1. INTRODUCTION
⌅The Apiaceae family consists of flowering and aromatic plants which are best known for their characteristic flowers, fruits (Heywood, 2007Heywood VH. 2007. The New Encyclopedia of Trees. Flowering Plant Families of the World. Royal Botanic Gardens, Ontorio. 35-38. https://doi.org/10.1111/j.1467-8748.2007.00585.x ) and volatile substances (Widodo et al., 2014Widodo W, Amin M, Al-Muhdar MHI. 2014. Morpho-Anatomical Analysis of Cosmostigma racemosum(Asclepiadoideae) Flowers. Biol. Med. Nat. Prod. Chem. 3, 35. https://doi.org/10.14421/biomedich.2014.31.35-46 ). 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, 2020Pakdemirli B. 2020. Economic importance of medicinal and aromatic plants in Turkey: the examples of thyme and lavender. Bahçe. 49, 51-58.). 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., 2020Duran A, Sağıroğlu M, Duman H. 2020. Prangos turcica (Apiaceae), a new species from South Anatolia , Turkey. Ann. Bot. Fennici. 42, 67-72. http://www.annbot.net/PDF/anbf42/anbf42-067.pdf ; Li et al., 2015Li G, Wang J, Li X. 2015. Two new sesquiterpene coumarins from the seeds of Ferula sinkiangensis. Phytochem Lett. 13, 123-126. http://dx.doi.org/10.1016/j.phytol.2015.06.002 ; Yang et al., 2006Yang JR, An Z, Li ZH. 2006. Sesquiterpene coumarins from the roots of Ferula sinkiangensis and Ferula teterrima. Chem. Pharm. Bull. 54, 1595-1598. https://doi.org/10.1248/cpb.54.1595 ).
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, 2011Brewer MS. 2011. Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr. Rev. Food Sci. Food Saf. 10 (4), 221-247. https://doi.org/10.1111/j.1541-4337.2011.00156.x ). 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., 2015Yehye WA, Rahman NA, Ariffin A, Abd Hamid SB, Alhadi AA, Kadir FA, Yaeghoobi M. 2015. Understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (BHT): A review. Eur. J. Med. Chem. 101, 295-312. http://dx.doi.org/10.1016/j.ejmech.2015.06.026 )
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., 2020Raoult D, Zumla A, Locatelli F. 2020. Coronavirus infections. Epidemiological, clinical and immunological features and hypotheses. Cell Stress. 4, 66-75. http://dx.doi:10.15698/cst2020.04.216 ). 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., 2014McBride R, van Zyl M, Fielding BC. 2014. The coronavirus nucleocapsid is a multifunctional protein. Viruses. 6, 2991-3018. https://doi.org/10.3390/v6082991 ). Plants are rich sources of natural compounds with antiviral effects (Sytar et al., 2021Sytar O, Brestic M, Hajihashemi S, Skalicky M, Kubeš J, Lamilla-Tamayo L, Ibrahimova U, Ibadullayeva S, Landi M. 2021. COVID-19 Prophylaxis Efforts Based on Natural Antiviral Plant Extracts and Their Compounds. Molecules. 26, 727. https://doi.org/10.3390/molecules26030727 ). The therapeutic potential of many phytochemicals has been reported with in silico techniques to combat coronavirus (Adem et al., 2020Adem Ş, Eyupoglu V, Sarfraz I, Rasul A, Zahoor AF, Ali M, Abdalla M, Ibrahim IM, Elfiky AA. 2021. Caffeic acid derivatives (CAFDs) as inhibitors of SARS-CoV-2: CAFDs-based functional foods as a potential alternative approach to combat COVID-19. Phytomedicine. 85, 153-310. https://doi.org/10.1016/j.phymed.2020.153310 ; Galanakis et al., 2020Galanakis CM, Aldawoud T, Rizou M, Rowan NJ, Ibrahim SA. 2020. Food ingredients and active compounds against the Coronavirus disease (COVID-19) pandemic: a comprehensive review. Foods. 9, 1701. https://doi.org/10.3390/foods9111701 ). 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.
2. MATERIALS AND METHODS
⌅2.1. 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.
