Chemical composition and radical scavenging activity of essential oil and methanolic extract of Eremostachys azerbaijanica Rech.f. from Iran.

In the present study, the chemical composition of the essential oil and methanol (MeOH) extract of aerials of E. azerbaijanica were identified. Furthermore, the free radical scavenging properties of the volatile oil as well as the MeOH extract of the plant were assessed. The essential oil of the air-dried aerial parts was obtained by hydro-distillation using a Clevenger-type apparatus. The oil was then analyzed by gas chromatography-mass spectrometry and gas chromatography with flame ionization detector. Soxhlet extraction was performed on the aerial parts using n-hexane, dichloromethane and MeOH. The MeOH extract was then subjected to solid-phase extraction using a C18 Sep-Pak cartridge. Isolation and structural elucidation of the pure components was accomplished by high-performance liquid chromatography and spectroscopic methods (UV, (1)H-NMR). The free radical scavenging properties were determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. A total of 59 components representing 95.9% of the oil constituents were identified which were primarily characterized as terpenoids or aliphatic skeletons. The major components of the oil were hexahydrofarnesyl acetone (27.1%), 2-methyl-6-propyl-dodecane (16.4%) and tricosane (9.3%). One flavonoid (luteolin-7-O-rutinoside) and one phenylethanoid (verbascoside) were also isolated and identified from the MeOH extract. The results of DPPH assays showed that the essential oil of E. azerbaijanica possessed weak free radical scavenging activity whereas the MeOH extract and its pure constituents showed significant scavenging activities in comparison with positive controls.

INTRODUCTION

The genus Eremostachys (Lamiaceae) is represented by about 60 species that occur mainly in central, middle-eastern and western Asian countries and the Caucasus. Of these, 15 species are endemic to Iran (123). Several studies have reported local analgesic and anti-inflammatory effects of the plants of this genus (45). Biological properties such as antioxidant and antibacterial activities have also been reported (46). Moreover, several pharma-cological studies have demonstrated the antidepressant and antinociceptive effects of these plants (6789). Phytochemical studies on the essential oil composition of species of the genus Eremostachys, such as E. laciniata (1011), E. laevigata (1213), E. macrophylla (141516), E. labiosa (15) and E. adenantha (16) have revealed that terpenoid structures, linear hydrocarbons and their derivatives are major components of these oils at different stages of growth. These reports showed that 1,8-cineol, germacrene-B and -D, α- and β-phellandrene, spathulenol and caryophyllene derivatives with terpenoid structures, 6,10,14-trimethyl 2 pentadecanone and dodecanal linear hydrocarbons are the most abundant constituents (10111213141516). Hexadecanoic acid is a common saturated fatty acid found in the volatile oil of some species of this genus (16). Other phytochemical studies on the extracts of plants of this genus have demonstrated the presence of different natural compounds. The rhizomes of E. laciniata have been identified as a source of iridoid glucosides, flavonoids, and phytosterols (678). Ferulic acid derivatives, furanolabdane diterpene glycoside, iridoid glycosides and phenylethanoid glycosides have been identified in the rhizomes of E. glabra (4171819).

The compounds loasin A and B and loasifolin with flavonoid structure, and eremoside A to C with iridoids skeleton have also been isolated from E. loasifolia (202122). In addition, other iridoid glycosides have been reported in E. moluccelloides and an isoflavone compound from E. vicaryi (2324). In the present study, the chemical composition of the essential oil and MeOH extract of the aerial parts of E. azerbaijanica were identified. The free radical scavenging properties of the volatile oil and MeOH extract were also assessed.

MATERIALS AND METHODS

Plant material

Aerial parts of E. azerbaijanica Rech.f. were collected during flowering stage from Bostan abad, Eastern Azerbaijan (37° 51’ N, 46° 51’ E), Iran, in July 2012. A voucher specimen (TBZ-fph-738) of the plant has been deposited in the Herbarium of the Faculty of Pharmacy, Tabriz University of Medical Sciences.

