This article was downloaded by: [Moskow State Univ Bibliote] On: 31 January 2014, At: 14:18 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties a
a
b
Jamal Kasaian , Milad Iranshahy , Milena Masullo , Sonia b
a
Piacente , Fatemeh Ebrahimi & Mehrdad Iranshahi
a
a
Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Giovanni Paolo II, 84084, Salerno, Italy Published online: 10 Dec 2013.
To cite this article: Jamal Kasaian, Milad Iranshahy, Milena Masullo, Sonia Piacente, Fatemeh Ebrahimi & Mehrdad Iranshahi (2014) Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties, Journal of Asian Natural Products Research, 16:3, 248-253, DOI: 10.1080/10286020.2013.866099 To link to this article: http://dx.doi.org/10.1080/10286020.2013.866099
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 3, 248–253, http://dx.doi.org/10.1080/10286020.2013.866099
Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties Jamal Kasaiana, Milad Iranshahya, Milena Masullob, Sonia Piacenteb, Fatemeh Ebrahimia and Mehrdad Iranshahia* a
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; bDipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno, Via Giovanni Paolo II, 84084, Salerno, Italy (Received 6 July 2013; final version received 11 November 2013) Two new sesquiterpene lactones, namely feruhodin A (1) and feruhodin B (2), together with six known compounds, daucoeudesmanolactone (4), dehydrooopodin (5), oopodin (6), badkhysin (3), 7-demethylplastochromenol (7), and scoparone (8), were isolated from the roots of Ferula oopoda. The structures of these compounds were elucidated by 1D and 2D NMR techniques as well as high-resolution mass spectrometry. Cytotoxic effects of these compounds were evaluated against two human cancer cell lines including MCF7and K562 using the Alamar blue assay. The results showed that dehydrooopodin (5) possessed significant cytotoxic effects with IC50 values of 15 and 5 mM against MCF7 and K562, respectively. Keywords: Ferula; Apiaceae; cytotoxic; Alamar blue; dehydrooopodin; sesquiterpene lactones
1.
Introduction
Ferula is one of the well-known plant species belongs to the family Apiaceae. This genus has been considered as a good source of biologically active compounds including sesquiterpene lactones and sesquiterpene coumarins [1 –6]. Sesquiterpene lactones are a class of terpenoid compounds with colorless, bitter, and stable characteristics. These plant secondary metabolites with a lipophilic character had been isolated from Asteraceae, Apiaceae, and some other plant families at concentrations often exceeding 1% of plant dry weight [7–9]. Sesquiterpene lactones are described as the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases [10]. Also, they are known to possess wide variety of biological and
pharmacological activities including antimicrobial, cytotoxic, antiinflammatory, antiviral, antibacterial, and antifungal activities [11,12]. Recently, in a high-throughput screening of antiplasmodial and cytotoxic properties of traditionally used iranian plants, Esmaeili et al. [13] found that Ferula oopoda had significant antiplasmodial and cytotoxic activities with IC50 values of 26.6 and 45.2 mg/ml, respectively. This study encouraged us to find out the cytotoxic compounds of F. oopoda. F. oopoda has distributed in Mediterranean area and central Asia, especially in Iran and its northern neighboring countries. It grows in central and eastern parts of Iran, particularly Kerman and Khorasan Razavi provinces [14,15]. In this study, we report the main constituents of the root extract of F. oopoda and their cytotoxic properties.
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Journal of Asian Natural Products Research Table 1. (CDCl3).
1
H NMR (500 MHz) and
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
C NMR (125 MHz) spectral data for compounds 1 and 2
1
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 100 200 300 400 500
13
249
2
dH
dC
dH
dC
5.41 d (9.0) 5.76 dd (9.0, 4.8) 5.73 d (4.8) – 2.32 d (11.4) 4.96 dd (11.4, 9.6) 3.35 m 1.87 ma 1.46 m, 1.60 ma – – – 1.57 s 1.99 s 0.82 s – – 6.13 q (7.2) 1.96 d (7.2) 1.86 s – – – – –
137.9 123.3 122.5 137.0 49.6 78.6 39.2 19.6 35.7 36.4 81.0 176.4 21.8 22.2 16.2 167.6 128.3 141.4 17.2 21.9
4.96 s – 6.00 s – 3.09 d (11.0) 4.76 dd (11.0, 8.0) 2.73 m 1.93 m 1.27 m, 2.06 m – – – 4.27 d (11.5), 4.44 d (11.5) 2.20 s 1.04 s – – 6.16 q (7.0) 1.98 d (7.0) 1.91 s – – 6.09 q (7.0) 1.95 d (7.0) 1.86 s
78.2 194.0 127.5 162.3 46.2 79.1 40.2 18.6 31.4 42.0 76.7 176.6 65.6 24.1 18.1 168.1 127.8 141.6 17.1 21.6 167.3 128.1 141.2 17.1 21.6
Note: J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by 1H– 1H COSY, HMQC, HMBC, and ROESY experiments. a Resonance partially obscured.
