structure of a target protein and a small molecule (ligand) and the most likely binding conformation of the ligands and its binding affinity through thermodynamic favorability [17]. The aims of this study are to identify the chemical composition of black cumin seeds' (N. sativa) methanolic extract and to clarify the ability of the major chemical constituents to bind and inhibit the trypsin-like serine protease from Fusarium oxysporum using molecular docking methods.

Analysis of Black Cumin Seeds' Methanolic Extract by HPLC-MS
The yield of the extraction was 30% (w/w). Forty-three peaks resulted from the HPLC-MS analysis of black cumin seeds' methanolic extract ( Figure 1). Seventeen peaks were identified and presented in Table 1, whereas the rest of the peaks were considered to be unknown. The identified peaks represent 83% of the peaks' area. Table 1 shows the retention time, m/z for the [M-H] − ion, m/z of the ESI-MS fragments, chemical formula, and the percentages of identified peaks.
binding affinity through thermodynamic favorability [17]. The aims of this study are to identify the chemical composition of black cumin seeds' (N. sativa) methanolic extract and to clarify the ability of the major chemical constituents to bind and inhibit the trypsin-like serine protease from Fusarium oxysporum using molecular docking methods.

Analysis of Black Cumin Seeds' Methanolic Extract by HPLC-MS
The yield of the extraction was 30% (w/w). Forty-three peaks resulted from the HPLC-MS analysis of black cumin seeds' methanolic extract ( Figure 1). Seventeen peaks were identified and presented in Table (1), whereas the rest of the peaks were considered to be unknown. The identified peaks represent 83% of the peaks' area. Table 1 shows the retention time, m/z for the [M-H] − ion, m/z of the ESI-MS fragments, chemical formula, and the percentages of identified peaks.

Molecular Docking Analysis
AutoDock Vina 1.2.0 was used for a docking study of eight compounds from black cumin seeds' methanolic extract and the known serine protease inhibitor benzamidine against the active site of the Fusarium oxysporum trypsin-like serine protease. The active site of the F. oxysporum trypsin consists of the catalytic site (His57, Ser195, and Asp102) and the specificity pocket S1 (Asp189, Gly216, Asn217, Gly219, and Ser225) [4].
To validate the obtained results of the AutoDock Vina protocol, we have re-docked the substrate that consists of single peptide GLY-ALA-LYS into the enzyme active site and compared the interaction with the X-ray structure of the F. oxysporum trypsin en- In comparison with the literature [33], peak 18 was identified as plumbagin ( Figure 2).

