Antibacterial Sesquiterpenoid Derivatives from Ferula ferulaeoides

• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Three new sesquiterpenoid derivatives 1, 2, and 3 were isolated from Ferula ferulaeoides. To confirm the structure, compound 2 was also synthesized via a condensation reaction between compound 1 and 2,2-dimethoxypropane. The structures of these three compounds were elucidated by means of spectroscopic and chemical methods. Their antibacterial activity against drug-resistant Staphylococcus aureus strains were evaluated with MIC values in the range of 0.5–128 µg/mL. Compounds 1 and 3 were capable of inhibiting efflux of ethidium bromide using an in vitro assay. The cytotoxicity of the compounds was evaluated on cultured HEK293 cells, and none of them showed toxicity to HEK293 cells at a concentration of 125 µg/mL.

The emergence of bacterial resistance to different classes of antibacterial agents such as β-lactams, quinolones, and macrolides is a major problem that seriously affects human health [1]. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread and virulent nosocomial pathogens in the world [2] and is usually resistant to multiple antibiotics, making infection with this organism difficult to treat. MRSA accounts for an increased proportion of staphylococcal infections among hospitalized patients in countries where it has become established. Despite new advances in antibiotic development with agents such as linezolid, daptomycin, and quinupristin/dalfopristin appearing over the last decade [3], [4], [5], there is still an urgent need to develop new classes of antibacterial agents in order to combat bacterial multidrug-resistance in a variety of both Gram-positive and gram-negative species.

The genus Ferula (Apiaceae) includes approximately 150 species growing over a vast geographical region ranging from Central Asia to the Mediterranean, and 26 species are distributed in China. As a traditional herb, several species of Ferula have a reputation in the treatment of various diseases, acting as stomachic, febrifuge, vermifuge, and carminative, as well as against skin infections and diabetes [6]. In terms of pharmacological research, Ferula species have been found to possess many biological effects including anti-inflammatory, cytotoxic, P-glycoprotein inhibitory, cancer chemopreventive, antibacterial, and antileishmanial activities [7]. Ferula ferulaeoides (Steud.) Korov. grows in western China [8] and has been reported to contain a series of sesquiterpene derivatives of a farnesyl moiety [9]. In our phytochemical research on this species, three new phenyl-substituted sesquiterpenoids, namely compounds 1, 2, and 3, were isolated and identified. The three compounds were assessed for their inhibitory activity against resistant bacterial strains, notably Staphylococcus aureus, by means of minimum inhibition concentration determinations, and 1 and 3 were tested for efflux pump inhibitory activity. The cytotoxicity of compounds 1 and 3 were also evaluated using human embryonic kidney cells.

The powdered roots of F. ferulaeoides were extracted with 95 % ethanol and fractionated with dichloromethane. The dichloromethane extraction was subjected to repeated column chromatography to afford three new natural sesquiterpenoid derivatives, 1, 2, and 3. Their structures were identified by spectroscopic and chemical methods.

Compound 1 was obtained as a white amorphous powder whose molecular formula was established as C₂₃H₃₄O₅ by HR-ESIMS showing an [M + Na]⁺ ion at m/z 413.2301. The ¹³C NMR spectrum exhibited 23 signals including seven quaternary carbons, six methines, six methylenes, and four methyl groups. The ¹H and ¹³C NMR spectra of 1 ([Table 1]) showed the existence of a 1′, 2′,4′-trisubstituted benzene ring (δ 6.39, 6.38, 7.65), two substituted double bonds (δ 5.12, 5.21), and a ketone (δ 204.7). Spectral data from the 1D NMR, HMQC, and HMBC spectra for compound 1 constructed a skeleton of a dihydroxyphenyl ethyl ketone connected to a farnesyl moiety. This skeleton was the same as that of dshamirone, a compound previously isolated from Ferula [10], [11]. In comparison with dshamirone, compound 1 showed an extra oxygen-bearing methine (δ 3.38) instead of an olefinic proton (ca. δ 5 ppm) as in dshamirone; two ¹³C signals of a double bond, resonating at ca. δ 124.1/CH and 135.1/C [11], were replaced with two oxygen-bearing carbons at δ 78.1 and 75.1 ppm, and a signal for a methyl group at 23.3 ppm was also evident ([Table 1]). Combining the molecular negative ion peak of 389 in its ESI-MS, the differences between the two compounds revealed that compound 1 was a dihydroxy derivative of dshamirone at the double bond ([Fig. 1]). The two hydroxyl groups were located at C-8 (CH) and C-9 (C) by careful inspection of the HMBC data of 1 ([Table 1]), and by reaction of the adjacent hydroxyl groups with a molecule of acetone with p-toluenesulfonic acid (PTSA), compound 1 was converted into 2 ([Fig. 2]). This condensation reaction also elucidated the relative configuration of the adjacent hydroxyl groups (C-8/C-9) as erythro ([Table 1]). This configuration was confirmed by NOESY and ROESY experiments for compounds 1 and 2, in which the signal enhancement of the 9-CH₃ protons was observed with irradiation of H-8. Therefore, compound 1 was identified as 1-(2′,4′-dihydroxy-phenyl)-8,9-dihydroxy-5,9,13-trimethyl-tetradeca-4,12-dien-1-one and was given the trivial name 8,9-dihydroxydshamirone.