2. Results and discussion Compound 1 was obtained as amorphous powder, and its molecular formula, C20H26O4, was established by high-resolution matrix-assisted laser desorption ionization time-of-flight (HR-MALDI-TOF) at m/z 331.1914 [M þ H]þ. The 1H and 13C NMR resonances (Table 1) of 1 were assigned by different 2D NMR experiments. The 1H NMR spectrum of 1 showed resonances characteristic for four methyl singlets at d 0.82, 1.57, 1.86, and 1.99, a methyl doublet at d 1.96, and four olefinic resonances at d 5.41 (1H, d, J ¼ 9.0 Hz), 5.76 (1H, dd, J ¼ 9.0 and 4.8 Hz), 5.73 (1H, d, J ¼ 4.8 Hz), and 6.13 (1H, q, J ¼ 7.2 Hz). In the HMBC spectrum of 1, the correlations of H-1 (dH 5.41) with C-10 (dC 36.4), C-5 (dC 49.6), and C-6 (dC 78.6);
H-5 (dH 2.32) with C-4 (dC 137.0), C-6 (dC 78.6), and C-10 (dC 36.4); H-6 (dH 4.96) with C-4 (dC 137.0), C-5 (dC 49.6), C-7 (dC 39.2), C-11 (dC 81.0), and C-12 (dC 176.4); and H-13 (dH 1.57) with C-7 (dC 39.2), C11 (dC 81.0), and C-12 (dC 176.4) confirmed the structure of compound 1 (Figure 1). In addition, ROESY cross-peaks between H-7, H-6, and H-15, between H-5, H-9 ax, and H-13 all supported the relative configuration of 1 (Figure 2). The configuration of the double bond at C-30 was determined as Z on the basis of the ROESY experiment, in which a cross-peak was observed from H-30 /H-50 pair (angeloyl). Other spectral data including 1H – 1H COSY and HSQC were also supported the assigned structure of compound 1. Therefore, the structure of compound 1 was
250
J. Kasaian et al. 5'' 1'' 15
O
O
1 9
O
5'
H
5
H
O 1' H 14
O 12
13
H
O 4'
O
O
O
OH
O
O
1 2
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Figure 1. Chemical structures of feruhodin A (1) and feruhodin B (2) from F. oopoda.
elucidated, as shown in Figure 1, and was named feruhodin A. Compound 2 was also obtained as amorphous powder, and its molecular formula, C25H32O8, was established by HRMALDI-TOF (m/z 461.2179 [M þ H] þ , calcd for C25H33O8, 461.2175). The structure of 2 was established from the analysis of 1H and 13C NMR spectra (Table 1). The downfield signals at dC 194.0, 176.6, 168.1, 167.3, and 162.3 were assigned to the carbons of C-2, C-12, C-10 , C-100 , and C-4, respectively. The 1H NMR spectrum of 2 showed resonances characteristic for four methyl singlets at dH 1.04 (H-15), 1.86 (H-500 ), 1.91 (H-50 ), and 2.20 (H-14), two methyl doublets at dH 1.95 (H-400 , J ¼ 7.0 Hz) and 1.98 (H-40 , J ¼ 7.0 Hz), and three olefinic resonances at dH 6.00 (H-3, s), 6.09 (H-300 , q, J ¼ 7.0 Hz), and 6.16 (H-30 , q, J ¼ 7.0 Hz). The downfield signals at dH 4.96 and 4.76 were assigned to the protons of H-1 and H-6, respectively. In the HMBC spectrum of 2, the correlations of H-1 (dH 4.96) with C-100 (dC 167.3), C-2 (dC 194.0), C-10 (dC 42.0), C-5 (dC 46.2), and C9 (dC 31.4); H-5 (dH 3.09) with C-4 (dC H
162.3), C-6 (dC 79.1), and C-10 (dC 42.0); H6 (dH 4.76) with C-11 (dC 76.7), C-5 (dC 46.2), C-7 (dC 40.2), and C-12 (dC 176.6); and H-13 (dH 4.27 and 4.44) with C-10 (dC 168.1), C-7 (dC 40.2), C-11 (dC 76.7), and C12 (dC 176.6) confirmed the structure of compound 2 (Figure 1). ROESY cross-peaks between H-7, H-6, H-13, and H-15, between H-5 and H-9 ax all supported the relative configuration of 2. The configuration of the double bonds at C-30 and C-300 was determined as Z on the basis of the ROESY experiment, in which cross-peaks were observed from H-30 /H-50 and H-300 /H-500 pairs (Figure 2). According to the analyses mentioned earlier, the structure of compound 2 was elucidated, as shown in Figure 1, and was named feruhodin B. The cytotoxic activities of all purified compounds (0.1, 1, 20 and 50 mM) against MCF7 and K562 cell lines were evaluated. The results of this screening are summarized in Table 2. Those compounds with IC50 values above 50 mM were considered inactive. Dehydrooopodin (5) showed a promising
O
CH3 H O
H
O
O H
O
H
H O
O 1
H
O
H
2
OH O
O O
HMBC ROESY
Figure 2. Selected HMBC and ROESY correlations of feruhodin A and feruhodin B.
