Investigation on Chemical Constituents of Foeniculum vulgare Essential Oil and the Molecular Docking Studies of its Components for Possible Matrix Metalloproteinase-13 Inhibition

The genus Foeniculum (Apiaceae) has two species in Iran, including Foeniculum carvi (L.) and Foeniculum vulgare Miller. The scientific synonyms are Carum carvi and Foeniculum officinale All., respectively. Foeniculum vulgare Miller. is recognized by “fennel”, “Razianeh”, and “Shamr” as English, Persian, and Arabic names, respectively. The plant is scattered around the Mediterranean region and central Europe. Due to the wide applications of the plant (e.g., culinary and medicinal uses), the plant is widely cultivated in Iran. It is noteworthy that it grows wild in Mazandaran province. Fennel can be an annual, perennial, or biennial herb. It has feathery leaves resembling Anethum and umbelliferous golden yellow flowers. The fruits become fully grown at the end of summer or early autumn (1). The fruit dimensions vary based on the plant growth region (2). Fennel fruit (FF) has an intense aroma such as the anise fruit and is used as a flavouring in alcoholic beverages, baked goods, ice cream, meat and seafood dishes, and herbal mixtures. The savor of the FF is used as a culinary supplement and its essential oil (EO) composition establishes therapeutic effects such as anti-inflammatory, analgesic, anti-spasmodic, antioxidant, and diuretic (4) and anti-thrombotic (5) effects. Avicenna Journal of Pharmaceutical Research

Moreover, these fennels can motivate menstruation and lactation, provide remarkable improvements in infantile colic (6), and is useful for the treatment of diarrhea and digestive complications. Additionally, FF is extensively used in folk medicine for different ailments such as cough remedies, digestive upset, and diarrhoea (7). According to previous research, EOFF may be considered as a potential candidate in studies on cognitive disorders such as depression, anxiety, dementia, and Alzheimer's (8). For example, Joshi et al found that Foeniculum vulgare had a memory-enhancing effect related to the involvement of the cholinergic system (9). EO composition between the plants in a species is not completely similar. Internal and external factors such as the genetic structure or the climate condition create these differences, leading to significant changes in the composition and the EO yield (10(. Thus, valuable pharmacognostic studies on the chemical constituents of EOs are considered necessary in order to reach-high quality EOs in terms of yields and compositions (11). Considering the wide range of the effects and uses and despite various reports on the chemical constituents of the EOFF, it remains a subject focus for researchers. Matrix metalloproteinase-13 (MMP-13) is a calciumcontaining enzyme, and zinc is a cofactor of the protein. MMP-13 is involved in the degradation of extracellular matrix proteins such as fibrillar collagen, fibronectin, tenascin-C, and aggrecan. Moreover, MMP-13 hydrolyses many other proteins including type I, type II, type III, type IV, type XIV, and type X collagen (12). The up-regulation of MMP-13 has been demonstrated in several disorders such as tooth decay, periodontitis, Alzheimer's disease, and cancer progression (1)(2)(3)(4)(13)(14)(15)(16). Therefore, the inhibition of MMP-13 may result in the prevention of mentioned disorders. In silico techniques such as molecular docking are fast and low-cost methods for predicting the affinity of small molecules to the active site of enzymes for therapeutic aims (17). In the present study, the molecular docking approach was performed to evaluate the binding affinity of four plant-based compounds, originated from Foeniculum vulgare, to the active site of MMP-13 using Autodock software. The results of the current study could be useful in the prevention approaches of a number of human disorders such as dental caries, cancer progression, periodontitis, and Alzheimer's disease. However, wetlab experiments are required to validate the results of the current study. Considering the importance of EOFF, variations in the type and amount of available compounds in different EOFF, the current study evaluated the chemical constituents of EOFF and the binding affinity of four compounds more than 1% in EFF to the active site of MMP-13 using Autodock software.

Preparation of the Essential Oil
Foeniculum fruits (FF) were purchased from a market in the Hamedan district. The authentication of the plant material was performed on the herbarium of the School of Pharmacy, Hamadan University of Medical Sciences. FF was washed and dried, and then the powdered FF was subjected to a Clevenger-type apparatus for 3 hours, and the oil was obtained by the hydrodistillation method (18). The oil was dried by anhydrous sodium sulfate and kept in a refrigerator until use.