2.2. 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/H2O (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) CHCl3, (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., 1988Khan NH, Rahman M, Kamal NE. 1988. Antibacterial activity of Euphorbia thymifolia Linn. Indian J. Med. Res. 87, 395-407. https://doi.org/10.1159/000067281 ).
2.3. 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.
2.4. 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, 2013Demirtas I, Sahin A. 2013. Bioactive volatile content of the stem and root of Centaurea carduiformis DC. subsp. carduiformis var. carduiformis. J. Chem. 2013, 1-7. https://doi.org/10.1155/2013/125286 ).
2.5. 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, 2013Demirtas I, Sahin A. 2013. Bioactive volatile content of the stem and root of Centaurea carduiformis DC. subsp. carduiformis var. carduiformis. J. Chem. 2013, 1-7. https://doi.org/10.1155/2013/125286 ).
2.6. 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., 2020Dinesh DC, Chalupska D, Silhan J. 2020 Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. PloS Pathog. 16, 12-e1009100. https://doi.org/10.1371/journal.ppat.1009100 ; Kang et al., 2020Kang S, Yang M, Hong Z. 2020. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm. Sin. B. 10, 1228-1238. https://doi.org/10.1016/j.apsb.2020.04.009 ). 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.
2.7. 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, 2013Demirtas I, Sahin A. 2013. Bioactive volatile content of the stem and root of Centaurea carduiformis DC. subsp. carduiformis var. carduiformis. J. Chem. 2013, 1-7. https://doi.org/10.1155/2013/125286 ; Abay G et al., 2015Abay G, Altun M, Koldas S, Riza Tufekci A, Demirtas I. 2015. Determination of antiproliferative activities of volatile contents and HPLC profiles of Dicranum scoparium (Dicranaceae, Bryophyta). Comb. Chem. High Throughput Screen. 18, 453-463. https://doi.org/10.2174/1386207318666150305112504 ).
2.8. 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., 2011Kedare SB, Singh RP. 2011. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol. 48 (4), 412-422. https://doi.org/10.1007/s13197-011-0251-1 ).
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., 2008Nabavi SM, Ebrahimzadeh MA, Nabavi SF. 2008. Free radical scavenging activity and antioxidant capacity of Eryngium caucasicum Trautv and Froripia subpinnata. Pharmacologyonline. 3, 19-25. Corpus ID: 45089683). 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 Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound.
3. RESULTS and DISCUSSION
⌅3.1. 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, 2001El-Feraly FS, Abourashed EA, Galal AM, Khan IA. 2001. Separation and quantification of the major daucane esters of Ferula hermonis by HPLC. Planta Med. 67, 681-682. https://doi.org/10.1055/s-2001-17354 ; Garg and Agarwal, 1988Garg SN, Agarwal SK. 1988. Further new sesquiterpenes from ferula jaeschkeana. J. Nat. Prod. 51, 771-774. https://doi.org/10.1021/np50058a020 ; Nagatsu et al., 2002Nagatsu A, Isaka K, Kojima K. 2002. New sesquiterpenes from Ferula ferulaeoides (STEUD.) KOROVIN. VI. Isolation and identification of three new dihydrofuro [2,3-b ]chromones. Chem. Pharm. Bull. 50, 675-677. https://doi.org/10.1248/cpb.50.675 ).