Journal of Asian Natural Products Research
251
Table 2. In vitro cytotoxicity of the isolated compounds from F. oopoda. Concentration % Inhibition Concentration 50 mM
20 mM
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Compound 1 2 3 4 5 6 8 Cþ
Compound name Feruhodin A Feruhodin B Badkhysin Daucoeudesmanolactone Dehydrooopodin Oopodin Scoparone Mitoxantrone
MCF7 0.00 5.36 8.20 0.00 45.39 0.00 4.69 100
K562 0.00 0.00 0.00 0.00 68.52 0.00 0.00 ND
MCF7 1.5 5.36 8.20 0.00 66 0.00 4.69 100
IC50 value (mM)
K562
MCF7
K562
1.91 29.36 23.03 8.06 70.05 20.53 19.96 ND
a
a
a
a
a
a
a
a
15
5
a
a
4
ND
a
a
Note: ND, not determined; C þ , positive control. IC50 . 50 mM. a
antiproliferative activity. IC50 values were , 20 mM in both evaluated cell lines. It showed the highest cytotoxic activity among the constituents of F. oopoda. 3. Materials and methods 3.1 General experimental procedures IR spectra for new compounds were recorded on a PerkinElmer Paragin 1000 FT-IR spectrometer (SpectraLab Scientific Inc, Ontario, Canada). NMR experiments were carried out on a Bruker DRX-600 spectrometer (Bruker BioSpinGmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe at 300 K. All 2D-NMR spectra were acquired in CD3OD (99.95%, Sigma-Aldrich, Munich, Germany) and standard pulse sequences and phase cycling were used for DQFCOSY, HSQC, HMBC, and ROESY spectra. The NMR data were processed using UXNMR software. Exact masses were measured by a Voyager DE mass spectrometer (Mass Spectrometry Laboratory, Urbana, IL, USA). Samples were analyzed by MALDI-TOF mass spectrometry (Bruker daltonics, Billerica, MA, USA). A mixture of analyte solution and a-cyano-4hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from ACTH (fragment 18 – 39) at 2465.1989 Da and a-cyano-4-hydroxycinnamic acid at 190.0504 Da as internal standard. Alamar blue and dimethyl sulfoxide (DMSO) were obtained from Sigma (Munich, Germany). Methanol, chloroform, and all other organic solvents (analytical grade) were purchased from Daejung (Siheung-city, Gyonggi-do, Korea). Column chromatography (CC) was conducted with Si gel 230–400 mesh (Merck, Darmstadt, Germany). 3.2
Plant material
The plant F. oopoda (Boiss. & Buhse) Boiss. was collected from Iran, Khorasan Razavi province, southwest of Sarakhs mountains, in May 2012. The plant material was identified by Mohammad Reza Joharchi, Ferdowsi University of Mashhad Herbarium (FUMH). A voucher specimen (No. 38717) has been deposited at FUMH. 3.3
Extraction and isolation
Total plant extracts were obtained by extraction of dried and milled roots of the plants with chloroform using maceration method for 3 days. After every 24 h, the
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
252
J. Kasaian et al.
mixture was filtered and new solvent was added to the plant powder. The combined extracts were concentrated to dryness under vacuum pressure. The extract (8 g) was subjected to CC on silica gel (mesh 230– 400, 5 £ 60 cm) using petrol with increasing volumes of EtOAc [petrol– EtOAc (1:0, 1 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 – methanol (9:1, 1 l). The fractions (200 ml each) were compared by TLC (silica gel using petrol– EtOAc, 3:1 ratio), and those giving similar spots were combined. Observation of plates was carried out under UV CAMAG spectrometer (CAMAG instruments, Berlin, Germany) (254 nm). Sixteen fractions were finally obtained. Fractions were subjected to reversed-phase HPLC using 20– 100% MeOH in H2O as the eluent including 0.05% trifluoroacetic acid (TFA). Purification of fractions was carried out using an ACE 5 C18 (Advanced Chromatography Technologies Limited, Aberdeen, Scotland) (5 mM, 250 £ 21.2 mm) at a flow rate 9 ml/min and linear gradient conditions of 20– 100% MeOH (0.05% TFA) within 20 min, followed by an isocratic condition of MeOH (0.05% TFA) for 5 min. Eight compounds were obtained including compounds 1 (15 mg), 2 (16.2 mg), 3 (69.1 mg), 4 (28.5 mg), 5 (11.2 mg), 6 (55.7 mg), 7 (9.9 mg), and 8 (216 mg). 3.3.1
Feruhodin A (1)
Amorphous powder; ½a25 D 2 33 (c 0.42, CH2Cl2); UV (MeOH) lmax (log 1) 226 (4.07), 264 (3.43) nm; IR nmax (CH2Cl2): 2922, 1784, 1715, 1455, 1382, 1234, and 1153 cm21. For 1H and 13C NMR spectral data, see Table 1. HR-MALDI-TOF-MS: m/z 331.1914 [M þ H] þ (calcd for C20H27O4, 331.1909). 3.3.2 Feruhodin B (2) Amorphous powder; ½a25 D 2 162 (c 0.14, CH2Cl2); UV (MeOH) lmax (log 1) 232 (4.29) nm; IR nmax (CH2Cl2): 3450, 2923,
1778, 1716, 1646, 1457, 1383, 1229, and 1151 cm21. For 1H and 13C NMR spectral data, see Table 1. HR-MALDI-TOF-MS: m/z 461.2179 [M þ H] þ (calcd for C20H27O4, 461.2175).
3.4 3.4.1
Biological activity Cell culture
MCF7 (human breast adenocarcinoma) and K562 (human acute myelocytic leukemia) cell lines were obtained from Biotechnology Research Center (Mashhad, Iran). Cell lines were cultured in RPMI-1640 supplemented with 10% fetal calf serum (Gibco, Invitrogen, Paisely, UK) and penicillin (100 units/ml) and streptomycin (100 mg/ml) for desired growth, completed with fetal bovine serum (10%) in a humidified incubator at 378C in an atmosphere of 5% CO2.
