Foetithiophenes C-F, thiophene derivatives from the roots of Ferula foetida

Abstract

Context: Ferula foetida Regel (Apiaceae) is an Iranian medicinal plant with various biological activities including antispasmodic and anthelmintic.

Objective: The sulfur compounds from the roots of F. foetida were isolated and characterized to test their cytotoxic and antimicrobial activities.

Materials and methods: The methanolic extract of the roots of F. foetida was fractionated using silica column chromatography. The components of each fraction were further purified using RP-HPLC. Their structures were elucidated by 1- and 2-D NMR spectroscopy as well as HREIMS. Their cytotoxic and antimicrobial activities were evaluated using Alamar Blue assay and broth microdilution method, respectively.

Results: Four new thiophene derivatives, namely foetithiophenes C-F (3–6), together with four known compounds, foetithiophenes A (1) and B (2), coniferaldehyde, and sinapic aldehyde, were isolated from the roots of F. foetida. Antimicrobial activities were observed in particular against the Gram-positive bacteria. The best antimicrobial activity was observed for compound 6 against B. cereus with a MIC value 50 µg/mL. The tested compounds did not show cytotoxic properties against MCF-7 and K562 cells.

Conclusion: Four new thiophene derivatives including foetithiophenes C-F (3–6) were characterized from the roots of F. foetida. Foetithiophene F (6) exhibited the most potent activity against the Gram-positive bacteria B. cereus.

• Alamar blue
• apiaceae
• cancer
• sulfur-containing compounds

Introduction

The genus Ferula (Apiaceae) consists of more than 170 species, widespread in central Asia, North Africa, and Mediterranean region. The compounds typically occurring in this genus include sesquiterpenes (Iranshahi et al., 2003; Kasaian et al., 2014; Meng et al., 2013; Sahebkar & Iranshahi, 2011), sesquiterpene coumarins (Abd El-Razek et al., 2003; Iranshahi et al., 2009, 2010a), sulfur-containing compounds (Iranshahi, 2012), and a few sesquiterpene coumarin glycosides (Iranshahi et al., 2008). Recent studies showed that some of these compounds are biologically active and have potential for pharmacological and therapeutical applications (Iranshahy & Iranshahi, 2011; Iranshahi et al., 2010b; Nazari & Iranshahi, 2011; Rassouli et al., 2011; Sahebkar & Iranshahi, 2010; Valiahdi et al., 2013). Most of the Ferula species are typically growing in mountains, while Ferula foetida Regel is found also in deserts, particularly in Iran and its eastern neighbor countries. Its aerial parts are edible and the plant has been used as an anthelminthic, antispasmodic, and diuretic in folk medicine (Zargari, 1997). Its roots were traditionally employed for the treatment of rheumatism and backache (Zargari, 1997). The chemistry of F. foetida has previously been studied and a number of compounds including sulfur-containing compounds and sesquiterpene coumarins were reported (Abd El-Razek et al., 2007; Duan et al., 2002). As a part of our ongoing investigation on anti-cancer natural products, we isolated and purified chemical constituents from the roots of F. foetida and evaluated their cytotoxic activities against certain cancer cell lines.

