Abstract
Context: Ferula cupularis (Boiss.) Spalik et S. R. Downie (Apiaceae) is a common plant in Iran that grows in the foothills of Dena Mountain. In traditional folk medicine, this plant has different applications, but there are no studies proving their uses.
Objective: This study is the first attempt to investigate the chemical composition and antibacterial effect of the essential oils of F. cupularis.
Materials and methods: The essential oils from flower, leaf, and stem of F. cupularis were analyzed by using GC and GC-MS. Antibacterial activity of essential oils was determined by microdilution method against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.
Results: The essential oil from flower of F. cupularis contained 15 monoterpene, 13 oxygenated monoterpene, and 2 sesquiterpene hydrocarbons. The leaf essential oil contained 12 monoterpene, 13 oxygenated monoterpene, 2 sesquiterpene, 6 oxygenated sesquiterpene hydrocarbons, and 3 non-terpenoid components. Stem essential oil contained one monoterpene, 23 oxygenated monoterpene, 2 sesquiterpene, and 6 oxygenated sesquiterpene hydrocarbons. The MIC value of stem essential oil was 2.85 mg/mL against both Gram-positive bacteria and Gram-negative bacteria except P. aeruginosa which was inhibited at 22.75 mg/mL. The MIC values of leaf and flower essential oils were higher than 5.69 and 22.75 mg/mL, respectively.
Discussion: This study highlighted the strong antibacterial effect of Ferula cupularis’s essential oil which might be due to its high content of oxygenated monoterpene hydrocarbons.
Conclusion: Our results suggested that this plant may be a good candidate for further biological and pharmacological investigations.
Introduction
The genus Ferula (Apiaceae) comprises about 170 species worldwide (Amin, Citation1991; Mozaffarian, Citation1998; Rechinger & Hedge, Citation1982) and, in Iran, is represented by about 30 species. Phytochemical analysis of Ferula species has confirmed the occurrence of biologically active compounds such as daucane sesquiterpenes, sulfur containing molecules, and coumarins (Dastan et al., Citation2012; Iranshahi et al., Citation2009, Citation2010; Sahebkar et al., Citation2010; Zedan et al., Citation2012). Some Ferula species are used in folk medicine as anticonvulsant, carminative, antispasmodic, diuretic, antihelmintic, tonic, laxative, anti-hysteric, decongestant, aphrodisiac, and some are applied for the treatment of neurological disorders, diabetes, rheumatism, and backache (Afifi & Abu-Irmaileh, Citation2000; Bagheri et al., Citation2010; Iranshahy & Iranshahi, Citation2011; Mahendra & Bisht, Citation2012; Zargari, Citation1996).
Recent investigations on Ferula genus have revealed many other valuable properties such as antimycobacterial (Appendino et al., Citation2004), anticonvulsant (Sayyah & Mandgary, Citation2003), antimicrobial (Asili et al., Citation2009; Maggi et al., Citation2009), antispasmodic (Fatehi et al., Citation2004), antifungal (Iranshahi et al., Citation2008a), and antioxidant (Dehghan et al., Citation2007; Kartal et al., Citation2007) activities which indicate the importance of the research on this genus.
Ferula cupularis (Boiss.) Spalik et S. R. Downie is a common plant in Iran that grows in the foothills of Dena Mountain in Yasuj, Kohkilooye Boyerahmad Province. The native peoples use this plant for the treatment of ulcer. It is also used for preservation of meat and oil (no published document). The literature survey showed that there is no previous study about biological effects or chemical constituents of this plant which prove their uses. Therefore, the aim of present study was to determine the chemical composition and antibacterial activity of the essential oils from flower, leaf, and stem of F. cupularis growing wild in Dena Mountain for the first time.
Materials and methods
Plant material
The flower, leaf, and stem parts of Ferula cupularis were collected by Ziba Alipour from Dena Mountain (1400 m height) in Yasuj, Kohkilooye Boyerahmad Province, in July 2012. The plant was identified and authenticated by Dr. A. Mazooji and the voucher specimen was deposited at the Herbarium of Iranian Research Institute of Plant Protection, Tehran, Iran (IRAN 56843).
Volatile oil extraction
The dried aerial parts of the plant were placed in a 2000 mL round bottomed flask with distilled, water (1000 mL for 100 g dry powder) and the essential oils were extracted by hydro-distillation using a Clevenger-type apparatus. The plant materials were exposed to temperature close to 100 °C. The essential oils were dried over anhydrous sodium sulfate and kept at 4 °C in sealed brown vials until required.