| No | Compoundsa | RT | % in oil | ||
|---|---|---|---|---|---|
| Root | Green | Stem | |||
| 1 | γ-cadinene | 17.742 | 0.35 | - | - |
| 2 | Acoradien | 19.762 | 0.54 | - | - |
| 3 | Bisabolene | 20.203 | 0.70 | - | - |
| 4 | Bicyclo [3.3.1 ]nonane-2,6-diol | 20.947 | 0.46 | - | - |
| 5 | Sesquisabinene hydrate | 22.475 | 1.04 | - | - |
| 6 | p-Mentha-2,8-diene, 1-hydroperoxide | 22.961 | 0.70 | - | - |
| 7 | 2,3-Dimethylhydroquinone | 23.562 | 0.93 | - | - |
| 8 | trans-8-Hydroxy-bicyclo(4,3,0)non-3-ene | 23.682 | 0.41 | - | - |
| 9 | 3-Hydroxy-2-(2-methylcyclohex-1-enyl)propionaldehyde | 24.380 | 1.03 | - | - |
| 10 | Pentadecanoic acid, methyl ester | 24.912 | 1.44 | - | - |
| 11 | 6- [1-(Hydroxymethyl)vinyl ]-4,8a-dimethyl-3,5,6,7,8,8a-hexahydro-2(1H)-naphthalenone | 25.301 | 2.85 | - | - |
| 12 | Palmitic acid methyl ester | 27.579 | 8.62 | 22.16 | 24.76 |
| 13 | Cyclododecane methanol | 31.092 | 0.53 | - | - |
| 14 | Linoleic acid, methyl ester | 32.173 | 70.37 | 35.38 | 53.58 |
| 15 | 9- octadecanoic acid, methyl ester | 32.248 | 9.30 | - | - |
| 16 | Linolenic acid, methyl ester | 32.276 | - | 25.04 | 12.47 |
| 17 | Octadecanoic acid, methyl ester | 32.562 | 0.74 | 4.26 | - |
| 18 | 3-Heptadecen-5-yne | 26.926 | - | 6.01 | - |
| 19 | Phytol | 32.431 | - | 7.15 | - |
| 20 | Stearic acid, methyl ester | 32.563 | - | - | 9.19 |
aCompounds are listed in order of their elution from the BPX-20 capillary column, RT-retention time. min.
3.2. 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%).
| No | Compoundsa | RT | % Composition |
|---|---|---|---|
| 1 | α-Thujene | 12.411 | 0.8±0.43 |
| 2 | α-Pinen | 12.711 | 1.41±0.05 |
| 3 | β-Phellandrene | 14.003 | 53.46±0.64 |
| 4 | beta-Myrcene | 14.328 | 0.91±0.05 |
| 5 | α-Terpinen | 15.387 | 1.46±0.06 |
| 6 | β-Cymene | 15.657 | 4.12±0.08 |
| 7 | α -Pinen | 15.887 | 1.89±0.13 |
| 8 | Ocimene | 16.295 | 6.79±0.01 |
| 9 | γ-Terpinen | 16.822 | 3.98±0.12 |
| 10 | 2,3-Heptadien-5-yne, 2,4-dimethyl- | 17.556 | 1.99±0.11 |
| 11 | 2,3,4,5-Tetramethylcyclopent-2-en-1-ol | 20.681 | 1.17±0.05 |
| 12 | 4-Terpineol | 21.163 | 5.94±0.01 |
| 13 | Santalol | 30.721 | 5.03±0.05 |
| 14 | Epiglobulol | 32.844 | 2.18±0.02 |
| 15 | alpha-Caryophyllene | 33245 | 2.47±0.08 |
| 16 | 6- [1-(Hydroxymethyl)vinyl ]-4,8a- dimethyl-4a,5,6,7,8,8a-hexahydro-2(1H)- naphthalenone 6-(1-Hydroxymethylvinyl)-4,8a-dimethyl- | 34.330 | 0.94±0.03 |
| 17 | 3,5,6,7,8,8a-hexahydro-1H-naphthalen-2one | 34.579 | 2.32±0.04 |
| 18 | alpha-Bisabolol | 37.069 | 3.13±0.01 |
| Total | 99.99 |
aCompounds are listed in order of their elution from the BPX-20 capillary column, RT-retention time. min.
After comparing the chemical composition of Ferula longipedunculata essential oil with other species of the ferula genus some differences and similarities were found. The main key components of the essential oil of Ferula persica were dillapiole (57.3%) and elemicine (5.6%) (Javidnia et al., 2005Javidnia K, Miri R, Kamalinejad M. 2005. Chemical composition of Ferula persica Wild. essential oil from Iran. Flavour Fragr J. 20, 605-616. https://doi.org/10.1002/ffj.1496 ). Guaiol (58.76%), (E)-nerolidol (10.16%) and α-eudesmol (3.05%) were found to be the major (key) compounds of the oil of Ferula ferulaoides (Shatar, 2005Shatar S. 2005. Essential oil of Ferula ferulaoides from western Mongolia. Chem. Nat. Compd. 41, 607-608. https://doi.org/10.1007/s10600-005-0222-8 ). These components were not present in Ferula longipedunculata essential oil.