3.4.2
Alamar blue assay
In vitro cytotoxicity tests were performed using a nonfluorescent substrate, Alamar blue (BioSource Invitrogen, Paisely, UK) as described by Haaften et al. [16] using some modification. In each well of 96-well plates, 1 £ 104 cells were seeded. Three wells for each concentration were seeded and triplicate plates were used for each cell line. Cell cultures, in triplicates, in exponential growth were treated with the different compounds of the plant, redissolved in DMSO and added at final concentrations of 0.1, 1, 20 and 50 mM. Then, the cells were incubated in CO2 incubator. After 72 h, Alamar blue reagent was added up to 10% of tissue culture volume, then incubated for further 1 – 4 h. The absorbance was measured at 600 nm using a microplate reader, and percent viability of the cells was investigated relative to the negative control that was exposed to the culture medium without extract. Mitoxantrone as positive control (final concentrations 0.5, 5, 50, 500, and
Journal of Asian Natural Products Research 5000 nM) was included and its IC50 was determined to be 4 mM.
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
4.
Conclusion
Two new sesquiterpene coumarins were reported in this paper, namely, feruhodin A and feruhodin B. The NMR spectral data of known compounds were in accordance with the previous literatures (6 [14,20], 4 and 5 [17 –19]). Cytotoxic activities of all purified compounds, except 7-demethylplastochromenol (7), was evaluated in this study. Study of the structure – activity relationship among sesquiterpene lactones has revealed that the presence of a C11 –C13 exocyclic double bond conjugated to the g-lactone was essential for cytotoxicity [7]. Our study was also in agreement with previous studies. According to the results of this study, dehydrooopodin (5) was suggested for further studies in order to identify its effect on other cell lines, in vivo studies, and its mechanism of action. Acknowledgments This research was financially supported by grants from the Mashhad University of Medical Sciences Research Council (No. 910421) and Iran National Science Foundation (No. 91057374). This study was also a part of the dissertation of Mrs Fatemeh Ebrahimi for the degree of Doctor of Pharmacy submitted to the School of Pharmacy, Mashhad University of Medical Sciences.
References [1] Z.E. Nazari and M. Iranshahi, Phytother. Res. 25, 315322 (2011). [2] M. Holub and M. Budesinsky, Phytochemistry 25, 20152026 (1986). [3] S. Sahranavard, F. Naghibi, M. Mosaddegh, S. Esmaeili, P. Sarkhail, M. Taghvaei, and S. Ghafari, Res. Pharm. Sci. 4, 133 (2009).
253
[4] H. Hajimehdipoor, S. Esmaeili, A. Ramezani, A. Jafari Anaraki, and M. Mosaddegh, Iran J. Pharm. Sci. 8, 115 (2012). [5] G. Anna and P.E. Brodelius, Phytochem. Rev. 11, 7796 (2012). [6] M. Iranshahi, P. Arfa, M. Ramezani, M.R. Jaafari, H. Sadeghian, C. Bassarello, S. Piacente, and C. Pizza, Phytochemistry 68, 554 (2007). [7] E. Rodriguez, G.H.N. Towersa, and J.C. Mftcheu, Phytochemistry 15, 1573 (1976). [8] S.V. Serkerov, A.N. Aleskerov, D.M. Akhmedov, and F.A. Rasulov, Chem. Nat. Comp. 28, 248 (1992). [9] M. Iranshahi, S.T. Hosseini, A.R. Shahverdi, K. Molazade, S. Suleman Khan, and V.U. Ahmad, Phytochemistry 69, 2753 (2008). [10] A. Ghantous, H. Gali-Muhtasib, H. Vuorela, N.A. Saliba, and N. Darwiche, Drug Discov. Today 15, 668 (2010). [11] D. Chaturvedi, Sesquiterpene lactones: structural diversity and their biological activities. Opportunity, challenge and scope of natural products in medicinal chemistry, 1st ed., (Research Singpost, Kerala, 2011). [12] M.R.O. Kreuger, S. Grootjans, M.W. Biavatti, P. Vandenabeele, and K. D’Herde, Anti-Cancer Drugs 23, 883 (2012). [13] S. Esmaeili, F. Naghibi, M. Mosaddegh, S.H. Sahranavard, S. Ghafari, and N.R. Abdullah, J. Ethnopharmacol. 121, 400 (2009). [14] S.V. Serkerov, Chem. Nat. Comp. 5, 314 (1969). [15] M. Sharifi Yazdi, A. Shahmoradi, S. Zarekia, and M. Khodashenas, Iran J. Range Desert Res. 15, 447 (2007). [16] C.V. Haaften, C.C. Duke, A.M. Weerheim, N.P.M. Smith, P.M.M. Van Haard, F. Darroudi, and B.J.M.Z. Trimbos, J. Exp. Clin. Cancer Res. 30, 29 (2011). [17] A.F. Barrero, M.M. Herrador, and P. Arteaga, Phytochemistry 37, 1351 (1994). [18] S.V. Serkerov, Chem. Nat. Compd. 16, 366 (1980). [19] S.V. Serkerov, Chem. Nat. Compd. 8, 57 (1972). [20] A.A. Sallam, Y. Hitptsuyanagi, E.S. Mansour, A.F. Ahmed, S. Gedara, H. Fukaya, and K. Takeya, Helv. Chim. Acta 93, 48 (2010).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties a
a
b
Jamal Kasaian , Milad Iranshahy , Milena Masullo , Sonia b
a
Piacente , Fatemeh Ebrahimi & Mehrdad Iranshahi
a
a
Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Giovanni Paolo II, 84084, Salerno, Italy Published online: 10 Dec 2013.
