Chemical Composition, Biological Activities and In Silico Analysis of Essential Oils of Three Endemic Prangos Species from Turkey

In this study, the essential oils (EOs) obtained from three endemic Prangos species from Turkey (P. heyniae, P. meliocarpoides var. meliocarpoides, and P. uechtritzii) were studied for their chemical composition and biological activities. β-Bisabolenal (12.2%) and caryophyllene oxide (7.9%) were the principal components of P. heyniae EO, while P. meliocarpoides EO contained sabinene (16.7%) and p-cymene (13.2%), and P. uechtritzii EO contained p-cymene (24.6%) and caryophyllene oxide (19.6%), as the most abundant components. With regard to their antioxidant activity, all the EOs were found to possess free radical scavenging potential demonstrated in both DPPH and ABTS assays (0.43–1.74 mg TE/g and 24.18–92.99 mg TE/g, respectively). Additionally, while no inhibitory activity was displayed by P. meliocarpoides and P. uechtritzii EOs against both cholinesterases (acetyl- and butyryl-cholinesterases). Moreover, all the EOs were found to act as inhibitors of tyrosinase (46.34–69.56 mg KAE/g). Molecular docking revealed elemol and α-bisabolol to have the most effective binding affinity with tyrosinase and amylase. Altogether, this study unveiled some interesting biological activities of these EOs, especially as natural antioxidants and tyrosinase inhibitors and hence offers stimulating prospects of them in the development of anti-hyperpigmentation topical formulations.


Introduction
Essential oils (EOs) are recognized for their exceptional medicinal value and are considered among the most attractive and potent plant-derived products. Eos, also referred to as ethereal oils, are volatile and odorous oils present in only 10% of the plant kingdom and are stored in plants in special brittle secretory structures, for instance glands, secretory hairs, secretory ducts, secretory cavities or resin ducts. EOs have been used as perfumes, flavors in foods and beverage ingredients, or to heal both the body and mind since ages and even today they continue to be of paramount importance [1].
In fact, many studies have focused on the pharmacological and cosmeceutical potentials of EOs such as antioxidant, antimicrobial, anticancer, anti-inflammatory, antiaging and antimelanogenic amongst many others [2][3][4]. Accordingly, growing evidences of the health benefits of these natural essences have prompted researchers to further investigate EOs and their individual components. Additionally, their mechanisms of action have been elucidated with respect to their biological activities [5][6][7][8], which so far are showing promising prospects.
The genus Prangos (Apiaceae) consists of 45 worldwide species and the genus has a wide distribution area ranging from Portugal to Tibet [9]. The genus is represented by 19 taxa, including 19 species in the flora of Turkey, of which 10 of them are endemic [10]. The members of this genus have been widely used in traditional medicine and are greatly valued as spices and medicinal plants in Asia, especially in Iran, Turkey, and Iraq. The above-ground parts, the roots as well as the EOs of different species of this genus have both internal and external applications. The most popular indications of these plants are the alleviation of gastrointestinal symptoms, but various other uses have also been reported [9]. For instance, they are used as carminative, tonic, and anthelmintic agents, to heal scars and in the treatment of external bleeding, gastric or digestive disorders, wounds, and leuckoplakia. Moreover, Prangos species are known to act as stimulants, aphrodisiacs and natural fertilizers [9,11]. Based on the ethnobotanical uses of the members of the Prangos genus, several phytochemical studies have been performed and the presence of different groups of bioactive compounds including coumarins [12][13][14], essential oils [15,16], flavonoids [17,18] and phenolic acids [19] have been reported.
P. uechtritzii Boiss & Hausskn is a rigid and perennial plant. The leaves have long lobes and the flowers are yellow. The plant prefers rock limestone slopes and roadsides. It is generally distributed in the southeastern region of Turkey [20]. It is known as "deli çakşır" in local area of Turkey and has reputed aphrodisiac properties. In addition, the aerial parts of P. uechtritzii have been used to treat hemorrohoids in Anatolian folk medicine [14]. This plant's antioxidant and antimicrobial effects as well as its chemical composition have been examined in earlier studies [14,21,22]. P. meliocarpoides Boiss. var. meliocarpoides is a perennial plant that ranges in height from 15 to 30 cm. It has yellow and glabrous flowers. It is distributed in the Inner Anatolia region of Turkey up to an altitude of 2000 m [20]. In earlier studies, the antioxidant properties of fruit extracts of the plant have been reported by several authors [21,23]. P. heyniae H. Duman & M.F. Watson is a perennial plant that reaches a height of 80 cm. Its flowers are yellow and glabrous. It is an endemic plant to the flora of Turkey and is distributed in the Central Anatolia region of Turkey, especially the calcareous slopes of Konya [24]. In recent studies, several coumarins and volatile compounds have been isolated from this plant, depending on the plant parts used [12,15].
In recent years, the number of studies reporting experimental data on the biological effects of Prangos species have increased considerably [17,22,[25][26][27] and significant information has been gathered on the therapeutic properties of different species. Nevertheless, there are still a few species that have remained largely ignored with regard to certain aspects of their biological potentials, thereby reducing the possibility of their exploitation as phytomedicines. Therefore, in this study, the chemical composition, antioxidant and enzyme inhibitory effect of EOs extracted from three endemic Prangos species from Turkey (P. heyniae, P. meliocarpoides var. meliocarpoides, and P. uechtritzi.) were investigated and the molecular docking technique was applied to elucidate the binding interactions of selected EOs' components with select enzymes.

