Analysis of the essential oil composition of three cultivated Nepeta species from Iran

2.1 Plant material

The research was conducted in the Experimental Garden of Agricultural Faculty of Ferdowsi University of Mashhad, Iran, in 2016–2018. The seeds of three Nepeta species were collected from Kerman and Mashhad provinces of Iran. The origin of plant material, year of collection, and herbarium voucher are presented in Table 1.

2.2 Cultivation conditions

The seeds were placed at 4 °C for 2 weeks, then sown under greenhouse-controlled conditions with a temperature range of 20°–25 °C and relative humidity of 60 °C (latitude: 36°16′N, longitude: 59°36′E) during the last week of March. The culture bed contained peat moss, cocopeat, and perlite (40:40:20, w:w:w). After about 30 days, seedlings were transplanted into pots of 30-cm height, with the same composition of the first bed; after about 45 days, seedlings were transplanted to the field, on the base of completely randomized design including three replications (1.5×2 m² plot) in the Experimental Garden of Ferdowsi, University of Mashhad. Based on the climatic conditions, the plants were irrigated to maintain near-field capacity. The characteristics of the field location are reported in Table 2.

2.3 Extraction of the EOs

During the summer, aerial parts of the plants were harvested at full flowering stage in the first and second year (about 120 and 360 days after cultivation) of their establishment on the farm. Plants were harvested at 5 cm above the soil level and then air-dried in the shade.

The EOs from the dry plants (30 g) of two cuts, during the first and second years, were extracted by hydro-distillation for 4 h using a Clevenger apparatus (Ashk shishe Co., Tehran, Iran), and the EO yield percentage was recorded as %v/w of plant dry weight. Sodium sulfate anhydrous purchased from Sigma (St Gallen, Switzerland) was added for removing water from the isolated EOs that was stored in a refrigerator in dark bottles until analyses.

2.4 Gas chromatography (GC) and gas chromatography coupled to mass spectroscopy (GC/MS) (GC/MS) analysis

The composition analysis of the essential oil samples of the first and second years was carried out using a GC–MS instrument with the following specifications: GC analysis was carried out using an Agilent-Technologies-7890A gas chromatograph equipped with an HP-5 column (30 m×0.32 mm ID) and film thickness of 0.25 μm. The oven temperature was from 60 °C and then programmed to 210 °C at a flow rate of 3 °C/min, then from 210 °C to 240 °C at 20 °C/min and held for 8.5 min; injector and detector (FID) temperature were 280 °C and 290 °C, respectively; N₂ was used as carrier gas with a linear velocity of 1 mL/min, split ratio was 1:50.

GC/MS analyses were carried out on GC/MS (Agilent Technologies-5975C-MS, 7890A-GC) equipped with HP-5MS capillary column (30 m×0.25 mm ID) and film thickness of 0.25 μm. The oven temperature was programmed as follows: from 60 °C to 210 °C with a rate of 3 °C/min, then increased to 240 °C with a rate of 20 °C/min, and the final temperature was kept for 8.5 min; run “time” was 60 min. The electron ionization energy was 70 eV in the electronic ionization (EI) mode, ion source 230 °C, detector MS, interface line temperature 280 °C, injector 280 °C, split ratio 1:50, carrier gas He 1 mL/min, mass range 50–480 amu.

The percentages of compounds were calculated by the area normalization method. The components of the oil were identified by comparison of retention indices (RI, HP-5) in their mass spectra with those of an Adams library and sorted in NIST and Wiley libraries or with authentic compounds reported in the literature. Retention indices were determined using retention times of n-alkane that were injected after the EO under the same chromatographic conditions [26].

2.5 Statistical analysis of EO yields

Mean values and variance analysis of EO yields were calculated according to a completely randomized design including three replications for each year. Statistical analysis of the obtained results was performed by (JMP software ve. 9.0.3). Significant differences among the samples were evaluated by Duncan test at the probability level (p≤0.05).

3.1 EO yields

Results of the EO yields from the three Nepeta species for 2 years are reported in Table 3. The EOs extracted from the N. assurgens and N. binaloudensis species were yellow, whereas the N. cataria was whitish-yellow in color. There were significant differences in the EO yield among the species. The highest EO yields were obtained from N. assurgens in 2 years (0.51% and 0.75% in the first and second years, respectively). The yields of N. cataria were 0.32% (first year) and 0.29% (second year), whereas N. binaloudensis yielded the lowest amounts (0.23% and 0.18% in the first and second years, respectively) (Table 3).