To cite this article: Jamal Kasaian, Milad Iranshahy, Milena Masullo, Sonia Piacente, Fatemeh Ebrahimi & Mehrdad Iranshahi (2014) Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties, Journal of Asian Natural Products Research, 16:3, 248-253, DOI: 10.1080/10286020.2013.866099 To link to this article: http://dx.doi.org/10.1080/10286020.2013.866099
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 3, 248–253, http://dx.doi.org/10.1080/10286020.2013.866099
Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties Jamal Kasaiana, Milad Iranshahya, Milena Masullob, Sonia Piacenteb, Fatemeh Ebrahimia and Mehrdad Iranshahia* a
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; bDipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno, Via Giovanni Paolo II, 84084, Salerno, Italy (Received 6 July 2013; final version received 11 November 2013) Two new sesquiterpene lactones, namely feruhodin A (1) and feruhodin B (2), together with six known compounds, daucoeudesmanolactone (4), dehydrooopodin (5), oopodin (6), badkhysin (3), 7-demethylplastochromenol (7), and scoparone (8), were isolated from the roots of Ferula oopoda. The structures of these compounds were elucidated by 1D and 2D NMR techniques as well as high-resolution mass spectrometry. Cytotoxic effects of these compounds were evaluated against two human cancer cell lines including MCF7and K562 using the Alamar blue assay. The results showed that dehydrooopodin (5) possessed significant cytotoxic effects with IC50 values of 15 and 5 mM against MCF7 and K562, respectively. Keywords: Ferula; Apiaceae; cytotoxic; Alamar blue; dehydrooopodin; sesquiterpene lactones
1.
Introduction
Ferula is one of the well-known plant species belongs to the family Apiaceae. This genus has been considered as a good source of biologically active compounds including sesquiterpene lactones and sesquiterpene coumarins [1 –6]. Sesquiterpene lactones are a class of terpenoid compounds with colorless, bitter, and stable characteristics. These plant secondary metabolites with a lipophilic character had been isolated from Asteraceae, Apiaceae, and some other plant families at concentrations often exceeding 1% of plant dry weight [7–9]. Sesquiterpene lactones are described as the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases [10]. Also, they are known to possess wide variety of biological and
pharmacological activities including antimicrobial, cytotoxic, antiinflammatory, antiviral, antibacterial, and antifungal activities [11,12]. Recently, in a high-throughput screening of antiplasmodial and cytotoxic properties of traditionally used iranian plants, Esmaeili et al. [13] found that Ferula oopoda had significant antiplasmodial and cytotoxic activities with IC50 values of 26.6 and 45.2 mg/ml, respectively. This study encouraged us to find out the cytotoxic compounds of F. oopoda. F. oopoda has distributed in Mediterranean area and central Asia, especially in Iran and its northern neighboring countries. It grows in central and eastern parts of Iran, particularly Kerman and Khorasan Razavi provinces [14,15]. In this study, we report the main constituents of the root extract of F. oopoda and their cytotoxic properties.
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Journal of Asian Natural Products Research Table 1. (CDCl3).
1
H NMR (500 MHz) and
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
C NMR (125 MHz) spectral data for compounds 1 and 2
1
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 100 200 300 400 500
13
249
2
dH
dC
dH
dC
5.41 d (9.0) 5.76 dd (9.0, 4.8) 5.73 d (4.8) – 2.32 d (11.4) 4.96 dd (11.4, 9.6) 3.35 m 1.87 ma 1.46 m, 1.60 ma – – – 1.57 s 1.99 s 0.82 s – – 6.13 q (7.2) 1.96 d (7.2) 1.86 s – – – – –
137.9 123.3 122.5 137.0 49.6 78.6 39.2 19.6 35.7 36.4 81.0 176.4 21.8 22.2 16.2 167.6 128.3 141.4 17.2 21.9
4.96 s – 6.00 s – 3.09 d (11.0) 4.76 dd (11.0, 8.0) 2.73 m 1.93 m 1.27 m, 2.06 m – – – 4.27 d (11.5), 4.44 d (11.5) 2.20 s 1.04 s – – 6.16 q (7.0) 1.98 d (7.0) 1.91 s – – 6.09 q (7.0) 1.95 d (7.0) 1.86 s
78.2 194.0 127.5 162.3 46.2 79.1 40.2 18.6 31.4 42.0 76.7 176.6 65.6 24.1 18.1 168.1 127.8 141.6 17.1 21.6 167.3 128.1 141.2 17.1 21.6
Note: J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by 1H– 1H COSY, HMQC, HMBC, and ROESY experiments. a Resonance partially obscured.