Isolation of the essential oil

Essential oil of the powdered air-dried aerial parts of the plant was obtained by hydrodistillation method using a Clevenger-type apparatus for 3 h. The oil was dried over anhydrous sodium sulphate and stored in sealed vials before chemical analyses.

Gas Chromatography–Mass Spectrometry

Gas Chromatography–mass spectrometry (GC–MS) and Gas Chromatography with Flame ionization detector (GC-FID) analysis were performed on a Shimadzu QP-5050A GC–MS system (Japan) and GC-17A equipped with a DB-1 fused silica column (60 m × 0.25 mm i.d., 0.25 μm film thickness); with an oven temperature of 50°C rising to 270°C at a rate of 2°C/min for a total run time of 114 min. Injector temperature was set at 240°C and transfer line temperature was 270°C. Helium was used as the carrier gas at a flow rate of 1.3 ml/min. Sample were diluted 1:10 in n-hexane and 1 μl was injectedinto the column. Split ratio, ionization energy, scan time, and acquisition mass range were 1:5, 70 eV, 1 s, and 30–600 amu, respectively.

Identification of components

Identification of the constituents was performed based on the direct comparison of the retention times and mass spectral data with standard alkanes (C8-C20) from Sigma-Aldrich (USA), and computer matching with the NIST 21, NIST 107 and WILEY229 library, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature (25).

Extraction, separation and identification of non-volatile compounds

The dried and ground aerial parts of E. azerbaijanica (100 g) were Soxhlet-extracted with n-hexane, dichloromethane (DCM) and MeOH (solvents were from Caledon, Canada), successively (1 L each). The MeOH extract (2 g) was subjected to solid-phase extraction (SPE) using a C18 Sep-Pak cartridge, eluting with a step gradient of MeOH-water mixture (10:90, 20:80, 40:60, 60:40, 80:20 and 100:0). All these extracts and fractions were separately concentrated using a rotary evaporator at a maximum temperature of 45°C.

The Sep-Pak fraction (40% MeOH in water) was further subjected to preparative reversed-phase HPLC (prep-HPLC) conducted on a Knauer HPLC (preparative pump 1800) fitted with a C18 column (250 mm length, 20 mm i.d, 10 μm particle size, Dr. Maisch, Germany) system. The mobile phase which consisted of 35%-55% MeOH in water for 40% methanol fraction in 70 min ran at flow rate of 8 ml/min and a detector set at 220 nm was used to detect the eluents. The isolated pure compounds were identified by a Bruker Spectrospin 400 MHz NMR-spectrometer. The spectroscopic data of the known compounds were also compared with the respective published data.

Free radical scavenging activity

Free radical scavenging activity of samples was assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH, obtained from Sigma Aldrich, Germany) free radical. Solutions of DPPH (0.08 mg/ml) were prepared in chloroform for the volatile oil and in MeOH for the extract.

The samples were dissolved in respective solvents to obtain the stock concentration of 1 mg/ml. Dilutions were made to obtain concentrations of 500, 250, 125, 62.5, 31.3 and 15.6 μg/ml. Diluted solutions (5 ml each) were mixed with DPPH solution (5 ml) and allowed to stand for 30 min for occurring any reaction. The UV-Visible absorbance was recorded at 517 nm by Spectronic Genesys 5 spectrophotometer. The experiment was performed in triplicate. Trolox and quercetin was used as positive controls (19).

RESULTS

The air-dried aerial parts of E. azerbaijanica were subjected to hydrodistillation for 3 h and exuded colorless oil with the yield of 0.1% (w/w), based on the dry mass. The study on the chemical composition of the essential oil was carried out on a non-polar DB-1 column using GC-MS, GC-FID and the qualitative-quantitative analytical results were compiled in Table 1.

Table 1

Volatile compounds identified in the aerial part of E. azerbaijanica.

