**1. Introduction**

Diabetes, or diabetes mellitus, is a chronic metabolic disease associated with high blood sugar levels over a prolonged period [1]. In 2017, according to the International Diabetes Federation report, approximately 425 million adults (20–79 years) had diabetes worldwide with 3.2 to 5.0 million deaths from the disease [2]. Unfortunately, these numbers are gradually increasing in most countries.

From ancient times, humans have used medicinal plants for the prevention and the therapy of diabetes mellitus. Medicinal plants serve as natural sources for the discovery of compounds with antidiabetic activities. In this relation, Tajikistan has a rich flora with around 4550 species of higher plants that represent grea<sup>t</sup> interest for the discovery of alternative medications for the treatment of diabetes [3]. Tajikistan is known for its diversity of environmental conditions, including climate, high altitudes, mountainous soil and minerals, and a relatively large number of sunny days per year, factors that can a ffect plant development as well as biosynthesis and accumulation of secondary metabolites [4,5]. Essential oils have applications in medicine, pharmaceutical, food, and cosmetic industries. They possess possible health benefits with antioxidant, antimicrobial, antitumor, anticarcinogenic, anti-inflammatory, anti-atherosclerosis, antimutagenic, antiplatelet aggregation, and angiogenesis-inhibitory activities. Therefore, extensive research has been directed toward the use of medicinal plants to control diabetes mellitus and its complications [6].

PTP-1B (protein-tyrosine phosphatase 1B) belongs to the protein tyrosine phosphatase (PTP) family. It is also known as tyrosine-protein phosphatase non-receptor type 1 that is encoded by the *PTPN1* gene [7,8]. PTP-1B is localized on the cytoplasmic face of the endoplasmic reticulum and contains the essential catalytic cysteine [9]. The PTP-1B enzyme catalyzes the hydrolysis of phosphotyrosine from specific proteins [10]. PTP-1B is considered to be a promising potential therapeutic target for treatment of various diseases, including diabetes, obesity, and cancer [11]. It inactivates the insulin signal transduction cascade by dephosphorylating phosphotyrosine residues in the insulin-signaling pathway [12]. Natural, synthetic, as well as semi-synthetic compounds have shown prominent antidiabetic activities by inhibiting PTP-1B activity [13].

*Prangos pabularia* Lindl., a member of the Apiaceae, is a widely distributed herb up to 150 cm high with a thick cylindrical root and has large reserves in Tajikistan [14]. *P. pabularia*, locally known as "Yugan", is a well-known species of the genus in Tajikistan. It typically grows in mountainous areas and limestone slopes at altitudes 780 to 3600 m above sea level [15]. Its roots and fruits are valued in Tajik traditional medicine and are widely used as general tonics as well as for treatment of vitiligo [4,15]. *P. pabularia* has been used to treat leukoplakia, digestive disorders, scars, and bleeding [16]. The root extracts of *P. pabularia* have been examined for cytotoxic activity; the dichloromethane extract of *P. pabularia* roots demonstrated notable cytotoxicity on the HeLa carcinoma cell line [17]. *P. ferulacea* root is used as an e ffective wound healing agen<sup>t</sup> in traditional medicine of the western north of Iran [18].

Members of the *Prangos* genus are natural sources of phytochemicals, including coumarins and terpenoids. Individual isolated pure compounds such as osthole and isoimperatorin showed the highest inhibitory potency against the growth of human carcinoma cell lines. Osthole exhibited the greatest cytotoxicity and was found to induce apoptosis in PC-3, H1299, and SKNMC cells at low micromolar concentrations. Thus, osthole can be considered to be a promising lead in anticancer drug discovery and development [17]. Several new compounds have been isolated from the essential oil of *Prangos* species. A new bisabolene derivative was isolated from essential oil of the fruits of Turkish endemic *Prangos uechtritzii* [19]. The 3,7(11)-Eudesmadien-2-one, a new eudesmane type sesquiterpene ketone was isolated from *Prangos heyniae* H. Duman & M.F. Watson essential oil [20]. The (2*S*)-3,5-Nonadiyne-2-yl acetate was isolated from *Prangos platychlaena* ssp. *platychlaena* fruit essential oils [21].

