Investigation on chemical composition, antioxidant activity and SARS-CoV-2 nucleocapsid protein of endemic Ferula longipedunculata Peşmen

A.Göçeria,, İ. Demirtaşb, M.H. Almac, Ş. Ademd, Z.A. Kasrae, F. Gülf and A. Uzung aDepartment of Bioengineering and Science, Scientific Research Center, Erbil Polytechnic University, KRG, Iraq bDepartment of Biochemistry, Faculty of Science and Arts, Igdir University, 76100, Igdir, Turkey cRector of Igdir University, 76100, Igdir, Turkey d Department of Chemistry, Faculty of Sciences, Çankırı Karatekin University, Çankırı, Turkey eDepartment of Bioengineering and Science, Salahaddin University-Erbil, Iraq fDepartment of Property, Protection and Security, Igdir Vocational School, Igdir University, 76000, Igdir, Turkey g Faculty of Forestry, Department of Forest Botany, Kahramanmaraş Sutcu İmam University, Turkey Corresponding author: goceriali@gmail.com


INTRODUCTION
The Apiaceae family consists of flowering and aromatic plants which are best known for their characteristic flowers, fruits (Heywood, 2007) and volatile substances (Widodo et al., 2014). Ferula longipedunculata Peşmen, Apiaceae, is a wild plant which is indigenous to Turkey. It grows in the central Anatolia region of the country. This plant has been used in Turkish folk medicine for stomach pain and as a wound healing remedy. Also, the roots and leaves of the Ferula plant are consumed as tea in Antolia in order to increase the aphrodisiac effect and sperm count. It has also been reported to be used to increase milk yield and fertility in goats and sheep (Pakdemirli, 2020). The Ferula species has been the subject of many studies on the chemicals often used in the characterization of compounds identified in the world as well as in the medical field. In the biochemical analysis, coumarins, methanolic, benzoic acid, antibacterial sesquiterpenes, ferulenol, terpenoids, steroidal esters, methanol, ethanol, sulfides, sinkiangenorin C have been found in many compounds and have been reported to be used in medicine (Duran et al., 2020;Li et al., 2015;Yang et al., 2006).
Antioxidants are gaining importance in the human health and food industry worldwide. Antioxidants are substances that prevent the easy degradation of the structure even in small quantities and the deterioration of the structure of oxidized substances (Brewer, 2011). Antioxidants are the main defense mechanism in the body and act as free-radical scavengers. They are manufactured inside the body and involve catalase, dismutase and peroxidase enzymes. BHT is the most widely used antioxidant and is a lipophilic organic compound, chemically a derivative of phenol, which is beneficial for antioxidant activity. Its aims to decelerate the effect of free-radical deterioration in several areas, especially the food, biomedical, rubber, plastic, oil, and petroleum industries (Yehye et al., 2015) SARS coronavirus-2 (SARS-CoV-2) is a pathogen which is easily transferred from human to human. It is the main cause of the worldwide pandemic with serious diseases and death rates (Raoult et al., 2020). The coronavirus nucleocapsids (N) play a delicate role in improving the activity of virus transcription and assembly. Therefore, they were suggested as targets for drugs to combat CoVs (McBride et al., 2014). Plants are rich sources of natural compounds with antiviral effects (Sytar et al., 2021). The therapeutic potential of many phytochemicals has been reported with in silico techniques to combat coronavirus (Adem et al., 2020;Galanakis et al., 2020). Molecular docking studies are actively used to describe biologically active compounds with the potential to bind the SARS-CoV-2 Nucleocapsid protein. However, no biotechnologically detailed studies on Ferula longipedunculata Peşmen plant have been found.
The aim of this study was to investigate the affinities of the phytochemicals found in the Endemic Ferula longipedunculata Peşmen towards SARS-CoV-2 nucleocapsid in silico. The constituents of the root, stem and green parts of the plant were investigated as the main reason for the chemical composition, antioxidant activities and SARS-CoV-2 nucleocapsid of Ferula longipedunculata Peşmen.

Plant Material
Parts of Ferula longipedunculata Peşmen were collected from the Berit mountain province, (Figure 1), central Anatolia, Turkey during the flowering stage (June 15, 2015). After identification of the plant by Prof. Dr. Ömer Saya, a voucher (No. 1416) was deposited in the KOSAF herbarium of Turkey. The collected plant materials were air-dried in the shade.

