Sahlep (Dactylorhiza osmanica): Phytochemical Analyses by LC-HRMS, Molecular Docking, Antioxidant Activity, and Enzyme Inhibition Profiles

Studies have shown an inverse correlation among age-related illnesses like coronary heart disease and cancer and intake of fruit and vegetable. Given the probable health benefits of natural antioxidants from plants, research on them has increased. Dactylorhiza osmanica is consumed as a food and traditional medicine plant in some regions of Turkey, so evaluation of the biological ability of this species is important. In this study, the amount of phenolic content (LC-HRMS), antioxidant activities and enzyme inhibitory properties of an endemic plant, D. osmanica, were investigated. The antioxidant capacities of an ethanol extract of D. osmanica aerial parts (EDOA) and roots (EDOR) were evaluated with various antioxidant methods. Additionally, the enzyme inhibitory effects of EDOA and EDOR were examined against acetylcholinesterase (AChE), α-glycosidase, and α-amylase enzymes, which are associated with common and global Alzheimer’s disease and diabetes mellitus. The IC50 values of EDOA against the enzymes were found to be 1.809, 1.098, and 0.726 mg/mL, respectively; and the IC50 values of EDOR against the enzymes were found to be 2.466, 0.442, and 0.415 mg/mL, respectively. Additionally, LC-HRMS analyses revealed p-Coumaric acid as the most plentiful phenolic in both EDOA (541.49 mg/g) and EDOR (559.22 mg/g). Furthermore, the molecular docking interaction of p-coumaric acid, quercitrin, and vanillic acid, which are the most plentiful phenolic compounds in the extracts, with AChE, α-glucosidase, and α-amylase, were evaluated using AutoDock Vina software. The rich phenolic content and the effective antioxidant ability and enzyme inhibition potentials of EDOA and EDOR may support the plant’s widespread food and traditional medicinal uses.


Introduction
The Orchidaceae family is one of the richest flowering plant groups in the world, and there are 24 genera and 170 taxa in Turkey [1]. Orchids have a very high economic value. Although it is known especially as an ornamental plant, many orchid species are commonly used in the foods and pharmacy industry. The vanilla flavor obtained from orchids is frequently traded [2]. Orchid species are used for treatment of Alzheimer's and Parkinson's diseases (AD and PD), anxiety, depression, cancer, chest pain, tuberculosis, intestinal disorders, dysentery, diarrhea, cough, cold, anemia, and are also used as an aphrodisiac in adults [2][3][4]. It has been reported that these medicinal properties are due to secondary compounds such as polyphenolic compounds, ascorbic acid, indole alkaloids, and saponins [4]. It has also been determined that orchid roots have many compounds such as polyphenols and glucomannan, which have strong antioxidant properties [3]. The flour
In the current study, the results of AD-related cholinesterase inhibition were evaluated and the IC 50 values for AChE were measured to be 1.809 µg/mL (r 2 : 0.9722) for EDOA; 2.466 µg/mL (r 2 : 0.9826) for EDOR; and 0.124 µM for tacrine (Table 5) [24]. p-Coumaric acid, quercitrin, and vanillic acid were detected to be the main phenolic acid of EDOA and EDOR. However, it was reported that p-coumaric acid has been shown to have no inhibition toward AChE [25]. The best binding-pose selection was performed for quercitrin and vanillic acid, the two main compounds of EDOA and EDOR, by placing them into the active site of the AChE. Additionally, the best binding-pose selection was performed for p-coumaric acid and vanillic acid to the active site of α-amylase and αglycosidase as other target enzymes. Docking studies were followed by analysis of binding modes to understand inhibition mechanisms. According to docking scores, quercitrin showed the highest binding affinity with AChE and p-coumaric acid showed the highest binding affinity with α-amylase and α-glycosidase enzyme targets (Table 6). Quercitrin and vanillic acid were placed in the active site of the enzyme AChE (PDB code: 4EY7). Figure 4B represents 3D and 2D interactions of Quercitrin-AChE and the docking score was calculated as −8.8 kcal/mol (Table 6). It is shown that the hydroxyl groups of quercitrin are linked to the active site through H-bond interactions with Gln-291, Arg-296, Glu-292, and Trp-286 active-site amino acids. Table 6. Molecular interactions of the AChE, α-amylase, and α-glycosidase with the major phenolic compounds of Dactylorhiza osmanica (vanillic acid, p-coumaric acid, and quercitrin).  p-Coumaric acid-α-glycosidase (5NN8) complex's docking score was calculated to be -6.5 kcal/mol (Table 6). A conventional H-bond of p-coumaric acid with α-glycosidase Asp-404 residue and two π anion interactions with Asp-518 and Arg-600 residues are shown in Figure 5B.