Compound 2 was obtained as a colorless colloid by repeated chromatography and exhibited extremely similar physical and spectral properties to those of 1. Comparing with 1, compound 2 showed signals for an extra acetonide group (106.7/C, 26.9/CH₃, 28.4/CH₃) in its ¹H and ¹³C NMR spectra ([Table 1]) rather than the hydroxyl groups; the two signals of two oxygen-bearing carbons, C-8 (78.1 ppm) and C-9 (75.1 ppm), were shifted downfield to δ 83.9 and 81.8 ppm, respectively. Correspondingly, the signals of δ 28.9 (C-7) and δ 35.6 ppm (C-10) of 1 were found to be δ 34.9 and 26.9 ppm in 2, respectively. Compound 2 was elucidated as a combination of compound 1 and a molecule of 2,2-dimethoxypropane via 8-O and 9-O. This connection was confirmed by the HMBC spectrum ([Table 1]) together with the HR-ESIMS which showed an [M + Na]⁺ ion at m/z 453.26123 of compound 2. Therefore, compound 2 was identified as 1-(2′,4′-dihydroxy-phenyl)-5-methyl-7-[2,2,5-trimethyl-5-(4-methyl-pent-3-enyl)-[1, 3]dioxolan-4-yl]-hept-4-en-1-one and was given the trivial name 8,9-oxoisopropanyldshamirone.

As mentioned above, compound 2 was also synthesized from 1 by the chemical transformation [12] ([Fig. 2]), which further confirmed its structural elucidation. We believe that 2 is a natural metabolite, because in the thin-layer chromatography (TLC) and Co-TLC along with freshly extracted plant material, the naturally occurring compound 2 was present. Moreover, compound 1 failed to give 2 when the isolated pure compound 1 was retreated by chromatography with acetone and silica gel for 24 hours.

Compound 3 was obtained as a colorless colloid. Its molecular formula was established as C₂₄H₃₄O₆ by an [M + Na]⁺ ion peak at m/z 441.22564 in the HR-ESIMS. The ¹³C NMR spectrum showed 24 signals including eight quaternary carbons, six methines, five methylenes, and five methyls. The ¹H-NMR and ¹³C-NMR spectra ([Table 2]) of 3 showed the existence of a 1′,2′,4′-trisubstituted benzene ring (δ 6.33, 6.37, 7.60) and a ketone (δ 195.7). The IR absorption of 1752 cm⁻¹ and the spectral signal at δ _C 172.1 revealed a lactone moiety in 3. These physical and spectral characteristics revealed that 3 had the same skeleton as the prenylated-benzoylfuranone compound 4, which has been previously isolated from the same plant [13]. In the sesquiterpene side chain on the structure of compound 3, C-7″ and C-8″ are a methylene and a hydroxyl bearing quaternary carbon (δ 71.8), respectively, instead of having a double bond as in 4. The double-bond configuration between C-3″ and C-4″ in 3 was deduced as E from its ROESY spectrum according to the cross-peaks of H-3″/H-5″. The relative stereochemistry of the dimethyldihydrofuran moiety at C-4 and C-5 were identified as 4R* and 5S* by its ROESY spectrum, in which cross-peaks between H-3/4-CH₃, 5-CH₃ and 4-CH₃/5-CH₃ were observed ([Table 2]). Compound 3 was therefore identified as 3-(2′,4′-dihydroxy-benzoyl)-5-(8″-hydroxy-4″,8″-dimethyl-non-3″(E)-enyl)-4,5-dimethyl-dihydro-furan-2-one and was given the trivial name ferulaeolactone A.