Extraction Procedure
122 g (root), 82 g (stem) and 75 g (green-aerial) parts of the plant were dried at room temperature and cut into small pieces before being macerated three times (24h each time) with methanol/H 2 O (80%). After filtration and evaporation, the obtained extract was partitioned with solvents in increasing polarity: chloroform, ethyl acetate and n-butanol. Each extract was evaporated under reduced pressure. The obtained extract contained (6.1 g root) CHCl 3 , (0.9 g stem) EtOAc and (1.3 g green part) n-BuOH. Antioxidant activity analyses were performed with 10 grams of each plant material set on a balloon flask and 100 ml methanol and acetone solvents were added to each one. Extraction was then carried out for two hours, using conventional extraction methods (Khan et al., 1988).

Isolation of the essential oils
The air-dried root of F. longipedunculata was subjected to methanol-distillation for 2 hours, using a Clevenger-type apparatus, according to the method recommended by the (European Pharmacopia procedure, 1983) to produce oils. The obtained essential oil was dried and after filtration, and stored at 4 °C until analysis.

Gas Chromatography (GC)
Fatty acids were analyzed by GC-MS (Agilent Technologies 7890A model GC system, 5975C inert MSD with Triple-Axis Detector/USA) using a BPX-20 capillary column (30 m x 0.25 mm, 0.25 µm film thickness; 5% phenyl polysilphenyl IN-siloxane), 70 eV ionization voltage, and FID detector. The oven temperature was between 50 and 120 ºC at 5 ºC/min and 120-240 ºC at 10 ºC/min and held for 5 minutes. 1.0 µL of diluted extracts 300:1 was injected in the split mode. The injector and detector temperatures were adjusted to 220 ºC and 290 ºC, respectively. Helium was used as carrier gas at a flow rate of 1 mL/min. The samples were determined with 1/1000 dilutions (Demirtas and Sahin, 2013).

Gas Chromatography/Mass spectrometry (GC/ MS)
GC/MS analysis was performed by gas chromatography-mass spectrometer using a BPX20 column with autosampler and column (30 m x 0.25 mm x 0.25 μm film). A GC/MS detection system was used for electron ionization (ionization energy 70 eV). Helium was used as carrier gas at a a flow rate of 1.3 mL/min and diluted to 1/1000 (Demirtas and Sahin, 2013).

Molecular Docking Study
The docking studies used Molegro Virtual Docker software. The Crystal Structure of the N-terminal RNA binding domain of the SARS-CoV-2 nucleocapsid protein (PDB ID:6M3M) was downloaded from the online PDB database (www. pdb.org), and prepared for molecular docking using Molegro Virtual Docker Tools. The score function used was the MolDock score with the coordinates of the position X: 8.50 Y: -34.91 and Z:-28.06 at 16 Å3 radius, and 0.30 grid resolution. The docking region of the protein was selected according to previously reported studies (Dinesh et al., 2020;Kang et al., 2020). The 3D structure of the phytochemicals was downloaded from the website https://www.ncbi.nlm.nih.gov/ pccompound, and geometrically optimized utilizing MarvinSketch 19.27 software.

Quantitative analysis by HPLC-TOF/MS
A HPLC analysis was performed with an Agilent Technology 1260 Infinity HPLC System equipped with 6210 Times of flight (TOF) LC/MS detector and ZORBAX SB-C18 (4.6 x100 mm, 3.5 µm) column. Mobile phases A and B were ultra-pure water with 0.1% formic acid and acetonitrile, respectively. The flow rate was 0.6 mL/min and column temperature was 35 ºC. Injection volume was 10 µL. The solvent program was as follow: 0-1 min 10% B; 1-20 min 50% B; 20-23 min 80% B; 23-30 min 10% B. Ionization mode of HPLC-TOF/MS instrument was negative and operated with a nitrogen gas at 325 ºC, nitrogen gas flow of 10.0 L/min, nebulizer of 40 psi, a capillary voltage of 4000 V and finally, fragmentor voltage of 175 V. For sample analysis, dried crude extracts (200 ppm) were dissolved in methanol at room temperature. Samples were filtered through a PTFE (0.45µm) filter with an injector to remove particulates (Demirtas and Sahin, 2013;Abay G et al., 2015).

