1.   Introduction

The genus Ferulago Koch (Apiaceae) is mainly growing in Anatolia and is represented by thirty-four species, among them nineteen species are endemic to Turkey. The Turkish name of the genus is “Şeytanteresi” [1]. Ferulago species are known under the names “kişniş” or “çakşır”. Many members of the genus are used as decoctions and infusions for alternative medical agents due to their sedative, tonic, and digestive properties as well as used for spice and flavoring agents. The occurrence of coumarins [2, 3],  sesquiterpenes [3], and flavonoids [4, 5] was previously reported in Ferulago species.

The essential oils are composed mainly of oxygenated and non-oxygenated forms of monoterpenes and sesquiterpenes. Due to the high lipophilicity of essential oils, normal-phase chromatography can be successfully applied to separate individual essential oil components. Preparative HPLC techniques can be used for the efficient separation of essential oils as well as natural extracts with low solvent consumption and timesaving. Purification of pure chemicals present in natural essential oils is critical for determining the molecules responsible for various biological actions. This paper reports the purification of the major components of Ferulago setifolia essential oil.

 

2. Materials and methods

2.1 Plant Material

Plant materials were collected at the inflorescence stage from Ergan Mountain, Erzincan/Turkey, in June 2021. The voucher specimens were authenticated by Prof. Dr. Ali Kandemir and deposited at the Herbarium of the Department of Science and Art Faculty, Erzincan Binali Yıldırım University, Erzincan, Turkey (ERZ-HERB No 9062).

2.2 Essential oil extraction

100 g of chopped fresh aerial parts of F. setifolia were subjected to Neo-Clevenger apparatus for 2 hours. The oil was obtained with 2.80% yield as pale-yellow liquid and dried by adding anhydrous sodium sulfate and stored in dark vials at +4°C until further analysis. 

2.3. Analysis of isolated components

GC/MS analyses were performed using a Thermo Scientific Trace 1310 GC/MS system, equipped with an HP5-MS capillary column (30 m x 0.25 mm and 0.25 µm). Helium (constant flow, 1.2 mL/min) was used as a carrier gas in split mode by 50:1. The injection site and mass transfer line temperature were set at 280 °C. The column oven temperature was programmed as follows: the initial column oven temperature was 60 °C, held for 3 min then ramped to 200 °C at a rate of 3 °C/min and held for 0 min, ramped to 240 °C at a rate of 5 °C/min and held for 5 minutes. The mass spectrometer conditions were as follows: the ion source temperature was 280 °C and the ionization energy was 70 eV in EI mode. NMR analysis was carried out on a Bruker Avance II, 400 MHz for 1H 100 MHz for 13C. The spectra were recorded in CDCl3.

2.4. Isolation of main components from essential oil

A Shimadzu semi-prep-HPLC instrument equipped with an LC-20AR dual pump, LC-20AT dual channel UV detector, and FRC-10A fraction collector was used for the preparative separation of main components. A silica-packed column (Dr. Maisch, Reprosil) was used for elution (250 mmX20 mm, 5 µm). n-hexane (A) and ethyl acetate (B) was used as mobile phase with a 4 ml/min flow rate. The mobile phase program was applied as follows: 90:10 (A: B) for 5 minutes, linear gradient to 50:50 (A: B) for 20 minutes, and finally linear gradient to 0:100 (A: B) for 10 minutes. The elution was monitored at 254 nm. 

2.5. Antimicrobial activity

Minimal inhibition concentration (MIC) values were calculated using the microdilution method. The selected microorganisms were adjusted to 0.5 McFarland in Muller Hamilton Broth (MHB). 50 μL of MHB was added to each well of the 96-well plate.  10 μL of isolated individuals were spiked and the mixture was incubated at 37 °C for 6 hours. 50 μL of resazurin solution (0.1%) was added and after 4 hours of additional incubation, the color changes were recorded. The first well where the resazurin blue color turned pink was recorded as the MIC value.

The antimicrobial activities of the isolated components were determined against ten bacterial strains. Five of the ten bacteria strains were gram-positive; Staphylococcus aureus (ATCC 6538), Listeria monocytogenes (ATCC 51774), Bacillus cereus (ATCC 10876), Clostridium perfringens (ATCC 13124), Enterococcus faecium (ATCC 8459), and other five strains were gram-negative; Pseudomonas fluorescens (ATCC 13525), Pseudomonas aeruginosa (ATCC 15442), Salmonella enteritica (ATCC 15442), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076). Tetracycline was used as positive control for bacterial strains.

 

3.   Results and discussion

The crude essential oil was eluted over NP-prep-HPLC to obtain main components. 500 µl essential oil was solved in 1500 µL 90:10 n-hexane: ethyl acetate and injected to instrument over the 2 ml sample loop. Four signals which monitored at 254 nm were collected. The separation process was repeated five times. The preparative collection pattern was given in Fig. 1 and GC/MS chromatograms of crude essential oil and isolated compounds were given in Fig. 2. 