Molecular Docking Analysis
AutoDock Vina 1.2.0 was used for a docking study of eight compounds from black cumin seeds' methanolic extract and the known serine protease inhibitor benzamidine against the active site of the Fusarium oxysporum trypsin-like serine protease. The active site of the F. oxysporum trypsin consists of the catalytic site (His57, Ser195, and Asp102) and the specificity pocket S1 (Asp189, Gly216, Asn217, Gly219, and Ser225) [4].
To validate the obtained results of the AutoDock Vina protocol, we have re-docked the substrate that consists of single peptide GLY-ALA-LYS into the enzyme active site and compared the interaction with the X-ray structure of the F. oxysporum trypsin enzyme and substrate complex (1GDN). The result showed that the substrate binds to the active site of the enzyme with a conformation very similar to the conformation reported in the X-ray structure ( Figure 5). In addition to forming all the hydrogen bonds reported in the X-ray structure ( Figure 5), Lys formed two hydrogen bonds with Ser190 and Gly193, Ala formed one hydrogen bond with Ser214, and Gly formed two hydrogen bonds with Gly216. The interactions between Lys and Asp189, His57, and Ser195 (water bridges) that are reported by Rypniewski et al. [4] in the X-ray structure were not possible within the docking method used in the current study. In the current study, the water molecules were deleted before the docking due to the limitation of AutoDock Vina in differentiating between the water atoms and the target protein atoms and treating the target protein atoms and the mediating water molecules as a one rigid structure which causes the water molecules to prevent the binding of the ligands.
bonds with Gly216. The interactions between Lys and Asp189, His57, and Ser195 (water bridges) that are reported by Rypniewski et al. [4] in the X-ray structure were not possible within the docking method used in the current study. In the current study, the water molecules were deleted before the docking due to the limitation of AutoDock Vina in differentiating between the water atoms and the target protein atoms and treating the target protein atoms and the mediating water molecules as a one rigid structure which causes the water molecules to prevent the binding of the ligands. Three compounds showed no affinity toward the active site of the F. oxysporum trypsin-like serine protease, Kudzusapongenol A-hexA-pen, tetrahydroxy-urs-12-en-28-O-[b-D-glucopyranosyl (1-2)-b-D-glucopyranosyl] ester, and procyanidin C2, as demonstrated by the positive binding energy, whereas the rest of the studied compounds were successfully docked to the enzyme. Table 2 shows the binding energy, type of bonds, bonds length, interacted residues, and 2D interactions between the compounds and the F. oxysporum trypsin-like serine protease. Amentoflavone, 5,7-dihydroxy-3,4-dimethoxyflavone, and quercetin3-O-sophoroside-7-O-rhamnoside showed the highest affinity toward the enzyme active site with a binding energy of −6.4, −6.5, and −6.5 Kcal/mol, respectively. Benzamidine and Borapetoside A bind to the active site of the enzyme with a binding energy of −5.5 and −4.2 Kcal/mol, respectively. All the compounds formed hydrogen bonds with the residues in the enzyme active site except for plumbigan, which binds to the enzyme active site through van der Waals forces only with a binding energy of 5.3 Kcal/mol. Amentoflavone formed two hydrogen bonds with His57 and Gly216, one carbon hydrogen bond with Asn217, and van der Waals forces with Ser195 and Gly219 in the active site of the enzyme and hydrophopic interaction (Pi-Pi stacked) with one residue outside the active site of the enzyme Trp215 (Figure 6), whereas 5,7-dihydroxy-3,4-dimethoxyflavone binds to the active site of the enzyme via two hydrogen bonds with Ser195 and one hydrogen bond with Asp189 and van der  Table 2 shows the binding energy, type of bonds, bonds length, interacted residues, and 2D interactions between the compounds and the F. oxysporum trypsin-like serine protease. Amentoflavone, 5,7-dihydroxy-3,4-dimethoxyflavone, and quercetin3-O-sophoroside-7-O-rhamnoside showed the highest affinity toward the enzyme active site with a binding energy of −6.4, −6.5, and −6.5 Kcal/mol, respectively. Benzamidine and Borapetoside A bind to the active site of the enzyme with a binding energy of −5.5 and −4.2 Kcal/mol, respectively. All the compounds formed hydrogen bonds with the residues in the enzyme active site except for plumbigan, which binds to the enzyme active site through van der Waals forces only with a binding energy of 5.3 Kcal/mol. Amentoflavone formed two hydrogen bonds with His57 and Gly216, one carbon hydrogen bond with Asn217, and van der Waals forces with Ser195 and Gly219 in the active site of the enzyme and hydrophopic interaction (Pi-Pi stacked) with one residue outside the active site of the enzyme Trp215 (Figure 6), whereas 5,7-dihydroxy-3,4-dimethoxyflavone binds to the active site of the enzyme via two hydrogen bonds with Ser195 and one hydrogen bond with Asp189 and van der Waals forces with Gly216, Gly219, and His57 and binds with two residues outside the active site Glu146 through a carbon hydrogen bond and Trp215 through hydrophobic interactions (Amid-Pi stacked) ( Figure 6). Quercetin3-Osophoroside-7-O-rhamnoside formed one hydrogen bond with Ser195, Pi-donor hydrogen bond with His57, and van der Waals forces with Asn217 in the active site of the enzyme while it formed hydrogen bonds with two residues outside the active site Ser214 and Asn99 and had a hydrophopic interaction (Pi-Pi stacked) with Trp215 ( Figure 6). Benzamidine formed two hydrogen bonds, one with Asp189 in the specificity bucket and one with Ser190 outside the active site and salt bridge with Asp189 (Figure 7).