2. Results and discussion Compound 1 was obtained as amorphous powder, and its molecular formula, C20H26O4, was established by high-resolution matrix-assisted laser desorption ionization time-of-flight (HR-MALDI-TOF) at m/z 331.1914 [M þ H]þ. The 1H and 13C NMR resonances (Table 1) of 1 were assigned by different 2D NMR experiments. The 1H NMR spectrum of 1 showed resonances characteristic for four methyl singlets at d 0.82, 1.57, 1.86, and 1.99, a methyl doublet at d 1.96, and four olefinic resonances at d 5.41 (1H, d, J ¼ 9.0 Hz), 5.76 (1H, dd, J ¼ 9.0 and 4.8 Hz), 5.73 (1H, d, J ¼ 4.8 Hz), and 6.13 (1H, q, J ¼ 7.2 Hz). In the HMBC spectrum of 1, the correlations of H-1 (dH 5.41) with C-10 (dC 36.4), C-5 (dC 49.6), and C-6 (dC 78.6);
H-5 (dH 2.32) with C-4 (dC 137.0), C-6 (dC 78.6), and C-10 (dC 36.4); H-6 (dH 4.96) with C-4 (dC 137.0), C-5 (dC 49.6), C-7 (dC 39.2), C-11 (dC 81.0), and C-12 (dC 176.4); and H-13 (dH 1.57) with C-7 (dC 39.2), C11 (dC 81.0), and C-12 (dC 176.4) confirmed the structure of compound 1 (Figure 1). In addition, ROESY cross-peaks between H-7, H-6, and H-15, between H-5, H-9 ax, and H-13 all supported the relative configuration of 1 (Figure 2). The configuration of the double bond at C-30 was determined as Z on the basis of the ROESY experiment, in which a cross-peak was observed from H-30 /H-50 pair (angeloyl). Other spectral data including 1H – 1H COSY and HSQC were also supported the assigned structure of compound 1. Therefore, the structure of compound 1 was
250
J. Kasaian et al. 5'' 1'' 15
O
O
1 9
O
5'
H
5
H
O 1' H 14
O 12
13
H
O 4'
O
O
O
OH
O
O
1 2
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Figure 1. Chemical structures of feruhodin A (1) and feruhodin B (2) from F. oopoda.
elucidated, as shown in Figure 1, and was named feruhodin A. Compound 2 was also obtained as amorphous powder, and its molecular formula, C25H32O8, was established by HRMALDI-TOF (m/z 461.2179 [M þ H] þ , calcd for C25H33O8, 461.2175). The structure of 2 was established from the analysis of 1H and 13C NMR spectra (Table 1). The downfield signals at dC 194.0, 176.6, 168.1, 167.3, and 162.3 were assigned to the carbons of C-2, C-12, C-10 , C-100 , and C-4, respectively. The 1H NMR spectrum of 2 showed resonances characteristic for four methyl singlets at dH 1.04 (H-15), 1.86 (H-500 ), 1.91 (H-50 ), and 2.20 (H-14), two methyl doublets at dH 1.95 (H-400 , J ¼ 7.0 Hz) and 1.98 (H-40 , J ¼ 7.0 Hz), and three olefinic resonances at dH 6.00 (H-3, s), 6.09 (H-300 , q, J ¼ 7.0 Hz), and 6.16 (H-30 , q, J ¼ 7.0 Hz). The downfield signals at dH 4.96 and 4.76 were assigned to the protons of H-1 and H-6, respectively. In the HMBC spectrum of 2, the correlations of H-1 (dH 4.96) with C-100 (dC 167.3), C-2 (dC 194.0), C-10 (dC 42.0), C-5 (dC 46.2), and C9 (dC 31.4); H-5 (dH 3.09) with C-4 (dC H
162.3), C-6 (dC 79.1), and C-10 (dC 42.0); H6 (dH 4.76) with C-11 (dC 76.7), C-5 (dC 46.2), C-7 (dC 40.2), and C-12 (dC 176.6); and H-13 (dH 4.27 and 4.44) with C-10 (dC 168.1), C-7 (dC 40.2), C-11 (dC 76.7), and C12 (dC 176.6) confirmed the structure of compound 2 (Figure 1). ROESY cross-peaks between H-7, H-6, H-13, and H-15, between H-5 and H-9 ax all supported the relative configuration of 2. The configuration of the double bonds at C-30 and C-300 was determined as Z on the basis of the ROESY experiment, in which cross-peaks were observed from H-30 /H-50 and H-300 /H-500 pairs (Figure 2). According to the analyses mentioned earlier, the structure of compound 2 was elucidated, as shown in Figure 1, and was named feruhodin B. The cytotoxic activities of all purified compounds (0.1, 1, 20 and 50 mM) against MCF7 and K562 cell lines were evaluated. The results of this screening are summarized in Table 2. Those compounds with IC50 values above 50 mM were considered inactive. Dehydrooopodin (5) showed a promising
O
CH3 H O
H
O
O H
O
H
H O
O 1
H
O
H
2
OH O
O O
HMBC ROESY
Figure 2. Selected HMBC and ROESY correlations of feruhodin A and feruhodin B.
