Original Papers
Bioactive Sesquiterpene Coumarins from Ferula pseudalliacea
Authors
Dara Dastan 1, Peyman Salehi 1, Ahmad Reza Gohari 2, Samad Nejad Ebrahimi 1, 3, Atousa Aliahmadi 4, Matthias Hamburger 3
Affiliations
The affiliations are listed at the end of the article
Key words " Ferula pseudalliacea l " Apiaceae l " sesquiterpene coumarin l " TDDFT l " cytotoxicity l " antibacterial l
Abstract !
One new and five known sesquiterpene coumarins were isolated from the roots of Ferula pseudalliacea. The structures were elucidated by 1D and 2D NMR, and HR-ESIMS data as 4′-hydroxy kamolonol acetate (1), kamolonol (2), szowitsiacoumarin A (3), farnesiferon B (4), farnesiferol C (5), and flabellilobin A (6). The absolute configuration of compounds 1, 2, and 4 was established by comparison of experimental and simulated electronic circular dichroism spectra using time
Introduction !
received revised accepted
April 13, 2014 July 1, 2014 July 17, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382996 Published online August 19, 2014 Planta Med 2014; 80: 1118–1123 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Peyman Salehi Department of Phytochemistry Medicinal Plants and Drugs Research Institute Shahid Beheshti University G. C. Evin 1983963113 Tehran Iran Phone: + 98 21 29 90 40 49 Fax: + 98 21 22 43 17 83 [email protected]
The genus Ferula (Apiaceae) counts approx. 180 species and is common throughout Central Asia, the Middle East, and Central Europe [1]. Several species, such as F. gummosa, F. asafetida and F. latisecta have been used in folk medicine to treat colitis in infants, stomachache, and asthma [2, 3]. The genus Ferula is a rich source for biologically active compounds such as coumarins, sesquiterpenes, sesquiterpene coumarins, sesquiterpene lactones, sulfur-containing derivatives, and daucane esters [4–8]. Among these, sesquiterpene coumarins are of particular interest due to their broad range of reported activities, such as antibacterial, anticoagulant, anti-inflammatory, antiviral (anti HIV), spasmolytic, P-glycoprotein (P‑gp) inhibitory, antiprotozoal, and cytotoxic properties [9–16]. Sesquiterpene coumarins are built up of a common coumarin group and a sesquiterpene moiety, therefore more extensive and promising biological properties can be expected from this class of natural compounds. Ferula pseudalliacea Rech. f. is an indigenous species of the Kurdistan (Sanandaj) mountains in western Iran. As part of an ongoing project on the discovery of new and active phytochemicals from the Iranian flora [17–19], extracts from roots
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dependence density function theory. 4′-Hydroxy kamolonol acetate and kamolonol showed antibacterial activity against Heliobacter pylori and Staphylococcus aureus at a concentration of 64 µg/mL. Kamolonol, 4′-hydroxy kamolonol acetate, and farnesiferon B displayed a cytotoxic activity in HeLa cells, with an IC50 of 3.8, 4.5, and 7.7 µM, respectively. Supporting information available online at http://www.thieme-connect.de/products
of F. pseudalliacea were investigated. We previously reported on the isolation and structure elucidation of several sesquiterpene coumarins and the first disesquiterpene coumarin [10]. In continuation of our studies on this species, we investigated an EtOAc extract from roots. We here report on the isolation and structure elucidation of a new and five known sesquiterpene coumarins, and on their in vitro antibacterial and cytotoxic activities. Structures including their absolute configuration were established by a combination of methods, such as 1D and 2D NMR, high-resolution mass spectrometry, and electronic circular dichroism (ECD) spectroscopy.
Results and Discussion !
The EtOAc extract obtained from roots of F. pseudalliacea was separated by normal and reversed phase chromatography to afford a new and five known sesquiterpene coumarins, namely, 4′-hydroxy kamolonol acetate (1), kamolonol (2), szowitsiacoumarin A (3), farnesiferon B (4), farnesi" Fig. 1). The ferol C (5), and flabellilobin A (6) (l molecular formula of C26H32O7 was deduced from 13 C and HRESIMS data (m/z 479.2032 [M + Na]+, calcd. 479.2046). Diagnostic signals in the 1H and
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Table 1
a
1
H and 13C‑NMR spectral data for 1 (CDCl3, J in Hz)a.
Position
δC
2 3 4 5 6 7 8 9 10 1′ 2′
161.0 113.4 143.4 128.6 112.9 162.2 101.7 155.9 112.4 22.5 36.7
3′ 4′ 5′ 6′ 7′
210.8 80.2 48.5 69.2 32.2
8′ 9′ 10′ 11′
35.8 38.8 35.4 75.7
12′ 13′ 14′ 15′ 1′′ 2′′
19.6 19.0 11.4 15.0 170.0 21.8
δH (J in Hz) 6.25, d (9.5) 7.63, d (9.5) 7.37, d (8.5) 6.85, dd (8.5, 2.3) 6.82, d (2.3)
1.82, m 2.32, m α 3.04, m β
Fig. 2 Key COSY, HMBC and NOESY correlations of 1. (Color figure available online only.)
5.59, dd (10.9, 4.8) 1.77, α 1.91, β 1.99 2.9, dd (11.4, 4.2) 3.81, d (9.1) 3.79, d (9.1) 1.14, s 1.18, s 1.00, s 1.13, d (7.2) 2.03, s
δ values were established from HMBC, COSY and HSQC experiments
13
Chemical structures of compounds 1–6.
C NMR spectra suggested a sesquiterpene coumarin. The 13C " Table 1), NMR spectrum of 1 displayed 26 carbon resonances (l assigned to five methyl groups (δc 11.4, 15.0, 19.0, 19.6, and 21.8), three aliphatic methylenes (δc 22.5, 32.2, and 36.7), a primary oxygenated carbon (δc 75.7), a secondary oxygenated carbon (δc 69.2), and ten quaternary carbons including three carbonyl groups (δc 210.8, 170.0, and 161.0). Nine signals were indicative of an umbelliferyl moiety, while the remaining 17 signals suggested a sesquiterpene residue and an acetyl group. Reso-
nances at δH 6.25 (d, J = 9.5 Hz), 6.82 (d, J = 2.3 Hz), 6.85 (dd, J = 8.5, 2.3 Hz), 7.37 (d, J = 8.5 Hz), and 7.63 (d, J = 9.5 Hz) in the 1 H NMR spectrum corroborated the coumarin moiety. Protonated carbons were assigned with the aid of a HSQC spectrum. In the sesquiterpene moiety, the location of carbonyl groups at C-3′ and C-1′′ was established by diagnostic HMBC correlations. Me13′ (δH 1.18), H-2′ eq (δH 2.32), and H-2′ ax (δH 3.04) showed crosspeaks with C-3′ (δC 210.8), while Me-2′′ (δH 2.03) and H-6′ (δH 5.59) were correlated with C-1′′ (δC 170.0). Positions of methyl groups in the sesquiterpene portion, and the linkage of the acetyl, sesquiterpene, and coumarin moieties were also established " Fig. 2). The relative configuration of by HMBC connectivities (l stereocenters at C-4′, C-5′, C-6′, C-8′, C-9′, and C-10′ was deter" Fig. 2). Crosspeaks of H-11′/H-10′, and mined by NOESY data (l H-8′/Me-12′ established a β-orientation for both H-10′ and Me15′, and an α-orientation for Me-12′. Crosspeaks between H-6′, Me-13′, and H-10′ established the α-orientation of Me-14′ and the acetyl group, and a β-orientation of Me-13′. The absolute configuration of 1 was established by comparison of experimental and calculated ECD spectra. The 3D structure of 1 was constructed on the basis of its relative configuration established by NMR and was subjected to conformational search using OPLS2005 molecular mechanics force field. Geometrical optimization and energy calculation using density function theory (DFT) with
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Fig. 1
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Fig. 3 Minimized conformers of 1 using with DFT at B3LYP/6–31 G** level in the gas phase. (Color figure available online only.)