Compounds 1, 2, and 3 were tested for their activity to inhibit drug-resistant S. aureus strains. The three compounds showed significant antibacterial activity with minimum inhibitory concentrations (MICs) in the range of 0.5–128 µg/mL ([Table 3]). Among the tested strains, SA-1199B overexpresses the NorA efflux protein that confers resistance to certain fluoroquinolones. Against this strain, compound 3 (8 µg/mL, 19.1 µM) was more active than the control antibiotic norfloxacin (32 µg/mL, 100.3 µM). For the MDR strain XU212, which possesses the TetK efflux transporter and is resistant to both tetracycline and methicillin, compounds 1 (64 µg/mL, 164.1 µM), 3 (2 µg/mL, 4.8 µM), and especially 2 (0.5 µg/mL, 1.2 µM) showed better inhibitory activity than the control antibiotic tetracycline (128 µg/mL, 288.0 µM). For erythromycin-resistant strain RN4220, compounds 2 (16 µg/mL, 37.2 µM) and 3 (16 µg/mL, 38.3 µM) showed better inhibitory activity than the control antibiotic erythromycin (64 µg/mL, 87.2 µM). For epidemic hospital MRSA strains EMRSA-15 and EMRSA-16, compound 3 showed the inhibitory activity against EMRSA-15 with an MIC of 2 µg/mL (4.8 µM), and compound 2 was active against EMRSA-16 with an MIC of 0.5 µg/mL (1.2 µM) showing stronger inhibitory activity than the control antibiotic oxacillin (> 128 µg/mL, 290.7 µM). The antibiotic vancomycin was employed as a positive control, presenting MIC values of 0.2–0.4 µM (0.25–0.5 µg/mL) for all six strains.

Compounds 1 and 3 showed potential inhibition of ethidium bromide (EtBr) efflux against drug-resistant strains RN4220 (MsrA) and EMRSA-16. In a time-dependent fashion, the fluorescent absorption of intracellular EtBr decreased in the absence of compounds 1 and 3 because of the efflux pump(s) of these strains. In the presence of compounds 1 and 3, the fluorescent absorption of intracellular EtBr decreased slowly within the experimental duration, and the absorption was at a higher level than that in the absence of compounds 1 and 3 ([Fig. 3]). These results indicate that compounds 1 and 3 may inhibit efflux of EtBr from resistant strains RN4220 (MsrA) and EMRSA-16.

To determine whether the isolated compounds were toxic, a cytotoxicity assay using a human embryonic kidney cell line (HEK293) was performed, and the cell viability was measured. Compounds 1 and 3 showed low toxicity for HEK293 cells at 125 µg/mL. Within the 2–32 µg/mL concentration of compounds 1 and 3, the viability of HEK293 was over 100 %; at a concentration of 62.5 µg/mL of 3, the viability of HEK293 was approximately 80 % ([Figs. 4] and [5]). These findings are encouraging and warrant further investigation of the characteristics of these compounds as anti-staphylococcal drug hits.

Materials and Methods

Roots of Ferula ferulaeoides were collected in Xinjiang Province, China. A voucher specimen (F001) was deposited at the Natural Medicine Chemistry Laboratory of the School of Pharmacy, Fudan University. The plant was identified by Mrs. Xie Hui-Qin, Associate Professor in the Department of Plant Protection, Shihezi University, China.

S. aureus ATCC 25923 and tetracycline resistant strain XU212, which possesses the TetK tetracycline efflux protein, were provided by Dr. Edet Udo [14]. Strain SA-1199B, which overexpresses the norA gene encoding the NorA MDR efflux pump, was the kind gift of Professor Glenn W. Kaatz [15]. Strain RN4220 (MsrA) was provided by Dr. Jon Cove [16]. EMRSA-15 and EMRSA-16, commonly occurring methicillin-resistant (MRSA) strains, were also used and were the generous gift of Dr. Paul Stapleton [17].

Efflux studies were carried out as previously described [14], with minor revisions. Cells were grown overnight in cation-adjusted Mueller Hinton broth (CAMHB). Organisms were then diluted into the same medium to an OD₆₀₀ of 0.9. Ethidium loading of cells was accomplished by adding ethidium bromide and reserpine (final concentrations of 10 and 50 µg/mL, respectively). After 30 min of incubation at room temperature, the OD₆₀₀ was adjusted to 0.7 using fresh cold (4 °C) CAMHB containing ethidium bromide and reserpine at the same concentrations. The cells in 1 mL of this suspension were pelleted by centrifugation. Cell pellets were then resuspended in fresh CAMHB containing compounds 1 and 3 (50 µg/mL), respectively, or lacking compounds (blank), and the fluorescence of the suspension was monitored continuously (excitation and emission wavelengths, 530 and 600 nm, respectively) for 60 minutes.

Supporting information

Detailed protocols for the extraction and isolation, minimum inhibitory concentration (MIC) assay, cytotoxicity assay, key HMBC correlations, possible biogenetic pathways, and information referring to the compounds 1–3 are available as Supporting Information.

