Article pubs.acs.org/JAFC
New Sesquiterpene Lactone Dimer, Uvedafolin, Extracted from Eight Yacon Leaf Varieties (Smallanthus sonchifolius): Cytotoxicity in HeLa, HL-60, and Murine B16-F10 Melanoma Cell Lines Yurika Kitai,† Kana Hayashi,‡ Moe Otsuka,‡ Hisashi Nishiwaki,† Tatsuya Senoo,§ Tomohiko Ishii,§ Genta Sakane,∥ Makoto Sugiura,⊥ and Hirotoshi Tamura*,†,‡ †
The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan The Graduate School of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa, 761-0795 Japan § Department of Advanced Materials Science, Faculty of Engineering, Kagawa University, 2217-20 Hayashicho, Takamatsu, Kagawa, 761-0396 Japan ∥ Department of Chemistry, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama, 700-0005 Japan ⊥ Western Region Agricultural Research Center, National Agriculture and Food Research Organization, 1-3-1 Butsuyuucho, Zentsuji, Kagawa, 765-8508 Japan ‡
S Supporting Information *
ABSTRACT: Uvedafolin, 1, a new sesquiterpene lactone dimer, was isolated from the leaves of Smallanthus sonchifolius with five related compounds, 2−6, and their cytotoxicity was assessed against three tumor cell lines (HeLa, HL-60, B16-F10 melanoma). The stereostructure of 1 was newly elucidated by ESI-TOF-MS, 1D/2D NMR, and single-crystal X-ray diffraction. Dimers 1 and 2 had the most effective IC50 values, 0.2−1.9 μM, against the three tumor cell lines when compared with monomers 3−6 (IC50 values 0.7−9.9 μM) and etoposide (IC50 values 0.8−114 μM). The ester linkages of two sets of monomers, uvedalin, 5, and sonchifolin, 6, for 1, and enhydrin, 4, and sonchifolin, 6, for 2, as well as the acetyl group at the C-9 position, were essential for the high cytotoxicity. Dimers 1 and 2 would have potential as anticancer agents. KEYWORDS: dimer sesquiterpene lactone, cytotoxicity, Smallanthus sonchifolius, ‘Sarada otome’
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INTRODUCTION
General Experimental Procedures. 1H NMR (600 MHz), 13C NMR (125 MHz), and 2D NMR were measured in acetone-d6 with 0.05% TMS as an internal standard by an ECA-600 spectrometer (JEOL, Tokyo, Japan). The HR-ESIMS spectrum was measured on a Xevo QTof MS spectrometer (Waters, Tokyo, Japan). The UV spectrum was recorded on a UV-975 Intelligent UV/vis detector (JASCO International, Tokyo, Japan). Optical rotations were measured on a JASCO P-1030 polarimeter. The IR spectrum was measured in a CHCl3 solution by a JASCO FT/IR-400 spectrometer. Preparative high-performance liquid chromatography (HPLC) was performed on a Develosil ODS-5 (250 mm × 20 mm i.d., 5 μm) column (Nomura Chemical Co., Aichi, Japan) equipped with a JASCO PU-980 Intelligent pump and an SPD-10Ai UV/vis detector (Shimadzu corporation, Kyoto, Japan). Plant Material. The dried leaves of S. sonchifolius (‘Sarada otome’) were donated by Nihonkaisui Co., Ltd. (Tokyo, Japan), in 2008. Four lines of yacon leaves, ‘SY11’, ‘SY23’, ‘SY102’, and ‘SY107’, and four cultivars, ‘Sarada otome’, ‘Sarada okame’, ‘Andesu no yuki’ and ‘Andesu no otome’, were provided by Western Region Agricultural Research Center, National Agriculture and Food Research Organization, Japan, in June 2015. Nine voucher specimens (YL12001TACW304-1, YL-TACW304-2-15001−YL-TACW304-2-15008) were
Yacon [Smallanthus sonchifolius (Poepp. and Endi.)] belongs to the Asteraceae family, is native to the Southern Andes, and has spread to European and Asian countries as a healthy food material.1 Yacon leaves have various physiological functions and warrant the increasing interest in their potential applications. The yacon leaf contains polyphenols,2 kaurene-type diterpenoids,3 geranylnerol derivatives,4 and melampolide-type sesquiterpene lactones.5,6 Yacon roots have been used as a folk medicine in the Andes region. In 2001, the leaf extracts were shown to exhibit a hypoglycemic activity in Sprague−Dawley rats, which suggested efficiency to control diabetes mellitus.7 Sesquiterpene lactones derived from yacon leaf extracts have been investigated, and various biological activities such as