DPPH radical-scavenging activity
Different methods can be used to evaluate antioxidant activity but a rapid, simple and inexpensive method to measure the antioxidant capacity of food is DPPH, which is widely used to test the ability of compounds to act as free-radical scavengers or hydrogen donors and to evaluate antioxidant activity (Kedare SB et al., 2011).
The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for the investigation of the free-radical scavenging activity of the extracts (Nabavi et al., 2008). Different concentrations of extract were added to the same volume of a methanol and acetone solution of DPPH (100 mM). Absorbance was recorded at 517 nm after 30 min in the dark at room temperature for reaction to take place. All tests were carried out three times. BHT was used for standard controls. The inhibition of free-radical DPPH in percent (I%) was calculated as follows: Where A blank is the absorbance of the control reaction (containing all reagents except the test compound), and A sample is the absorbance of the test compound.

Chemical composition of the fatty acids
The analysis of fatty acid compositions of root, green and stem parts of F. longipedunculata plant was performed using gas chromatography (GC-MS).
The results obtained from the GC and GC-MS analysis of the fatty acids of the plant are presented in Table 1. 16, 6 and 4 components of the root, green and stem parts, respectively, were identified as fatty acids representing 100%. For all parts (root, green and stem) the major compound was linoleic acid at 70.37, 35.38 and 53.58%, respectively. Our research showed that the stem part had more fatty acid than the root and green parts. A literature search showed that Ferula oils are rich in fatty acids (El-feraly and Khan, 2001;Garg and Agarwal, 1988;Nagatsu et al., 2002).

Chemical composition of the essential oil
The GC-MS analysis of the essential oil of the F. longipedunculata root part is presented in Table 2. Eighteen compounds, representing 99.9% of the essential oil, were identified and characterized. Monoterpene β-phellandrene (53.46%) was the major compound in this plant. Other major monoterpene compounds included ocimene (6.79%), 4-terpineol (5.94%) and sesquiterpene santalol (5.03%).

Identification and quantification of phenolic acids by HPLC-TOF/MS
The n-BuOH extract was obtained from the root, green and stem parts of Ferula longipedunculata and analyzed by HPLC-TOF/MS. The identification was performed based on their retention times and mass spectrometry by comparison with those of different standards. The results show the presence of 43 compounds including 17 organic and phenolic acids (Table 3), 26 flavonoids and phenolics (Table  4). Some phenolics were detected in a very small amount and barely reached detection limits (trace) because their concentration had not been seen. The main compounds of F. longipedunculata were fu-maric acid, quercetin-3-β-D-glucoside, quercetin, ferulic acid, vanillic acid, and 4-hydroxybenzoic acid. The highest amounts were determined as vanillic acid in the root part, quercetin-3-β-D-glucoside in the green part and fumaric acid in the stem part. The green part of the plant contains more flavonoids than other parts of the plant. In terms of the phenolic acid richness of the plant parts, it was determined as stem, green and root part, respectively. As a result, F. longipedunculata is rich in flavonoids and phenolic compounds.

DPPH radical-scavenging Activity
The antioxidant activity may be due to different mechanisms, such as the decomposition of peroxides, prevention of chain initiation, reducing capacity, prevention of continued hydrogen abstraction, free-radical scavenging and binding of transition metal ion catalysts (Mao et al., 2006). The radical scavenging activity of organic extracts was determined from the reduction in the optical absorbance at 517 nm due to the scavenging of stable DPPH free radicals. The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen contribution ability. DPPH is a stable free radical and accepts an elec-  (Soares et al., 1997). The DPPH radical-scavenging activity of F. Longipedunculata root oil and its methanol and acetone extract are shown in Table 5. The methanol root extract at 0.1 mL concentration had the highest antioxidant value (98.5%). In the acetone solvent, it was found that the parts of green and stem at 0.3 mL concentration had the highest antioxidant value (86.8%). Among the solvent extracts from different parts of F. longipedunculata, the lowest concentration of methanol extract had the best antioxidant activity, whereas the stem part of the acetone extract showed the lowest activity. Interestingly, the results of the DPPH free-radical scavenging assay showed that the extracts had higher activities than the positive control (BHT) in all concentrations and higher activities in lower concentrations in methanol extracts as seen in Table 5. The reason for the high antioxidant activity is due to the phenolic compounds it possesses. The extract of F. assafoetida exhibited a good antioxidant activity in all models studied. The extracts had good Fe 2+ chelating ability, DPPH radical and nitric oxide scavenging activity (Dehpour et al., 2009). Ferula-assafoetida leaves are free-radical scavengers and may act as primary antioxidants, which react with free radicals by donating hydrogen (Nabavi et al., 2011). Research shows that the ferula-assa-foetida leaves have different kind of flavonoides, phenolic compounds (Dehpour et al., 2009). All these compounds probably contribute to the main reason for its significant radical-scavenging activity. Research- ers recently obtained better results regarding natural antioxidant compounds like gallic acid, coenzyme Q10, rosmarinic acid, tannins and flavonoids from medicinal herbs rather than artificial antioxidants (Tavafi and Ahmadvand, 2011). Natural antioxidants compared to artificial antioxidants are much safer and more beneficial and also have fewer side effects (Craft et al., 2010).