In the essential oil analysis of Ferula elaeochytris with GC-MS, nonane (27.1%), α-pinene (12.7%) and germacrene B (10.3%) were obtained as the main compounds (Başer et al., 2000Başer KHC, Özek T, Demirci B. 2000. Composition of the essential oils of Zosima absinthifolia (Vent.) Link and Ferula elaeochytris Korovin from Turkey. Flavour Fragr J. 15, 371-372. https://doi.org/10.1002/1099-1026(200011/12)15:6<371::AID-FFJ919>3.0.CO;2-Z ). In a study conducted in Iran, the essential compounds of the Ferula szowitsiana plant were obtained as α-pinene (12.6%), germacrene D(12.5%) and β-pinene (10.1%) (Rustaiyan et al., 2006Rustaiyan A, Aghaie HR, Ghahremanzadeh R. 2006. Composition of the Essential Oils of Ferula szowitsiana DC., Artedia squamata L. and Rhabdosciadium petiolare Boiss. & Hausskn.ex Boiss. Three Umbelliferae Herbs Growing Wild in Iran. J. Essent. Oil Res. 18, 503-505. https://doi.org/10.1080/10412905.2006.9699153 ). As expected, compounds such as α-pinene and β-pinene were not obtained as the main compounds for the F. longipedunculata plant. In addition, α-pinene was identified in the Ferula longipedunculata.
Moreover, the major components in the oil of F. gummosa were found to be β-pinene (50.1%), α-pinene (18.3%), 3-carene (6.7%), α-thujene (3.3%) and sabinene (3.1%) (Eftekhar et al., 2004Eftekhar F, Yousefzadi M, Borhani K. 2004. Antibacterial activity of the essential oil from Ferula gummosa seed. Fitoterapia. 75, 758-9. https://doi.org/10.1016/j.fitote.2004.09.004 ).
The genera Ferula is rich in its essential content, which is also named Ferula oil. Of the genera, F. assafoetida, F. gummosa and F. badrakema contain essential oils. Those essential oils give a strong aromatic smell to the plant species. Furthermore, these oils have been documented to possess antifungal and antibacterial activities (Sahebkar and Iranshahi, 2011Sahebkar A, Iranshahi M. 2011. Volatile constituents of the genus ferula (apiaceae): A review. J. Essent. Oil-Bear. Plants. 14, 504-531. https://doi.org/10.1080/0972060X.2011.10643969 ). Among the components of the essential oil, alpha-pinene and beta-pinene are of the major compounds (Benevides et al., 2001Benevides PJC, Young MCM, Giesbrecht AM. 2001. Antifungal polysulphides from Petiveria alliacea L. Phytochem. 57, 743-7. https://doi.org/10.1016/S0031-9422(01)00079-6 ; Kim et al., 2006Kim S, Kubec R, Musah RA. 2006. Antibacterial and antifungal activity of sulfur-containing compounds from Petiveria alliacea L. J. Ethnopharmacol. 104, 188-192. https://doi.org/10.1016/j.jep.2005.08.072 ).
3.3. 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 fumaric 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.