Fig. 4 Experimental (blue) and calculated (black) ECD spectra of compound 1. Calculated spectra were obtained in MeCN using TDDFT at B3LYP/ 6–31 G** level. (Color figure available online only.)
the B3LYP function and 6–31 G** in the gas-phase indicated the " Fig. 3). The key difference of presence of 11 stable conformers (l conformers was in the orientation of the coumarin moiety. Experimental and weighted ECD spectra in MeCN are shown in " Fig. 4. The ECD spectrum of compound 1 showed two negative l Cotton effects (CEs) around 330 and 215 nm, together with a positive CE at 290 nm. The calculated spectrum was in good agreement, and the differences between calculated and experimental spectra most likely resulted from minor differences in solution conformation and computed conformers due to the high flexibility of the compound. Thus, the absolute configuration was established as 4′S,5′S,6′S,8′S,9′S,10′R. Kamolonol (2) had been previously reported from F. asafetida, but no spectral data were recorded at that time [20]. We report here full NMR spectral assignments and determination of the absolute configuration of 2. The relative configuration of 2 was established on the basis of NOESY data. Crosspeaks of H-10′ with H11′ and Me-15′ established an α-orientation for both H-10′ and Me-15′, and a β-orientation for Me-12′. Crosspeaks of H-6′ with H-4′ and H-10′ established β-orientation of Me-13′, Me-14′, and Dastan D et al. Bioactive Sesquiterpene Coumarins …
Fig. 5 Experimental ECD spectra of compound 2 in MeCN. (Color figure available online only.)
the hydroxyl group. The experimental ECD spectrum showed two negative and successive CEs at 323 and 235 nm, two positive CEs at 290 and 210 nm, and a shoulder between 220–230 nm " Fig. 5). The spectrum was in good agreement with previously (l reported data for kamolonol acetate [10], thus the absolute configuration of 2 was similar and established as 4′S,5′R,6′S,8′S,9′ S,10′R. Farnesiferon B (4) had been previously reported from F. flabelliloba, and we attempted to establish its absolute configuration [21]. In the experimental ECD spectrum, two negative CEs around 295 and 210 nm and one positive CE around 325 nm were observed. Since compound 4 possesses one chiral center, two stereoisomers were expected. The 5′S stereoisomer was selected for ECD calculation. Comparison of experimental and calculated ECD are " Fig. 6. The calculated ECD spectrum did not match shown on l with the experimental data, but the mirror-inversion of the computed ECD spectra was in agreement with the experimental data, especially with respect to CEs at higher wavelengths. Thus, the absolute configuration of 4 was established as 5′R.
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Materials and Methods !
Fig. 6 Comparison of experimental and calculated ECD spectra of compound 4. (Color figure available online only.)
Szowitsiacoumarin A (3), farnesiferol C (5) and flabellilobin A (6) had been previously reported from F. szowitsiana [7], F. kopedaghensis [21] and F. flabelliloba [22], respectively. Compounds 1–6 were tested for in vitro antibacterial activity against seven pathogenic bacterial strains, and for cytotoxicity in " Table 2). 4′-Hydroxy kamolothe human HeLa cancer cell line (l nol acetate (1) and kamolonol (2) inhibited growth of Staphylococcus aureus and Heliobacter pylori at a concentration of 64 µg/ mL, whereas the MIC for chloramphenicol and clarythromycin were 4 µg/mL and 16 µg/mL, respectively. The highest cytotoxicity was observed for kamolonol (2), followed by 4′-hydroxy kamolonol acetate (1), farnesiferon B (4), szowitsiacoumarin A (3), flabellilobin A (6), and farnesiferol C (5), respectively. Other sesquiterpene coumarins have previously been reported to possess antiproliferative activity. Interaction with microtubules has been invoked as mechanism of action for ferulenol [23], while farnesiferol C was shown to act via inhibition of vascular endothelial growth factor signaling [16].
Melting points were determined on a melting point apparatus (Electrothermal IA9000). Optical rotation was measured on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV‑PC 2501 spectrometer. IR spectra were measured on a Bruker Tensor 27 FT‑IR spectrometer. ECD spectra were recorded in MeCN with a Chirascan™ CD spectrometer. NMR spectra were recorded at a target temperature of 18 °C on a Bruker Avance III 500 MHz spectrometer operating at 500.13 MHz for 1 H, and 125.77 MHz for 13C. A 1 mm TXI microprobe with z-gradient was used for 1H-detected experiments. 13C NMR spectra were recorded with a 5 mm BBO probe head with z-gradient. Spectra were analyzed using Bruker TopSpin 3.0 software. CDCl3 (100 Atom % D) for NMR was purchased from Armar Chemicals. HR-ESIMS spectra in positive mode were recorded on a Bruker microTOF ESI‑MS system with a scan range of m/z 150–1500. MS calibration was performed using a reference solution of sodium formate 0.1 % in isopropanol-water (1 : 1) containing 5 mM sodium hydroxide. The typical mass accuracy was ± 3 ppm. HyStar 3.0 software (Bruker Daltonics) was used for data acquisition and processing. Semi-preparative HPLC was carried out with an Eurospher 100–7 RP C18 (250 × 20 mm; Macherey Nagel) column with a MeOH‑H2O gradient. Silica gel (70–230 and 230–400 mesh, Merck) and Sephadex LH-20 (25–100 µm, Fluka) were used for column chromatography. Silica TLC was performed on Merck F254 silica gel plates (10 × 10 cm).
Plant material Roots of F. pseudalliacea were collected from Sanandaj (Senadezh) mountains, Kurdistan Province, Iran, in September 2008, at an altitude of 1500 meter. The plant material was identified by Mr. Hossein Maroofi, and a voucher specimen (MPH-1197) was deposited at the Herbarium of the Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Iran.
Table 2 Antibacterial and cytotoxic activities of isolated compounds.