Antioxidant Activity
Oxidative stress has been identified as the root cause of the development and evolution of several diseases. Supplementation of exogenous antioxidants or increasing endogenous antioxidant defenses of the body is a promising way of fighting the undesirable effects of reactive oxygen species (ROS) induced oxidative damage [33]. The plant kingdom is a rich source of health-promoting compounds especially as natural antioxidants [34]. Several studies have accentuated on the high antioxidant capacity of plants, their derivatives such as EOs and isolated compounds in the recent years, thus highlighting their usefulness in pharmaceutical, cosmetics, food and beverage industries, especially as some synthetic antioxidants such as BHA and BHT are now suspected to be potentially harmful to human health [35][36][37][38][39].
In the present investigation, the EOs extracted from all the three Prangos species were found to possess free radical scavenging potential in both DPPH and ABTS assays (0.43-1.74 mg TE/g and 24.18-92.99 mg TE/g). While P. uechtritzii EO showed the highest scavenging activity in DPPH assay, P. heyniae EO demonstrated the most significant scavenging activity in ABTS assay. Reducing potential was also noted by all EOs in CUPRAC and FRAP assays (103.15-113.43 mg TE/g and 47.98-61.20 mg TE/g, respectively) ( Table 3). In FRAP assay also, P. heyniae EO displayed the highest activity while in CUPRAC assay P. meliocarpoides var. meliocarpoides EO showed higher reducing activity compared the other EOs. Metal chelating activity was demonstrated as well in the range of 28.66-30.94 mg EDTAE/g. Total antioxidant capacity was revealed by the phosphomolybdenum assay and the order of the potency of the EOs were as follows: P. meliocarpoides var. meliocarpoides > P. heyniae > P. uechtritzii (15.64-24.37 mmol TE/g) ( Table 3). Interestingly, several previous studies have also reported other members of the Prangos genus to exhibit strong antioxidant abilities [40,41]. In another study, the antioxidant activities of the water and methanol extracts obtained from the root, herb, and fruits of P. ferulacea, including the three species studied herein (P. heyniae, P. meliocarpoides var. meliocarpoides, and P. uechtritzii) from Konya Province (Turkey) were also evaluated using DPPH and thiobarbituric acid assays [21].

Enzyme Inhibitory Effects
Enzyme inhibitors play a significant role in the drug discovery process. An understanding of diseases at the molecular level has revealed the root cause of many to be the dysfunction, overexpression, or hyperactivation of enzymes. This hyperactivation or overexpression of enzymes can be treated by using suitable enzyme inhibitors. These efforts have provided several enzyme inhibitors in the clinic, including some from a natural origin [42]. Hence, in this present study, an attempt was made to assess the inhibitory effects of the EOs against some enzymes of clinical interest, notably cholinesterases, tyrosinase, amylase and glucosidase.
Cholinesterases are a group of serine hydrolases that split the neurotransmitter acetylcholine (ACh) and terminate its action. While acetylcholinesterase (AChE) plays the key role in ending cholinergic neurotransmission Butyrylcholinesterase (BChE) is a nonspecific cholinesterase enzyme that hydrolyzes choline-based esters. BChE plays a critical role in maintaining normal cholinergic function like AChE through hydrolyzing ACh [43]. Thus, cholinesterase inhibitors are useful substances that help to interfere with the break-down of ACh and prolong its action [44].
In this study, while no inhibitory activity was demonstrated by P. meliocarpoides var. meliocarpoides and P. uechtritzii EOs against both cholinesterases (AChE and BChE), while P. heyniae EO displayed only anti-BChE activity (9.85 mg GALAE/g) ( Table 4). The inhibition of α-glucosidase and α-amylase, enzymes involved in the digestion of carbohydrates, can significantly diminish the post-prandial surge of blood glucose and consequently can be an important strategy in the management of blood glucose level in type 2 diabetic and borderline patients. Presently, there is renewed interest in plant-based medicines and functional foods modulating physiological effects in the prevention and cure of diabetes and obesity. The plant kingdom is a wide field to search for natural effective oral hypoglycaemic agents that have minor or no side effects [45]. In the present study, the EOs were found to inhibit amylase (0.09-0.61 mmol ACAE/g) only, although the activity was not prominent, whereas they showed no activity against glucosidase (Table 4).
Tyrosinase plays a vital role because it is the critical enzyme and restriction enzyme in the course of melanin composition. Pigment spots and melanoma are markedly increased by cumulative tyrosinase activity and quantity. Consequently, tyrosinase inhibitors have received broad consideration owing to their use as hypopigmented agents in recent years [46].
Herein, all the EOs were found to act as tyrosinase inhibitors with inhibitory activity ranging from 46.34 to 69.56 mg KAE/g ( Table 4). The anti-tyrosinase potency of the EOs was obtained in the following order: P. meliocarpoides var. meliocarpoides > P. heyniae > P. uechtritzii.