Recently, we reported that the roots of *P. pabularia* are good sources of biologically active secondary metabolites (coumarins) such as heraclenol, heraclenin, imperatorin, osthole, yuganin A, and others. Yuganin A showed potent effects on the proliferation of B16 melanoma cells [22]. In a continuation of this investigation, the current report presents the promising antidiabetic activity and the chemical composition of volatile secondary metabolites of the underground parts of *P. pabularia* growing wild in Tajikistan. There are several reports on the composition of the essential oils isolated from leaves, fruits, and umbels of *P. pabularia* growing in Iran and Turkey [23–25], but, until now, there has been no published reports on antidiabetic activity and volatile secondary metabolites of the underground parts of *P. pabularia*.

#### **2. Results and Discussions**

#### *2.1. Chemical Composition of Essential Oils*

Volatile secondary metabolites were obtained by hydrodistillation of *P. pabularia* roots growing wild in Tajikistan and were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Identification of the oil components was based on their Kovats retention indices (RI) 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 [26] and stored in the MS library. Forty-two compounds were identified in the volatile oil accounting for 97.3% of the composition; 5-Pentylcyclohexa-1,3-diene (44.6%), menthone (12.6%), 1-tridecyne (10.9%), and osthole (6.0%) were identified as major constituents of the volatile oil obtained from roots of *P. pabularia* (Table 1). The structure of the osthole was established on the basis one-dimensional (1D) NMR and electrospray ionization (ESI)-MS spectroscopic studies, respectively [22]. The chemical structures of the main components of the essential oil from the roots of *P. pabularia* are presented in Figure 1. The GC-MS chromatogram of the volatile oil of *P. pabularia* is presented in Figure 2.

**Figure 1.** Structures of the main components of *Prangos pabularia* essential oil.





\*RI: retention indices.

**Figure 2.** The GC-MS chromatogram of the volatile oil of *Prangos pabularia*. 1. menthone; 2. 5-pentylcyclohexa-1,3-diene; 3. 1-tridecyne; 4. osthole.

#### *2.2. NMR Data of Osthole*

1H NMR (400 MHz, CDCl3): δ 6.23 (1H, d, *J* = 9.4 Hz, H-3), 7.61 (1H, d, *J* = 9.4 Hz, H-4), 7.28 (1H, d, *J* = 8.6 Hz, H-5), 6.83 (1H, d, *J* = 8.6 Hz, H-6), 3.53 (1H, d, *J* = 7.3 Hz, H-11), 5.22 (1H, m, H-12), 1.67 (3H, s, H-14), 1.84 (3H, s, H-115), 3.92 (3H, s, OCH3-7); 13C NMR (100 MHz, CDCl3): δ 161.49 (C-2), 113.11 (C-3), 143.87 (C-4), 126.32 (C-5), 107.47 (C-6), 160.34 (C-7), 118.10 (C-8), 152.95 (C-9), 113.14 (C-10), 22.06 (C-11), 121.25 (C-12), 132.75 (C-13), 18.06 (C-14), 25.91 (C-15), 56.17 (OCH3-7).

Acorenone, (*E*)-anethol, β-bisabolenal, β-bisabolenol, β-bisabolene, bicyclogermacrene, δ-3-carene, chrysanthenyl acetate, β-caryophyllene, elemol, 3,7(11)-eudesmadien-2-one, geranial, germacrene D, α-humulene, kessane, limonene, *p*-menth-3-ene, nerolidol, (*Z*)-β-ocimene, (*E*)-β-ocimene, α-pinene, β-pinene, α-phellandrene, β-phellandrene, sabinene, γ-terpinene, α- terpinolene, *m*-tolualdehyde, and 2,3,6-trimethyl benzaldehyde were reported as major components (≥10%) in the essential oil of *Prangos* species (Table 2).