a
Compounds
S. aureus
B. cereus
E. faecium
K. pneumoniae
P. aeruginosa
H. pylori
E. coli
Hela cells
4′-hydroxy kamolonol acetate (1) Kamolonol (2) Szowitsiacoumarin A (3) Farnesiferon B (4) Farnesiferol C (5) Flabellilobin A (6) Chloramphenicol Clarithromycin Paclitaxel
64a
NAb
NA
NA
NA
64
256
4.5 ± 0.1c
64
256
256
NA
NA
64
NA
3.8 ± 0.1
256
NA
256
NA
NA
128
NA
8.4 ± 0.1
NA
128
128
NA
NA
NA
256
7.7 ± 0.2
NA
NA
NA
NA
NA
NA
NA
9.4 ± 0.1
NA
NA
256
NA
NA
NA
NA
9.1 ± 0.2
4
0.5
32
16
32
–
8
–
–
–
–
–
16
– 0.004 ± 0.002
b
c
MIC value are in µg/mL (values are mean of triplicate); NA: not active (MIC > 256 µg/mL); IC50 in µM with standard deviation
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General experimental procedures
Original Papers
Extraction and isolation The dried roots of F. pseudalliacea (1 kg) were powdered and extracted successively with n-hexane (3 × 4 L, rt for 24 h) and EtOAc (3 × 4 L, rt for 24 h) to obtain 28 and 60 g of dry extracts, respectively. A portion (20 g) of EtOAc extract was fractionated on a silica gel column (5 × 70 cm, mesh 70–230) eluted with mixtures of n-hexane-EtOAc (100 : 0, 90 : 10, 80 : 20, 70 : 30, 50 : 50, 20 : 80, 0 : 100, 500 mL each) to afford eight fractions (A1–A8). Fraction A3 (2 g) was further purified on a silica gel column (3 × 70 cm, mesh 230–400) [n-hexane-EtOAc (9 : 1) → (0 : 10)] to afford six fractions (A3.1-A3.6, 50 mL each). Fraction A3.3 (700 mg) was separated on a Sephadex LH-20 column [MeOH‑CHCl3 (8 : 2)] to obtain three fractions (A3.3.1-A3.3.3, 70 mL each). Flabellilobin A (6, 6 mg, 97 %) was obtained from fraction 3.3.2 (250 mg) on a silica gel column (1 × 100 cm, mesh 230–400) [CHCl3-MeOH (9 : 1)] and Sephadex LH-20 column [MeOH]. Silica gel chromatography (1 × 100 cm, mesh 230–400) [CHCl3-MeOH (9 : 1)] of fraction A3.3.3 (300 mg) afforded four fractions (A3.3.3.1–A3.3.3.4, 100 mL each). Sephadex LH-20 column chromatography of fraction A3.3.3.2 (150 mg) with MeOH‑CHCl3 (8 : 2) gave 3 (10 mg, 98%). Semi-preparative RPHPLC (MeOH in H2O (70% to 100 % MeOH in 40 min; flow rate 20 mL/min) of fraction A3.3.3.4 (100 mg) gave 2 (20 mg, > 98 %) and 1 (6 mg, > 98%). Fraction A7 (1 g) was further purified on a Sephadex LH-20 column [MeOH‑CHCl3 (8 : 2)] to obtain seven fractions (A7.1-A7.7, 50 mL each). Farnesiferon B (4, 8 mg, > 98 %) was obtained from fraction A7.2 (300 mg) on a silica gel column (1 × 120 cm, mesh 230–400) [CHCl3-MeOH (9 : 1 to 3 : 7), 10 mL each], Sephadex LH-20 column [MeOH], and semi-preparative RP-HPLC (MeOH in H2O (80% to 100% MeOH in 35 min; flow rate 20 mL/min), respectively. Sephadex LH-20 column chromatography of fraction A7.5 (150 mg) with MeOH gave 5 (12 mg, 98 %).
Computational details Conformational analysis of compounds 1 and 4 were performed with MacroModel 9.1 software (Schrödinger, LLC) using the OPLS 2005 (optimized potential for liquid simulations) force field in H2O. Conformers occurring within a 2 kcal/mol energy window from the global minimum were chosen for geometrical optimization and energy calculation using DFT with the B3LYP functional and the 6–31 G** basis set in the gas-phase with the Gaussian 09 program [24]. Vibrational analysis was done at the same level to confirm minima. TD‑DFT/B3LYP/6–31 G** in MeCN using the selfconsistent reaction field (SCRF) method with the conductor-like polarizable continuum model (CPCM). ECD curves were constructed on the basis of rotational strength; dipole velocity (Rvel) and dipole length (Rlen) were calculated with a half-band of 0.2 eV using SpecDis v1.61 [25].
Antibacterial assay In vitro antibacterial activity of isolated compounds was assessed by determination of their MIC values against S. aureus ATCC 25 923, Bacillus cereus PTCC 1015, clinical isolate of vancomycin resistant Enterococcus faecium [26], β-lactamase producing clinical strain of Klebsiella pneumoniae, Pseudomonas aeruginosa PTCC1430, clinical isolate of H. pylori, and Escherichia coli ATCC 25 922. The standard protocol of CLSI (Clinical Laboratory and Standards Institute) was used with some modifications [27]. The inoculum was prepared from freshly cultured strains by using sterile normal saline and was adjusted to 0.5 McFarland standard turbidity. Further dilutions (1 : 100) were prepared with sterile Mueller-Hinton broth (MHB), immediately prior to adding to the
Dastan D et al. Bioactive Sesquiterpene Coumarins …
wells containing test samples in the final concentration range of 250 to 0.0625 µg/mL. Inoculated trays were incubated for 20 h at 37 °C. MICs were recorded as the lowest concentrations that could inhibit the visible growth of microorganisms. In the case of H. pylori, a clinical strain was included in the study. The isolate was from gastric biopsy. The procedure was the same as other bacteria except that MHB was supplemented by 10 % FBS, and that a bacterial suspension with turbidity equal to 2 McFarland standard units was used for inoculation of wells contacting serial diluted samples. All experiments were done in triplicate.
Evaluation of cytotoxicity Cytotoxicity of compounds was assessed by determination of their IC50s in HeLa-60 cells. The ovarian cancer cell line (HeLa60) was obtained from the Pasteur Institute of Iran and was cultured in RPMI 1640 medium supplemented with 10 % FBS, 100 U/ mL penicillin, and 100 µg/mL streptomycin. Stock solutions of compounds were prepared in DMSO. The final DMSO concentration in the assay was kept below 0.05 %. Cells were incubated at 37 °C with 5 % CO2 in 96-well plates. After incubation for 24 h, cells were treated with the compounds at different concentrations (2.0, 5.0, 10.0, 25.0, 50.0, 100.0 µM). After incubation for 48 h, cell growth was measured by MTT assay. The percentage of cell growth inhibition was calculated as follows: Inhibition % of cancer cells growth = [A–B/A] × 100 where A was optical density of control cancer cells, and B optical density of compound-treated HeLa cells [28].