Identification of Oil Components
EOFF was injected into the gas chromatography-mass spectrometry system (GC-MS) and the system was ThermoQuest GC-coupled to the Thermo Finnigan mass system. The GC apparatus was equipped with a DB-5 column ((5%-Phenyl)-methylpolysiloxane, 30 m * 0.25 mm * 0.25 μm)) and the carrier gas was He by a flow rate of 1.1 mL/min. The split ratio and the run time were 1:50 and 40 minutes, respectively. The oven temperature was 60°C which increased to 250°C by a ramp of 5°/minute and continued for 2 minutes at 250°C. The temperature of the detector and the injector was 250°C. The mass analyzer was the Quadropole system operating at 70 eV ionization energy. Accurate mass spectra and electron ionization mass spectra (EIMS) were recorded at m/z and m/e ranges of 50-700 and 40-460 amu in the scan mode, respectively. Further, n-alkanes were injected into GC-MS at the same condition as the oil, and their retention times were used for calculating retention indices (RIs). Then, the EIMS fragmentation pattern of constituents was compared by the published data and those held in the Wiley library of mass spectra. For accurate identification, the calculated RI of compounds was compared by previous reports (19).

Molecular Structure Preparing
The three-dimensional (3-D) structure of MMP-13 was attained from the Research Collaboratory for Structural Bioinformatics (https://www.rcsb.org, PDB ID: 5b5o) at 1.2 Å x-ray resolution (18). Chain A in the 5b5o file was selected for molecular docking analysis. Moreover, the Swiss-PdbViewer, version 4.1.0 (http://www.expasy.org/ spdbv) was utilized for the energy minimization (EM) of MMP-13 (19). Four herbal compounds were considered as the candidates for MMP-13 inhibition, including thymol (PubChem ID: 6989), fenchone (PubChem ID: 14525), trans-anethole (PubChem ID: 637563), and methyl chavicol (PubChem ID: 8815). The 3-D structures of all small molecules were obtained as a structure data file (SDF) format from the PubChem database ( cactus webtool (http://cactus.nci.nih.gov/chemical/structure) was utilized to convert the SDF to PDB format. All ligands were structurally and geometrically optimized by EM and using the HyperChem software, version 8.0.10.

Physical and Chemical Properties of Small Molecules
The PubChem database (https://pubchem.ncbi.nlm.nih. gov) was applied to evaluate the drug-likeness of herbal compounds according to the theoretical method of pharmacokinetic parameters in consonance with the Rule of Five (RO5) defined by Lipinski et al (20). The molecular mass and oil/water partition coefficient in addition to the number of donors and the acceptors of hydrogen bonds were studied as well.

Pharmacokinetic and Toxicological Properties (e.g., absorption, distribution, metabolism, and excretion, ADMET)
The SwissADME webserver (http://www.swissadme.ch/) was used to predict the pharmacokinetic properties of herbal compounds, including ADME (21). Furthermore, the toxicological features of small molecules were considered using the PreADMET (https://preadmet.bmdrc. kr/) webserver in order to predict the carcinogenicity of compounds in the mouse and rat models, in addition to estimating the possible inhibition of the human ether-ago-go-related gene (hERG) channel in the heart.

Molecular Docking Analysis
A windows-based operating system was used to carry out bioinformatic analyses. The basic information of the system included installed memory (32 GB), processor (Intel Core i7), and system type (64-bit). The molecular docking analysis was performed using AutoDock software, version 4.0 (http://autodock.scripps.edu). The AutoDock tool predicts the conformation of the ligand bounded to the receptor with a predicted Gibbs free energy of the connection. Furthermore, it estimates the free energy of binding (∆G binding ) from the following formulas (22):

∆G binding = Intermolecular Energy + Total Internal Energy + Torsional Free Energy -Energy of Unbound System
The catalytic domain of the MMP-13 was considered as the receptor of the ligands. The grid box options were set as spacing (0.375 Å), X-dimension (52), Y-dimension (52), and Z-dimension (60). Based on the study by Nara et al (23), the main amino acids are located in the active site of MMP-13, in addition to the 3-D view of the interaction between the MMP-13 active site and the inhibitor of the protein (https://www.rcsb.org/structure/5B5O). These key amino acids were selected as the docking box containing 16 residues such as His187, Ala186, Leu185, Val219, Leu218, Tyr244, Thr245, Ile243, Pro242, Ala238, His232, Phe241, His226, His222, Leu239, and Glu223 (23). The number of conformations for each ligand was set as 50. Finally, the BIOVIA Discovery Studio Visualizer (version 19.1.0.18287) was used for visualizing the interaction modes between ligands and the residues within the MMP-13 active site.

Results
The EO of Foeniculum vulgare was extracted by the hydrodistillation method. The EFF yield was 0.5% w/w, and the oil was studied by GC-MS (Figure 1). The list of EFF components is reported in Table 1. Based on the results, 14 compounds were identified representing 99.2% of EFF. The major components of the oil were e-anethole (86.86%), fenchone (7.43%), estragole (1.65%), and thymol (1.21%). Other ten compounds have an amount of less than 1%. Based on the obtained data (Table 1), the main components of EFF belongs to phenylpropanoids (88.51%), monoterpene ketones (7.53%), monoterpene phenols (1.21%), and monoterpene hydrocarbons (1.17%).