Materials and methods

General experimental procedures

The optical rotation was measured on a JASCO 1030 polarimeter (JASCO, Gross-Umstadt, Germany). UV spectra were obtained in MeOH on a Shimadzu UV-1650 PC spectrophotometer. IR spectra were recorded on a Unicam SP1100 infrared spectrophotometer and a Thermo Nicolet 5700 FT-IR spectrometer (JASCO, Gross-Umstadt, Germany). ¹H- and ¹³C NMR, DEPT, ¹H–¹H COSY, HSQC, HMBC, and ROESY spectra were recorded on a Bruker DRX 500 spectrometer operating at 500.13 MHz for ¹H and 125.76 MHz for ¹³C. The spectra were referenced to the residual solvent signal of CDCl₃ (¹H: δ 7.24; ¹³C: δ 77.23). Electron impact (EIMS) and high-resolution (HREIMS) mass spectra were obtained on a Micromass MasSpec (Micromass, Manchester, UK) sector-field mass spectrometer in a positive ion mode using 70 eV ionisation energy. LC-high resolution ESIMS (LC-HRESIMS) data of compound 5 were recorded on a LC-MS/MS system consisting of an Ultimate 3000 series RSLC system (Dionex, Sunnyvale, CA), and an Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). HRESIMS spectra were measured in positive ionization mode on the Orbitrap mass analyzer. Column chromatography was conducted with silica gel 230–400 mesh, Merck (Darmstadt, Germany). The chemical ionization mass spectrum (CIMS) of compound 5 was measured on a Micromass MS02 mass spectrometer (Waters, Manchester, UK) in positive mode. Isobutane was used as the ionization gas. The source temperature was set to 150 °C. Direct insertion probe was used with a temperature program starting at 60 °C (0.5 min), 20 °C min⁻¹ to 300 °C and kept at this temperature for 5 min. For the HPLC work, a Knauer Smartline Pump (Waters, Manchester, UK) (incl. 50 mL pump) equipped with a Knauer UV detector 2500 (Waters, Manchester, UK) was used.

Plant material

The plant material (F. foetida) was collected from the mountains of Dorouneh village, Khorasan Razavi province, Iran, in May 2012 and was identified by Mohammad Reza Joharchi, Ferdowsi University of Mashhad Herbarium (FUMH). A voucher specimen (No. 12612) was deposited at the herbarium of the Faculty of Pharmacy, Mashhad University of Medical Sciences.

Extraction and isolation

The air-dried roots (300 g) were ground into a powder and extracted exhaustively by maceration at room temperature with petroleum ether. The petroleum ether extract was discarded. Afterwards, the plant material was extracted with MeOH exhaustively. After filtration, the extract was concentrated under vacuum to yield 6 g of a brown residue. The extract (5.5 g) was subjected to column chromatography on Si gel (5 × 50 cm, 588 g) using petroleum ether with increasing volumes of EtOAc and MeOH [petroleum ether: EtOAc (1:0, 2 L), (9:1, 2 L), (8:2, 3 L), (3:1, 1 L), (2:1, 1 L), (1:1, 1 L), (0:1, 1 L), and EtOAc:MeOH (9:1, 1 L), (8:2, 2 L), (3:1, 1 L), 2:1, 1 L), (1:1, 1 L), (0:1, 2 L)]. The fractions (50 mL each) were compared by TLC (Si gel using petroleum ether/EtOAc, 4:1), and those giving similar spots were combined. Twenty-four fractions were obtained. The fractions were subjected to reversed-phase HPLC using 20–100% MeOH in H₂O as an eluent including 0.1% TFA. Purification of compounds was carried out using a C18 Eurospher column (Thermo Finnigan, Dreieich, Germany) (5 μM, 250 × 16 mm) at a flow rate 8.5 mL/min and linear gradient conditions of 20–100% MeOH (0.1% TFA) within 20 min, followed by an isocratic condition of MeOH (0.1% TFA) for 5 min. Compounds 3 (22.7 mg) and 4 (4.8 mg) were obtained from fraction 2. Compounds 1 (10.9 mg), coniferaldehyde (3.3 mg), 2 (4.2 mg), sinapic aldehyde (11.9 mg), 5 (38.3 mg), and 6 (17.7 mg) were obtained from fractions 11, 12, 13, 14, 16, and 17, respectively.

Cell culture

MCF-7 and K562 cell lines obtained from ATCC. The MCF-7 and K562 cells were grown on 25 cm² cell culture flasks to approximately 80–85% confluence and maintained in an incubator with 5% CO₂ atmosphere at 100% humidity and 37 °C. RPMI-1640 medium containing 10% FBS was used in the experiments. After reaching confluence, the cells were washed with phosphate-buffered saline (PBS), detached from the flasks by a brief treatment with trypsin–EDTA (PAA).