Spectroscopic data of compounds 4′-Hydroxy kamolonol acetate (1): White amorphous powder; mp 224–225 °C; [α]25 D : – 13.0 (c = 0.2, CHCl3); UV (CHCl3) λmax (log ε) 323 (3.9), 238 (3.5) nm; IR (KBr) νmax 3300, 1720, 1555, 1460, 1220 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, " Table 1; ECD (MeCN, c = 0.9 mM, 0.1 cm path 125 MHz), see l length) [θ]232 = − 2080, [θ]295 = + 1545, [θ]324 = − 2165; HR-ESIMS m/z 479.2032 [M + Na]+ (calcd. for C26H32O7Na, 479.2046). Kamolonol (2): White amorphous powder; mp 215–217 °C; [α]25 D : + 11.7 (c = 0.2, CHCl3); UV (CHCl3) λmax (log ε) 325 (4.0), 238 (3.5) nm; IR (KBr) νmax 3350, 1721, 1610, 1564, 1350, 1200 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.63 (1H, d, J = 9.5 Hz, H-4), 7.37 (1H, d, J = 8.5 Hz, H-5), 6.85 (1H, dd, J = 8.5, 2.3 Hz, H-6), 6.80 (1H, d, J = 2.3 Hz, H-8), 6.25 (1H, d, J = 9.5 Hz, H-3), 3.95 (1H, dd, J = 11.2, 4.5 Hz, H-6′), 3.79–3.76 (2H, d, J = 8.9 Hz, H-11′), 2.46 (1H, H-2′β), 2.52 (1H, m, H-4′), 2.35 (1H, H-2′α), 2.17 (1H, dd, J = 11.6, 4.2 Hz, H-10′), 1.99 (1H, H-8′), 1.91–1.61 (2H, H-7′), 1.26 (2H, m, H-1′), 1.17 (3H, d, J = 6.6 Hz, H-13′), 1.11 (3H, s, H-12′), 1.10 (3H, d, J = 7.2 Hz, H-15′), 0.85 (3H, s, H-14′); 13C NMR (CDCl3, 125 MHz) δ 210.8 (C, C-3′), 162.2 (C, C-7), 161.0 (C, C-2), 155.8 (C, C-9), 143.2 (CH, C-4), 128.6 (CH, C-5), 113.5 (CH, C-3), 112.8 (CH, C-6), 112.4 (C, C-10), 101.6 (CH, C-8), 75.6 (CH2, C-11′), 72.8 (CH, C-6′), 58.4 (CH, C-4′), 47.3 (C, C-5′), 43.7 (CH, C-10′), 41.4 (CH2, C-2′), 39.8 (C, C-9′), 36.6 (CH, C-8′), 36.5 (CH2, C-7′), 29.6 (CH2, C-1′), 19.7 (CH3, C-12′), 15.6 (CH3, C-15′), 10.4 (CH3, C-13′), 9.2 (CH3, C14′); ECD (MeCN, c = 0.5 mM, 0.1 cm path length) [θ]238 = − 1390, [θ]292 = + 5539, [θ]328 = − 2023; HR-ESIMS m/z 421.1954 [M + Na]+ (calcd. for C24H30O5Na, 421.1991). Farnesiferon B (4): White amorphous powder; mp 102–103 °C; [α]25 D : − 36.0 (c = 0.2, CHCl3); UV (CHCl3) λmax (log ε) 324 (4.0), 292 (3.7), 245 (3.5) nm; IR (KBr) νmax 1725, 1660, 1615, 1550, 1210 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.64 (1H, d, J = 9.5 Hz, H-
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4), 7.37 (1H, d, J = 8.5 Hz, H-5), 6.84 (1H, dd, J = 8.5, 2.3 Hz, H-6), 6.81 (1H, d, J = 2.3 Hz, H-8), 6.25 (1H, d, J = 9.5 Hz, H-3), 5.43 (1H, t, J = 8.5 Hz, H-10′), 5.03–4.83 (2H, brs, H-15′), 4.59 (2H, d, J = 6.5 Hz, H-11′), 2.62 (1H, dt, J = 13.7, 10.6 Hz, H-2′β), 2.46 (2H, dd, J = 9.9, 4.3 Hz, H-1′), 2.30 (1H, dt, J = 14.0, 4.4 Hz, H-2′α), 2.13 (1H, dd, J = 12.1, 3.8 Hz, H-5′), 1.99–1.85 (2H, m, H-8′), 1.71 (3H, s, H-12′), 1.69 (2H, m, H-7′), 1.19 (3H, s, H-13′), 1.04 (3H, s, H-14′); 13 C NMR (CDCl3, 125 MHz) δ 215.1 (C, C-3′), 162.0 (C, C-7), 161.2 (C, C-2), 155.8 (C, C-9), 144.6 (C, C-6′), 143.4 (CH, C-4), 141.9 (C, C9′), 128.6 (CH, C-5), 118.7 (CH, C-10′), 113.5 (CH2, C-15′), 113.3 (CH, C-6), 112.9 (CH, C-3), 112.4 (C, C-10), 101.5 (CH, C-8), 65.3 (CH2, C-11′), 55.9 (CH, C-5′), 49.0 (C, C-4′), 37.6 (CH2, C-8′), 37.2 (CH2, C-2′), 30.6 (CH2, C-2′), 27.1 (CH3, C-14′), 25.4 (CH2, C-7′), 21.3 (CH3, C-13′), 16.8 (CH3, C-12′); ECD (MeCN, c = 0.7 mM, 0.1 cm path length) [θ]226 = − 2729, [θ]288 = − 1357, [θ]326 = + 1340; HR-ESIMS m/z 403.1894 [M + Na]+ (calcd. for C24H28O4Na, 403.1885).
Supporting information 1D and 2D NMR spectra for compounds 1, 2, and 4 are available as Supporting Information.
Acknowledgments !
We are grateful to Shahid Beheshti University Research Council and Iran National Science Foundation (INSF; Grant No. 86023.07) for financial support of this work. ECD spectra were measured at the Biophysics Facility, Biozentrum, University of Basel.
Conflict of Interest !
The authors declare no conflict of interest.
Affiliations 1
2
3 4
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C. Evin, Tehran, Iran Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Division of Pharmaceutical Biology, University of Basel, Basel, Switzerland Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C. Evin, Tehran, Iran
References 1 Pimenov MG, Leonov MV. The Asian Umbelliferae biodiversity database (ASIUM) with particular reference to South-West Asian taxa. Turk J Bot 2004; 28: 139–145 2 Zargari A. Medicinal plants, Vol 2. Tehran: Tehran University Publications; 1997: 602 3 Eigner D, Scholz D. Ferula asa-foetida and Curcuma longa in traditional medical treatment and diet in Nepal. J Ethnopharmacol 1999; 67: 1–6 4 Kapoor LD. Handbook of Ayurvedic medicinal plants. Boca Raton: CRC Press; 1990: 184 5 Kajimoto T, Yahiro K, Nohara T. Sesquiterpenoid and disulphide derivatives from Ferula assa-foetida. Phytochemistry 1998; 28: 1761–1763 6 Abd El-Razek MH, Ohta S, Ahmed AA, Hirata T. Sesquiterpene coumarins from the roots of Ferula assa-foetida. Phytochemistry 2001; 58: 1289– 1295 7 Iranshahi M, Arfa P, Ramezani M, Jaafari MR, Sadeghian H, Bassarello C, Piacente S, Pizza C. Sesquiterpene coumarins from Ferula szowitsiana and in vitro antileishmanial activity of 7-prenyloxycoumarins against promastigotes. Phytochemistry 2007; 68: 554–561 8 Kanani MR, Rahiminejad MR, Sonboli A, Mozaffarian V, Kazempour Osaloo S, Ebrahimi SN. Chemotaxonomic significance of the essential oils of 18 Ferula species (Apiaceae) from Iran. Chem Biodivers 2011; 8: 503– 517 9 Abd El-Razek MH, Ohta S, Hirataa T. Terpenoid coumarins of the genus Ferula. Heterocycles 2003; 60: 689–716
10 Dastan D, Salehi P, Gohari AR, Zimmermann S, Kaiser M, Hamburger M, Khavasi HR, Ebrahimi SN. Disesquiterpene and sesquiterpene coumarins from Ferula pseudalliacea, and determination of their absolute configurations. Phytochemistry 2012; 78: 170–178 11 Nazari ZE, Iranshahi M. Biologically active sesquiterpene coumarins from Ferula Species. Phytother Res 2011; 25: 315–323 12 Shahverdi AR, Saadat F, Khorramizadeh MR, Iranshahi M, Khoshayand MR. Two matrix metalloproteinases inhibitors from Ferula persica var. persica. Phytomedicine 2006; 13: 712–717 13 Shahverdi AR, Fakhimi A, Zarrini G, Dehghan G, Iranshahi M. Galbanic acid from Ferula szowitsiana enhanced the antibacterial activity of penicillin G and cephalexin against Staphylococcus aureus. Biol Pharm Bull 2007; 30: 1805–1807 14 Appendino G, Mercalli E, Fuzzati N, Arnoldi L, Stavri M, Gibbons S, Ballero M, Maxia A. Antimycobacterial coumarins from the Sardinian giant fennel (Ferula communis). J Nat Prod 2004; 67: 2108–2110 15 Barthomeuf C, Lima S, Iranshahi M, Chollet P. Umbelliprenin from Ferula szowitsiana inhibits the growth of human M4Beu metastatic pigmented malignant melanoma cells through cell-cycle arrest in G1 and induction of caspase dependent apoptosis. Phytomedicine 2008; 15: 103–111 16 Lee JH, Choi S, Lee Y. Herbal compound