Chemical composition, antioxidant and antibacterial activities of essential oils from Ferulago angulata.

Context: Ferulago angulata Boiss. (Apiaceae), a perennial aromatic herb, grows wild in Iran. The aerial parts of F. angulata are used as a flavouring in foods, especially dairy foods by indigenous people in western and southwestern Iran. Objective: This study investigates variation in chemical compositions, antioxidant and antibacterial activities of the essential oils from F. angulata collected from natural habitats in the alpine regions of southwestern Iran. Materials and Methods: The antimicrobial activity, minimum inhibitory concentration (MIC) and minimum bactericidal (MBC) of the essential oils were evaluated against four bacteria (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus and Salmonella typhimurium). Antioxidant activity of the oils was determined by DPPH assay. Results: The essential oils were analyzed by GC-FID and GC/MS, which 49 volatile components were identified. There were significant differences between the various populations for oil yield and some main compounds. The major constituents of the essential oils from F. angulata were α-pinene, and cis-β-ocimene. The MICs of the essential oils were within concentration ranges from 62 to 250 μg/mL and the respective MBCs were 125 to > 500 μg/mL. Generally, the oils from F. angulata indicated weak to moderate inhibitory activities against bacteria, especially against Listeria monocytogenes. The highest antioxidant activity was obtained from the oil of the Kallar population (IC₅₀ value=488 μg/mL) and BHT as positive control (IC₅₀ value =321 μg/mL). Discussion and conclusion: The essential oil of F. angulata could be serving as a potential source of α-pinene and cis-β-ocimene for use in the food, cosmetic and pharmaceutical industries.

Keywords: α-Pinene; biological activity; cis-β-ocimene; sabinene; volatile oil

Due to concern about the safety of synthetic compounds, numerous bioactive secondary metabolites of plants are being used in various industries, including pharmaceutical, chemical, cosmetic and food production (Gourine et al. [14]). The products of plant secondary metabolites, such as essential oils, aromatics, and volatile constituents, have a wide range of applications in folk medicine, food flavouring and food preservative as well as in food industries (Ghasemi Pirbalouti [5]), leading to an increase use of natural substances and encouraging more detailed studies on the plant materials being used. Essential oils with antioxidant and antibacterial activities could be promising agents in the food and flavouring industries (Imelouane et al. [17]).

The genus Ferulago (Apiaceae) is represented by 35–40 species in the world that about eight species exist in flora of Iran (Mozaffarian [23]). This genus grows in different areas of the world, including Turkey, Greece, Yugoslavia, Macedonia, Australia and Iran. Ferulago angulata (Schlecht.) Boiss. ("Chavil" or "Chavir" in Persian) is an important medicinal and aromatic plant in Iran. Ferulago angulata grows in the alpine regions of Iran, especially western and southwestern (Mozaffarian [23]). Results of an ethnobotanical study (Ghasemi Pirbalouti et al. [9]) indicated that the aerial parts of F. angulata have been used as spice plant, antiseptic and air fresher by the people of Abdanan and Dehloran districts, western Iran. In addition, results of other studies demonstrated that the herb is used as an anti-diabetics, tonic, sedative, aphrodisiac, anti-haemorrhoids (Ghasempour et al. [13]; Taran et al. [26]) antiulcer, and also treatment for snake bites, spleen and headache (Javidnia et al. [18]). The aerial parts of F. angulata harvested before the flowering stage are used as flavouring in foods, especially dairy foods by the indigenous people in southwestern Iran (Ghasemi Pirbalouti [5]). Previous phytochemical studies indicated that the aerial parts of the Ferulago species have various coumarins (Jimenez et al. [19]) and volatile oils (Erdurak et al. [4]).

Results of several studies (Ghasemi Pirbalouti et al. [8],[9][7], [6]) have shown that genetic and ecological factors and their interaction effects can affect some characteristics of herbs. To our knowledge, any documents on chemical diversity of the essential oils, antibacterial and antioxidant activities of various populations of F. angulata are not available. Therefore, this study was done to investigate variation in chemical compositions, antioxidant and antibacterial activities of the essential oils of F. angulata collected from different natural habitats in the alpine regions of southwestern Iran.

Samples of F. angulata wild populations collected during the mid-flowering stage from various regions of southwestern Iran were used in this study. A total of three replicated samples of each plant were gathered from four natural habitats from 20 May to 20 June 2012. The aerial parts (100 g) of the plants (10–15 cm above ground level) were harvested. Selected geographic and characteristics of accessions differed (Table 1). Plant identities were confirmed by Prof. V. Mozaffarian, and a representative voucher specimen (No. 1712) was been placed in the Herbarium of RCANR, Chaharmahal va Bakhtiari province, Shahrekord, Iran.