Molecular Docking
The molecular docking approach was performed to evaluate the binding affinity of four compounds more than 1% to the active site of MMP-13. Table 2 presents the details of binding energies between the MMP-13 and the tested compounds in this study, along with the inhibition constant estimated for each ligand obtained from docking results. All four compounds were identified with an inhibition constant at the micromolar (μM) scale.

Physical and Chemical Properties of the Compounds
The physical and chemical features of the compounds were studied based on the RO5 as described by Lipinski et al (20). According to RO5, an appropriate compound must illustrate a molecular mass of ≤500 g/mol, logarithm of the partition coefficient between n-octanol and water (LogP) ≤ 5, a hydrogen bond acceptor count ≤10, and a hydrogen bond donor count ≤ 5 (12)(13)(14)(15)(24)(25)(26). According to the results, all four compounds were in agreement with the RO5 and considered to be drug-like molecules for oral use. Table 3 provides the details of characteristics related to the RO5 for each compound.

Pharmacokinetic and Toxicological Properties (ADME/ Tox) of Compounds
Further analyses focused on the compounds for predicting the ADMET of each ligand. The pharmacokinetic features evaluated by the SwissADME webserver were gastrointestinal (GI) absorption, blood-brain barrier (BBB) permeability, P-gp (P-glycoprotein) substrate, cytochrome   P-450 inhibition, and the skin permeation coefficient (k p ). The possible inhibition of the human cardiac potassium channel hERG and the carcinogenicity of the compounds in mouse and rat models were considered as the toxicological properties of the compounds. Thymol and fenchone were predicted to have lower toxicity effects as compared with other compounds. The ADMET results of the current study are presented in Table 4.
E-anethole and estragole belonging to phenylpropanoids constitute 86.86% and 1.65% of EOFF, respectively. Bahmani et al studied the oils of FFs from 12 regions in Iran and they found that the content of e-anethole in Khash and Sari was 88.455% and 1.47%, respectively, whereas that of estragole was 0.221% and 55.09% in the EFF of Khash and Sari, respectively. The amounts of e-anethole and estragole were 14.7% and 59.1% in Kalibar (27). Likewise, Mehrpour et al studied FF growing in Hamedan and concluded that anethole and estragole constituted 78.6% and 3.2% of the EFF. They used the Note. RI: Retention index; GC-MS: Gas chromatography-mass spectrometry.   (30). According to previous evidence, they reported that there are estragole, estragole/anethole, and estragole fenchone chemotypes in different geographical regions. They also found that the isolation procedure did not affect the chemotype in one distinct growing population.
In the present study, the computational screening approach was carried out using AutoDock software to identify compounds originated from Foeniculum vulgare capable of inhibiting the MMP-13. Thymol, as an oxygenated monoterpene (31) was estimated to be the most effective compound from the tested ligands in this study, followed by trans-anethole, methyl chavicol, and fenchone.
In the present study, thymol demonstrated one hydrogen interaction and seven hydrophobic interactions with Leu185, Ala186, Val219, His222, His226, His232, and Tyr244 residues within the MMP-13 active site. Methyl chavicol revealed three hydrophobic interactions with Leu185, Val219, and His222 residues within the MMP-13 active site. Moreover, fenchone showed one hydrogen interaction and five hydrophobic interactions with Gly183, Leu185, His222, His226, His232, and Pro242 residues within the MMP-13 active site. Moreover, transanethole formed three hydrophobic interactions with Leu185, Val219, and His222 residues within the MMP-13 active site. The interaction modes between each of the compounds and the MMP-13 active site are presented in Table 5. Figure 2 shows the images of ligand-amino acid interactions for the four studied compounds.
According to the results of the present study, no significant correlation was found between the structures of phenylpropanoid compounds (i.e., trans-anethole and methyl chavicol) and monoterpene compounds (i.e., thymol and fenchone) in the inhibition of MMP13.

Conclusion
Considering the wide diversity in the type of EFF components, it seems that the geographical growth region of F. vulgare is one of the most important affected factors. Accordingly, despite the large number of studies conducted in this regard, it is the subject of interest for researchers. The present study successfully found that all four compounds in the EO of F. vulgare are capable of inhibiting the MMP-13 according to their estimated ∆G binding and the inhibition constant. In addition, they all revealed drug-like features according to Lipinski's RO5. Furthermore, thymol and fenchone showed lower toxicity effects as compared with other compounds. Therefore, they may be considered as potential drug candidates for the next steps of drug discovery. The findings of the present study may be helpful for the prevention and/ or therapeutic procedures of several human disorders  such as dental caries, Alzheimer's disease, periodontitis, and tumor cell invasion and migration. Nevertheless, validation is necessary in the future.