farnesiferol C exerts antiangiogenic and antitumor activity and targets multiple aspects of VEGFR1 (Flt1) or VEGFR2 (Flk1) signaling cascades. Mol Cancer Ther 2010; 9: 389–399 17 Farimani M, Ebrahimi SN, Salehi P, Bahadori BM, Sonboli A, Khavasi HR, Zimmermann S, Kaiser M, Hamburger M. A novel triterpenoid with a εlactone in ring E, from Salvia urmiensis. J Nat Prod 2013; 76: 1806– 1809 18 Ebrahimi SN, Zimmermann S, Zaugg J, Smiekso M, Brun R, Hamburger M. Abietane diterpenoids from Salvia sahendica – Antiprotozoal activity and determination of their absolute configurations. Planta Med 2013; 79: 150–156 19 Ayyari M, Salehi P, Ebrahimi SN, Zimmermann S, Portmann L, KrauthSiegel RL, Kaiser M, Brun R, Rezadoost H, Rezazadeh S, Hamburger M. Antitrypanosomal isothiocyanate and thiocarbamate glycosides from Moringa peregrine. Planta Med 2014; 80: 86–89 20 Hofer O, Widhalm M, Greper H. Circular dichroism of sesquiterpene umbelliferone ethers and structure elucidation of a new derivative isolated from the gum resin assa-foetida. Monatsh Chem 1984; 115: 1207–1218 21 Nabiev AA, Khassanov TK, Malikov VM. A new terpenoid coumarin from Ferula kopedaghensis. Khim Prir Soedin 1982; 18: 48–50 22 Iranshahi M, Kalategi F, Sahebkar A, Sardashti A, Schneider B. New sesquiterpene coumarins from the roots of Ferula flabelliloba. Pharm Biol 2010; 48: 217–220 23 Bocca C, Gabriel L, Bozzo F, Miglietta A. Microtubule interacting activity and cytotoxicity of prenylated coumarin ferulenol. Planta Med 2002; 68: 1135–1138 24 Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian 09, Revision A.02. Wallingford: Gaussian Inc.; 2009 25 Bruhn T, Schaumlöffel A, Hemberger Y, Bringmann G. SpecDis version 1.61. Würzburg: University of Würzburg; 2013 26 Feizabadi MM, Asadi S, Aliahmadi A, Parvin M, Parastan R, Shayegh M, Etemadi G. Drug resistant patterns of enterococci recovered from patients in Tehran during 2000–2003. Int J Antimicrob Agents 2004; 24: 521–522 27 Jorgensen JH, Turnidge JD. Manual of clinical microbiology, 9th edition. Washington DC: ASM Press; 2007: 1152–1172 28 Meng H, Li G, Huang J, Zhang K, Wang H, Wang J. Sesquiterpene coumarin and sesquiterpene chromone derivatives from Ferula ferulaeoides (Steud.) Korov. Fitoterapia 2013; 86: 70–77
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Original Papers
Bioactive Sesquiterpene Coumarins from Ferula pseudalliacea
Authors
Dara Dastan 1, Peyman Salehi 1, Ahmad Reza Gohari 2, Samad Nejad Ebrahimi 1, 3, Atousa Aliahmadi 4, Matthias Hamburger 3
Affiliations
The affiliations are listed at the end of the article
Key words " Ferula pseudalliacea l " Apiaceae l " sesquiterpene coumarin l " TDDFT l " cytotoxicity l " antibacterial l
Abstract !
One new and five known sesquiterpene coumarins were isolated from the roots of Ferula pseudalliacea. The structures were elucidated by 1D and 2D NMR, and HR-ESIMS data as 4′-hydroxy kamolonol acetate (1), kamolonol (2), szowitsiacoumarin A (3), farnesiferon B (4), farnesiferol C (5), and flabellilobin A (6). The absolute configuration of compounds 1, 2, and 4 was established by comparison of experimental and simulated electronic circular dichroism spectra using time
Introduction !
received revised accepted
April 13, 2014 July 1, 2014 July 17, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382996 Published online August 19, 2014 Planta Med 2014; 80: 1118–1123 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Peyman Salehi Department of Phytochemistry Medicinal Plants and Drugs Research Institute Shahid Beheshti University G. C. Evin 1983963113 Tehran Iran Phone: + 98 21 29 90 40 49 Fax: + 98 21 22 43 17 83 [email protected]
The genus Ferula (Apiaceae) counts approx. 180 species and is common throughout Central Asia, the Middle East, and Central Europe [1]. Several species, such as F. gummosa, F. asafetida and F. latisecta have been used in folk medicine to treat colitis in infants, stomachache, and asthma [2, 3]. The genus Ferula is a rich source for biologically active compounds such as coumarins, sesquiterpenes, sesquiterpene coumarins, sesquiterpene lactones, sulfur-containing derivatives, and daucane esters [4–8]. Among these, sesquiterpene coumarins are of particular interest due to their broad range of reported activities, such as antibacterial, anticoagulant, anti-inflammatory, antiviral (anti HIV), spasmolytic, P-glycoprotein (P‑gp) inhibitory, antiprotozoal, and cytotoxic properties [9–16]. Sesquiterpene coumarins are built up of a common coumarin group and a sesquiterpene moiety, therefore more extensive and promising biological properties can be expected from this class of natural compounds. Ferula pseudalliacea Rech. f. is an indigenous species of the Kurdistan (Sanandaj) mountains in western Iran. As part of an ongoing project on the discovery of new and active phytochemicals from the Iranian flora [17–19], extracts from roots
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dependence density function theory. 4′-Hydroxy kamolonol acetate and kamolonol showed antibacterial activity against Heliobacter pylori and Staphylococcus aureus at a concentration of 64 µg/mL. Kamolonol, 4′-hydroxy kamolonol acetate, and farnesiferon B displayed a cytotoxic activity in HeLa cells, with an IC50 of 3.8, 4.5, and 7.7 µM, respectively. Supporting information available online at http://www.thieme-connect.de/products
of F. pseudalliacea were investigated. We previously reported on the isolation and structure elucidation of several sesquiterpene coumarins and the first disesquiterpene coumarin [10]. In continuation of our studies on this species, we investigated an EtOAc extract from roots. We here report on the isolation and structure elucidation of a new and five known sesquiterpene coumarins, and on their in vitro antibacterial and cytotoxic activities. Structures including their absolute configuration were established by a combination of methods, such as 1D and 2D NMR, high-resolution mass spectrometry, and electronic circular dichroism (ECD) spectroscopy.
Results and Discussion !
The EtOAc extract obtained from roots of F. pseudalliacea was separated by normal and reversed phase chromatography to afford a new and five known sesquiterpene coumarins, namely, 4′-hydroxy kamolonol acetate (1), kamolonol (2), szowitsiacoumarin A (3), farnesiferon B (4), farnesi" Fig. 1). The ferol C (5), and flabellilobin A (6) (l molecular formula of C26H32O7 was deduced from 13 C and HRESIMS data (m/z 479.2032 [M + Na]+, calcd. 479.2046). Diagnostic signals in the 1H and
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Table 1
a
1
H and 13C‑NMR spectral data for 1 (CDCl3, J in Hz)a.