Table 1. Geographical and climate of natural habitats of F. angulata populations.

1 P, annual precipitation (mm); T, average temperature (°C); E.C.., electrical conductivity (dS m−1); O.C., organic carbon (%), and sand, silt and clay in %.

• 2 Meteorological information was obtained from weather stations located within the study area and the surrounding zone; each value in the mean of 10–15 year data.
• 3 Soil characteristics are based on average of samples taken from three farms in each region.

The fresh aerial parts of F. angulata were dried inside for 5 d at room temperature (30 ± 5 °C), and then ground to a fine powder using Moulinex food processor and passed through a 20 mesh sieve to remove large pieces of debris. The essential oil was extracted from 100 g of ground tissue in 1 L of water contained in a 2 L flask and heated by heating jacket at 100 °C for 3 h in a Clevenger-type apparatus, according to producers outlined British Pharmacopoeia. The collected essential oils were dried over anhydrous sodium sulphate and stored at 4 °C until analyzed.

Composition of the essential oils was determined by gas chromatography (GC) and mass spectrophotometry (GC/MS). The GC analysis was done on an Agilent Technologies 7890 GC (Agilent Technologies, Santa Clara, CA) equipped with a single injector and a flame ionisation detector (FID) using a HP-5MS capillary column (30.00 m × 0.25 mm, 0.25 μm film thicknesses) coated with 5% phenyl and 95% methyl polysiloxane. The carrier gas was helium (99.999% pure) at a flow rate of 0.8 mL/min. The initial column temperature was 60 °C and programmed to increase at 4 °C/min to 280 °C. The injector temperature was set at 280 and 300 °C. Split injection was conducted with a ratio split of 1:40. Essential oil samples of 0.1 μL were injected neat (directly).

GC-MS analyses of aromatic oil samples were performed on an Agilent Technologies 7890 gas chromatograph coupled to Agilent 5975 C mass selective detector (MSD) and quadrupole EI mass analyzer (Agilent Technologies, Palo Alto, CA). A HP-5MS 5% column (coated with methyl silicone) (30 m × 0.25 mm, 0.25 μm film thicknesses) was used as the stationary phase. The temperature was programmed from 60 to 280 °C at 4 °C/min ramp rate. The injector and the GC-MS interface temperatures were maintained at 290 °C and 300 °C, respectively. Mass spectra were recorded at 70 eV. Mass range was from m/z 50 to 550. The ion source and the detector temperatures were maintained at 250 and 150 °C, respectively.

Oil constituents were identified based on their retention indices (determined with reference to homologous series of C5–C24n-alkanes), by comparison of their mass spectra with those reported in the literature (Adams [1]; McLafferty [21]) and stored in NIST 08 (National Institute of Standards and Technology) and Willey (ChemStation data system) libraries. The peak area percentages were computed from BP-5 column without the use of FID response factors.

Clinical isolates of three Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and a Gram-negative bacterium (Salmonella typhimurium) were obtained from Food Microbiology Laboratory, Veterinary Medicine Faculty, (I.A.U.) Iran and had been positively identified using PCR-RFLP. The population of each bacterial strain was increased by culturing in an overnight Mueller Hinton broth (MHB) at 37 °C. The density of bacteria culture required for the test was adjusted to 0.5 McFarland standards (1.0 × 107 CFU/mL) and measured using a spectrophotometer. Minimum inhibitory concentrations (MIC) were determined using the broth-serial dilution method following standardized methods (CLSI [3]). The essential oils and the antimicrobial agents (ampicillin and flumequine) were dissolved in 5% DMSO were first diluted to the highest concentration (1 mg/mL) to be tested, and then series of two-fold dilutions were made in a concentration range from 0.032 to 1.00 mg/mL in 10 mL sterile test tubes containing nutrient broth. A population of bacteria was subsequently added to each tube containing an essential oil or antimicrobial agent, and then incubated at 37 °C for 24 h. After the incubation period, the absorbance of each incubated solution was measured at 630 nm using a spectrophotometer as a measure of bacterial growth to indicate MIC values (CLSI [3]). The minimum bactericidal concentrations (MBC) of each essential oil was determined according to the MIC values by transferring 5 μL from MIC tubes to agar plates and incubating at 37 °C for 24 h. The MBC were referred to the minimum concentration of essential oil with no viable bacteria. Experiments were performed at three different times.