**Table 2.** The major compounds reported as chemical composition of essential oil from *Prangos* species.


**Table 2.** *Cont.*

Razavi reported that the composition of the essential oils isolated from leaves, fruits, and umbels of *P. pabularia* collected from Iran were dominated by spathulenol, α-bisabolol, and α-pinene [25]. Bicyclogermacrene, (*Z*)-β-ocimene, α-humulene, α-pinene, and spathulenol were reported as the main constituents of the essential oil of *P. pabularia* fruits collected from Turkey [24]. The chemical composition of the root essential oil of *P. pabularia* differed from those from leaves, fruits, and umbels with regard to predominance of sesquiterpenes and monoterpenes. In 2016, Tabanca and co-authors reported that suberosin (1.8%) was identified in the essential oil obtained from fruits of *P. pabularia*. In present work, 5-pentylcyclohexa-1,3-diene (44.6%), menthone (12.6%), 1-tridecyne (10.9%), and osthole (6%) (an isomer of suberosin) were identified as the dominant constituents of the volatile oil of the roots of *P. pabularia.* These major volatile compounds were not identified from the other *Prangos* species (Table 2). Therefore, it confirms the different chemical composition from *P. pabularia*. Recently, we reported that osthole was isolated from the chloroform extract of the roots of *P. pabularia,* and its structure was elucidated by spectroscopic means, namely, high resolution electrospray ionisation mass spectrometry (HR-ESIMS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy [22]. In addition, osthole was isolated from the hexane extract of the fruits of *P. asperula* [30].

Both osthole and suberosin were found in *Arracacia tolucensis* var. *multifida* volatile oil [45]. The essential oil with the high coumarin content showed moderate in-vitro antibacterial activity against representative Gram-positive and Gram-negative bacteria [45].

#### *2.3. Antidiabetic Activity of Essential Oil and Isolated Compound (Osthole)*

The effect of the obtained essential oil and the pure compound (osthole) from *P. pabularia* roots for its *in vitro* inhibition of the enzyme PTP-1B was determined. The essential oil induced a PTP-1B enzymatic inhibition in a concentration-dependent manner with IC50 values 0.06 ± 0.01 μg/mL (*p* < 0.02), which is more than 25 times more potent than the positive control (3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethylbenzofuran-6-sulfonic acid-(4-(thiazol-2-ylsulfamyl)- phenyl)-amide) with IC50 1.46 ± 0.4 μg/mL (*p* < 0.05). The individual compound (osthole) also exhibited strong inhibitory activity against PTP-1B, with IC50 values 0.93 ± 0.1 μg/mL (*p* < 0.01); it was also more effective than the positive control. The dose response curves of the inhibition of the PTP-1B enzyme of *P. pabularia* essential oil and osthole are shown in Figure 3.

Wang and co-authors presented a strategy based on GC-MS coupled with molecular docking for analysis, identification, and prediction of PTP-1B inhibitors in the Himalayan cedar essential oil. β-Pinene (49.3%), α-pinene (29.4%), α-terpineol (4.1%), and β-caryophyllene (3.7%) were the main components of Himalayan cedar oil that inhibited PTP-1B with IC50 value 120.71 ± 0.26 μg/mL. The docking results of the PTP-1B inhibitory activity of caryophyllene oxide was also in agreemen<sup>t</sup> with its *in vitro* activity [46]. The IC50 value for PTP-1B inhibition for caryophyllene oxide was in the range 25.8–31.3 μM [46,47]. New terpenoids cedrodorols A-B from *Cedrela odorata* showed inhibitory PTP-1B activity with IC50 values 13.09 and 3.93 μg/mL, respectively [48]. In another study, Bharti and co-authors reported the *in vivo* antidiabetic activity of *Cymbopogon citratus* essential oil with major compounds, geranial (42.4%), neral (29.8%), myrcene (8.9%), and geraniol (8.5%), which were fully supported by molecular docking predictions [49].

**Figure 3.** Dose response curve of inhibition of protein-tyrosine phosphatase 1B (PTP-1B) enzyme for *Prangos pabularia* essential oil ( **A**) (IC50 = 0.06 ± 0.01 μg/mL) and the pure compound osthole (**B**) (IC50 = 0.93 ± 0.01 μg/mL).

Hong-Jen Liang investigated the hypoglycemic e ffects of osthole in diabetic db/db mice, and the main mechanisms of these e ffects were elucidated using an *in vitro* cell-based assay and *in vivo* assays using a diabetic db/db mouse model. Results showed that osthole significantly alleviated hyperglycemia by activating PPAR α/γ in a dose-dependent manner based on the results of the transition transfection assay [50,51]. Wei-Hwa Lee reported that the western blot analysis revealed osthole to significantly induce phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) as well as increase translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner [50]. These results sugges<sup>t</sup> that the increase in the AMP:ATP ratio by osthole had triggered activation of the AMPK signaling pathway, leading to increases in plasma membrane GLUT4 concentration and glucose uptake level [52]. Other research has clearly shown that osthole lowered fasting blood glucose (FBG) and improved insulin secretion. This may indicate partial recovery from pancreatic damage, as indicated from histological characteristics [53].