Figure 1. NP-prep-HPLC chromatogram of crude essential oil monitored at 254 nm

Figure 2. GC/MS chromatograms of crude essential oil and isolated main component

A: Essential oil, B: sabinene, C: α-pinene, D: 2,3,4-trimethyl benzaldehyde.

After removing solvents under nitrogen atmosphere, the liquid residues for 1 and 4 fractions and white solid crystalline residue for 12th fraction were obtained. The fractions were identified as α-pinene (79 mg) (1), sabinene (2) (39 mg) and 2,3,4-trimethyl benzaldehyde (3) (133 mg), respectively, by comparing MS ionization patterns with those authentic libraries (Nist2005 and Wiley) for 1 and 2 and by NMR analysis for 3.  The 1H-NMR spectra of 3 were recorded in CDCl3. An aldehyde signal at δH 10.28 ppm (1H, s, CHO), an AB system δH 7.74 (d, J=7.8 Hz, 1H, H6) and δH 7.13 (d, J=7.8 Hz, 1H, H5), and three methyl signals δH 3.57 (3H, 4-CH3), 3.45 (3H, 3- CH3) and 3.32 (3H, 2-CH3) ppm related to 2,3,4-trimethyl benzaldehyde (Fig. 3). 13

Figure 3. 1H-NMR spectra 3 (400 MHz, in CDCl3)

C-NMR of 3 contain 10 carbons including four quaternaries [128.4 (C1), 138.64 (C2), 136.67 (C3) 141.60 (C4)], three methyl [15.78 (2-CH3), 17.23 (3-CH3), 20.81 (4-CH3), three methine one of aldehydic [192.57 (CHO), 127.21 (C5), 127. 16 (C6)] corresponding to C10H12O. (Fig. 4). The NMR assignments were fully coincident with literature [6]. Several studies reported that the essential oils of some Ferulago species contain benzaldehyde derivatives including 2,3,4-trimethyl benzaldehyde, 2,4,5-trimethyl benzaldehyde, and 2,4,6-trimethyl benzaldehyde as a major component. These aldehydes were found in different plant parts of eight Ferulago species ranging from %5.25 % to 92.7 [7-12].

Figure 4. 13C-NMR spectra 3 (100 MHz, in CDCl3)

A literature search showed that the essential oils of Ferulago setifolia have not previously been subjected to any biological activity evaluation. In this study, the antibacterial activity of purified main components of essential oil was evaluated by the resazurin based microdilution method. The results are given in Table 1.

Table 1. Antimicrobial activity of main components of Ferulago setifolia essential oil.

 

Microorganisms	Minimal inhibition concentration (MIC) in µg/ml
	α-pinene	Sabinene	2,3,4-trimethtyl benzaldehyde	Tetracycline
Pseudomonas fluorescence	7.81	31.25	250	<1.95
Staphylococcus aureus	15.63	31.25	250	<1.95
Listeria monocytogenes	7.81	31.25	250	3.91
Pseudomonas aeruginosa	31.25	31.25	125	3.91
Salmonella enteritica	7.81	15.63	125	1.95
Escherichia coli	31.25	62.5	250	7.81
Bacillus cereus	7.81	31.25	125	3.91
Clostridium perfringens	125	125	250	1.95
Enterococcus faecium	15.63	31.25	250	<1.95
Salmonella enteritis	15.63	62.5	62.5	<1.95

 

According to our results, pure α-pinene (1) was found to be most active compound against Pseudomonas fluorescence, Listeria monocytogenes, Salmonella enteritica, and Bacillus cereus with the 7.81 µg/ml MIC value. Sabinene (2) has a moderate activity against all tested microorganisms. 2,3,4-trimethyl benzaldehyde was found to have a less activity when compared the 1 and 2. It seems from the antimicrobial activity of isolated main components was evaluated against the selected microorganisms; the activities of pure compounds (1, 2 and 3) can be sequenced as follows α-pinene (1) > sabinene (2)> 2,3,4-trimethyl benzaldehyde (3).

Although there are some studies on the antibacterial activity of pure or α-pinene [13, 14] and sabinene-dominated essential oils [15, 16], there are no data on the activity of  pure 2,3,4-trimethyl benzaldehyde. This is the first report on the antimicrobial activity of 3. 

 

4. Conclusions

The essential oil of F. setifolia could be serving as a potential source of α-pinene, sabinene, and 2,3,4-trimethyl benzaldehyde for use in the food, cosmetic and pharmaceutical industries. The results presented in this study showed the antimicrobial activity of 2,3,4-trimethyl benzaldehyde obtained from essential oil of Ferulago setifolia growing in Turkey for the first time. The occurrence of 2,3,4-trimethyl benzaldehyde in the essential oil contributed to a better knowledge of this species.

Also, here we report the one-step purification of the major components of essential oils using NP-prep-HPLC. This technique can be recommended to other researchers interested in the purification of essential oil components.

 

Authors’ contributions

All authors contributed equally.

 

Acknowledgements

We thank Özlem POLAT (Scientific and Technological Application and Research, University of Tokat Gaziosmanpaşa) for recording NMR spectra.

 

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

Conflicts of interest

The authors declare no conflict of interest.

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