The DPPH radical scavenging activity of essential oils was determined using the method proposed by Hung et al. ([16]). The essential oil (100 μL) at concentrations of 8–500 μg/mL was mixed with 3.9 mL an equal volume of 0.2 mM ethanol solution of DPPH. The disappearance of the DPPH after 30 min of incubation at room temperature was determined using a Perkin-Elmer Lambda UV/Vis spectrophotometer (Perkin-Elmer Inc., Waltham, MA) at 515 nm against a blank, i.e., without DPPH. The amount of sample necessary to decrease the absorbance of DPPH by 50% (IC50) was calculated graphically and the percentage inhibition was determined according to the equation:

Graph

where AC0 is the absorbance of the control at t = 0 min and AAt is the absorbance of the antioxidant at t = 30 min. The food preservative butylhydroxyanisole (BHT) was used as a positive control. All measurements were replicated three times.

Data were analyzed by one-way analysis of variance with three replications using the SPSS 19.0 statistical software (SPSS Inc., Chicago, IL). The significance of differences among treatment means was tested using Duncan's multiple range test at p ≤ 0.05 level.

Statistical analysis results indicated that there was a significant difference (p ≤ 0.05) among various populations for the essential oil yield. The highest and lowest oil yields were obtained from the Rig population and the Saldaran population with 0.61 and 0.32% (v/w), respectively (Figure 1). Ghasempour et al. ([13]) reported that the oil yields obtained from the leaves and seeds of F. angulata were 3.2 and 0.63% of fresh plants, respectively. Results of a study by Taran et al. ([26]) indicated that the essential oil yields of the aerial parts and seeds of F. angulata subsp. carduchorum collected from western Iran (Kermanshah province) were 0.63 and 3.2% (v/w) based on the dry weight, respectively.

Graph: Figure 1. The essential oil yield of F. angulata populations (significant different at p < 0.05 have been indicated with different letters).

In total, 49 volatile constituents were identified representing 90–95% of total oils (Table 2 and Figure 2). The analysis of essential oils by GC-FID and GC/MS detected the major compounds, α-pinene, cis-β-ocimene, sabinene, trans-β-ocimene, α-phellandrene, β-phellandrene, thymol and myrcene (Table 2 andFigure 2). Generally, monoterpene hydrocarbons (61.8–77.3%) were the main chemical groups in the volatile oils from the F. angulata populations tested in this study (Table 2 and Figure 2). Earlier studies have identified β-ocimene, α-pinene, α-phellandrene, β-phellandrene, sabinene and terpinolene as the major constituents of the essential oils from the aerial parts of F. angulata collected from different regions of Iran (Rustaiyan et al. [24]; Javidnia et al. [18]). Ghasempour et al. ([13]) reported the major constituents of essential oils from the leaves of F. angulata collected at two different habitats in western Iran were α-pinene (25–27%), cis-β-ocimene (22–27%) and bornyl acetate (3.9–8.5%). A comparison of our results with the previous reports on the chemical composition of F. angulata suggests differences in the volatile composition of the plant material could be attributed to genetic (species, ecotype and chemotype), distinct environmental and climatic conditions, seasonal sampling periods, geographic origins, plant populations, type of the plant part, drying methods and extraction and quantification methods (Asghari et al. [2]; Ghasemi Pirbalouti et al. [8],[10],[12]; Ghasemi Pirbalouti et al. [6]; Memarzadeh et al. [22]). Our results indicated significant differences (p ≤ 0.01) among the four studied F. angulata populations for percentages of α-pinene, sabinene, myrcene, α-phellandrene, β-phellandrene and trans-β-ocimene (Table 2), while no significant difference among the studied populations for percentage of cis-β-ocimene was apparent (Table 2). The highest percentage of α-pinene was achieved from the Saldaran population, while the lowest percentage was obtained from the Olya population (Table 2). Probably, higher percentage of organic carbon in soil of Saldaran region providing a better growing conditions which led to a higher accumulation of α-pinene in the essential oil. The higher amount of thymol (6.9%) in the essential oil from the Kallar population is attributed to chemotype, and distinct environmental.

Graph: Figure 2. The aerial parts of F. angulata and the chromatogram found in a sample (for peak identification see Table 2).

Table 2. Chemical compositions of the essential oils from F. angulata populations.

• 4 Not significant; *significant at p ≤0.05; **significant at p ≤0.01.
• 5 Retention indices (RI) relative to C5–C24n-alkanes on HP-5MS capillary column. cal = determined values; lit = literature values.
• 6 % GC peak, the percentage composition was computed from the GC peak areas.
• 7 Values of major compounds are given as means ± SD.