antimicrobial,8 antifungal,5 antioxidant,9,10 anti-inflammatory,11 anti-tumor promotion,12 and trypanocidal activity13 were demonstrated. Recently significant cytotoxicity and cell death induction of monomer sesquiterpene lactones, polymatin B, 3, enhydrin, 4, uvedalin, 5, and sonchifolin, 6 (Figure 1), against some cancer cell lines have been reported.14,15 To date, there has been no investigation on the cytotoxicity of sesquiterpene lactone dimer from the yacon leaf. In this study, we offer the first report on the enhancement of cytotoxicity against three tumor cell lines (HeLa, HL-60, and B16-F10 melanoma) by utilizing a new sesquiterpene lactone dimer, uvedafolin, 1. © 2015 American Chemical Society
MATERIALS AND METHODS
Received: Revised: Accepted: Published: 10856
August 29, 2015 November 17, 2015 November 18, 2015 November 18, 2015 DOI: 10.1021/acs.jafc.5b05229 J. Agric. Food Chem. 2015, 63, 10856−10861
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of sesquiterpene lactones from yacon leaves. (1) Uvedafolin. (2) Enhydrofolin. (3) Polymatin B. (4) Enhydrin. (5) Uvedalin. (6) Sonchifolin. (7) Parthenolide. (8) Costunolide. (1.7um, 2.1 × 50 mm) column (Waters, Tokyo, Japan). The instrument was operated with ESI source in negative ion mode. Mass determination was performed using the following MS conditions: capillary voltage, 2.5 kV; desolvation temperature, 400 °C; flow rate of desolvation gas, 1,000L/h. High-purity argon (99.99%) was used as a collision gas. Mass spectra were collected in the range m/z 100−1,000. Data acquistion and proceeding were performed using MassLynx software (Waters, Tokyo, Japan). Single-Crystal X-ray Crystallography Analysis. A crystal of 1 (size 0.25 mm × 0.04 mm × 0.01 mm) was selected for X-ray diffraction experiment. The crystal was mounted on glass fibers. Data was collected on a VariMax Saturn 724 diffractometer (Rigaku, Tokyo, Japan) using Mo Kα radiation with λ = 0.71075 Å. The program SHELXS2013 was used to refine structure. (+)-Methyl(1(10)Z,4E)-8β-[(1(10)Z,4E)-2-hydroxy-3-oxy-(8βangeloxygermacra-1(10),4,11(13)-trien-6α,12-olide)]-9α-acetoyloxygermacra-1(10),4,11(13)-dien-6α,12-olide-14-oate. Compound 1 was obtained as colorless needles: 110 mg; mp 182−184 −1 3536, °C; [α]20 D +13.4° (c 1.75, acetone); UV λmax 219 nm; IR λ cm 2952, 1762, 1714, 1437, 1382, 1303, 1244, 1142, 1096, 1014; 1H and 13 C NMR in Table 1; ESIMS m/z 809.3461 [M + H]+ (calcd for C43H53O15, 809.3384). Crystal data of uvedafolin: size 0.25 mm × 0.04 mm × 0.01 mm, orthorhombic, space group P212121, Z = 4, a = 10.012(4) Å, b = 15.246(6) Å, c = 27.796(11) Å, α = β = γ = 90°. V = 4245(3) Å3, Dcal = 1.266 Mg m−3, R [F2 > 2σ(F2)] = 0.107. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre: Deposition number CCDC 1420037. Copies of the data can be obtained free of charge from http://www.ccdc.cam.ac.uk/retrieving. html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, U.K.; fax + 44 1223 336033; e-mail
[email protected]). Cell Culture. The HeLa and HL-60 cell lines were purchased from RIKEN Bioresource Center (Ibaraki, Japan). The murine B16-F10 melanoma cell line was provided from Prof. Katsuichiro Okazaki (Kagawa University). The HeLa and B16-F10 melanoma cells were cultured in E-MEM (Wako Pure Chemical Industries Ltd., Osaka, Japan) medium supplemented with 4.2 mM HEPES (Dojindo Molecular Inc., Kumamoto, Japan), 10% (v/v) fetal bovine serum (Equitech-Bio, Kerrville, TX), 100 units/mL penicillin, and 100 μg/ mL streptomycin (Invitrogen, Carlsbad, CA) at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The HL-60 cells were