Discussion
It is important to choose the most appropriate method when determining the antioxidant capacity of plants. In the present study, Fe 3+ reducing, Cu 2+ reducing and Fe 3+ -TPTZ reducing effects, ABTS and DPPH radical removal methods, and Fe 2+ binding ability were used to determine the antioxidant capacity of the extract [26]. The DPPH method is based on the DPPH • scavenging percentage of antioxidants in the plant extract. On the other hand, ABTS assay is based on the percentage of antioxidants in the plant extract to scavenge ABTS •+ radicals [27]. Ferrozine is known to form complexes with Fe 2+ ions. In the presence of chelating agents in the environment, the generation of the complex is disrupted and leads to a decrease in the red complex color. In this way, the estimation of color reduction allows for estimating the chelating ability of the chelator [28].
Ascorbic acid was found to be the compound with the most effective DPPH • scavenging activity. EDOA and EDOR were found to have a free radical scavenging ability close to standard compounds. In one study, DPPH • scavenging activity of methanol extract of D. osmanica aerial parts was investigated and the IC 50 value was found to be 0.1838 ± 0.0015 mg/mL [1]. In another study, DPPH • scavenging activity of aqueous ethyl alcohol extract (70%) of Dactylorhiza maculata was investigated and the IC 50 value was found to be 217.89 ± 10.89 mg ascorbic acid [29]. In a study conducted in 2020, extracts prepared from Dactylorhiza romana plant roots using different solvents were investigated, and the DPPH test IC 50 of ethanol extract was calculated to be 1.53 ± 0.004 mg/mL [20]. When all these results were interpreted, it was determined that EDOA and EDOR did not exhibit a very strong DPPH • scavenging activity. According to the ABTS •+ scavenging activity method, a stable form of the radical is produced in the experiment and forms blue-green ABTS •+ by reacting with an antioxidant, and decolorization specifies the rate of ABTS •+ inhibition [30,31].
Additionally, the results indicate that EDOA and EDOR have very strong metal chelating activity. In the literature search, there were no publications about Fe 2+ chelating activity measured in D. osmanica extracts, so this study is the first. The antioxidant profile of EDOA and EDOR, characterized by using the ferric ion (Fe 3+ ) reduction and CUPRAC and FRAP assays, are shown in Table 2 and Figure 2. Reduction capacity is an important factor in determining whether a molecule has antioxidant activity [32]. The first method used was to reduce Fe 3+ to Fe 2+ in Fe[(CN) 6 ] 3+ solution, which is one of the common methods. The reaction system is based on the reduction of Fe +3 in potassium ferricyanide to Fe 2+ with the addition of an antioxidant agent and the formation of the Prussian blue color at 700 nm [19]. According to the results, it was determined that EDOA and EDOR have strong ability to reduce ferric ions (Fe 3+ ) (p < 0.001) ( Table 2). However, this value was found to be lower than standard antioxidants. In the CUPRAC test, the absorbance measurement of the stable complex occurred between neocuproine and Cu 2+ ions, observed at 450 nm. High absorbance values indicate high reducing ability [19]. The Cu 2+ ion reducing ability of EDOA, EDOR, and positive controls are demonstrated in Table 2 and Figure 2b. The FRAP assay is based on measuring the power of a sample with antioxidant properties to reduce oxidant ferric iron to ferrous form [33]. According to the method, higher absorbance values represent the higher reduction ability of the Fe 3+ -TPTZ complex. Furthermore, EDOA and EDOR demonstrated effective FRAP ability (p < 0.001) ( Table 2). In one study, a methanol extract of aerial parts of D. osmanica was investigated and the Fe 3+ -TPTZ reducing value was found to be 804 ± 8.6217 (µM TE/g) [1]. Dactylorhiza chuhensis ethanol extracts were examined in a study and the FRAP values of tubers and flowers were found to be 85.3 ± 8.6 and 511.6 ± 252 µmol Fe 2+ /g DW, respectively [2].
The plants exhibited effective antioxidant capacity due to their secondary metabolites, including a large spectrum of phenolic and flavonoids [34]. Phenolic compounds are among the plant's main secondary metabolites. It was determined that a diet rich in phenolic compounds has protective effects against cancer and cardiovascular diseases. Phenolic and flavonoids have many biological effects including anticancer, antibacterial, antiallergic, anti-inflammatory, and free radical scavenger [35]. Flavonoids form an important chemical class of secondary compounds in plants. Numerous phenolic hydroxyl groups attached to the ring structures of flavonoids give them antioxidant ability [36,37]. Owing to their strong free radical scavenging properties, flavonoids show antioxidant activities such as metal chelation and reduction [38]. In another study, total phenolic and flavonoid quantities in Dactylorhiza hatagirea tuber extract were found to be 11.42 ± 0.48 mg GAE/g and 11.46 ± 0.28 mg QE/g, respectively [4]. Dactylorhiza chuhensis ethanol extracts were examined in a study and the total phenolics of tubers and flowers were found to be 13.9 ± 0.6 and 44.2 ± 2.0 mg GAE/g DW, respectively [2]. In another study, the ethanol extract of Dactylorhiza romana plant roots was investigated and total phenolic and flavonoid contents were determined as 24.91 ± 0.95 mg GAE/g and 3.58 ± 0.08 QE/g, respectively [20]. The results obtained in these previous studies were found to be sometimes higher and sometimes lower than our results. The reason for this is thought to be due to differences in ecological and soil structure of the region where the plant is grown, analysis methods, solvents used, and extraction conditions.