A hierarchical cluster analysis of the percentages of the main compounds and biological activity of the essential oils could be grouped into two distinctive clusters (Figure 3). The first cluster formed by the oils from three samples, including the Olya, Rig and Kallar populations of F. angulata. The second cluster was formed by the essential oil from the Saldaran population. Reason of difference of this population with other populations probably can cause highest latitude in comparison other regions.

Graph: Figure 3. Dendrogram obtained by hierarchical cluster analysis (HCA).

The antibacterial activity of essential oils from the various populations of F. angulata was tested against the four pathogenic bacteria by using the serial-dilution method. The MICs of the essential oils were within concentration ranges from 62 to 250 μg/mL and the respective MBCs were 125 to >500 μg/mL (Table 3). Generally, the essential oils from F. angulata indicated weak to moderate inhibitory activities against four bacteria. Similarly, results from a study by Khalighi-Sigaroodi et al. ([20]) indicated that the essential oil from F. bernardii had weak to moderate inhibitory activities against Staphylococcus aureus (MIC values = 250 μg/mL), Bacilus subtilis (MIC values <125 μg/mL), Escherichia coli (MIC values <125 μg/mL), Candida albicans (MIC values = 500 μg/mL) and Aspergillus niger (MIC values = 250 μg/mL). Taran et al. ([26]) reported that the essential oils from the aerial parts and seeds of F. angulata subsp. carduchorum had the antimicrobial effects against S. aureus, and L. monocytogenesis, and C. albicans. Probably, in this study, the antibacterial activity of the essential oils from F. angulata against L. monocytogenes could be attributed to the relatively high level of α-pinene, a constituent with known antimicrobial properties (Stojkovic et al. [25]). Lipophilic constituents, including terpenoid derivatives, have been shown to disrupt cellular membranes in bacteria and fungi, thus inhibiting cellular respiration and ionic transport (Hayouni et al. [15]). The antimicrobial activity of essential oils may be due to the presence of synergy between the major constituents and other components of the oils leading to various degrees of antimicrobial activity (Ghasemi Pirbalouti et al. [11]).

Table 3. Antibacterial activity of essential oils from the studied populations of F. angulata.

The antioxidant activity of the essential oils from the studied populations of F. angulata was expressed as IC50 value. A low IC50 value indicates an active ability of the oil to act as a DPPH scavenger (Figure 4). The highest antioxidant activity was obtained from the essential oil of the Kallar population and BHT (positive control). Probably, the highest antioxidant activity of this population oil could be attributed to the relatively high level of thymol, as a phenolic component, in comparison with other populations. In total, results of this study indicated that the essential oils from F. angulata had weak-to-good antioxidant (DPPH) radical scavenging activity.

Graph: Figure 4. The antioxidant activity of the essential oils from the studied populations of F. angulata and a chemical antioxidant (BHT) using DPPH assay (IC50 value = μg/mL) (significant different at p < 0.05 have been indicated with different letters).

Ferulago angulata is an aromatic edible plant which wild grows in the alpine regions of Iran, especially western and southwestern. The dried/fresh aerial parts of F. angulata before the flowering stage are used as flavouring in foods, especially dairy foods and also used as seasoning agent of foodstuffs in Iran. In Iranian traditional medicine (Unani medicine), the antidiabetics, antiseptic, antiulcer, air fresher, sedative and aphrodisiac properties of the herb or its essence have been accepted among Iranians. The results of this study provide, for the first time, data on variation of phytochemical, and antibacterial and antioxidant activities of the essential oils from various populations of F. angulata. The present study indicates the essential oil components of wild populations of F. angulata vary with chemotypes, environmental conditions and geographic origin. The essential oil of F. angulata is effective for inhibition or control of bacteria pathogens, especially L. monocytogenes and so could be used as a natural antibacterial agent. It is difficult to attribute the antibacterial and antioxidant effects of an essential oil to one or a few active compounds, such as α-pinene and β-ocimene, because in general they contain a mixture of different chemical compounds. Among the tested populations, the essential oils from the Saldaran and Kallar populations showed the highest antibacterial and antioxidant activities, respectively. In final, the use of F. angulata essential oil in foods, cosmetics and drugs, requires the identification of the bioactive compounds to perform further studies on their mechanism of action.

There is no conflicts of interest among the author who contributed to this study.

This study was supported by Research Center for Medicinal Plants & Ethno-veterinary, Technology, Islamic Azad University of Shahrekord Branch, Iran.