According to Table 1, a total of 59 volatile components were identified in the aerial parts of E. azerbaijanica Rech.f. accounting for 95.9% of the total oil. As can be seen in Fig. 1, the essential oil of E. azerbaijanica was mostly constituted by linear hydrocarbons (43.6%) with 2-methyl-6-propyl-dodecane (16.4%) and tricosane (9.3%) as the most abundant compounds. The oil also contained high levels of ketones (30.6%), represented by high levels of ketones (30.6%), represented by hexahydrofarnesyl acetone or Phytone (27.1%). Furthermore, the oil presented moderate level of esters (6.7%) and alcohols (6.3%) with 2, 2, 4-trimethyl-1, 3-pentanediol diisobutyrate (6.1%) and 1-dodecanol (1.7%) as the most abundant representative, respectively. Apart from the main components reported above, only hexacosane (4.4%) and heneicosane (2.1%) exceeded a content of 2% of the total oil composition, while the remaining components (52) showed low amount, most of them presenting lower than 1%.

Fig. 1

Identified chemical groups from the essential oil of E. azerbaijanica.

Reversed-phase preparative HPLC analysis of 40% fraction of MeOH extract of E. azerbaijanica aerial parts afforded one phenylethanoid glycoside and a flavonoid structure, which were identified unequivocally as verbascoside (11mg, tR = 20.1 min) and Luteolin-7-O-rutinoside (9 mg, tR = 27.8 min) on the extensive 1D H-NMR data analyses. The spectroscopic data of the known compounds were also compared with the respective published data.

Verbascoside [1]: brown amorphous powder, UV λmax (MeOH) 220, 300, 330 nm,1 H- NMR (400MHz, DMSO-d6): δ 7.48 (d, 1H, J = 15.8 Hz, H-β’), 7.02 (bs, 1H, H-2’’’), 6.99 (d, 1H, J = 8.2 Hz, H-6’’’), 6.77 (d, 1H, J = 8.0, H-5’’’), 6.64 (s, 1H, H-2), 6.62 (bs, 1H, H-5), 6.50 (d, 1H, J = 8.0, H-6), 6.22 (d, 1H, J = 15.9, H-α’), 5.02 (bs, 1H, H-1’), 4.73 (t, 1H, J = 9.6 Hz, H-4’), 4.36 (d, 1H, J = 7.8 Hz, H-1’’), 3.73 (m, 1H, H-3’), 3.67 (s, 1H, H-2’’), 3.62 (m, 2H), 3.23 (dd, 1H, J = 8.4, 8.5, H-2’), 3.12 (t, 1H, J = 9.6, 9.3, H-4’’), 2.72 (m, 2H, H-α), 2.50 (bs, 2H, H-β), 1.03 (d, 3H, J = 6Hz, H-6’’), 3.26- 3.45 (remaining sugar protons). Data were in agreement with the published data (2627).

Luteolin-7-O-rutinoside or scolymoside [2]: brown amorphous powder, UV λmax (MeOH) nm: 210, 255, 350 nm (NaOMe) 225, 270, 400 nm (AlCl3) 215, 275, 420 nm, (AlCl3/HCl) 215, 275, 360, 390 nm, (NaOAc) 240, 260, 370 nm, (NaOAc/H3 BO3) 240, 260, 370 nm,1 H- NMR (400 MHz, DMSO-d6): δ 7.45 (d, 1H, J = 8.0 Hz, H-6’), 7.42 (bs, 1H, H-2’), 6.91 (d, 1H, J = 8.1Hz, H-5’), 6.78 (s, 1H, H-8), 6.75 (s, 1H, H-3), 6.44(s, 1H, H-6), 5.13 (bs, 1H, H-1’’’) 5.09 (d, 1H, J = 7.2, H-1’’), 1.21 (d, 3H, J = 6.1 Hz, H-6’’’), 3.17-3.50 (remaining sugar protons) (2829).

The free radical scavenging activities of the essential oil, MeOH extract and its pure compounds were evaluated using the DPPH method and the results are displayed in Table 2.

Table 2

Free radical scavenging activity of essential oil, MeOH extract and its pure compounds.