Gas chromatography-Mass spectroscopy
The essential oil was analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The analytical GC was carried out on a Hewlett–Packard (HP) model 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA) equipped with a flame ionization detector (FID) and DB-1 fused silica column (60 m × 0.25 mm i.d., film thickness 0.25 µm). Injector and detector temperatures were 250 and 265 °C, respectively. Oven temperature was adjusted to 250 °C at a rate of 4 °C/min. Helium gas was used as the carrier gas at a constant flow rate of 1 mL/min and 1 µL of the dissolved sample in CH2Cl2 was injected.
GC/MS analysis was carried out on the same chromatograph equipped with a HP 5973 mass selective detector (Agilent Technologies, Santa Clara, CA) with an ionization energy of 70 eV. Identification of the essential oil components was based on retention indices relative to normal alkanes and computer watching with the Wiley 275 library, as well as by comparison of the fragmentation patterns of mass spectra with those reported in the literature (Adams, Citation1995; Sandra & Bicchi, Citation1987).
Determination of minimum inhibitory concentrations (MICs)
Antibacterial activity of the essential oils from flower, leaf, and stem of F. cupularis was evaluated against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Eshcerichia coli ATCC 8739, Klebsiella pneumoniae ATCC 10031, and Pseudomonas aeruginosa ATCC 9027. The antibacterial susceptibility test was determined by the microdilution method using 96 U-shaped wells plates (NCCLS, Citation2006).
Aliquot of 100 µL pure DMSO and 10 µL Tween 80 was used to dissolve 20 µL of essential oil in 870 µL Mueller–Hinton broth (MHB). A 200 µL aliquot of stock solution of essential oil was transferred into the first well in each row and serially diluted by mixing with 100 µL of MHB in subsequent wells. Then turbidity of the actively growing bacterial culture was adjusted photometrically (600 nm) comparable with that of a 0.5 McFarland turbidity standard (1.5 × 108 CFU/mL). The adjusted bacterial suspension was diluted in sterile broth (1:50) and 100 µL aliquots were added to each well so that after inoculation, each well contained approximately 5 × 105 CFU/mL. After 24 h incubation at 35 °C, the microdilution trays were tested for the absence or presence of bacterial turbidity by visible examination and using ELISA reader in 570 nm. The endpoint MIC was the lowest concentration of the essential oil at which the test strain did not demonstrate visible growth.
Results
The dried flowers, leaves, and stems of Ferula cupularis yielded 0.36, 0.45, and 0.39% w/w of essential oil, respectively. GC-MS analysis revealed 30, 36, and 32 components in the essential oils from flower, leaf, and stem parts representing 98.57, 93.73, and 91.93% of the total essential oil, respectively. The major constituents of flower essential oil () were dl-limonene (25.04%), δ-2-carene (15.81%), sabinene (7.96%), β-phellandrene (6.89%), α-terpinolene (5.61%), δ-3-carene (5.21%), p-mentha-1-en-9-ol (2.75%), and γ-terpinene (2.19%).
Table 1. Chemical composition of the essential oil from flowers of Ferula cupularis.
As shown in , the profile of essential oil from leaf exhibited β-pinene (13.87%) as the main compound; additionally other major compounds were β-ocimene (9.05%), bornyl angelate (6.55%), allo-ocimene (6.06%), trans-isolimonene (5.78%), dihydro-linalool acetate (5.02%), β-phellandrene (4.18%), p-mentha-1,5,8-triene (4.05%), α-terpinyl isobutyrate (3.7%), terpin-4-ol (3.45%), cis-dihydro-α-terpinyl acetate (3.11%), δ-2-carene (2.9%), camphene (2.69%), neo-allo-ocimene (2.68%), citronellyl n-butyrate (2.63%), decane (2.37%), and α-phellandrene (2.36%).
Table 2. Chemical composition of the essential oil from leaves of Ferula cupularis.
The main components of essential oil from stem were α-terpinyl isobutyrate (8.69%), δ-3-carene (8.38%), bornyl angelate (7.45%), trans-sabinol (6.87%), sothol (5.96%), p-cymen-9-ol (5.53%), terpinyl acetate (5.19%), linalool isobutyrate (3.41%), camphor (3.03%), β-bourbonene (2.7%), p-menth-1-en-9-ol acetate (2.65%), citronellyl butyrate (2.57%), myrcenone (2.39%), trans-sabinyl acetate (2.19%), and iso-verbanol acetate (2.18%) ().
Table 3. Chemical composition of the essential oil from stems of Ferula cupularis.