Alamar blue assay

To determine IC₅₀ values, MCF-7 and K562 cell lines were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). Cells were grown under 5% CO₂ in a humidified environment at 37 °C. Compound toxicity was measured after 72 h incubation using an Alamar Blue proliferation assay. The medium (200 µL) containing 10 000 cells (MCF-7 and K562) was added to a 96-well microtiter plate. Plates were incubated overnight at 37 °C under an atmosphere of 5% CO₂ and 80% humidity to allow cells to adhere. Stock concentrations of pure compounds were prepared at 100, 50, 5, and 0.5 mM in 100% DMSO. A part (100 µL) of the medium of each well was discarded and replaced by 100 µL fresh medium containing 200, 100, 10, and 1 μM of pure compounds. Final compound concentration range tested was 0.5–100 μM (a final DMSO concentration of 0.1%). Each concentration in media was tested in triplicate. Cells and compounds were then incubated for 72 h at 37 °C 5% CO₂ and 80% humidity. Cell proliferation was measured with the addition of 20 μL of an Alamar blue solution in media to each well of the microtiter plate. The plates were incubated at 37 °C under an atmosphere of 5% CO₂ and 80% humidity for 8 h. The absorbance of each well was read at 570 and 600 nm on a BioTech plate reader (BioTek Instruments, Winooski, VT). Paclitaxel was used during each screening run as a positive control compound.

Microbiological assay

Bacillus cereus PTCC 1247 (B. cereus), Candida albicans ATCC 10231 (C. albicans), and Escherichia coli ATCC 8739 (E. coli) were used as test microorganisms. Minimum inhibitory concentrations (MICs) were determined by the standard broth microdilution method (National Committee for Clinical Laboratory Standards, 2003). Each isolated compound was prepared from serial two-fold dilutions (from 400 to 50 µg/mL). The inoculum was prepared by pipetting 0.5 mL of an overnight culture (37 °C) of each strain into 9.5 mL of the Müller–Hinton broth (MHB) medium. Aliquot solutions (100 mL) of compounds were added to the wells of a microtiter plate. This was followed by the addition of 10 µL of test organism inoculum. Microplate was incubated for 24 h. According to definitions, the MIC was the lowest antibiotic concentration that yielded no visible growth. The test medium was MHB, and the inoculum was 5 × 10⁵ CFU/mL. The inoculated microplates were incubated at 37 °C for 24 h before being read. The MIC was then determined by adding TTC and was incubated for 30 min at 37 °C. The minimum bactericidal concentrations (MBCs) values were defined as the lowest concentration at which no growth was observed. MBCs were determined by plating out 5 µL of each of the broth cultures from the MIC tests onto each suitable agar plate and incubating those plates at 37 °C for 24 h. Ketoconazole (Sigma, St. Louis, MO) and gentamicin (Sigma, St. Louis, MO) were used as standard antimicrobial agents.

Results and discussion

A thorough fractionation of the methanolic extract of the dried roots of F. foetida resulted in the isolation and structure elucidation of four new thiophene derivatives named foetithiophenes C-F (3–6) along with two already reported derivatives (Duan et al., 2002), foetithiophenes A (1) and B (2), and two known compounds, coniferaldehyde and sinapic aldehyde. The structures of the thiophene derivatives isolated in this study are reported in Figure 1. ¹H and ¹³C NMR data for foetithiophenes C-F (3–6) are given in Table 1. The structures of the known compounds were confirmed by direct comparison of their spectral data with those reported in the corresponding literature.

Figure 1. The chemical structures of thiophene derivatives from F. foetida.

Table 1. ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectroscopic data of compounds 3–6 (CDCl₃)^a.

	3		4		5		6
Position	δH	δC			δH	δC	δH	δC
2	–	129.5	–	127.9	–	119.3	–	71.8
3	–	134.8	–	136.3	–	137.9	3.36 q (7.5)	52.3
4	–	133.0	–	133.6	–	134.0	–	147.5
5	–	130.2	–	131.5	–	132.8	–	135.4
6	3.75 s	30.6	3.84 s	36.5	4.00 d (13.8) 4.18 d (13.8)	53.5	1.68 s	17.3
7	2.03 s	12.9	2.04 s	13.3	2.08 s	13.7	1.14 d (7.5)	12.9
8	1.97 s	12.4	1.98 s	12.8	1.99 s	12.8	1.71 s	14.1
9	2.28 s	13.2	2.28 s	13.5	2.29 s	13.4	2.08 s	12.2
10	2.07 s	15.2	–	–	2.48 s	37.6	2.13 s	12.7

^aCoupling constants are given in parentheses and reported in Hz; multiplicity is defined as follows: s, singlet; d, doublet; q, quartet; assignments were confirmed by HSQC, HMBC, and ROESY experiments.