| Organic and phenolic acids | RT | Root | Green | Stem |
|---|---|---|---|---|
| Gallic acid | 2.4 | Trace | Trace | Trace |
| Fumaric acid | 3.2 | 75.49±2.14a | 6.63±0.51a | 2100.21±5.15a |
| Gentisic acid | 4.5 | 24.73±1.04 | 120.21±2.89 | 16.51±1.53 |
| Chlorogenic acid | 5.5 | 5.13±0.01 | 72.35±1.95 | 16.19±1.06 |
| 4-Hydroxybenzoic acid | 7.0 | 119.92±0.29 | 183.42±1.24 | 34.20±0.84 |
| Protocatechuic acid | 7.1 | 22.05±0.39 | 74.22±1.79 | 23.43±0.85 |
| Caffeic acid | 7.6 | 6.72±0.27 | 82.00±1.25 | 6.42±0.47 |
| Vanillic acid | 7.9 | 141.35±0.68 | 239.88±1.34 | 57.59±0.96 |
| Syringic acid | 8.1 | 116.57±0.52 | 214.24±1.14 | 50.87±0.38 |
| 4-Hydroxybenzaldehyde | 9.4 | 13.25±0.57 | 89.11±1.01 | Trace |
| Ellagic acid | 9.7 | Trace | 127.58±2.08 | Trace |
| Sinapic Acid | 10.5 | Trace | 2.04±0.28 | Trace |
| Ferulic Acid | 10.6 | 126.19±0.72 | 246.22±1.7 | nd |
| p-Coumaric acid | 12.1 | Trace | Trace± | Trace |
| Protocatechuic acid ethyl ester | 12.8 | Trace | Trace | Trace |
| Salicylic acid | 13.1 | 51.14±1.88 | 185.69±1.3 | 11.13±1.23 |
| Cinnamic acid | 15.2 | 9.62±0.04 | 10.77±1.26 | nd |
RT-retention time. min, aValues expressed are means ± S.D. of three parallel measurements
nd: not detected
| Flavonoids and phenolics | RT | Root | Green | Stem |
|---|---|---|---|---|
| Catechin | 5.8 | 10.36±0.66 | 11.02±1.01 | nd |
| Rutin | 9.2 | Trace | 10.08±0.51 | Trace |
| Polydatine | 9.6 | Trace | Trace | Trace |
| Scutellarin | 9.7 | Trace | 16.45±1.80 | Trace |
| Quercetin-3-β-D-Glucoside | 9.8 | 7.39±0.53 | 1737.70±36.5 | 163.24±2.97 |
| Naringin | 10.5 | Trace | 213.29±3.67 | 19.02±2.04 |
| Diosmin | 10.6 | 38.70±0.82 | 46.38±1.63 | 47.57±1.28 |
| Taxifolin | 10.6 | Trace | Trace | Trace |
| Hesperidin | 10.8 | Trace | 278.38±1.07 | Trace |
| Apigetrin | 10.9 | Trace | Trace | Trace |
| Neohesperidin | 11.1 | Trace | 2.55±0.16 | Trace |
| Myricetine | 11.9 | Trace | Trace | Nd |
| Baicalin | 12.0 | Trace | Trace | Trace |
| Fisetin | 12.1 | Trace | Trace | Trace |
| Morin | 13.0 | 12.95±1.97 | 17.20±0.31 | 14.51±0.97 |
| Resveratrol | 13.0 | Trace | Trace | Trace |
| Quercetin | 14.0 | 10.54±0.67 | 531.35±2.45 | 3.93±0.6 |
| Silibinin | 15.1 | Trace | Trace | nd |
| Apigenin | 15.6 | Trace | Trace | Trace |
| Naringenin | 15.7 | Trace | Trace | Trace |
| Kaempferol | 15.7 | Tr | 60.51±2,51 | tr |
| Diosmetin | 16.1 | Trace | Trace | Trace |
| Neochanin | 17.7 | Trace | Trace | Trace |
| Eupatorin | 18.9 | Trace | Trace | Trace |
| Wogonin | 19.8 | Trace | Trace | Trace |
| Biochanin A | 20.5 | Trace | Trace | nd |
RT-retention time. min. aValues expressed are means ± S.D. of three parallel measurements
nd: not detected
3.4. 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., 2006Mao LC, Pan X, Que F. 2006.Antioxidant properties of water and ethanol extracts from hot air-dried and freeze-dried daylily flowers. Eur. Food. Res. Technol. 222, 236-241. https://doi.org/10.1007/s00217-005-0007-0 ). 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 electron or hydrogen radical to become a stable diamagnetic molecule (Soares et al., 1997Soares JR, Dinis TCP, Cunha AP. 1997. Antioxidant activities of some extracts of Thymus zygis. Free Radic. Res. 26, 469-478. https://doi.org/10.3109/10715769709084484 ).
