INTRODUCTION

The genus Ferula L. is a member of the family Apiaceae and has been found to be a rich source of gum resin.¹ Ferula species are known in Turkey as “çakşır”, “asaotu”, “kıngor”, “heliz” etc.,² and Ferula orientalis is known as “heliz”,³ and they have been used as a carminative, sedative, laxative, antispasmodic, digestive, expectorant, diuretic, aphrodisiac, antiseptic, anthelmintic, analgesic,⁴ and stimulant.⁵ Ferula species have been found to contain sesquiterpenes and sesquiterpene coumarins.⁶ Fresh peeling stems of F. orientalis L., known as “at kasnisi” are used by local people to give flavor to pickles.⁵ It is 100-150 cm high, grows on rocky slopes at 1600-2900 m, and has distinguished yellow flowers, with a flowering time in late May and June.⁷ Ferulago W. Koch. is represented by approximately 83 taxa throughout the world and is a perennial genus of Apiaceae.⁸ Ferulago species are known as “çakşır”, “şeytanteresi”, and “kişniş” in Turkey and Ferulago sandrasica is known as “kuzu kişnişi”.² Since ancient times Ferulago species have been used for the treatment of intestinal worms and hemorrhoids; as a tonic, aphrodisiac, digestive, and sedative; and against ulcers, snake bites, spleen diseases, and headache. These species have been found to contain coumarins, quinones, flavonoids, and sesquiterpenes.⁹ F. sandrasica Peşmen and Quézel is an endemic glabrous species, 30-35 cm high; it grows on rocky serpentine slopes at 2000 m and its flowering time is in June and July.⁷

The genus Hippomarathrum link is a member of the family Apiaceae and it has five species. Hippomarathrum is an erect, much-branched perennial genus, 50-100 cm high, and distributed on rocky slopes and in fields. Hippomarathrum microcarpum is also used as food and is known as “çakşır” or “çaşır” by local people in Eastern Anatolia in Turkey.¹⁰ The species of this genus have long been used as spices in ethnobotany.¹¹ H. microcarpum Petrov is a gray shrub with yellowish flowers⁷ and it is reported that coumarins and furanocoumarins are found in the roots and fruits of the genus Hippomarathrum.¹² Essential oils or their components have been shown to exhibit antimicrobial, antiviral, antimycotic, antitoxigenic, antiparasitic, and insecticidal properties. It is considered that these characteristics are related to the function of these compounds in plants.¹³

The aim of the present study was to present and compare the chemical compositions of the essential oils of the aerial parts of F. orientalis, roots of F. sandrasica, and aerial parts of H. microcarpum growing wild in Turkey. We determined the chemical composition of the essential oils by gas chromatography (GC) and GC/mass spectrometry (MS) analysis and examined the antimicrobial activities of the essential oils by thin-layer chromatography (TLC)-bioautography assay. To the best of our knowledge, this is the first report on the chemical composition and antimicrobial activity of the essential oils in F. orientalis, F. sandrasica, and H. microcarpum.

MATERIALS AND METHODS

Plant material

The plant materials were collected from different parts of Turkey and were identified by Prof. Dr. Hayri Duman (Gazi University, Faculty of Science, Department of Biology) and the voucher specimens are kept in AEF (Herbarium of Ankara University Faculty of Pharmacy). The localities where these species were found are given in Table 1.

Isolation of the essential oil

The roots and aerial parts were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus in accordance with the method recommended in the European Pharmacopoeia. The oils obtained were dried in anhydrous sodium sulfate and stored in sealed vials at +4°C in the dark until analyzed and tested. All oils were pleasant smelling and transparent with a faint yellow and greenish color. The essential oil % yields of the aerial parts of F. orientalis, roots of F. sandrasica, and aerial parts of H. microcarpum were 0.022%, 0.019%, and 0.048%, respectively.

GC/MS analysis

GC/MS analysis was performed with an Agilent 5975 GC-MSD system. An Innowax FSC column (60 m×0.25 mm, 0.25 mm film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min, and kept constant at 220°C for 10 min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted to 40:1 and injector temperature was set to 250°C. Mass spectra were recorded at 70 eV and mass range was from m/z 35 to 450.

GC analysis

GC analysis was performed with an Agilent 6890N GC system. The temperature of the flame ionization detector (FID) detector was 300°C. In order to obtain the same elution order as GC/MS, simultaneous auto-injection was done on a duplicate of the same column conforming with the same operational conditions. Relative percentage quantities of the separated compounds were calculated from FID chromatograms. The results of the analysis are given in Table 2. Identification of the essential oil components was performed by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index to series of n-alkanes. Computer matching against commercial sources^14,15 and the in-house “Başer Library of Essential Oil Constituents” established with genuine compounds and components of known oils, alongside MS literature data,^16,17 was used for the identification.

Determination of antimicrobial compounds of the essential oils by TLC-bioautography assay

Chromatography was carried out on 0.2 mm silica gel 60 F₂₅₄ aluminum sheet TLC plates. To the plates was applied 10 µL of essential oils with a minicaps capillary pipette. The plates were then developed with toluene:ethyl acetate, 93:7, as a mobile phase and another TLC plate for bioautography was prepared in parallel. After the development, the TLC plates were evaluated at UV 254 nm and 366 nm for determination of fluorescent compounds. Alcoholic vanillin–sulfuric acid reagent was used to visualize the separated compounds and they were heated for 3 min at 110°C.