DISCUSSION

Previous studies on the essential oil of the aerial parts of E. azerbaijanica from the Khalkhal mountains in northwestern Iran identified 64.5% carvone as its major component (30). Similar studies on the other Iranian species revealed the presence of dodecanol (72.5%) in the aerial parts of E. laciniata (11) and germacrene D (47.1%) in the aerial parts of E. macrophylla (14). Esmaeili (12) found 1, 8−cineole, benzaldehyde and piperitenone oxide to be the primary components of the oil of the flowers, stems and roots of E. laevigata. The variations in the essential oil components of E. azerbaijanica in the present study in comparison with the findings of previous studies could result from various factors such as geographical location, climatic conditions (variations in temperature, humidity, atmospheric pressure), harvesting season and extraction method (3132).

Solid phase extraction of the polar extract from E. azerbaijanica followed by reversed-phase preparative HPLC analysis of the 40% fraction of MeOH extract led to identification of a phenylethanoid [1] and a flavonoid [2] structure. The compound [1] was identified on the basis of its 1H-NMR and UV spectrum. The 1H-NMR spectrum showed the presence of a tri-substituted phenyl moiety characterized by signals appearing at δH 7.02 (bs, 1H, H-2’’’), 6.99 (d, 1H, J = 8.2 Hz, H-6’’’) and 6.77 (d, 1H, J = 8.0, H-5’’’), a p-hydroxyphenethyl alcohol moiety with proton resonances at δH 6.64 (s, 1H, H-2), 6.62 (bs, 1H, H-5), 6.50 (d, 1H, J = 8 Hz, H6), 2.72 (m, 2H, H-α) and 2.50 (bs, 2H, H-β), and olefinic protons (δH 7.48, d, 1H, J = 15.8 Hz; 6.22, d, 1H, J = 15.9 Hz, AX system) ascribable to H-β’ and H-α’ in the caffeic acid derivative. Anomeric proton signals were observed at δH 4.36 (d, 1H, J = 7.8 Hz, H-1’’) and δH 5.02 (bs, 1H, H-1’) that were consistent with the β-glucopyranose unit and α-rhamnopyranosyl moiety, respectively. The results identified the structure [1] as phenylethanoid, verbascoside or acteoside and are consistent with published spectral data (2627). Verbascoside has been isolated from other species of the genus Eremostachys and the rhizomes of E. azerbaijanica (1933).

UV spectra of the compounds [2] were identical with a flavone moiety (34); this was confirmed by the 1H-NMR spectrum, which showed characteristic signals at δH 6.75 (s, 1H, H-3) and demonstrates that the chromophores are flavones. The anomeric proton signals at δH 5.13 (bs, 1H, H-1’’’) and 5.09 (d, 1H, J = 7.2, H-1’’) indicated the presence of α-rhamnopyranosyl and β-glucopyranose units, respectively. The structure of the compound [2] was identified by comparison of the NMR with published data (2829) as the diglycosylated flavone luteolin-7-O-rutinoside (scolymoside). To the best of our knowledge, this is the first time that scolymoside has been isolated from the Eremostachys genus. Previous studies on the rhizomes of E. azerbaijanica have also demonstrated the presence of iridoid and phenylehanoid glycosides (3335).

DPPH free radical scavenging assay is based on DPPH as a stable free radical capable of changing color from purple to yellow in the presence of antioxidant agents (36). The free radical scavenging properties of the essential oil, MeOH extract, and isolated compounds are summarized in Table 2. MeOH extract showed a high level of free radical scavenging activity with an RC50 value of 0.141 mg/ml. It appears that the compounds responsible for this activity are flavonoids and phenylethanoids such as the luteolin-7-O-rutinoside and verbascoside that were isolated from the 40% fraction of the MeOH extract. The results indicate a notable decrease in capacity of these two pure compounds in comparison with the positive control because of the presence of phenolic hydroxyl groups and the number of ortho-dihydroxy positions (3738). The essential oil of E. azerbaijanica showed weak free radical scavenging properties, with an RC50 value of 15.431mg/ml because of the presence of aliphatic hydrocarbons and the absence of free phenolics and other susceptible scavenging groups in the oil composition.

CONCLUSION

Phytochemical investigation of the aerial parts of E. azerbaijanica demonstrated that this plant is a good source of flavonoids and phenylethanoids that show antioxidant activity.