Multivariate Analysis
To gain more insights between the detected compounds and biological activities of the tested essential oils, we performed a PLS analysis. The results are given in Figure 1. R 2 X and Q 2 values are indicator the quality of PLS parameters and the values were 0.87 and 0.97, respectively. Apparently, good connections were established between the identified compounds and biological activities. For example, p-cymene (compound 16) made the main contribution to DPPH scavenging ability and this fact was confirmed by several researchers in previous studies [47,48]. As another example, β-elemene (compound 28) was very close to the ability of phosphomolybdenum (PBD) and this compound has already been described as an agent for antioxidant therapy [49]. In metal chelating ability (MCA), α-copaene (compound 20) was a major contributor. β-Pinene was very close in cupric reducing ability. Principal component analysis (PCA) was also performed to determine similarities or differences between the tested Prangos species based on their biological activities. The tested species were different and two components (PC1: 53% and PC2: 33.9%) were obtained in PCA (Figure 2). PC1 was mainly contributed by ABTS, BChE, FRAP and CUPRAC, while PBD, tyrosinase, DPPH and amylase were the main players in PC2. The obtained results could be useful for further application by using the tested species in future studies.

Molecular Docking
Molecular modelling has been used for the predicting the ligand-target affinity as well as interaction [50,51]. In this study, molecular docking investigation of 12 components (selected from five or the five most abundant components of each EO) from the three EOs of PH (P. heyniae), PM (P. meliocarpoides var. meliocarpoides) and PU (P. uechtritzii) against tyrosinase and amylase were investigated. The reason for selecting these two targeted enzymes was that these three EOs have demonstrated better inhibitory activity against these two enzymes. These EO components were selected to decipher the inhibition pattern against tyrosinase and amylase enzyme. Detailed docking scores of all selected compounds against tyrosinase and amylase are shown in Table 5 and details of the binding interactions of best docked pose of α-bisabolol and elemol against tyrosinase and amylase are reported in Table 6. Analysis of docking score revealed elemol and α-bisabolol to be the most effective in binding with tyrosinase and amylase based on their ChemGauss scores of −8.10 and −8.94, respectively. Detailed 3-D binding interactions of α-bisabolol against tyrosinase and elemol against amylase are depicted in Figure 3.

Plant Materials
Prangos species were collected in the city of Konya. The location information is given in Table 7 below. The plants' identity were confirmed by one of co-authors (Evren Yıldıztugay) and voucher specimens have been deposited in Selcuk University. The aerial parts of the plant samples were dried under shade conditions for 10 days at room temperature. The plant samples were then powdered using a laboratory mill and the powdered plant samples were stored in the dark at room temperature.

Essential Oil Extraction and GC-MS Analysis
The essential oil was obtained by using the hydro-distillation technique. One hundred g dried plant samples were distilled in a Clevenger-type apparatus for 5 h. The essential oil was dried over sodium sulphate (anhydrous) and then the obtained essential oils were stored in an amber vial at +4 • C until analysis.
The obtained essential oil was characterized by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrophotometry (GC-MS) techniques. GC-MS analysis was performed by using a 5975 GC-MS system (Agilent, city, state abbreviation if USA, country) coupled to an Agilent 7890 A GC. To separate chemical components, a HP-Innowax column (60 m × 0.25 mm, 0.25 µm film thickness) was used. Other analytical parameters were reported in our earlier paper [52]. All analytical details are given in the Supplementary Materials.
The retention index (RI) calculated by co-injection with reference to a homologous series of n-alkanes (C 8 -C 30 ) under identical experimental circumstances was used to identify the components. By comparing their mass spectra to those from the NIST 05 and Wiley 8th edition libraries, as well as comparing their RIs to literature values, we were able to make more accurate identifications.