Preparation of microorganisms and the TLC-bioautography assay

After TLC separation, the antimicrobial activity of the essential oils was determined by direct bioautography.^18,19 Pseudomonas aeruginosa ATCC 13388, Staphylococcus aureus ATCC BAA 1026, Candida albicans ATCC 24433, and Escherichia coli NRRL B-3008 strains were used for bioautography. Microbial suspensions were grown overnight in double strength Mueller-Hinton broth standardized to 10⁸ CFU mL^-1 (corresponding to McFarland no. 0.5). TLC plates were placed on nutrient agar plates and molten agar culture medium containing inocula was overlaid on the TLC plates and they were incubated at 37°C for 24 h. Then, by incubation, 2,3,5-triphenyl-2H-tetrazolium chloride solution was sprayed on the TLC plates. The treated plates were incubated at 37°C for 2 h and after incubation the inhibition zones were visible as pale spots against a red background.

RESULTS

Thirteen compounds were identified in the essential oil of the aerial parts of F. orientalis, representing 96.6% of the oil. α-Pinene (75.9%), β-pinene (3.4%), trans-verbenol (3.0%), and β-caryophyllene (2.5%) were the major components. The analysis on the roots of F. sandrasica resulted in the identification of 69 essential compounds representing 96.0% of the oil. Limonene (28.9%) was the most abundant compound in the essential oil, followed by α-pinene (15.6%), terpinolene (13.9%), camphene (2.6%), myrcene (2.8%), p-cymene (2.8%), and 2,3,6-trimethylbenzaldehyde (3.2%).

Twenty-one compounds were characterized in the oil of the roots of H. microcarpum, representing 98.7% of the oil. The major constituents were β-caryophyllene (31.4%), caryophyllene oxide (23.1%), bornyl acetate (9.1%), α-humulene (4.9%), germacrene D (4.2%), β-phellandrene (4.6%), α-pinene (3.0%), and caryophylla-2(12),6-dien-5β-ol (=caryophyllenol II) (3.0%). The essential oils obtained from these species did not show much qualitative and quantitative similarity. α-Pinene, camphene, β-pinene, limonene, β-phellandrene, p-cymene, and β-caryophyllene were the main compounds in the three species. Trans-verbenol was the main compound in the essential oils of F. orientalis and F. sandrasica. Thuja-2,4(10)-diene, pinocarvone, trans-pinocarveol, myrtenol, and cuparene were only found in the essential oils of the aerial parts of F. orientalis.

Sabinene, α-phellandrene, (Z)-β-ocimene, γ-terpinene, (E)-β-ocimene, terpinolene, α-copaene, bornyl acetate, α-humulene, germacrene D, δ-cadinene, caryophyllene oxide, and humulene epoxide-II were the main compounds in the essential oils of F. sandrasica and H. microcarpum. Caryophylla-2(12),6-dien-5β-ol (=Caryophyllenol II) was only found in the essential oils of the aerial parts of H. microcarpum. The composition of the essential oils obtained from these species and their relative percentages are given in Table 2.

The results for antimicrobial activity by bioautography showed that essential oils from the aerial parts of F. orientalis and roots of F. sandrasica were active against S. aureus and C. albicans strains. However, they were not active against the E. coli strain. Similarly, essential oil from the aerial parts of H. microcarpum was found to contain compounds active against S. aureus and C. albicans. The essential oil was more effective against C. albicans than against S. aureus. However, it did not have good activity against E. coli. The essential oils did not give any inhibition zone against P. aeruginosa. The TLC evaluation of the essential oils is shown in Figure 1.

DISCUSSION

Monoterpene hydrocarbons (P-cymene, myrcene, γ-terpinene, limonene, terpinolene, and (Z)-β-ocimene), oxygenated monoterpenes (carvacrol methyl ether, 2,5-dimethoxy-p-cymene, trans-chrysanthenyl acetate, cis-chrysanthenyl acetate, and ferulagone), aldehydes (like 2,3,6-trimethylbenzaldehyde, (E)-2-decenal, and octanal), alkane derivatives (hexadecanoic acid), sesquiterpene hydrocarbons (α-humulene, 4,6-guaiadiene, and 7-epi-1,2-dehydrosesquicineole), and oxygenated sesquiterpenes (like cubenol, humuleneepoxide II, and spathulenol) were the major components of some Ferulago species.

Some Ferula species contain monoterpene hydrocarbons [β-pinene, sabinene, camphene, β-phellandrene, and (E)-β-ocimene], alkane derivatives (nonane), sesquiterpene hydrocarbons (germacrene D, germacrene B, δ-cadinene, (Z)-β-farnesene, dehydrosesquicineole, and eremophilene), and oxygenated sesquiterpenes (germacrene D-4-ol, α-cadinol, shyobunone, epi-shyobunone, 6-epi-shyobunone, β-eudesmol, and α-eudesmol) were the major components of some Ferula species.