deposited at The Research Institute of Food Safety and Nutraceutical Science, Kagawa University, Japan. Extraction and Isolation. The dried leaves of S. sonchifolius (1.6 kg) were extracted three times with acetone (2 L × 3) at room temperature. The acetone extracts were filtered and the solvent was evaporated under vacuum to afford 40.6 g of the crude extracts. The crude extracts were dissolved in 70% MeOH/H2O (7:3 v/v, 1 L). The insoluble precipitates, which contain chlorophyll as major coloring material, were completely removed, and then the soluble liquid was evaporated under vacuum to afford 22.3 g of dried solid. The solid extracts were dissolved again in 40 mL of hexane and mixed with 20 g of Celite. The slurry was dried and then applied to a 40 cm × 4.5 cm i.d. silica gel 60 column (Merck, Darmstadt, Germany). The chromatography technique for the separation of sesquiterpene lactones was conducted as described in a previous report.14,16 The fractions containing six sesquiterpene lactones were processed by recrystallization and/or preparative HPLC to yield 110 mg (0.007% yield) of 1, 30 mg (0.002% yield) of 2, 20 mg (0.001% yield) of 3, 1300 mg (0.08% yield) of 4, 100 mg (0.006% yield) of 5, and 25 mg (0.002% yield) of 6. HPLC Analysis. High-performance liquid chromatography (HPLC) analysis was performed on an Inertsil ODS-3 (150 mm × 4.6 mm i.d., 4 μm) column (GL Sciences, Tokyo, Japan), equipped with in a JASCO HPLC system with two PU-980 Intelligent pumps and two detectors, UV-975 Intelligent UV−vis and MD 2010puls. The mobile phase was consisted of 40% aqueous acetonitrile (solvent A) and 80% aqueous acetonitrile (solvent B). The injection volume was 10 μL, and the analytical temperature was set at 40 °C. The HPLC elution conditions were as follows: 0.0−2.0 min, 0% B, 2.0−40.0 min, 100% B, held at 100% B for a further 5 min. The flow rate was 1.0 mL/ min. The chromatograms were recorded at a wavelength of 230 nm. Compounds 1, 2, and 4−6 were used to create standard curves. Standard stock solutions were prepared at 1 mg/mL in acetone. Five standard solutions were prepared by diluting those of the standard stock solution (compounds 4 and 5 were 0.025−1.0 mg/mL, and 1, 2, and 6 were 0.005−0.5 mg/mL). Compound 3 was quantitated by using the calibration data for 6. LC/ESI-Q-TOF-MS Analysis. Liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS) analysis was conducted using an Acquity UPLC/Xevo QTof MS system (Waters, Tokyo, Japan). Chromatographic separations were conducted using an Acquity UPLC BEH C18 10857
DOI: 10.1021/acs.jafc.5b05229 J. Agric. Food Chem. 2015, 63, 10856−10861
Article
Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Data of Uvedafolin in Acetoned6a A unit
B unit
δC
δH
1
148.61
2a
25.92
2b 3
36.43
7.00 dd (7.56, 9.66) 2.45 dd (6.18, 7.56, 11.64) 2.70 d (6.18) 2.39 br q (6.18, 4.08)
4 5 6
137.94 126.94 75.04
7 8
50.30 71.46
5.00 d (10.32) 4.95 t (9.6, 10.32) 2.91 d (9.6) 6.55 d (8.28)
9
71.68
5.34 d (8.28)
position
10 11 12 13a 13b 14 15 16 17 18 1′ 2′ 3′ 4′ 5′
130.68 135.04 168.41 119.64 165.79 16.29 169.86 20.20 51.63 173.76 76.13 74.02 13.01 22.36
cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. All test samples were dissolved in DMSO (Wako Pure Chemical Industries Ltd., Osaka, Japan) and stored at −20 °C until further experiments. The final concentrations of DMSO did not exceed more than 0.1%. The cell viability was examined by cell counting Kit-8 (Dojindo Molecular Inc., Kumamoto, Japan). Etoposide, parthenolide, 7, and costunolide, 8, were purchased from Sigma-Aldrich Co. Ltd. (Tokyo, Japan). Cytotoxicity Assay. The cytotoxic activities of sesquiterpene lactones were evaluated using the 2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt (WST-8) assay. HeLa and B16-F10 melanoma cells (2 × 103 cell/well) and HL-60 cells (2 × 104 cell/well) were exposed to test samples for 48 h at 37 °C. Control and blank (no cells) were exposed to 0.1% DMSO. The test concentrations were 1−20 μM for monomer sesquiterpene lactones, costunolide, and parthenolide (reference compounds), and 0.5−5 μM for dimer sesquiterpene lactones and etoposide (positive control). After exposure was completed, 10 μL of WST-8 reagent was added to each well and then incubated for 4 h. The OD values were measured at 450 nm wavelength using a Multiskan FC Microplate Photometer (Thermo Scientific Inc., MA). The cytotoxicity percentages in treated populations were determined by comparing with that of the control treatment using eq 1.