Journal of Asian Natural Products Research
251
Table 2. In vitro cytotoxicity of the isolated compounds from F. oopoda. Concentration % Inhibition Concentration 50 mM
20 mM
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
Compound 1 2 3 4 5 6 8 Cþ
Compound name Feruhodin A Feruhodin B Badkhysin Daucoeudesmanolactone Dehydrooopodin Oopodin Scoparone Mitoxantrone
MCF7 0.00 5.36 8.20 0.00 45.39 0.00 4.69 100
K562 0.00 0.00 0.00 0.00 68.52 0.00 0.00 ND
MCF7 1.5 5.36 8.20 0.00 66 0.00 4.69 100
IC50 value (mM)
K562
MCF7
K562
1.91 29.36 23.03 8.06 70.05 20.53 19.96 ND
a
a
a
a
a
a
a
a
15
5
a
a
4
ND
a
a
Note: ND, not determined; C þ , positive control. IC50 . 50 mM. a
antiproliferative activity. IC50 values were , 20 mM in both evaluated cell lines. It showed the highest cytotoxic activity among the constituents of F. oopoda. 3. Materials and methods 3.1 General experimental procedures IR spectra for new compounds were recorded on a PerkinElmer Paragin 1000 FT-IR spectrometer (SpectraLab Scientific Inc, Ontario, Canada). NMR experiments were carried out on a Bruker DRX-600 spectrometer (Bruker BioSpinGmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe at 300 K. All 2D-NMR spectra were acquired in CD3OD (99.95%, Sigma-Aldrich, Munich, Germany) and standard pulse sequences and phase cycling were used for DQFCOSY, HSQC, HMBC, and ROESY spectra. The NMR data were processed using UXNMR software. Exact masses were measured by a Voyager DE mass spectrometer (Mass Spectrometry Laboratory, Urbana, IL, USA). Samples were analyzed by MALDI-TOF mass spectrometry (Bruker daltonics, Billerica, MA, USA). A mixture of analyte solution and a-cyano-4hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from ACTH (fragment 18 – 39) at 2465.1989 Da and a-cyano-4-hydroxycinnamic acid at 190.0504 Da as internal standard. Alamar blue and dimethyl sulfoxide (DMSO) were obtained from Sigma (Munich, Germany). Methanol, chloroform, and all other organic solvents (analytical grade) were purchased from Daejung (Siheung-city, Gyonggi-do, Korea). Column chromatography (CC) was conducted with Si gel 230–400 mesh (Merck, Darmstadt, Germany). 3.2
Plant material
The plant F. oopoda (Boiss. & Buhse) Boiss. was collected from Iran, Khorasan Razavi province, southwest of Sarakhs mountains, in May 2012. The plant material was identified by Mohammad Reza Joharchi, Ferdowsi University of Mashhad Herbarium (FUMH). A voucher specimen (No. 38717) has been deposited at FUMH. 3.3
Extraction and isolation
Total plant extracts were obtained by extraction of dried and milled roots of the plants with chloroform using maceration method for 3 days. After every 24 h, the
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
252
J. Kasaian et al.
mixture was filtered and new solvent was added to the plant powder. The combined extracts were concentrated to dryness under vacuum pressure. The extract (8 g) was subjected to CC on silica gel (mesh 230– 400, 5 £ 60 cm) using petrol with increasing volumes of EtOAc [petrol– EtOAc (1:0, 1 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 – methanol (9:1, 1 l). The fractions (200 ml each) were compared by TLC (silica gel using petrol– EtOAc, 3:1 ratio), and those giving similar spots were combined. Observation of plates was carried out under UV CAMAG spectrometer (CAMAG instruments, Berlin, Germany) (254 nm). Sixteen fractions were finally obtained. Fractions were subjected to reversed-phase HPLC using 20– 100% MeOH in H2O as the eluent including 0.05% trifluoroacetic acid (TFA). Purification of fractions was carried out using an ACE 5 C18 (Advanced Chromatography Technologies Limited, Aberdeen, Scotland) (5 mM, 250 £ 21.2 mm) at a flow rate 9 ml/min and linear gradient conditions of 20– 100% MeOH (0.05% TFA) within 20 min, followed by an isocratic condition of MeOH (0.05% TFA) for 5 min. Eight compounds were obtained including compounds 1 (15 mg), 2 (16.2 mg), 3 (69.1 mg), 4 (28.5 mg), 5 (11.2 mg), 6 (55.7 mg), 7 (9.9 mg), and 8 (216 mg). 3.3.1
Feruhodin A (1)
Amorphous powder; ½a25 D 2 33 (c 0.42, CH2Cl2); UV (MeOH) lmax (log 1) 226 (4.07), 264 (3.43) nm; IR nmax (CH2Cl2): 2922, 1784, 1715, 1455, 1382, 1234, and 1153 cm21. For 1H and 13C NMR spectral data, see Table 1. HR-MALDI-TOF-MS: m/z 331.1914 [M þ H] þ (calcd for C20H27O4, 331.1909). 3.3.2 Feruhodin B (2) Amorphous powder; ½a25 D 2 162 (c 0.14, CH2Cl2); UV (MeOH) lmax (log 1) 232 (4.29) nm; IR nmax (CH2Cl2): 3450, 2923,
1778, 1716, 1646, 1457, 1383, 1229, and 1151 cm21. For 1H and 13C NMR spectral data, see Table 1. HR-MALDI-TOF-MS: m/z 461.2179 [M þ H] þ (calcd for C20H27O4, 461.2175).
3.4 3.4.1
Biological activity Cell culture
MCF7 (human breast adenocarcinoma) and K562 (human acute myelocytic leukemia) cell lines were obtained from Biotechnology Research Center (Mashhad, Iran). Cell lines were cultured in RPMI-1640 supplemented with 10% fetal calf serum (Gibco, Invitrogen, Paisely, UK) and penicillin (100 units/ml) and streptomycin (100 mg/ml) for desired growth, completed with fetal bovine serum (10%) in a humidified incubator at 378C in an atmosphere of 5% CO2.