^bSignals of two identical subunits. ¹³C NMR chemical shifts of compounds 3 and 4 were extracted from HSQC and HMBC spectra.

Compound 3 (foetithiophene C) was obtained as a yellow oil and its molecular formula, C₉H₁₄S₂, was established by HREIMS ([M]⁺, m/z 186.0533, calcd 186.0537). Its ¹H NMR spectrum resembled that of 2 with resonances characteristic for four methyl singlets at δ 1.97 (H-8), 2.03 (H-7), 2.07 (H-10), and 2.28 (H-9), and a methylene singlet (H-6) at δ 3.75. In the HMBC spectrum, the correlations of H-6 with C-2 (δ_C 129.5) and C-3 (134.8); H-7 with C-2 (δ_C 129.5), C-3 (134.8), and C-4 (133.0); H-8 with C-3 (134.8), C-4 (133.0), and C-5 (130.2); and H-9 with C-4 (133.0) and C-5 (130.2) along with the correlation between H-10 and C-6 (δ_C 30.6) and H-6 with C-10 (15.2) confirmed the structure of compound 3 (Figure 1).

Compound 4 (foetithiophene D) was obtained as a yellow oil and its molecular formula, C₁₆H₂₂S₃, was established by HREIMS ([M]⁺, m/z 310.0873, calcd 310.0884). Its ¹H and ¹³C NMR resonances were similar to those of 3 (Table 1) with only slight differences in their chemical shift values. However, the absence of one of the signals previously assigned to a methyl group together with the given molecular formula led to its identification as a dimer of the hypothetical S-demethyl foetithiophene C. HSQC and HMBC correlations are in accordance with the assignment.

Compound 5 (foetithiophene E) was obtained as needle crystals with molecular formula C₉H₁₄OS₂ ([M+H]⁺; m/z 203.0554, calcd 203.0558). The ¹H NMR spectrum showed resonances of four methyl singlets at δ 1.99 (H-8), 2.08 (H-7), 2.29 (H-9), and 2.48 (H-10), and a diastereotopic methylene with two proton doublets (H-6) at δ 4.00 (J = 13.8 Hz) and 4.18 (J = 13.8 Hz). In the HMBC spectrum, the correlations of H-6 with C-2 (δ_C 119.3), C-3 (137.9), and C-10 (37.6); H-7 with C-2 (δ_C 119.3), C-3 (137.9), and C-4 (134.0); H-8 with C-3 (137.9), C-4 (134.0), and C-5 (132.8); H-9 with C-4 (134.0) and C-5 (132.8); and H-10 with C-6 (δ_C 53.5) confirmed the structure of compound 5. The shift of the signals of C/H-6 and C/H-10 to the lower field compared with the corresponding signals of compound 3, together with mass spectrometric data, constitutes a further evidence of the presence of an S oxide in the side chain of the molecule.

Compound 6 (foetithiophene F) obtained as a yellow oil was assigned the molecular formula, C₉H₁₆S₂, as determined by HREIMS ([M]⁺, m/z 188.0690, calcd 188.0693). The ¹³C NMR spectrum displayed only two deshielded ¹³C resonances at δ_C 135.4 and 147.5 indicating a change of the thiophene ring reported for the other compounds described here. In the ¹H NMR spectrum, resonance characteristics for four methyl singlets were present at δ 1.68 (H-6), 1.71 (H-8), 2.08 (H-9), and 2.13 (H-10), along with one methyl doublet at δ 1.14 (H-7, J = 7.5 Hz) and a methine quartet at δ 3.36 (H-3, J = 7.5 Hz). In the HMBC spectrum, the correlations of H-6 with C-2 (δ_C 71.8), C-3 (52.3); H-7 with C-2 (δ_C 71.8), C-3 (52.3), and C-4 (147.5); H-8 with C-3 (52.3), C-4 (147.5), and C-5 (135.4); and H-9 with C-4 (147.5) and C-5 (135.4) together with the additional correlation between H-10 and C-2 (71.8) confirmed the structure of compound 6 (Figure 1). In addition, ROESY cross-peaks between H-3 and H-6, and no ROE between H-6 and H-7 supported the relative configuration of 6.