It is known that antioxidant compounds including phenolics, flavonoids, and phenolic acids have a wide variety of pharmacological effects such as anti-inflammatory, anticarcinogenic and antiatherosclerotic activity [39]. Phenolic compounds, which have many beneficial effects on human health, are found in plants, vegetables, fruits, and cereals [40,41]. p-Coumaric acid is a hydroxyl derivative of cinnamic acid, and p-coumaric acid is one of its most abundant isomers in nature [42]. p-Coumaric acid is a natural phenolic acid found in many edible plants and exhibits various biological effectiveness as an antioxidant, antimicrobial, anti-inflammatory, and analgesic. It has also been determined to act as a tyrosinase inhibitor [42][43][44][45]. Few studies have been found to determine the phenolic content of D. osmanica. Only in 2018, a study was conducted with D. osmanica, which was collected from a different part of Turkey, and according to this, the most plentiful phenolics were determined by HPLC measurement to be syringaldehyde, p-coumaric acid, ferulic acid, synaptic acid, and benzoic acid [1]. The results of this research were found to be in agreement with the present study.
Diabetes mellitus (DM) is a metabolic disease caused by a disorder in insulin secretion. It causes chronic hyperglycemia and irregularity in carbohydrate, fat, and protein metabolism [46,47]. In the treatment of DM, compounds that inhibit the enzymes involved in carbohydrate absorption and metabolism are used, especially for the inhibition of pancreatic α-amylase and α-glucosidase enzymes, which are among the main enzymes involved in the intestinal absorption of glucose and play a key role in treatment. Inhibition of these enzymes reduces the absorption of sugars from the intestine and provides regulation of postprandial blood glucose level in Type 2-DM (T2DM) patients [48,49]. However, these drugs used for treatment have side effects. For this reason, natural compounds obtained from medicinal plants are being investigated for the treatment of T2DM, as they create better glycemic control and show fewer side effects [50]. Orchid species are among the plants widely used in traditional medicine due to their medicinal properties [48]. The determination of the inhibition of antidiabetic enzymes, α-glycosidase and α-amylase, was conducted for determination of antidiabetic capability of D. osmanica.
According to the results obtained, it was shown that ethanolic extracts of D. osmanica effectively inhibited α-amylase and α-glycosidase activities. These inhibitory effects were compared with Acarbose. In particular, EDOR had a very high affinity for α-amylase and α glycosidase. In the literature search, no data were found on the inhibitory properties of D. osmanica for α-glycosidase and α-amylase enzymes. However, there are studies carried out in different Dactylorhiza species. In a study conducted in 2020, the extracts prepared from the roots of D. romana with different solvents were investigated and the α-glucosidase and α-amylase inhibition IC 50 values of the ethanol extract was determined to be 4.368 ± 0.053 and 76.554 ± 0.303 mmol/g, respectively [20]. In another study, αglycosidase and α-amylase inhibition were investigated and the IC 50 values of the methanol extract of Dactylorhiza hatagireas leaves were investigated and found to be 199.8 ± 4.7 and 210.28 ± 5.4 µg/mL, respectively [48]. In another study, α-glycosidase and α-amylase percentage inhibition values of D. hatagirea tuber extract were determined to be 46.80% and 27.97%, respectively [4]. The results of this study were consistent with the results of previous studies on the inhibition ability of different orchid species on α-amylase and α-glucosidase activities.
Alzheimer's disease (AD) presents with memory loss and other behavioral abnormalities. In the treatment of AD, one of the most important methods is to control the level of acetylcholine by blocking the breakdown of acetylcholinesterase inhibitors [51]. AChE is an important enzyme that hydrolyzes neurotransmitter acetylcholine at cholinergic synapses in the central nervous system and peripheral nervous system [52]. Several compounds such as donepezil, galantamine, tacrine, and rivastigmine are used as AChE inhibitors in the treatment [53]. However, current AChE inhibitors have side effects and are only used to treat mild to moderate symptoms [52]. Medicinal plants are rich in different bioactive compounds including flavonoids and alkaloids, which are can be used in the treatment of some diseases including AD. Therefore, there is increasing interest in studies to obtain new drugs from plant extracts or compounds of plant origin [51,53]. It was determined that EDOA effectively inhibited AChE enzyme. In the literature search, no data were found on the inhibitory properties of D. osmanica for AChE enzyme. This work constitutes an initial reference for this. However, there are some studies carried out in several Dactylorhiza species. In a study, the extracts prepared from the roots of Dactylorhiza iberica and different solvents were investigated and AChE inhibition value of the methanol extract determined as 28.9% [51,54].
In this research study, data on the phytochemical bioactivity and properties, phenolic and flavonoid contents, antioxidant capacity, and enzyme inhibition potential of an endemic plant, D. osmanica, are presented for the first time. It was determined that D. osmanica contains a good level of phenolic and flavonoid contents and exhibits antioxidant activity close to standard compounds. In addition, it was also determined that there was a statistically significant difference between the averages of the groups. As a result of LC-HRMS analysis, it was determined that the main phenolic compounds of D. osmanica extracts are very rich in p-coumaric acid, vanillic acid, and quercitrin. Additionally, possible inhibition of D. osmanica extracts against α-glucosidase, α-amylase, and AChE enzymes was measured and it was determined that both EDOA and EDOR showed effective enzyme inhibition according to the results, which were also supported by molecular docking studies. The results obtained from this study with D. osmanica may provide a basis for studies to obtain secondary compounds of the species in pure form.