Position
δC
2 3 4 5 6 7 8 9 10 1′ 2′
161.0 113.4 143.4 128.6 112.9 162.2 101.7 155.9 112.4 22.5 36.7
3′ 4′ 5′ 6′ 7′
210.8 80.2 48.5 69.2 32.2
8′ 9′ 10′ 11′
35.8 38.8 35.4 75.7
12′ 13′ 14′ 15′ 1′′ 2′′
19.6 19.0 11.4 15.0 170.0 21.8
δH (J in Hz) 6.25, d (9.5) 7.63, d (9.5) 7.37, d (8.5) 6.85, dd (8.5, 2.3) 6.82, d (2.3)
1.82, m 2.32, m α 3.04, m β
Fig. 2 Key COSY, HMBC and NOESY correlations of 1. (Color figure available online only.)
5.59, dd (10.9, 4.8) 1.77, α 1.91, β 1.99 2.9, dd (11.4, 4.2) 3.81, d (9.1) 3.79, d (9.1) 1.14, s 1.18, s 1.00, s 1.13, d (7.2) 2.03, s
δ values were established from HMBC, COSY and HSQC experiments
13
Chemical structures of compounds 1–6.
C NMR spectra suggested a sesquiterpene coumarin. The 13C " Table 1), NMR spectrum of 1 displayed 26 carbon resonances (l assigned to five methyl groups (δc 11.4, 15.0, 19.0, 19.6, and 21.8), three aliphatic methylenes (δc 22.5, 32.2, and 36.7), a primary oxygenated carbon (δc 75.7), a secondary oxygenated carbon (δc 69.2), and ten quaternary carbons including three carbonyl groups (δc 210.8, 170.0, and 161.0). Nine signals were indicative of an umbelliferyl moiety, while the remaining 17 signals suggested a sesquiterpene residue and an acetyl group. Reso-
nances at δH 6.25 (d, J = 9.5 Hz), 6.82 (d, J = 2.3 Hz), 6.85 (dd, J = 8.5, 2.3 Hz), 7.37 (d, J = 8.5 Hz), and 7.63 (d, J = 9.5 Hz) in the 1 H NMR spectrum corroborated the coumarin moiety. Protonated carbons were assigned with the aid of a HSQC spectrum. In the sesquiterpene moiety, the location of carbonyl groups at C-3′ and C-1′′ was established by diagnostic HMBC correlations. Me13′ (δH 1.18), H-2′ eq (δH 2.32), and H-2′ ax (δH 3.04) showed crosspeaks with C-3′ (δC 210.8), while Me-2′′ (δH 2.03) and H-6′ (δH 5.59) were correlated with C-1′′ (δC 170.0). Positions of methyl groups in the sesquiterpene portion, and the linkage of the acetyl, sesquiterpene, and coumarin moieties were also established " Fig. 2). The relative configuration of by HMBC connectivities (l stereocenters at C-4′, C-5′, C-6′, C-8′, C-9′, and C-10′ was deter" Fig. 2). Crosspeaks of H-11′/H-10′, and mined by NOESY data (l H-8′/Me-12′ established a β-orientation for both H-10′ and Me15′, and an α-orientation for Me-12′. Crosspeaks between H-6′, Me-13′, and H-10′ established the α-orientation of Me-14′ and the acetyl group, and a β-orientation of Me-13′. The absolute configuration of 1 was established by comparison of experimental and calculated ECD spectra. The 3D structure of 1 was constructed on the basis of its relative configuration established by NMR and was subjected to conformational search using OPLS2005 molecular mechanics force field. Geometrical optimization and energy calculation using density function theory (DFT) with
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Fig. 1
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Fig. 3 Minimized conformers of 1 using with DFT at B3LYP/6–31 G** level in the gas phase. (Color figure available online only.)
Fig. 4 Experimental (blue) and calculated (black) ECD spectra of compound 1. Calculated spectra were obtained in MeCN using TDDFT at B3LYP/ 6–31 G** level. (Color figure available online only.)
the B3LYP function and 6–31 G** in the gas-phase indicated the " Fig. 3). The key difference of presence of 11 stable conformers (l conformers was in the orientation of the coumarin moiety. Experimental and weighted ECD spectra in MeCN are shown in " Fig. 4. The ECD spectrum of compound 1 showed two negative l Cotton effects (CEs) around 330 and 215 nm, together with a positive CE at 290 nm. The calculated spectrum was in good agreement, and the differences between calculated and experimental spectra most likely resulted from minor differences in solution conformation and computed conformers due to the high flexibility of the compound. Thus, the absolute configuration was established as 4′S,5′S,6′S,8′S,9′S,10′R. Kamolonol (2) had been previously reported from F. asafetida, but no spectral data were recorded at that time [20]. We report here full NMR spectral assignments and determination of the absolute configuration of 2. The relative configuration of 2 was established on the basis of NOESY data. Crosspeaks of H-10′ with H11′ and Me-15′ established an α-orientation for both H-10′ and Me-15′, and a β-orientation for Me-12′. Crosspeaks of H-6′ with H-4′ and H-10′ established β-orientation of Me-13′, Me-14′, and Dastan D et al. Bioactive Sesquiterpene Coumarins …
Fig. 5 Experimental ECD spectra of compound 2 in MeCN. (Color figure available online only.)
the hydroxyl group. The experimental ECD spectrum showed two negative and successive CEs at 323 and 235 nm, two positive CEs at 290 and 210 nm, and a shoulder between 220–230 nm " Fig. 5). The spectrum was in good agreement with previously (l reported data for kamolonol acetate [10], thus the absolute configuration of 2 was similar and established as 4′S,5′R,6′S,8′S,9′ S,10′R. Farnesiferon B (4) had been previously reported from F. flabelliloba, and we attempted to establish its absolute configuration [21]. In the experimental ECD spectrum, two negative CEs around 295 and 210 nm and one positive CE around 325 nm were observed. Since compound 4 possesses one chiral center, two stereoisomers were expected. The 5′S stereoisomer was selected for ECD calculation. Comparison of experimental and calculated ECD are " Fig. 6. The calculated ECD spectrum did not match shown on l with the experimental data, but the mirror-inversion of the computed ECD spectra was in agreement with the experimental data, especially with respect to CEs at higher wavelengths. Thus, the absolute configuration of 4 was established as 5′R.
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Materials and Methods !
Fig. 6 Comparison of experimental and calculated ECD spectra of compound 4. (Color figure available online only.)
Szowitsiacoumarin A (3), farnesiferol C (5) and flabellilobin A (6) had been previously reported from F. szowitsiana [7], F. kopedaghensis [21] and F. flabelliloba [22], respectively. Compounds 1–6 were tested for in vitro antibacterial activity against seven pathogenic bacterial strains, and for cytotoxicity in " Table 2). 4′-Hydroxy kamolothe human HeLa cancer cell line (l nol acetate (1) and kamolonol (2) inhibited growth of Staphylococcus aureus and Heliobacter pylori at a concentration of 64 µg/ mL, whereas the MIC for chloramphenicol and clarythromycin were 4 µg/mL and 16 µg/mL, respectively. The highest cytotoxicity was observed for kamolonol (2), followed by 4′-hydroxy kamolonol acetate (1), farnesiferon B (4), szowitsiacoumarin A (3), flabellilobin A (6), and farnesiferol C (5), respectively. Other sesquiterpene coumarins have previously been reported to possess antiproliferative activity. Interaction with microtubules has been invoked as mechanism of action for ferulenol [23], while farnesiferol C was shown to act via inhibition of vascular endothelial growth factor signaling [16].
