*2.4. Molecular Docking*

A molecular docking analysis was carried out on the 12 most abundant components from the root essential oil of *P. pabularia* using the Molegro Virtual Docker program [54]. The MolDock "rerank" docking energies as well as the scaled molecular docking energies are summarized in Table 3. There are two ligand binding regions in human PTP-1B—the catalytic site and an allosteric site (Figure 4). Nearly all of the ligands examined docked preferentially to the allosteric binding site in PDB 1T48, and the best docking ligand was osthole. In the allosteric binding site, the coumarin rings are located in a hydrophobic sandwich formed by Phe280 and Leu192 (Figure 5). Additionally, the aromatic residues Trp291 and Phe196 form face-to-edge π interactions with the coumarin moiety. Residues Ala189 and Glu276 surround the isopropylidene group of osthole. There are no apparent hydrogen-bonding interactions in the docked osthole in the allosteric binding site.

**Figure 4.** Ribbon structure of human protein tyrosine phosphatase 1B (PTP-1B, PDB 1T48). The catalytic site (**A**) and the allosteric binding site (**B**) are shown as green cross-hatched areas.

The active site of PTP-1B is composed of highly polar residues, including Arg24, Lys41, Arg47, and Asp48, as well as the phosphate-binding loop, Cys215-Arg22 [55]. Therefore, the active site of PTP-1B is not a likely target for the hydrophobic essential oil components of *P. pabularia*. Nevertheless, the lowest-energy docked pose of osthole with the active site of PTP-1B (PDB 2HB1) has a scaled docking energy of −103.0 kJ/mol. This docking pose of osthole shows π-stacking of the coumarin moiety with Phe182 and Tyr46 and is held close to the catalytic site residues of Cys215 and Arg221 (Figure 6). In addition, there are hydrogen-bonding interactions between the osthole carbonyl oxygen and the side chains of Lys120 and Arg221.



#### *Appl. Sci.* **2019** , *9* , 2362

**Figure 5.** The allosteric binding site of human protein tyrosine phosphatase 1B (PTP-1B, PDB 1T48) with the lowest-energy docked pose of osthole.

**Figure 6.** The active site of human protein tyrosine phosphatase 1B (PTP-1B, PDB 2HB1) with the lowest-energy docked pose of osthole. Hydrogen-bonding interactions are indicated by the blue dashed lines.

Ala and co-workers noted that " ... the active site of PTP-1B possesses very few desirable drug-design features" and that the highly charged portions of the active site " ... significantly increases the difficulty of designing potent inhibitors with acceptable membrane permeability" [55]. Thus, we conclude that the likely binding site for the *P. pabularia* essential oil components is the hydrophobic allosteric binding site, which is more consistent with the greater exothermic docking energies with the allosteric binding site (PDB 1T48, Figure 4B) than with the enzyme active site (Figure 4A). Furthermore, the docking energy for osthole is the most exothermic of the ligands examined, and this compound represents 6.0% of the essential oil composition.

The most abundant component, 5-pentylcyclohexa-1,3-diene (44.6%), is a hydrocarbon, and although the docking energies are somewhat lower for the allosteric site than those for other essential oil components, the abundance of this compound may be a factor in the PTP-1B inhibitory activity of *P. pabularia* root essential oil. Wiesmann and co-workers pointed out that allosteric inhibitors " ... prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop" [56].

#### **3. Materials and Methods**

#### *3.1. Plant Material*

The roots of *P. pabularia* Lindl. were collected from the Yovon region (38 1847 N, 69 02 35 E and 950 m above sea level) of Tajikistan in April 2017. The plant was authenticated by Doctor Farukh Sharopov, and the voucher sample (No. TAS 23659-1) was deposited in the herbarium of the Xinjiang Technical Institute of Physics and Chemistry Urumqi, Chinese Academy of Science. The air-dried sample was chopped into small pieces and hydrodistilled for 3 h to give the yellow essential oil with 0.1% yield. Osthole was isolated from the roots of *Prangos pabularia* by silica gel column (100–200 mesh) by elution of hexane ethyl acetate (20:1) [22].