Lyophilized Water Extract
The extraction procedures were performed according to the procedures previously described [55,56]. For the preparation of the ethanol extracts of D. osmanica aerial parts (EDOA) and roots (EDOR), the aerial parts and roots (each 25 g) of the shade-dried D. osmanica were first pulverized in a grinder. Then, the ground plant materials were soaked separately with 0.5 L of ethanol. The ethanol was evaporated (Heidolph Hei-VAP HL, Schwabach, Germany) and stored at −20 • C [57].

Radical Scavenging Methods
For evaluating the DPPH radical removing effect of the EDOA and EDOR, extracts and standards were prepared at different concentrations (10-30 µg/mL) and 1 mL of DPPH radicals (0.1 mM) was added to each sample tube. After 30 min of incubation, absorbance was recorded at 517 nm, as described previously [58]. To determine the ABTS radical scavenging effects of EDOA and EDOR, the method in a prior study was used [58]. For determination of ABTS +• scavenging effects of EDOA and EDOR, the previously given method was used [59][60][61]. First, 2.45 mM persulfate solution was added to 2 mM ABTS solution to generate ABTS radicals. The absorbance of the ABTS •+ radical as control sample containing a 0.1 M phosphate buffer (pH 7.4) was adjusted to 0.750 ± 0.025 nm at 734 nm. Then, one mL of ABTS •+ solution was added to different EDOA and EDOR concentrations and after 30 min incubation absorbance were recorded at 734 nm [17,62]. Metal chelating ability was measured by inhibiting the formation of Fe 2+ -Ferrozine complex after treatment of test material with Fe 2+ [63] with minor modification [64]. Fe 2+ -chelating effect was determined by the absorbance of the Fe 2+ -Ferrozine complex at 562 nm [65]. To summarize, different concentrations of EDOA and EDOR in 0.5 mL ethanol were transferred to a 0.1 mL of FeCl 2 (0.6 mM). The reaction was started by adding 0.4 mL of Ferrozine (5 mM), which prepared in ethanol. The absorbance was measured spectrophotometrically at 562 nm [66].