3.2 The composition of EOs

The constituents of the Nepeta EOs analyzed by GC and GC-MS are reported in Table 4. In total, 89 constituents were identified. During 2 years of cultivation, a number of constituents were identified in the EOs of N. binaloudensis in the first and second years (26 and 37, respectively), N. cataria (25 and 32, respectively) and N. assurgens (45 and 50, respectively) The results indicated that there were differences between the species in the first and second years in EO composition and number of compounds. The EOs were rich in monoterpenoids, as other Nepeta species previously reported. In the oils of N. binaloudensis, 4a-α,7-α,7a-α-NL (1.2–59.7%) and 1,8-cineole (14.8–19.6%) were the main components in the 2 years. Other abundant constituents included β-pinene (10.4%), p-cymene (9.7%), α-terpineol (7%), γ-terpinene (4.5%), α-pinene (4.33%), terpinen-4-ol (4.2%), δ-terpineol (3.61%), and thymol (1.7%) in the first year. Thymol, carvacrol (0.6%), α-copaene (0.2%), β-copaene (0.1%), β-bourbonene (1.0%), 4a-α,7-α,7a-β-NL (NL2) (0.9%), germacrene-D (0.2%), γ-muurolene (0.3%), and γ-terpinene were identified only in the first year, whereas 4a-α,7-β,7a-α-NL (0.9%) was detected only during the second year. The EOs of N. cataria were rich in NL isomers, while in the second year, the main compound was 4a-α,7-α,7a-β-NL (72.8%). In general, 4a-α,7-β,7a-α-NL (2.5–73.9%) and 4a-α,7-α,7a-α-NL (17.9–19.8%) in the first and second years, respectively, were quantified as the main components in this species. In the EOs of N. assurgens, 4a-α,7-α,7a-α-NL (34.7–55.5%) and 1,8-cineole (24.1–16.8%) were the main constituents in the first and second years, respectively. Other compounds included β-pinene (3.9–4.6%), trans-carveol (6.7–2.2%), NL2 (5.7% only in the first year), and 4a-α,7-β,7a-α–NL (2.7%) only in the second year.

4.1 EO yields

The yields of EOs from the studied Nepeta species were from 0.18% (N. binaludensi) to 0.79% (N. assurgens). In previous studies, EO yields of N. cataria were in the range of 0.1–2.5%, in plants from different regions (Table 5), whereas the yield of N. binaludensis collected in Iran was 0.74% [11]. Therefore, the previously reported EO yields from N. cataria were remarkably higher: 2.5% [19] and 1.02% [8] from plants cultivated in Iran and Morocco, respectively. To the best of our knowledge, the yields of EO from N. assurgens were not reported previously. Noteworthy, the variability of EO yields during the 2 years may be attributed to environmental factors possibly modifying photosynthate allocation to secondary metabolic pathways [20], [21], [22], [23], [24], [25].

4.2 The composition of EOs

Biological activities of NLs were thoroughly investigated, and some Nepeta species were previously introduced into cultivation due to their horticultural and medicinal values [16], [19], [20], [21]. Most EOs of the Nepeta species contain NLs as the main components, though different oil compositions were identified among the different species. In the present study, three types of NL isomers, 4a-α,7-α,7a-α-NL, 4a-α,7-α,7a-β-NL, and 4a-α,7-β,7a-α-NL, were identified. All these NL isomers were the dominant components of the oils of N. cataria, whereas in N. assurgens and N. binaloudensis, other constituents were significantly present besides 4a-α,7-α,7a-α-NL, in particular, 1,8-cineole.

The EO of the composition of various Nepeta species grown in Iran was extensively investigated [29], [30], [31], [32], [33]. The essential oil of N. binaludensis, an endemic species to Iran, was studied by Mohammadpour et al. [30] who identified 65 components including 1,8-cineol (68.31%), α-terpineol (5.24%), β-pinene (4.7%), δ-terpineol (2.57%), and α-pinene (1.54%) [30]. The most abundant constituents in N. binaludensis investigated by Rustaiyan and Nadji [32] were 1,8-cineole (42%), nepetalactone (25%), linalol (4%), α-terpineol (4%), and β-pinene (3%). According to the literature, the EO composition of N. assurgens, an Iranian endemic species was previously reported only by Moradalizadeh et al. [31] who identified 4aα,7α,7aα-NL, 4aα,7α,7aβ-NL, 1, 8-cineole, α-pinene, β-pinene, and α-terpineol as the main components.

The differences between wild and cultivated medicinal plants growing in various regions were reported in terms of EO composition. The chemical profiles shown in the present study are quite similar to the ones previously reported in other studies, except for the amounts of some compounds. For instance, the main EO constituents of N. cataria were 4a-α,7-α,7a-α-NL, and 4a-α,7-β,7a-α-NL, in the range of 78–91%, in plants cultivated in Iran [19], and 20.81–35.15%, in plants cultivated in Egypt [20], [21]. In the EO from N. cataria cultivated in Lithuania, 4aα,7α,7aβ-NL (50.16%) was the dominant constituent, in addition to 4aα,7α,7aα-NL (35.64%) and 4aα,7β,7aα-NL (1.80%), which represented 87.60% of the total EO volatiles [12]. Again, NLs were the major compounds in the EO of N. cataria from Turkey, Poland, and Morocco, but their percentages were different [19].

It is well known that variability in quality and quantity of secondary metabolites in plants is mainly influenced by the environmental factors possibly modifying the expression levels of the key genes/enzymes involved in the biosynthesis of the EO constituents. Therefore, introduction of Nepeta species into the cultivation under different growing conditions represents a promising perspective in the field of medicinal plants. Mashhad province is a region with semi-arid weather and an annual rainfall less than 200 mm; in these conditions, water deficit and high temperatures represent the limiting factors for Nepeta grown in the summer. Therefore, timing of sowing as well as management of nutrients and water could improve the economic Nepeta production.

In addition, the phytochemical differences could also be due to the existence of different Nepeta chemotypes [18], [19], [20]. Accordingly, on the basis of our results, N. binaloudensis and N. assurgens are characterized by high percentages of and NLs and 1,8-cineole, whereas N. cataria EO was composed mainly of NLs.