Melting points were determined on a melting point apparatus (Electrothermal IA9000). Optical rotation was measured on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV‑PC 2501 spectrometer. IR spectra were measured on a Bruker Tensor 27 FT‑IR spectrometer. ECD spectra were recorded in MeCN with a Chirascan™ CD spectrometer. NMR spectra were recorded at a target temperature of 18 °C on a Bruker Avance III 500 MHz spectrometer operating at 500.13 MHz for 1 H, and 125.77 MHz for 13C. A 1 mm TXI microprobe with z-gradient was used for 1H-detected experiments. 13C NMR spectra were recorded with a 5 mm BBO probe head with z-gradient. Spectra were analyzed using Bruker TopSpin 3.0 software. CDCl3 (100 Atom % D) for NMR was purchased from Armar Chemicals. HR-ESIMS spectra in positive mode were recorded on a Bruker microTOF ESI‑MS system with a scan range of m/z 150–1500. MS calibration was performed using a reference solution of sodium formate 0.1 % in isopropanol-water (1 : 1) containing 5 mM sodium hydroxide. The typical mass accuracy was ± 3 ppm. HyStar 3.0 software (Bruker Daltonics) was used for data acquisition and processing. Semi-preparative HPLC was carried out with an Eurospher 100–7 RP C18 (250 × 20 mm; Macherey Nagel) column with a MeOH‑H2O gradient. Silica gel (70–230 and 230–400 mesh, Merck) and Sephadex LH-20 (25–100 µm, Fluka) were used for column chromatography. Silica TLC was performed on Merck F254 silica gel plates (10 × 10 cm).
Plant material Roots of F. pseudalliacea were collected from Sanandaj (Senadezh) mountains, Kurdistan Province, Iran, in September 2008, at an altitude of 1500 meter. The plant material was identified by Mr. Hossein Maroofi, and a voucher specimen (MPH-1197) was deposited at the Herbarium of the Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Iran.
Table 2 Antibacterial and cytotoxic activities of isolated compounds.
a
Compounds
S. aureus
B. cereus
E. faecium
K. pneumoniae
P. aeruginosa
H. pylori
E. coli
Hela cells
4′-hydroxy kamolonol acetate (1) Kamolonol (2) Szowitsiacoumarin A (3) Farnesiferon B (4) Farnesiferol C (5) Flabellilobin A (6) Chloramphenicol Clarithromycin Paclitaxel
64a
NAb
NA
NA
NA
64
256
4.5 ± 0.1c
64
256
256
NA
NA
64
NA
3.8 ± 0.1
256
NA
256
NA
NA
128
NA
8.4 ± 0.1
NA
128
128
NA
NA
NA
256
7.7 ± 0.2
NA
NA
NA
NA
NA
NA
NA
9.4 ± 0.1
NA
NA
256
NA
NA
NA
NA
9.1 ± 0.2
4
0.5
32
16
32
–
8
–
–
–
–
–
16
– 0.004 ± 0.002
b
c
MIC value are in µg/mL (values are mean of triplicate); NA: not active (MIC > 256 µg/mL); IC50 in µM with standard deviation
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General experimental procedures
Original Papers
Extraction and isolation The dried roots of F. pseudalliacea (1 kg) were powdered and extracted successively with n-hexane (3 × 4 L, rt for 24 h) and EtOAc (3 × 4 L, rt for 24 h) to obtain 28 and 60 g of dry extracts, respectively. A portion (20 g) of EtOAc extract was fractionated on a silica gel column (5 × 70 cm, mesh 70–230) eluted with mixtures of n-hexane-EtOAc (100 : 0, 90 : 10, 80 : 20, 70 : 30, 50 : 50, 20 : 80, 0 : 100, 500 mL each) to afford eight fractions (A1–A8). Fraction A3 (2 g) was further purified on a silica gel column (3 × 70 cm, mesh 230–400) [n-hexane-EtOAc (9 : 1) → (0 : 10)] to afford six fractions (A3.1-A3.6, 50 mL each). Fraction A3.3 (700 mg) was separated on a Sephadex LH-20 column [MeOH‑CHCl3 (8 : 2)] to obtain three fractions (A3.3.1-A3.3.3, 70 mL each). Flabellilobin A (6, 6 mg, 97 %) was obtained from fraction 3.3.2 (250 mg) on a silica gel column (1 × 100 cm, mesh 230–400) [CHCl3-MeOH (9 : 1)] and Sephadex LH-20 column [MeOH]. Silica gel chromatography (1 × 100 cm, mesh 230–400) [CHCl3-MeOH (9 : 1)] of fraction A3.3.3 (300 mg) afforded four fractions (A3.3.3.1–A3.3.3.4, 100 mL each). Sephadex LH-20 column chromatography of fraction A3.3.3.2 (150 mg) with MeOH‑CHCl3 (8 : 2) gave 3 (10 mg, 98%). Semi-preparative RPHPLC (MeOH in H2O (70% to 100 % MeOH in 40 min; flow rate 20 mL/min) of fraction A3.3.3.4 (100 mg) gave 2 (20 mg, > 98 %) and 1 (6 mg, > 98%). Fraction A7 (1 g) was further purified on a Sephadex LH-20 column [MeOH‑CHCl3 (8 : 2)] to obtain seven fractions (A7.1-A7.7, 50 mL each). Farnesiferon B (4, 8 mg, > 98 %) was obtained from fraction A7.2 (300 mg) on a silica gel column (1 × 120 cm, mesh 230–400) [CHCl3-MeOH (9 : 1 to 3 : 7), 10 mL each], Sephadex LH-20 column [MeOH], and semi-preparative RP-HPLC (MeOH in H2O (80% to 100% MeOH in 35 min; flow rate 20 mL/min), respectively. Sephadex LH-20 column chromatography of fraction A7.5 (150 mg) with MeOH gave 5 (12 mg, 98 %).
Computational details Conformational analysis of compounds 1 and 4 were performed with MacroModel 9.1 software (Schrödinger, LLC) using the OPLS 2005 (optimized potential for liquid simulations) force field in H2O. Conformers occurring within a 2 kcal/mol energy window from the global minimum were chosen for geometrical optimization and energy calculation using DFT with the B3LYP functional and the 6–31 G** basis set in the gas-phase with the Gaussian 09 program [24]. Vibrational analysis was done at the same level to confirm minima. TD‑DFT/B3LYP/6–31 G** in MeCN using the selfconsistent reaction field (SCRF) method with the conductor-like polarizable continuum model (CPCM). ECD curves were constructed on the basis of rotational strength; dipole velocity (Rvel) and dipole length (Rlen) were calculated with a half-band of 0.2 eV using SpecDis v1.61 [25].
Antibacterial assay In vitro antibacterial activity of isolated compounds was assessed by determination of their MIC values against S. aureus ATCC 25 923, Bacillus cereus PTCC 1015, clinical isolate of vancomycin resistant Enterococcus faecium [26], β-lactamase producing clinical strain of Klebsiella pneumoniae, Pseudomonas aeruginosa PTCC1430, clinical isolate of H. pylori, and Escherichia coli ATCC 25 922. The standard protocol of CLSI (Clinical Laboratory and Standards Institute) was used with some modifications [27]. The inoculum was prepared from freshly cultured strains by using sterile normal saline and was adjusted to 0.5 McFarland standard turbidity. Further dilutions (1 : 100) were prepared with sterile Mueller-Hinton broth (MHB), immediately prior to adding to the
Dastan D et al. Bioactive Sesquiterpene Coumarins …
wells containing test samples in the final concentration range of 250 to 0.0625 µg/mL. Inoculated trays were incubated for 20 h at 37 °C. MICs were recorded as the lowest concentrations that could inhibit the visible growth of microorganisms. In the case of H. pylori, a clinical strain was included in the study. The isolate was from gastric biopsy. The procedure was the same as other bacteria except that MHB was supplemented by 10 % FBS, and that a bacterial suspension with turbidity equal to 2 McFarland standard units was used for inoculation of wells contacting serial diluted samples. All experiments were done in triplicate.