BE (Kcal/mol)
Amentoflavone formed one hydrogen bond with Ser195, Pi-donor hydrogen bond with His57, and van der Waals forces with Asn217 in the active site of the enzyme while it formed hydrogen bonds with two residues outside the active site Ser214 and Asn99 and had a hydrophopic interaction (Pi-Pi stacked) with Trp215 ( Figure 6). Benzamidine formed two hydrogen bonds, one with Asp189 in the specificity bucket and one with Ser190 outside the active site and salt bridge with Asp189 (Figure 7). Benzamidine formed one hydrogen bond with Ser195, Pi-donor hydrogen bond with His57, and van der Waals forces with Asn217 in the active site of the enzyme while it formed hydrogen bonds with two residues outside the active site Ser214 and Asn99 and had a hydrophopic interaction (Pi-Pi stacked) with Trp215 ( Figure 6). Benzamidine formed two hydrogen bonds, one with Asp189 in the specificity bucket and one with Ser190 outside the active site and salt bridge with Asp189 ( Figure 7). Benzamidine and 5,7-dihydroxy-3,4-dimethoxyflavone were the only compounds that formed hydrogen bonds with the Asp189 residue which consider the residue responsible for the substrate binding and the specificity of the enzyme for the amino acids with a positively charged side chain. Quercetin3-O-sophoroside-7-O-rhamnoside and 5,7-dihydroxy-3,4dimethoxyflavone were the only compounds that formed hydrogen bonds with the Ser195 residue in the catalytic site of the enzyme; Ser195 is the residue responsible for starting the cleavage of the peptide bond through a nucleophilic attack on the carbonyl group carbon. The results of the docking indicate that flavonoids of black cumin seeds' methanolic extract, amentoflavone, 5,7-dihydroxy-3,4-dimethoxyflavone, and quercetin3-O-sophoroside-7-Orhamnoside could be effective inhibitors for the F. oxysporum trypsin-like serine protease, specially 5,7-dihydroxy-3,4-dimethoxyflavone, which occupies the specificity pocket S1 of the enzyme and in the same time binds to the catalytic site serine residue, which makes it a perfect potential inhibitor for trypsin-like serine proteases. In addition, in comparison with the docking output of benzamidine, amentoflavone, 5,7-dihydroxy-3,4-dimethoxyflavone, and quercetin3-O-sophoroside-7-O-rhamnoside have been found to be more effective as inhibitors for F.oxysporum trypsin than benzamidine whose effectiveness as a serine protease inhibitor has been proven [34]. Several in vitro and in silico [35] studies had suggested that flavonoids could be effective inhibitors for different types of proteases including serine proteases; according to the results reported by [36], amentoflavone caused the inhibition of 50% of the cysteine protease Cathepsin B at 1.75 µM. Furthermore, it has been reported that quercetin and amentoflavone inhibited 50% of human thrombin activity at 57.77 and 19.5 µM, respectively [37].  Benzamidine and 5,7-dihydroxy-3,4-dimethoxyflavone were the only compound that formed hydrogen bonds with the Asp189 residue which consider the residue re sponsible for the substrate binding and the specificity of the enzyme for the amino acid with a positively charged side chain. Quercetin3-O-sophoroside-7-O-rhamnoside and 5,7-dihydroxy-3,4-dimethoxyflavone were the only compounds that formed hydrogen bonds with the Ser195 residue in the catalytic site of the enzyme; Ser195 is the residue responsible for starting the cleavage of the peptide bond through a nucleophilic attack on the carbonyl group carbon. The results of the docking indicate that flavonoids o black cumin seeds' methanolic extract, amentoflavone 5,7-dihydroxy-3,4-dimethoxyflavone, and quercetin3-O-sophoroside-7-O-rhamnoside could be effective inhibitors for the F. oxysporum trypsin-like serine protease, specially 5,7-dihydroxy-3,4-dimethoxyflavone, which occupies the specificity pocket S1 of the en zyme and in the same time binds to the catalytic site serine residue, which makes it a perfect potential inhibitor for trypsin-like serine proteases. In addition, in comparison with the docking output of benzamidine, amentoflavone 5,7-dihydroxy-3,4-dimethoxyflavone, and quercetin3-O-sophoroside-7-O-rhamnoside have been found to be more effective as inhibitors for F.oxysporum trypsin than ben zamidine whose effectiveness as a serine protease inhibitor has been proven [34]. Severa in vitro and in silico [35] studies had suggested that flavonoids could be effective inhibi tors for different types of proteases including serine proteases; according to the result reported by [36], amentoflavone caused the inhibition of 50% of the cysteine protease Cathepsin B at 1.75 µM. Furthermore, it has been reported that quercetin and amen toflavone inhibited 50% of human thrombin activity at 57.77 and 19.5 µM, respectively [37].
Borapetoside A binds to the active site via two hydrogen bonds with Gly216, a hy drophobic interaction (Pi-Pi T-shaped) with His57, and van der Waals forces with Ser195, Gly219, and Asn217 in the active site of the enzyme, and binds to four residue outside the active site Asn99 via a hydrogen bond, Cys42 and Trp41 via hydrophobi interactions, Pi alkyl and Pi-Pi T-shaped, respectively, and Gln192 via an unfavorabl interaction (unfavorable acceptor-acceptor); however, Borapetoside A showed the low est affinity toward the enzyme active site. The low binding energy was attributed to the unfavorable interaction with the Gln192 residue outside the active site of the enzyme Despite the relatively low binding energy, the result of the docking suggests tha Borapetoside A binds to the active site via two hydrogen bonds with Gly216, a hydrophobic interaction (Pi-Pi T-shaped) with His57, and van der Waals forces with Ser195, Gly219, and Asn217 in the active site of the enzyme, and binds to four residues outside the active site Asn99 via a hydrogen bond, Cys42 and Trp41 via hydrophobic interactions, Pi alkyl and Pi-Pi T-shaped, respectively, and Gln192 via an unfavorable interaction (unfavorable acceptor-acceptor); however, Borapetoside A showed the lowest affinity toward the enzyme active site. The low binding energy was attributed to the unfavorable interaction with the Gln192 residue outside the active site of the enzyme. Despite the relatively low binding energy, the result of the docking suggests that Borapetoside A could be a potential inhibitor for the F. oxysporum trypsin-like serine protease. The inhibitory effect of diterpenoids and their derivatives against the serine protease has been reported [38].
Plumbagin was the only compound that does not bind to the catalytic site or the specificity pocket of the enzyme through hydrogen bonds or hydrophopic inertactions but interacts with a relatively high affinity with two residues in the specificity pocket, Gly216 and Asn217, and four residues outside the active site of the enzyme, Asn99, Trp215, Try172, and Ala175. It is unknown whether plumbagin interactions would affect the enzyme activity or not.