Reducing Activity Methods
The Fe 3+ reducing effects of EDOA and EDOR were measured depending on different concentrations (10-30 µg/mL). According to this method, the reducing capacity of an active molecule can be directly measured by reduction of Fe[(CN) 6 ] 3 to Fe[(CN) 6 ] 2 [67]. As a result, the Perl-Prussian blue complex, which exhibits absorbance at 700 nm, leads to the formation of Fe 4 [Fe(CN − ) 6 ] 3 [68]. To determine the CUPRAC of EDOA and EDOR, a previous method with some changes was applied [69]. The FRAP method is based on the reduction of the TPTZ-Fe 3+ complex [70].

Total Phenolic and Flavonoid Concentration
The quantity of phenolics in EDOA and EDOR was performed as described in previous studies [71,72]. The total amount of flavonoids found in EDOA and EDOR was determined as described before [73] and as given in a prior study [74].

Enzyme Inhibition Assay
The AChE enzyme inhibition properties of the extracts were determined according to a prior study [75]. The α-amylase and α-glycosidase inhibition effects of both extracts were estimated according to a method from previous studies [76,77]. The IC 50 value is defined as the concentration of antioxidant compound causing 50% enzyme inhibition and was obtained from activity (%) against compound concentrations [78,79].

LC-HRMS Procedure and Optimization of HPLC Methods
The final mobile phase included an acidified methyl alcohol and water gradient by the HPLC method [83]. The identification of the phenolics was made by comparing the retention times of the standard phenolics (in the purity range 95-99%; see section chemicals) and HRMS data of ILMER in Bezmialem Vakıf University. Dihydrocapsaicin (95%, purity) was used as the internal standard (IS) for LC-HRMS for reducing repeatability caused by external effects such as ionization repeatability in mass spectrometry measurements; 0.1 g/L dihydrocapsaicin (97%, Sigma-Aldrich) solution was used as the IS. The linear range of the standard solutions is given as mg/kg in Table 7. The mass parameters related to target compounds are summarized in Table 4 [82][83][84].

Method Validation
Validation of the LC-HRMS method was performed by using analytical standards of corresponding compounds (see Section 4.1) as the target ions (Table 7). Considering the EURACHEM/CITAC Guide [84] and purpose of the method, the validation parameters were selected as linearity, recovery, repeatability, LOD, and LOQ for the applied method. The limit of detection (LOD) of the method for each compound was determined according to the following equation: LOD or LOQ = κSDa/b, where 3 is used for LOQ and κ = 3 for LOD; SDa represents the standard deviation of the intercept and b represents the slope (Table 7) [83].

Molecular Docking Studies
The 3D version of compound chemical structures was downloaded from pubChem [84]. The 3D X-ray crystal structures of acetylcholinesterase (PDB ID: 4EY7) [85], α-glycosidase (PDB ID: 5NN8) [86] and α-amylase (PDB ID: 2QV4) [87] were downloaded from the "Protein Data Bank" website, with resolutions 2.35, 2.45, and 1.97 Å, respectively [88]. The structures of these enzymes were optimized in AutoDockTools 1.5.7 [89]. The most stable conformations and structure optimization of ligands were determined with AutoDockTools; the PDBQT file of the ligands was then prepared. The optimized enzyme and ligand structures were loaded into AutoDockTools and the same program was used for docking. The best scores of docking energy and binding interactions were analyzed with BIOVIA Discovery Studio.

Statistical Analysis
Statistical analyses employed the unpaired Student's t-test by using the statistical program of IBM SPSS Statistics 20. The results obtained were recorded as means with their standard deviation (SD); p < 0.05 was established as the minimum significance level.

Conclusions
In conclusion, in this study, EDOA and EDOR demonstrated effective antioxidant ability when compared to the standards, including BHA, BHT, α-Tocopherol, and Trolox. Additionally, EDOA and EDOR showed a value close to the standard compounds in all antioxidant activity tests. The ABTS •+ scavenging test showed better results than standard compounds. Both extracts possessed a wide spectrum of biological activities and can neutralize ROS and free radicals. EDOA and EDOR can be used to prevent or delay the formation of lipid autoxidation. Additionally, EDOA and EDOR were tested against enzymes such as acetylcholinesterase, butyrylcholinesterase, and α-glycosidase, which are associated with common diseases such as diabetes, Alzheimer's disease, and glaucoma. Finally, the results indicated that EDOA and EDOR have some biological effects, including anticholinergic and antidiabetic effects. Thus, EDOA and EDOR may provide beneficial outcomes for treatment of diseases, following approval by further clinical and in vivo studies .