5.63 d (3.42) 5.91 d (3.42) 1.95 s 2.01 s 3.69 s
position
δC
1
143.86
2a
25.74
2b 3
36.65
4 5 6
138.39 125.78 75.80
7 8
49.78 66.37
9
30.00
10 11 12 13a 13b 14 15 1″ 2″ 3″ 4″ 5″
131.28 136.51 168.96 119.14 165.67 16.40 166.09 127.48 138.48 15.23 19.99
δH 6.75 t (5.46, 5.51) 2.31 dd (4.13, 5.46) 2.03 m
5.07 d (5.46) 5.07 d (5.46) 2.5 m 6.36 t (7.56, 8.28) 2.80 br q (5.45, 8.28, 7.56)
5.52 d (3.48) 6.06 d (3.48)
cytotoxicity (%) =
1.87 s
A sample − A blank Acontrol − A blank
× 100
(1)
The IC50 value was calculated by using Graphpad prism software 6.0.
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RESULTS AND DISCUSSION Isolation and Structure Elucidation of a Sesquiterpene Lactone Dimer, Uvedafolin, 1. Compound 1 gave colorless crystals (110 mg) with 95% or higher purity. The molecular formula of 1 was determined to be C43H52O15 based on the ESI-TOF-MS molecular ion peak [M + H]+. The IR spectrum of 1 indicated the presence of a hydroxy group (3536 cm−1) and α,β-unsaturated-γ-lactone (1762 cm−1). The 13C NMR data of 1 (Table 1) showed the presence 13 methine
6.11 m 1.92 s 1.83 s
5.22 q (6.18, 6.24) 1.19 d (6.18) 1.25 s
The assignments were based on DEPT and 2D NMR data. δ in ppm, mult (J in Hz).