As shown in , the stem essential oil had the MIC value of 2.85 mg/mL against Gram-positive bacteria (S. aureus, S. epidermidis, and B. subtilis) and some Gram-negative bacteria (E. coli), while P. aeruginosa was inhibited at higher concentration (22.75 mg/mL). The MIC values of essential oils from leaf and flower parts of F. cupularis were higher than 5.69 and 22.75 mg/mL, respectively.
Table 4. In-vitro antibacterial activities of essential oils from stem, leaf, and flower parts of Ferula cupularis (MIC values in mg/mL for essential oils and µg/mL for ciprofloxacin).
Discussion
Ferula cupularis has many applications in Iranian folk medicine such as preservation of meat and oil and treatment of ulcer, but there are no studies proving their uses.
The essential oil from flower of F. cupularis contained 15 monoterpene (81.19%), 13 oxygenated monoterpene (17.25%), and 2 sesquiterpene (0.31%) hydrocarbons. The leaf essential oil contained 12 monoterpene (55.09%), 13 oxygenated monoterpene (29.17%), 2 sesquiterpene (0.68%), and 6 oxygenated sesquiterpene (4.00%) hydrocarbons. Three non-terpenoid components (4.79%) also identified in the leaf essential oil. The results of the analysis of stem essential oil revealed one monoterpene (8.38%), 23 oxygenated monoterpene (63.69%), 2 sesquiterpene (3.19%), and 6 oxygenated sesquiterpene (18.67%) hydrocarbons. The total amounts of monoterpene fractions in essential oils from flower, leaf, and stem were 98.44%, 84.26%, and 72.07%, respectively, which were much higher than sesquiterpene fractions (0.31%, 4.68%, and 21.86%). Previous studies on chemical composition of essential oils of other species of genus Ferula including F. ovina (Ghannadi et al., Citation2002), F. cummunis (Chibani et al., Citation2011), and F. gummosa (Sayyah et al., Citation2001) similarly indicated that monoterpene fractions were the main constituents of essential oils (76.2, 81.7, and 75.9% of essential oils, respectively). The main component in flower, leaf, and the stem are dl-limonene (25.04%), β-pinene (13.87%), and α-terpinyl isobutyrate (8.69%), respectively, which are different from the main compounds of other Ferula species essential oils like F. ovina, F. cummunis, and F. gummosa. It was found that monoterpene hydrocarbons were the most abundant compounds in the essential oils from flower (81.19%) and leaf (55.09%), while oxygenated monoterpenes were the main constituents of the essential oil from stem (61.69%).
Ozek et al. (Citation2008) showed that the essential oil of F. szowitsiana inhibited the growth of both Gram-positive and Gram-negative bacteria and its major constituents were β-eudesmol, α-eudesmol, and α-pinene. Abedi et al. (Citation2008) reported that F. gummosa essential oil was contained about 88% monoterpene hydrocarbons and the main constituents were sabinene, β-pinene, and α-pinene. The MIC values of F. gummosa essential oil against some pathogenic bacteria (S. aureus 3.125 µL/mL and P. aeruginosa 50 µL/mL) were comparable with the MICs of F. cupularis essential oils. Previous studies showed that the essential oils from F. badrakema (Asili et al., Citation2009), F. latisecta (Iranshahi et al., Citation2008b), and Ferula assafoetida (Kavoosi et al., Citation2013) were moderately active against Gram-positive bacteria (S. aureus and Bacillus cereus), but Gram-negative bacteria (E. coli and P. aeruginosa) were not susceptible to the essential oils of these plants. Results of this study showed that the essential oil from stem of F. cupularis inhibited the growth of all test bacteria at lower MIC values than essential oils of flower and leaf parts. This may be due to its high content of oxygenated monoterpene hydrocarbons. The previous investigation (Kotan et al., Citation2007) also concluded that essential oils rich in oxygenated monoterpenes are potent antimicrobial agents.
Further studies on antimicrobial activity of each component of F. cupularis essential oils are needed to define specific compounds that are responsible for this biological effect. However, there are some limitations in collecting this species as the plant grows in impassable mountainous areas of Iran which makes it difficult to gather enough amounts.
Conclusions
Despite many scientific studies on Ferula genus, this study is the first attempt to investigate the chemical composition and antibacterial effect of essential oils from different aerial parts F. cupularis. This study highlighted the strong antibacterial effect of Ferula cupularis’s essential oil which might be due to its high content of oxygenated monoterpene hydrocarbons. Our results suggested this plant may be a good candidate for further biological and pharmacological investigations.
Acknowledgements
We thank Dr. Mazooji and Mrs. Akhoondi for all their technical support.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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