Cytotoxic activity of compounds 1–6, coniferaldehyde, and sinapic aldehyde was tested against MCF-7 and K562 cells using the Alamar Blue assay. The compounds did not show cytotoxic activity and were considered inactive (IC₅₀ > 100 μM). Microbiological assay of compounds 1–6 was carried out on C. albicans (yeast), E. coli (Gram-negative bacteria), and Bacillus cereus (Gram-positive bacteria). Our findings revealed that the compounds were almost inactive on E. coli. The compounds 3, coniferaldehyde and sinapic aldehyde, showed the best antifungal activity against C. albicans with a minimum inhibitory concentration (MIC) value of 200 µg/mL. However, the best antimicrobial activities were observed against the Gram-positive bacteria B. cereus where foetithiophene F (6) exhibited the most potent activity with an MIC value of 50 µg/mL (Table 2).

Table 2. The results of antimicrobial activities of the compounds from Ferula foetida roots.

	C. albicans		E. coli		B. cereus
Compound no.	MIC (µg/mL)	MBC (µg/mL)	MIC (µg/mL)	MBC (µg/mL)	MIC (µg/mL)	MBC (µg/mL)
1	INA	INA	INA	INA	INA	INA
2	INA	INA	INA	INA	200	INA
3	400	INA	INA	INA	100	INA
4	200	INA	400	INA	400	INA
5	400	INA	INA	INA	400	INA
6	400	INA	INA	INA	50	INA
Coniferaldehyde	200	INA	INA	INA	200	INA
Sinapic aldehyde	200	INA	INA	INA	400	INA
Gentamicin			10	10	10	10
Ketoconazole	25	50

INA donates inactive (MIC and MBC were >400 µg/mL).

Foetithiophene C (3): Yellow oil; UV (MeOH) λ_max (log ɛ) 248 (2.79) nm; IR ν_max (CH₂Cl₂) 2916, 2851, 1503, 1466, 1250, and 1210. For ¹H and ¹³C NMR data, see Table 1.

Foetithiophene D (4): Yellow oil; UV (MeOH) λ_max (log ɛ) 248 (3.17) nm; IR ν_max (CH₂Cl₂) 2922, 2853, 1652, 1559, 1458, and 1252. For ¹H and ¹³C NMR data, see Table 1.

Foetithiophene E (5): Needle crystals; Mp. 91–93 °C; UV (MeOH) λ_max (log ɛ) 248 (3.94) nm; IR ν_max (CH₂Cl₂) 2914, 2854, 1644, 1413, 1003, and 950. For ¹H and ¹³C NMR data, see Table 1.

Foetithiophene F (6): Yellow oil; +76.9 (c 0.39, CH₂Cl₂); UV (MeOH) λ_max (log ɛ) 248 (2.29) nm; IR ν_max 2920, 1684, 1653, 1559, 1436, 1202, 1132, and 1029. For ¹H and ¹³C NMR data, see Table 1.

Conclusions

Four new thiophene derivatives including foetithiophenes C-F (3–6) together with four known compounds, foetithiophenes A (1) and B (2), coniferaldehyde, and sinapic aldehyde, were characterized from the roots of F. foetida. No significant cytotoxic activity (IC₅₀ > 100 μM) was observed for the tested compounds. However, the tested compounds showed moderate antimicrobial activity. Foetithiophene F (6) exhibited the most potent activity against the Gram-positive bacteria B. cereus (MIC value = 50 µg/mL).

Acknowledgements

The authors are very grateful to M. R. Joharchi of the Ferdowsi University of Mashhad Herbarium, for help with the identification of the plant.

Declaration of interest

The authors declare that they have no conflict of interest to report. This research was supported by a grant from the Mashhad University of Medical Sciences (Grant no. 900558).