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 Fe2+ chelating ability, DPPH radical and nitric oxide scavenging activity (Dehpour et al., 2009Dehpour AA, Ebrahimzadeh MA, Fazel N. 2009. Antioxidant activity of the methanol extract of Ferula assafoetida and its essential oil composition. Grasas Aceites. 60, 405-12. https://doi.org/10.3989/gya.010109 ). Ferula-assafoetida leaves are free-radical scavengers and may act as primary antioxidants, which react with free radicals by donating hydrogen (Nabavi et al., 2011Nabavi SM, Ebrahimzadeh MA, Nabavi SF. 2011. Antioxidant and antihaemolytic activities of Ferula foetida regel (Umbelliferae). Eur. Rev. Med. Pharmacol. Sci. 15, 157-164. PMID: 21434482). Research shows that the ferula-assa-foetida leaves have different kind of flavonoides, phenolic compounds (Dehpour et al., 2009Dehpour AA, Ebrahimzadeh MA, Fazel N. 2009. Antioxidant activity of the methanol extract of Ferula assafoetida and its essential oil composition. Grasas Aceites. 60, 405-12. https://doi.org/10.3989/gya.010109 ). All these compounds probably contribute to the main reason for its significant radical-scavenging activity. Researchers 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, 2011Tavafi M, Ahmadvand H. 2011. Effect of rosmarinic acid on inhibition of gentamicin induced nephrotoxicity in rats. Tissue Cell. 43, 392-397. https://doi.org/10.1016/j.tice.2011.09.001 ). Natural antioxidants compared to artificial antioxidants are much safer and more beneficial and also have fewer side effects (Craft et al., 2010Craft BD, Kosińska A, Amarowicz R. 2010. Antioxidant Properties of Extracts Obtained from Raw, Dry-roasted, and Oil-roasted US Peanuts of Commercial Importance. Plant Foods Hum Nutr. 65, 311-8. http://dx.doi.org/10.1007/s11130-010-0174-4 ).
| Parts of Plant | Solvents | DPPH free radical scavenging (%) | |||||
|---|---|---|---|---|---|---|---|
| F. longipedunculata extract | BHT | ||||||
| Concentration (ml) | Concentration (ml) | ||||||
| 0.1 | 0.2 | 0.3 | 0.1 | 0.2 | 0.3 | ||
|
Root part Green part Stem part Root part Green part Stem part |
Methanol Methanol Methanol Acetone Acetone Acetone |
98.5 97.2 98.1 79.5 80.6 79.1 |
98 97 97.9 80.6 82 85.4 |
98.3 96.6 97.7 82.8 86.8 86.8 |
90.7 90.7 90.7 77.6 77.6 77.6 |
92.1 92.1 92.1 75.1 75.1 75.1 |
97.9 97.9 97.9 74.8 74.8 74.8 |
BHT (Butylated hydroxytoluene): as control, DPPH: 1,1-diphenyl-2-picryl hydrazyl
3.5. 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., 2020Raoult D, Zumla A, Locatelli F. 2020. Coronavirus infections. Epidemiological, clinical and immunological features and hypotheses. Cell Stress. 4, 66-75. http://dx.doi:10.15698/cst2020.04.216 ). 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., 2020Dinesh DC, Chalupska D, Silhan J. 2020 Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. PloS Pathog. 16, 12-e1009100. https://doi.org/10.1371/journal.ppat.1009100 ; Kang et al., 2020Kang S, Yang M, Hong Z. 2020. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm. Sin. B. 10, 1228-1238. https://doi.org/10.1016/j.apsb.2020.04.009 ). 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 protein. 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 significant binding affinity with this target. Some of the flavonoids and phenolics are silibinin, rutin, neohesperidin, naringin, diosmin, hesperidin, scutellarin, apigetrin, and polydatine. Table 6 presents the binding score and amino acid residues that make their hydrogen bond. Figure 4 demonstrates the possible binding modes of some phytochemicals. Silibinin exhibited the highest binding energy at the active site of SARS-CoV-2 nucleocapsid protein. It formed hydrogen bond interactions Arg 150, Tyr 112, Asn 49, Asn 48, Gly 117, Thr 149. Active site residues Gln 192, Thr 190, Arg 188, His 164, Gln 189, Glu 166, Gly 143, Ser 144, and Cys 145 participated in hydrogen bond interactions with rutin. Chlorogenic acid and sinapic acid with -106.120 and -85.529 MolDock scores exhibited the most effective phenolic acids against the target as in silico. The computer analysis results suggest that two phenolic acids had electrostatic potential in the interaction. The results of the prepared study shown that Ser 52, Thr 50, Gly 117, Thr 149, Arg 89, and Tyr 112 were critical residues in the hydrogen bonding of chlorogenic acid with protein. It also interacts electrostatically with Arg 150. The docking results in Table 6 demonstrate that chlorogenic acid interacted with the region where UMP was connected. Arg 89, Arg 90, Asp 129 amino acids were responsible for sinapic acid-binding in the SARS-CoV-2 nucleocapsid protein. It acted electrostatically with the Arg 89 amino 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.