Determination of Antioxidant and Enzyme Inhibitory Effects
The antioxidant activity of the essential oils tested in this study was determined using a variety of assays [53]. The assays used were 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical scavenging capacity (CUPRAC), ferric ion reducing antioxidant power (FRAP), metal chelating ability (MCA), and phosphomolybdenum assay (PDA). The data for the DPPH, ABTS, CUPRAC, and FRAP assays were expressed in mg Trolox equivalents (TE)/g essential oils whereas the data for MCA and PDA were expressed in mg EDTA equivalents (EDTAE)/g essential oils and mmol TE/g essential oils, respectively. Previously, we provided the experimental components for the acetylcholinesterase, butyrylcholinesterase, tyrosinase, α-amylase, and α-glucosidase assays. In cholinesterase assays, galanthamine was used as a positive control, and data were expressed as mg galanthamine equivalents (GALAE)/g essential oils. In the tyrosinase inhibitory assay, kojic acid was used as a standard inhibitor, and the results were expressed as mg kojic acid equivalents (KAE)/g essential oils [53,54]. In the anti-diabetic assays, acarbose was chosen as an inhibitor of both amylase and glucosidase, and the results are expressed as mmol acarbose equivalents (ACAE)/g essential oils. The experimental procedures of the assays are given in Supplementary Materials. The assays were performed in triplicate, and ANOVA (Tukey's test) was used to determine the differences in the essential oils (p < 0.05).

Multivariate Analysis
A partial least squared (PLS) regression was performed for the relation between bioactive components and biological activities of the tested essential oils. A principal component analysis (PCA) was also performed to detect differences between the tested species. The multivariate analysis was done with the SIMCA 14.0 Software (Umetrics, Umeå, Sweden).

Molecular Docking
A molecular docking investigation of the 12 most abundant components from the three essential oils of Prangos heyniae, Prangos meliocarpoides var. meliocarpoides and Prangos uechtritzii against tyrosinase and amylase were carried out. A list of these compounds is summarized in Table 1.
The 2D structures of these selected compounds were retrieved from PubChem and then imported into Discovery studio client for making the 3D structures. OMEGA tool of OpenEye software was used to generate molecular structures of these compounds with their optimized stereoisomers, ring conformations, tautomers, and ionization states to make broad structural and chemical diversity from a single input compound. The default settings of OMEGA were used for the generation of multiconformers of each compound, as the generation of conformer is a prerequisite for subsequent molecular docking.
Three-dimensional X-rays crystallographic structures of the target enzymes tyrosinase and amylase were retrieved from the Enzyme Data Bank utilizing the PDB ID: 2Y9X and 4W93, respectively [55]. To prepare the enzyme structures, the Discovery Studio Client software was utilized, which eliminates the heteroatoms and water molecules, inserts hydrogens and missing residues and assigns charges. The binding site of each enzyme was located based on the co-crystalized ligand within each targeted enzyme. Prior to docking of compounds, optimization of docking calculations was performed by re-docking the cocrystal ligand within the active site of the respective enzyme. Binding site coordinates was obtained using a co-crystal ligand in the binding site of tyrosinase and amylase. In case of tyrosinase, active site was adjusted upon the coordinate X = −10.021, Y = −28.82, and Z = −43.596111 in XYZ dimensions. While in case of amylase, coordinate was selected using X = −9.63, Y = 4.34 and Z = −23.10 dimensions. In both cases of redocking co-crystal ligands within the target protein, the RMSD was found to be less than 2 Å which shows the reliability of the docking protocol [50].
After optimization of the docking protocol, molecular docking of all selected compounds (as listed in Table 1) were performed using the FRED tool in OEDOCKING of the OPENEYE Software. For each compound, ten poses were generated and sorted out based on the corresponding ChemGauss 4 score. The best-docked pose was selected based on the lowest ChemGauss 4 score for deciphering the binding interactions between the compound and the amino acid residues of the targeted enzyme. Discovery Studio Visualizer was used for the visualization of the binding interactions of the best compounds with the amino acid residues of tyrosinase and amylase.

Conclusions
In this study, the chemical profile of the EOs obtained from three endemic Prangos species from Turkey was revealed and analyzed. Moreover, the in vitro antioxidant and enzyme inhibitory properties of the EOs were investigated in an attempt to assess the biological potentials of the plant-derived products of these Prangos species. While all the EOs showed moderate to good antioxidant potentials, as evidenced by various assays, their activity as enzyme inhibitors was not as prominent. While all EOs demonstrated anti-tyrosinase and anti-amylase activity, only P. heyniae EO showed anti-BChE activity. Overall, the findings from this study unveiled the varying biological potentials of the studied EOs and some interesting prospects of the investigated EO components as enzyme inhibitors. Particularly, their antioxidant and anti-tyrosinase potentials offer suggestions for their use in the development of topical anti-hyperpigmentation formulations. However, further research is warranted to assess the potency and safety of these EOs and their active components as cosmeceutical agents in vivo.