Materials
Black cumin seeds (Nigella sativa) were purchased from Ragab El Attar herbs shop at Cairo, Egypt. All the solvents utilized in the current study were of analytical grade and purchased from Sigma-Aldrich, Cairo, Egypt.

Preparation of Extract
Black cumin seeds were ground to fine powder and extracted using step-wise extraction technique. The ground seeds were extracted by macerating 50 gm of the resulted powder in 500 mL of solvent and hexane, followed by methylene chloride, and then methanol 70% (v/v) was prepared as 70 parts of absolute methanol to 30 parts of distilled water at room temperature (28 • C). Every step of the extraction was repeated 3 times. The extracts were recovered from solvents by air drying the solvents at room temperature (28 • C) [39].

High-Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS) Analysis
Chemical composition of black cumin seeds' methanolic extract was determined by HPLC-MS. Shimadzu HPLC-2040 (Shimadzu, Kyoto, Japan) equipped with LC 2024 controller, LC-2040 Pump, LC-2040 autosampler, and LC2030/2040 PDA detector at 254 nm was used. Shimadzu uplcms 8045 C-18 column (1.7 mm × 2.1 mm × 50 mm) was used for compound separation, and Bruker triple quadrupole LC-mass spectrometer was employed. The mobile phase was gradient of water and acetonitrile, ranging from 5% acetonitrile to 95% at flow rate of 0.2 mL/min, and the injected volume was 2 µL. The interface was set to 4.00 kv voltage and 300 • C temperature, and the flow rate of the heating and drying gases were 10.00 L/min. Negative electrospray ionization mood was used and the mass spectra were recorded at 3000 u/s scan speed in the range of 100-1200 m/z. The compounds were identified by comparing the resulting spectrums with WILEY 09 and NIST 11 mass spectral databases and literature, and the structure of identified compounds was drawn using King Draw 3.0 software (King Draw Business Corporation, Qingdao, China).

Ligand Preparation
Ligand structure was drawn using Chem Draw 21.0.0 (PerkinElmer, Waltham, MA, USA) and saved as SDF files. Ligand energy was minimized by MM2 calculation, logP was calculated, and the structures of the ligands were converted to pdb file format using Chem3D 21.0.0. Nonpolar hydrogen atoms were deleted, Gasteiger charges were calculated, torsion root was detected, and the structures were saved as pdbqt file format using AutoDockTools-1.5.6.

Target Protein Preparation
The target protein, Fusarium Oxysporum trypsin-like serine protease enzyme structure encoding 1GDN [4], was downloaded from Protein Data Bank "www.rcsb.org/. (accessed on 24 July 2022)". The target protein structure was prepared by deleting water and solvent molecules and ligands, adding polar hydrogen atoms, calculating kollman charges, and saving the structure of target protein as pdbqt using AutoDockTools-1.5.6 (The Scripps Research Institute, San Diego, CA, USA).

Molecular Docking Procedures
The molecular interactions between the active site of Fusarium oxysporium trypsin-like serine protease and eight compounds of black cumin seeds' methanolic extract and the known serine protease inhibitor, benzamidine, were determined using AutoDock Vina 1.2.0 (The Scripps Research Institute, San Diego, CA, USA) [40]. A random seed number was used and the exhaustiveness function was increased to 32. A 18 × 15 × 22 Å grid box with 2.663 × −1.211 × −9.636 grid point spacing of 1 Å was used for docking the ligand into the enzyme active site.

Analysis and Visualization of Protein-Ligand Interactions
The conformers of each ligand were separated using vina_split command. The conformer with the highest affinity was analyzed and visualized using Discovery Studio-21 software (Dassault Systems BIOVIA San Diego, CA, USA). In silico interactions between the substrate and F. oxysporum trypsin-like serine protease was analyzed and visualized using PyMOL software (Schrödinger, Inc., Broadway, NY, USA).