| Name | MolDock Score | Hydrogen Bond | Amino acids involved in hydrogen bonding | Electrostatic | Nucleotides bound to the same region |
|---|---|---|---|---|---|
| Silibinin | -126.107 | -10.155 | Arg150, Tyr 112, Asn 49, Asn 48, Gly117, Thr 149 | UMP, CMP, GMP | |
| Rutin | -126.039 | -16.549 | Arg108, Tyr110, Thr58, Tyr 173, Gln 161, Leu 160, Aln 161, Ala174 | AMP, UMP, CMP, GMP | |
| Neohesperidin | -124.043 | -13.436 | Arg 89, Tyr112, Ser52, Tyr110, Thr 92, Thr 149 | UMP, CMP, GMP | |
| Naringin | -118.464 | -12.895 | Tyr 112, Thr 50, Thr 149, Asn 48, Asn 49 | UMP, CMP, GMP | |
| Diosmin | -111.507 | -9.955 | Arg 150, Tyr 110, Tyr 112, Ser 52, Ala 56 | UMP, CMP, GMP | |
| Hesperidin | -111.133 | -17.762 | Asn 49, Ser 52, Phe 54, Arg 150, Arg 89, Tyr 112, Arg103, Tyr 110 | UMP, CMP, GMP | |
| Scutellarin | -107.010 | -9.490 | Tyr 112, Thr 149, Asn 48, Thr 50, Asn 49, Arg89 | UMP, CMP, GMP | |
| Apigetrin | -106.803 | -18.390 | Tyr 112, Ser 52, Phe54, Arg150, Tyr110, Arg89, Thr 149, Thr 50 | UMP, CMP, GMP | |
| Polydatine | -106.120 | -14.276 | Leu 160, Ala 174, Gln 161, Thr 58, His 60, Tyr 173, Gln 161 | AMP | |
| Chlorogenic acid | -102.058 | -14.141 | Ser 52, Thr 50, Gly117, Thr149, Arg 89, Tyr 112 | Arg 150 | UMP, CMP, GMP |
| Sinapic Acid | -85.529 | -12.355 | Arg 89, Arg 90, Asp 129 | Arg 89 | CMP, GMP |
| Linolenic acid | -114.959 | -1.023 | Arg 89, Tyr 112 | Arg 89 | GMP, CMP |
| 9- octadecanoic acid | -113.834 | -2.468 | Tyr 110, Arg 108 | Arg 108, Arg 93 | AMP, UMP |
| 6-(1-Hydroxymet hylvinyl)-4,8adimethyl | -98.624 | -5.959 | Gly 117, Thr 149, Thr 50 | GMP, CMP | |
| 6 [1(Hydroxymethyl) vinyl ]4,8adimethyl-4a,5,6,7,8,8ahexahydro-2(1H)-naphthalenone | -96.182 | -7.907 | Tyr 173, Thr 58, Gln 161, Lue 160 | AMP | |
| AMP | -121.197 | -13.876 | Arg93, Arg108, Ala 56, Thr 58, Tyr 173, His 60 | Arg 108, Arg 93 | |
| GMP | -114.272 | -3.803 | Arg89, Tyr112, Thr50, Thr 149 | Arg 89 | |
| UMP | -100.274 | -7.321 | Tyr 110, Arg 150, Tyr 112, Ser 52, Phe 54 | Arg 93, Arg 108 | |
| CMP | -99.163 | -14.516 | Tyr 112, Asn49, Ala51, Thr 149 | Arg 89 |
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.
4. 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.