## *3.2. Gas Chromatography*

The quantification of the essential oil of *P. pabularia* was carried out by gas chromatography using Shimadzu GC-2010 (Shimadzu, Kyoto, Japan) plus gas chromatograph, non-polar Phenomenex ZB-5 fused bonded column (30 m length × 0.25 mm inner diameter and 0.25 μm film thickness) and flame ionization detector (FID). Helium was the carrier gas, and the flow rate = 1.5 mL/min with split mode. The following temperature program was used: Initial temperature 120 ◦C held for 2 min, temperature increased at a rate of 8 ◦C/min until 320 ◦C and then held for 10 min at 320 ◦C. Injector and detector and injector temperatures were 310 ◦C and 320 ◦C, respectively. GC Solution software (version 2.53, Shimadzu, Kyoto, Japan) was used for recording and integration. The percentages of each component are reported as raw percentages based on peak area without standardization.

#### *3.3. Gas Chromatographic-Mass Spectral Analysis*

Compound identification of *P. pabularia* essential oil was carried out by gas chromatography-mass spectrometry using Agilent 6890 GC, Agilent 5973 (Agilent Technologies, Palo Alto, CA, USA) mass selective detector with electron ionization mass spectrometry (EIMS), (electron energy = 70 eV, scan range = 45–400 amu, and scan rate = 3.99 scans/s), with HP-5ms capillary column (30 m length × 0.25 mm inner diameter and 0.25 μm phase thickness). Helium was the carrier gas with a flow rate of 1 mL/min. Oven temperature program: Hold at 40 ◦C for 10 min, increase at 3 ◦C/min up to 200 ◦C, and then increase at 2 ◦C/min to 220 ◦C. The injector and the interface temperatures were 200 ◦C and 280 ◦C, respectively. A 1% *w*/*v* solution of the essential oil in CH2Cl2 was prepared, and 1 μL was injected with a splitless injection mode. Identification of the oil components was based on their Kovats indices determined by reference to a homologous series of *n*-alkanes and by comparison of their mass spectral fragmentation patterns with those reported in the literature (Adams 2007) and stored in the MS databases (NIST 17, WILEY 10, FFNSC versions 1.2, 2, and 3).

#### *3.4. NMR and HR-ESIMS Analysis*

NMR spectra were recorded on a Varian MR-400 (400 MHz for 1H and 100 MHz for 13C) spectrometer (Palo Alto, CA, USA) in CDCl3. TMS (δ 0.00) signal was used as an internal standard for 1H NMR shifts, and CDCl3 (77.160 ppm vs. TMS) signal was used as a reference for 13C NMR shifts. The HR-ESIMS data were collected with a QStar Elite mass spectrometer (AB SCIEX, Framingham, MA, USA).

#### *3.5. Antidiabetic Activity: PTP-1B Enzymatic Assay*

The *P. pabularia* essential oil was screened for PTP-1B inhibition using pNPP (*p*-nitrophenyl phosphate disodium salt) as the substrate. Both the essential oil sample and the enzyme were pre-incubated at room temperature for 5 min before use. A bu ffer solution (178 μL of 20 mM HEPES, 150 mM NaCl, and 1 mM EDTA) was added to each well of a 96-well plate. The PTP-1B protein solution (1 μL at a concentration 0.115 mg/mL) was added to the bu ffer solution, and then 1 μL of the test solution and the positive control solution were added. The pNPP substrate (20 μL of 35 mM) was added and mixed for 10 min. The plate was incubated for 30 min in the dark, and the reaction then terminated by adding 10 μL of 3 M NaOH. The absorbance was then determined at 405 nm wavelength using a Spectra Max MD5 plate reader (Molecular Devices, USA). The system without the enzyme solution was used as a blank. Inhibition (%) = [(OD405 − OD405 blank)/OD405 blank] × 100. The IC50 was calculated from the percent inhibition values.