a
Figure 2. 1H−1H COSY (bold line) and selected HMBC correlations (arrow) of 1. 10858
DOI: 10.1021/acs.jafc.5b05229 J. Agric. Food Chem. 2015, 63, 10856−10861
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Journal of Agricultural and Food Chemistry
no otome’, and ‘Andesu no yuki’) were analyzed (Figure 4). We found that dimer 2 was present in all varieties up to 0.005 to 0.03 mg/g fresh weight (Table 2), while the newly identified dimer, 1, was found in only ‘Sarada otome’ and ‘Sarada okame’ cultivars, 0.064 and 0.045 mg/g fresh weight, respectively. Some of the varieties (‘Sarada otome’ and ‘Sarada okame’) contained dimers 1 and 2. Cytotoxicity of Two Dimer Sesquiterpene Lactones. The cytotoxic activity of two dimer sesquiterpene lactones from yacon leaves was determined using WST-8 assay against HeLa (cervical cancer), HL-60 (leukemia), and B16-F10 melanoma (skin melanoma) cell lines. The results (Table 3) showed that the dimers, 1 and 2, had the most effective IC50 values against the three tumor cell lines ranging from 0.2 to 1.9 μM, compared with those of monomers 3−6, which ranged from 0.7 to 9.9 μM. As a positive control, etoposide, which is widely used in several human cancers such as lung cancer, breast cancer, and leukemia,18−20 had IC50 values of 0.8−114 μM. 1 and 2 showed significant cytotoxicity with IC50 values 1.69 and 1.93 μM for HeLa cells, 0.53 and 0.51 μM for HL-60 cells, and 0.21 and 0.31 μM for B16-F10 melanoma cells, respectively. The dimers caused high cytotoxicity in the B16-F10 melanoma cells, which was indicated by the low IC50 values, up to 8-fold lower than the IC50 values for HeLa cells. The common functional group of these eight sesquiterpene lactones is the α-methylene-γ-lactone skeleton. Among two dimers, 1 and 2, four monomers, 3−6, parthenolide, 7, and costunolide, 8, only 4 possesses two epoxide rings on the molecule, while 2, 5, and 7 have a single epoxy group. Compounds 1, 3, 6, and 8 are lipophilic substances without an epoxide ring in the structures. An acetyl group at the C-9 position is found in compounds 1−5 but not in 6−8. As the activities of 1−5 are much greater than those of 6−8, the acetyl group at the C-9 position is an essential functional group, required for higher cytotoxicity. Compounds 1 and 2, which are composed of two monomers, 4 and 6 for 1, and 5 and 6 for 2, exhibited the greatest cytotoxic activity among the eight sesquiterpene lactones on a mole basis. Moreover, 1:1 mixtures of 4 + 6 and 5 + 6 showed about 3−40 times lower cytotoxicity than 1 and 2 (Table 3). Cytotoxicity of both mixtures showed similar values to compounds 4 and 5. This means that the contribution of 6 to the cytotoxicity of both mixtures was weak. Interestingly, for the B16-F10 melanoma cell line, the mixtures (4 + 6 and 5 + 6) were suppressed 4−5 times more than individual components even though the concentration of 1:1 mixtures was expressed as chemical concentration of either side of either compound, 4 or 6, and 5 or 6. Therefore, a cross linkage of both monomer sesquiterpene lactones with 2′R and 3′S configuration is another important functional group for the cytotoxicity, which may be due to higher lipophilicity and disorder of functional structure of cell membrane. It is known that the α-methylene-γ-lactone moiety forms a Michael-type addition reaction with glutathione and other proteins bearing many SH groups.21 The α-methylene-γ-lactone fragment has been reported to be effective for antibacterial, anti-inflammatory, antitumor, and anti-HIV activities,22−26 and is common in compounds from the Asteraceae. Moreover, the epoxy group also is vulnerable to nucleophilic attack from cysteine side chain of glutathione, a biologically important peptide.27−29 These reactions may cause the disorder of functions of the important proteins and also show higher cytotoxicity. In conclusion, the stereostructure of uvedafolin, 1, a dimer sesquiterpene lactone, isolated from yacon leaves has been
carbons (including five olefin methine carbons at δC 125.7, 126.9, 138.4, 143.8, 148.6), seven methylene carbons (including two olefinic methylene carbons at δC 119.1, 119.6), eight methyl carbons, and 15 quaternary carbons signals (including one oxygenated carbon signal at δC 76.1). These results suggested that 1 was a sesquiterpene lactone dimer. The 1H NMR data of 1 (Table 1) showed the typical signals of two α-methylene-γ-lactone skeletons (δC/H13A 119.64/5.63 and 5.91, δC12A 168.41, and δC/H7A 50.30/2.91 for the A unit, and δC/H13B 119.14/5.22 and 6.06, δC12B 168.96, and δC7B 49.78 for the B unit).5 From the typical proton signals of H7AB on αmethylene-γ-lactone, 1H−1H COSY spectra and HMBC spectra indicated the existence of two sets of ten-membered rings (Figure 2). This was similar to those of compound 2, a sesquiterpene lactone dimer which was previously isolated from yacon leaves.6 The difference in chemical shift between 1 and 2 was due to the lack of an epoxide at C4 and C5 positions (δC 137.94 and 126.94) on the A unit of 1 of the ten-membered rings. This was determined by comparison to 2 at C4 and C5 positions (δC 58.99 and 62.61) on the A unit. The linkage between the A and B units was proven by cross peaks between δH8A 6.55 and δC1′ 173.76, and also between δH3′ 5.22 and δC14B 165.67 (Figure 2). Consequently, the chemical structure of 1 is explained by the esterification of sonchifolin, 6, to uvedalin, 5, through the opening of the epoxy ring of 5 by bimolecular nucleophilic substitution reaction (SN2 reaction) of the carboxylate oxygen at C14B of 6. This reaction reverses the stereochemistry at the 3′-epoxide oxygen. As the absolute chemical structure of enhydrin bromohydrin has been determined by X-ray crystallographic analysis to be 4R, 5R, 6S, 7S, 8S, 9S, 20S, 22S,17 all of the configurations of 1 were expected to be the 6S, 7S, 8S, 9S, 2′R, 3′S form. An X-ray crystallographic analysis of 1 supported the prospective ORTEP structure (Figure 3). In the same way,
Figure 3. ORTEP diagram of 1.