Evaluation of cytotoxicity Cytotoxicity of compounds was assessed by determination of their IC50s in HeLa-60 cells. The ovarian cancer cell line (HeLa60) was obtained from the Pasteur Institute of Iran and was cultured in RPMI 1640 medium supplemented with 10 % FBS, 100 U/ mL penicillin, and 100 µg/mL streptomycin. Stock solutions of compounds were prepared in DMSO. The final DMSO concentration in the assay was kept below 0.05 %. Cells were incubated at 37 °C with 5 % CO2 in 96-well plates. After incubation for 24 h, cells were treated with the compounds at different concentrations (2.0, 5.0, 10.0, 25.0, 50.0, 100.0 µM). After incubation for 48 h, cell growth was measured by MTT assay. The percentage of cell growth inhibition was calculated as follows: Inhibition % of cancer cells growth = [A–B/A] × 100 where A was optical density of control cancer cells, and B optical density of compound-treated HeLa cells [28].
Spectroscopic data of compounds 4′-Hydroxy kamolonol acetate (1): White amorphous powder; mp 224–225 °C; [α]25 D : – 13.0 (c = 0.2, CHCl3); UV (CHCl3) λmax (log ε) 323 (3.9), 238 (3.5) nm; IR (KBr) νmax 3300, 1720, 1555, 1460, 1220 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, " Table 1; ECD (MeCN, c = 0.9 mM, 0.1 cm path 125 MHz), see l length) [θ]232 = − 2080, [θ]295 = + 1545, [θ]324 = − 2165; HR-ESIMS m/z 479.2032 [M + Na]+ (calcd. for C26H32O7Na, 479.2046). Kamolonol (2): White amorphous powder; mp 215–217 °C; [α]25 D : + 11.7 (c = 0.2, CHCl3); UV (CHCl3) λmax (log ε) 325 (4.0), 238 (3.5) nm; IR (KBr) νmax 3350, 1721, 1610, 1564, 1350, 1200 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.63 (1H, d, J = 9.5 Hz, H-4), 7.37 (1H, d, J = 8.5 Hz, H-5), 6.85 (1H, dd, J = 8.5, 2.3 Hz, H-6), 6.80 (1H, d, J = 2.3 Hz, H-8), 6.25 (1H, d, J = 9.5 Hz, H-3), 3.95 (1H, dd, J = 11.2, 4.5 Hz, H-6′), 3.79–3.76 (2H, d, J = 8.9 Hz, H-11′), 2.46 (1H, H-2′β), 2.52 (1H, m, H-4′), 2.35 (1H, H-2′α), 2.17 (1H, dd, J = 11.6, 4.2 Hz, H-10′), 1.99 (1H, H-8′), 1.91–1.61 (2H, H-7′), 1.26 (2H, m, H-1′), 1.17 (3H, d, J = 6.6 Hz, H-13′), 1.11 (3H, s, H-12′), 1.10 (3H, d, J = 7.2 Hz, H-15′), 0.85 (3H, s, H-14′); 13C NMR (CDCl3, 125 MHz) δ 210.8 (C, C-3′), 162.2 (C, C-7), 161.0 (C, C-2), 155.8 (C, C-9), 143.2 (CH, C-4), 128.6 (CH, C-5), 113.5 (CH, C-3), 112.8 (CH, C-6), 112.4 (C, C-10), 101.6 (CH, C-8), 75.6 (CH2, C-11′), 72.8 (CH, C-6′), 58.4 (CH, C-4′), 47.3 (C, C-5′), 43.7 (CH, C-10′), 41.4 (CH2, C-2′), 39.8 (C, C-9′), 36.6 (CH, C-8′), 36.5 (CH2, C-7′), 29.6 (CH2, C-1′), 19.7 (CH3, C-12′), 15.6 (CH3, C-15′), 10.4 (CH3, C-13′), 9.2 (CH3, C14′); ECD (MeCN, c = 0.5 mM, 0.1 cm path length) [θ]238 = − 1390, [θ]292 = + 5539, [θ]328 = − 2023; HR-ESIMS m/z 421.1954 [M + Na]+ (calcd. for C24H30O5Na, 421.1991). Farnesiferon B (4): White amorphous powder; mp 102–103 °C; [α]25 D : − 36.0 (c = 0.2, CHCl3); UV (CHCl3) λmax (log ε) 324 (4.0), 292 (3.7), 245 (3.5) nm; IR (KBr) νmax 1725, 1660, 1615, 1550, 1210 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.64 (1H, d, J = 9.5 Hz, H-
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4), 7.37 (1H, d, J = 8.5 Hz, H-5), 6.84 (1H, dd, J = 8.5, 2.3 Hz, H-6), 6.81 (1H, d, J = 2.3 Hz, H-8), 6.25 (1H, d, J = 9.5 Hz, H-3), 5.43 (1H, t, J = 8.5 Hz, H-10′), 5.03–4.83 (2H, brs, H-15′), 4.59 (2H, d, J = 6.5 Hz, H-11′), 2.62 (1H, dt, J = 13.7, 10.6 Hz, H-2′β), 2.46 (2H, dd, J = 9.9, 4.3 Hz, H-1′), 2.30 (1H, dt, J = 14.0, 4.4 Hz, H-2′α), 2.13 (1H, dd, J = 12.1, 3.8 Hz, H-5′), 1.99–1.85 (2H, m, H-8′), 1.71 (3H, s, H-12′), 1.69 (2H, m, H-7′), 1.19 (3H, s, H-13′), 1.04 (3H, s, H-14′); 13 C NMR (CDCl3, 125 MHz) δ 215.1 (C, C-3′), 162.0 (C, C-7), 161.2 (C, C-2), 155.8 (C, C-9), 144.6 (C, C-6′), 143.4 (CH, C-4), 141.9 (C, C9′), 128.6 (CH, C-5), 118.7 (CH, C-10′), 113.5 (CH2, C-15′), 113.3 (CH, C-6), 112.9 (CH, C-3), 112.4 (C, C-10), 101.5 (CH, C-8), 65.3 (CH2, C-11′), 55.9 (CH, C-5′), 49.0 (C, C-4′), 37.6 (CH2, C-8′), 37.2 (CH2, C-2′), 30.6 (CH2, C-2′), 27.1 (CH3, C-14′), 25.4 (CH2, C-7′), 21.3 (CH3, C-13′), 16.8 (CH3, C-12′); ECD (MeCN, c = 0.7 mM, 0.1 cm path length) [θ]226 = − 2729, [θ]288 = − 1357, [θ]326 = + 1340; HR-ESIMS m/z 403.1894 [M + Na]+ (calcd. for C24H28O4Na, 403.1885).
Supporting information 1D and 2D NMR spectra for compounds 1, 2, and 4 are available as Supporting Information.
Acknowledgments !
We are grateful to Shahid Beheshti University Research Council and Iran National Science Foundation (INSF; Grant No. 86023.07) for financial support of this work. ECD spectra were measured at the Biophysics Facility, Biozentrum, University of Basel.
Conflict of Interest !
The authors declare no conflict of interest.
Affiliations 1
2
3 4
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C. Evin, Tehran, Iran Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Division of Pharmaceutical Biology, University of Basel, Basel, Switzerland Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C. Evin, Tehran, Iran
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