In addition, esters like bornyl acetate were major components of some Ferula and Ferulago species.²⁰

Previous studies demonstrated that the major components of the essential oil of leaves from F. sandrasica were ocimene (30.5%), carene-δ-3 (27.4%), and α-pinene (17.8%).¹⁹ Baser and Kırımer²⁰ studied 12 Ferulago species (F. asparagifolia Boiss., F. aucheri Boiss., F. confusa Velen, F. galbanifera (Mill.) W. D. J. Koch, F. humilis Boiss., F. idaea Özhatay and Akalın, F. macrosciadia Boiss. and Balansa, F. mughlae Peşmen, F. sandrasica Peşmen and Quézel, F. silaifolia (Boiss.) Boiss., F. sylvatica (Besser) Rchb., and F. trachycarpa Boiss.) growing in Turkey and that study showed that the major components of essential oils were 2,3,6-trimethylbenzaldehyde (38.9%) and myrcene (18.2%), α-pinene (35.9%), 2,5-dimethoxy-p-cymene (63.4%), a-pinene (31.8%) and sabinene (15.8%), (Z)-β-ocimene (32.4%), p-cymene (18.4%), carvacrol methyl ether (78.1%), α-pinene (25.4%), α-pinene (40.8%), trans-chrysanthenyl acetate (83.5%), p-cymene (45.8%), and (Z)- β-ocimene (30.7%).²¹

The major components of essential oils of some Ferula species were reported as phenol, 2-methyl-5-(1-methylethyl) (18.2%), cyclopropa [α] naphthalene-octahydro-tetramethyl (6.6%), and α-bisabolol (10.4%) (3); α-pinene (18.3%), β-pinene (50.1%), and Δ-3-carene (6.7%).²² Comparing these results with previous studies of F. orientalis showed that the major components were nonane (45.6%) and 2-methyloctane (19.4%).²³ Furthermore, essential oils from the aerial parts of F. orientalis were obtained: β-phellandrene (24%), (E)-β-ocimene (14%), α-pinene (13%), α-phellandrene (12%), and dehydrosesquicineole (10%),²⁴ but α-pinene (75.9%) was a major component in our study.

Comparison of these results with previous studies of Hippomarathrum boissieri from Turkey¹⁸ showed that the major component of the essential oils from both species was β-caryophyllene (31.4% for aerial parts oil of H. microcarpum, 25.6% for aerial parts oil of H. boissieri). Another study showed that the major components of essential oils of the leaf and flower of H. microcarpum were a-caryophyllene (26.4%), γ-muurolene (19.0%), and linalool (6.1%); and β-caryophyllene (18.5%), γ-muurolene (19.2%), thymol (6.9%), and linalool (5.9%), respectively.¹⁰ The results gained in this investigation suggest that this chemical diversity may be useful in taxonomic classification.

There are not enough data on antimicrobial activity for these species. In a previous survey, the essential oil of F. sandrasica was tested against E. coli MC 400, E. coli ATCC 25922, E. coli 0157 H7, Enterobacter colaecea ATCC 23355, Enterococcus faecalis ATCC 19433, P. aeruginosa NRRL B-2679, S. aureus ATCC 25923, S. aureus ATCC 33862, Bacillus cereus NRRL B-3711, Bacillus subtilis ATCC 6633, B. subtilis NRRL B-209, Bacillus licheniformis NRRL B-1001, Micrococcus luteus NRRL B-1013, and Listeria monocytogenes ATCC 7644 by disk diffusion method. The results showed that the essential oil was active against all tested microorganisms.¹⁸ It was previously reported that the essential oil of H. microcarpum was studied for antimicrobial and antifungal activity. The results showed that the essential oil of H. microcarpum had antimicrobial activity against C. albicans A117 and S. aureus ATCC-29213 but had no activity against E. coli A1 or Pseudomonas sp.¹⁰ Our finding concur with this study.

Bioautography is a suitable method for evaluating essential oils because they contain mixtures of compounds. Therefore, there is a need for the detection of common antimicrobial compounds in essential oils. Additionally, this method is rapid, easy, economical, and inexpensive.²⁵ In the present study, our aim was the chemical characterization of the essential oils of F. orientalis, F. sandrasica, and H. microcarpum and the detection of antimicrobial activity of essential oils and their main components against some pathogenic bacteria and yeast by TLC-bioautography. The antimicrobial activity test performed against four different microorganisms showed that the essential oils were active against S. aureus and C. albicans strains; however, they were not active against P. aeruginosa or E. coli strains.

CONCLUSIONS

These data provide an abundance of information on the essential oil compositions of F. orientalis, F. sandrasica, and H. microcarpum and their antimicrobial activities against some pathogenic microorganisms. As far as we know, this is the first report on the antimicrobial activity of essential oils by TLC-bioautography. The antimicrobial activities against S. aureus and C. albicans of these species may be attributed to the presence of the main components in the essential oils. A comprehensive study should be conducted including the main compounds isolated from the essential oils or their combinations against different pathogenic microorganisms.

Conflict of Interest: No conflict of interest was declared by the authors.