3.4.2
Alamar blue assay
In vitro cytotoxicity tests were performed using a nonfluorescent substrate, Alamar blue (BioSource Invitrogen, Paisely, UK) as described by Haaften et al. [16] using some modification. In each well of 96-well plates, 1 £ 104 cells were seeded. Three wells for each concentration were seeded and triplicate plates were used for each cell line. Cell cultures, in triplicates, in exponential growth were treated with the different compounds of the plant, redissolved in DMSO and added at final concentrations of 0.1, 1, 20 and 50 mM. Then, the cells were incubated in CO2 incubator. After 72 h, Alamar blue reagent was added up to 10% of tissue culture volume, then incubated for further 1 – 4 h. The absorbance was measured at 600 nm using a microplate reader, and percent viability of the cells was investigated relative to the negative control that was exposed to the culture medium without extract. Mitoxantrone as positive control (final concentrations 0.5, 5, 50, 500, and
Journal of Asian Natural Products Research 5000 nM) was included and its IC50 was determined to be 4 mM.
Downloaded by [Moskow State Univ Bibliote] at 14:18 31 January 2014
4.
Conclusion
Two new sesquiterpene coumarins were reported in this paper, namely, feruhodin A and feruhodin B. The NMR spectral data of known compounds were in accordance with the previous literatures (6 [14,20], 4 and 5 [17 –19]). Cytotoxic activities of all purified compounds, except 7-demethylplastochromenol (7), was evaluated in this study. Study of the structure – activity relationship among sesquiterpene lactones has revealed that the presence of a C11 –C13 exocyclic double bond conjugated to the g-lactone was essential for cytotoxicity [7]. Our study was also in agreement with previous studies. According to the results of this study, dehydrooopodin (5) was suggested for further studies in order to identify its effect on other cell lines, in vivo studies, and its mechanism of action. Acknowledgments This research was financially supported by grants from the Mashhad University of Medical Sciences Research Council (No. 910421) and Iran National Science Foundation (No. 91057374). This study was also a part of the dissertation of Mrs Fatemeh Ebrahimi for the degree of Doctor of Pharmacy submitted to the School of Pharmacy, Mashhad University of Medical Sciences.
References [1] Z.E. Nazari and M. Iranshahi, Phytother. Res. 25, 315322 (2011). [2] M. Holub and M. Budesinsky, Phytochemistry 25, 20152026 (1986). [3] S. Sahranavard, F. Naghibi, M. Mosaddegh, S. Esmaeili, P. Sarkhail, M. Taghvaei, and S. Ghafari, Res. Pharm. Sci. 4, 133 (2009).
253
[4] H. Hajimehdipoor, S. Esmaeili, A. Ramezani, A. Jafari Anaraki, and M. Mosaddegh, Iran J. Pharm. Sci. 8, 115 (2012). [5] G. Anna and P.E. Brodelius, Phytochem. Rev. 11, 7796 (2012). [6] M. Iranshahi, P. Arfa, M. Ramezani, M.R. Jaafari, H. Sadeghian, C. Bassarello, S. Piacente, and C. Pizza, Phytochemistry 68, 554 (2007). [7] E. Rodriguez, G.H.N. Towersa, and J.C. Mftcheu, Phytochemistry 15, 1573 (1976). [8] S.V. Serkerov, A.N. Aleskerov, D.M. Akhmedov, and F.A. Rasulov, Chem. Nat. Comp. 28, 248 (1992). [9] M. Iranshahi, S.T. Hosseini, A.R. Shahverdi, K. Molazade, S. Suleman Khan, and V.U. Ahmad, Phytochemistry 69, 2753 (2008). [10] A. Ghantous, H. Gali-Muhtasib, H. Vuorela, N.A. Saliba, and N. Darwiche, Drug Discov. Today 15, 668 (2010). [11] D. Chaturvedi, Sesquiterpene lactones: structural diversity and their biological activities. Opportunity, challenge and scope of natural products in medicinal chemistry, 1st ed., (Research Singpost, Kerala, 2011). [12] M.R.O. Kreuger, S. Grootjans, M.W. Biavatti, P. Vandenabeele, and K. D’Herde, Anti-Cancer Drugs 23, 883 (2012). [13] S. Esmaeili, F. Naghibi, M. Mosaddegh, S.H. Sahranavard, S. Ghafari, and N.R. Abdullah, J. Ethnopharmacol. 121, 400 (2009). [14] S.V. Serkerov, Chem. Nat. Comp. 5, 314 (1969). [15] M. Sharifi Yazdi, A. Shahmoradi, S. Zarekia, and M. Khodashenas, Iran J. Range Desert Res. 15, 447 (2007). [16] C.V. Haaften, C.C. Duke, A.M. Weerheim, N.P.M. Smith, P.M.M. Van Haard, F. Darroudi, and B.J.M.Z. Trimbos, J. Exp. Clin. Cancer Res. 30, 29 (2011). [17] A.F. Barrero, M.M. Herrador, and P. Arteaga, Phytochemistry 37, 1351 (1994). [18] S.V. Serkerov, Chem. Nat. Compd. 16, 366 (1980). [19] S.V. Serkerov, Chem. Nat. Compd. 8, 57 (1972). [20] A.A. Sallam, Y. Hitptsuyanagi, E.S. Mansour, A.F. Ahmed, S. Gedara, H. Fukaya, and K. Takeya, Helv. Chim. Acta 93, 48 (2010).