Docking Results
The SARS-CoV-2 nucleocapsid is a vital protein in the RNA genomic packing, viral transcription, and assembly in an infectious cell (Raoult et al., 2020). Therefore, it is considered an excellent target to battle against SARS-CoV-2. The possible interaction areas with nucleotides and RNA of the SARS-CoV-2 N protein N-terminal domain were previously determined (Dinesh et al., 2020;Kang et al., 2020). The site selected for docking, the binding sites of nucleotides and some amino acids are shown in Figure 2. Uridine 5'-monophosphate (UMP), adenosine 5'-monophosphate (AMP), cytidine 5'-monophosphate (CMP), and guanosine 5'-monophosphate (GMP) were used to compare the binding domain and affinity scores of  phytochemicals. Our study shows that several phytochemicals present in the endemic Ferula longipedunculata Peşmen presented significant predicted binding activity towards the SARS-CoV-2 nucleocapsid pro-tein. Figure 3 shows the binding affinity information of our phytochemicals, and details of their estimated binding scores were demonstrated in Table 6. Also, many of the phenolics present in endemic plant have  Table 6 presents the binding score and amino acid residues that make their hydrogen bond. Figure 4   no acid, in which GMP and GMP interacted as electrostatic. Two compounds, linolenic acid and 9-octadecanoic acid, showed the highest docking scores (-114.959 and -113.834, respectively) among all the fatty acids. Linolenic acid formed hydrogen bonds with Arg 89 and Tyr 112, and made an electrostatic interaction with Arg 89. This phytochemical was found to share the same region with CMP and GMP in the target protein. 9-octadecenoic acid showed a hydrogen bond with Tyr 110 and Arg 108, and was found to have electrostatic interaction with Arg 108 and Arg 93. It interacted with the same amino acids as AMP and UMP nucleotides. 6-(1-Hydroxymethylvinyl)-4,8a-dimethyl-, the most active compound in the essential oils, formed hydrogen bonds with Gly 117, Thr 149, and Thr 50. The hydrogen bond interaction of 6-[1-(Hydroxymethyl)vinyl]-4,8a-dimethyl-4a,5,6,7,8,8a-hexahydro-2(1H)-naphthalenone was formed with Tyr 173, Thr 58, Gln 161 and Lue 160 residues of protein.
Both compounds made hydrogen bonds with similar amino acids to nucleotides GMP, UMP, and CMP.

CONCLUSIONS
F. longipedunculata flowers were investigated for their chemical composition. The extracts from the different plant parts exhibited well. The results of the present work indicate that the antioxidant activity of the methanol and acetone extracts of Ferula longipedunculata is higher than the control, such as BHT. The methanol and acetone extracts of the plant might be an alternative additive in foods, medicine and cosmetics, instead of toxic artificial antioxidants. The different results achieved in this study may be caused by factors such as the use of different parts of the plant, environmental and genetic differences and species diversity. These results interestingly encourage to continue the work to isolate the active molecules responsible for the antioxidant and assessment of biological activity of each compound individually and the need for in-depth studies on the plant extract.
The study also provided important insights into the first step of the COVID-19 infection, viral entry into cells, and defined potential phytochemicals for antiviral intervention. Although confirmation with an infectious virus is pending, our results indicate that natural compound responses raised against SARS-S could offer some protection against COVID-19 infection, which may have implications for outbreak control.