Acknowledgements

This work was financed by the Royal Society International Joint Project (Sino-UK Joint Project, JP091083/NSFC81011130165) and partly supported by the NSFC grant (21172041) of China. We also thank the support from the Mindao Project for medical graduate students of the Fudan University (MDJH2012010).

Conflict of Interest

The authors declare no conflict of interest.

• References

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Correspondence

• References

• 1 Fisher JF, Meroueh SO, Mobashery S. Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chem Rev 2005; 105: 395-424
  CrossrefPubMedSearch in Google Scholar
• 2 Crowcroft NE, Catchpole M. Mortality from methicillin resistant Staphylococcus aureus in England and Wales: analysis of death certificates. Br Med J 2002; 325: 1390-1391
  CrossrefPubMedSearch in Google Scholar
• 3 Akins RL, Rybak MJ. Bactericidal activities of two daptomycin regimens against clinical strains of glycopeptide intermediate-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and methicillin-resistant Staphylococcus aureus isolates in an in vitro pharmacodynamic model with simulated endocardial vegetations. Antimicrob Agents Chemother 2001; 45: 454-459
  CrossrefPubMedSearch in Google Scholar
• 4 Patel R. Clinical impact of vancomycin-resistant enterococci. J Antimicrob Chemother 2003; 51: 13-21
  PubMedSearch in Google Scholar
• 5 Wesson KM, Lerner DS, Silverberg NB, Weinberg JM. Linezolid, quinupristin/dalfopristin, and daptomycin in dermatology. Dis Mon 2004; 50: 395-406
  CrossrefPubMedSearch in Google Scholar
• 6 Jabrane A, Ben jannet H, Skhiri FH, Miyamoto T, Lacaille-Dubois MA. Phytochemical study from the roots of Ferula lutea L. Planta Med 2008; 74: 1073
  PubMedSearch in Google Scholar
• 7 Nazari ZE, Iranshahi M. Biologically active sesquiterpene coumarins from Ferula species. Phytother Res 2011; 25: 315-323
  PubMedSearch in Google Scholar
• 8 Xinjiang Institute of Biology, Pedology and Desert Research (XIBPDR). Flora of medicinal plant in Xinjiang. Wulumuqi: Xinjiang Peopleʼs Press; 1977: 116
  Search in Google Scholar
• 9 Nagatsu A, Isaka K, Kojima K, Ondognii P, Zevgeegiin O, Gombosurengyin P, Davgiin K, Irfan B, Iqubal CM, Ogihara Y. New sesquiterpenes from Ferula ferulaeoides (Steud.) Korovin. VI. Isolation and identification of three new dihydrofuro[2,3-b]chromones. Chem Pharm Bull 2002; 50: 675-677
  CrossrefPubMedSearch in Google Scholar
• 10 Kamilov KM, Niknov GK. Dshamirone, a new ketone from Ferula dshaudshamyr roots. Khim Prir Soedin 1976; 6: 817-818
  PubMedSearch in Google Scholar
• 11 Kojima K, Isaka K, Purev O, Jargalsaikhan G, Suran D, Mizukami H, Ogihara Y. Sesquiterpenoid derivatives from Ferula ferulioides . Chem Pharm Bull 1998; 46: 1781-1784
  CrossrefPubMedSearch in Google Scholar
• 12 Lange GL, Humber CC, Manthorpe JM. [2 + 2] Photoadditions with chiral 2,5-cyclohexadienone synthons. Tetrahedron Asymm 2002; 13: 1355-1362
  CrossrefPubMedSearch in Google Scholar
• 13 Kojima K, Isaka K, Purev O, Jargalsaikhan G, Suran D, Mizukami H, Ogihara Y. Sesquiterpenoid derivatives from Ferula ferulioides. II. Chem Pharm Bull 1999; 47: 1145-1147
  CrossrefPubMedSearch in Google Scholar
• 14 Gibbons S, Udo EE. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytother Res 2000; 14: 139-140
  CrossrefPubMedSearch in Google Scholar
• 15 Kaatz GW, Seo SM, Ruble CA. Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus . Antimicrob Agents Chemother 1993; 37: 1086-1094
  CrossrefPubMedSearch in Google Scholar
• 16 Ross JI, Farrell AM, Eady EA, Cove JH, Cunliffe WJ. Characterization and molecular cloning of the novel macrolide-streptogramin B resistance determinant from Staphylococcus epidermidis . J Antimicrob Chemother 1989; 24: 851-862
  CrossrefPubMedSearch in Google Scholar
• 17 Richardson JF, Reith S. Characterisation of a strain of methicillin-resistant Staphylococcus aureus (EMRSA-15) by conventional and molecular methods. J Hosp Infect 1993; 25: 45-52
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material