the chemical structure of 2 was confirmed to be the 4R, 5R, 6S, 7S, 8S, 9S, 2′R, 3′S form. Therefore, total stereochemistry of compounds 1 and 2 was clearly determined (Figure 1). Thus, sesquiterpene lactone dimer 1 from compounds 5 and 6, and 2 from compounds 4 and 6, are, therefore, named as uvedafolin, 1, and enhydrofolin, 2, and the molecular structure is generated by the linkage of the two monomer sesquiterpene lactones (Figure 1). Dimer Sesquiterpene Lactones from Eight Varieties of Yacon Leaf. The dimer sesquiterpene lactones in the eight varieties of yacon leaf (lines, ‘SY11’, ‘SY23’, ‘SY102’, and ‘SY107’; and cultivars, ‘Sarada otome’, ‘Sarada okame’, ‘Andesu 10859
DOI: 10.1021/acs.jafc.5b05229 J. Agric. Food Chem. 2015, 63, 10856−10861
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Journal of Agricultural and Food Chemistry
Figure 4. HPLC chromatograms of four cultivars of yacon leaf extracts (230 nm): (A) ‘Sarada otme’; (B) ‘Sarada okame’; (C) ‘Andesu no yuki’; (D) ‘Andesu no otome’. (1) Uvedafolin. (2) Enhydrofolin. (3) Polymatin B. (4) Enhydrin. (5) Uvedalin. (6) Sonchifolin. Peak at 2.1 min is acetone.
Table 2. Quantitation of Sesquiterpene Lactones (mg/g fresh weight) in Eight Varieties of Yacon (S. sonchifolius) Leaf Extractsb
Table 3. Cytotoxicicty of Sesquiterpene Lactones on Three Tumor Cell Lines IC50 (μM)d
compound variety
1
2
3
4
5
6
Sarada otome Sarada okame Andesu no yuki Andesu no otome SY11 SY23 SY102 SY107
0.064 0.045 −c − − − − −
0.027 0.018 0.005 0.006 0.012 0.014 0.030 0.004
0.004 0.003 0.006 0.008 0.011 0.005 0.010 0.010
0.177 0.166 0.084 0.202 0.577 0.272 0.739 0.132
0.072 0.070 0.160 0.048 0.149 0.084 0.207 0.055
0.004 0.003 0.006 0.008 0.011 0.005 0.010 0.010
b
Quantitation of sesquiterpene lactones was carried out using standard curves. cNot detected.
compound
HeLa
HL-60
B16-F10 melanoma
uvedafolin (1) enhydrofolin (2) polymatin B (3) enhydrin (4) uvedalin (5) sonchifolin (6) 5 + 6e 4 + 6e etoposidef parthenolide (7)g costunolide (8)g
1.69 1.93 5.51 6.55 5.99 9.92 9.74 (4.87) 10.25 (5.12) 3.2 16.55 23.34
0.53 0.51 1.99 1.39 1.62 4.52 3.00 (1.50) 2.36 (1.18) 0.88 10.08 13.58
0.21 0.31 3.93 0.74 1.22 7.59 10.64 (5.32) 8.28 (4.14) 114.3 8.47 61.13
d
IC50 values were calculated from dose−response curves after samples were exposed for 48 h. e5 + 6 and 4 + 6 were prepared as 1:1 mol ratios of 5 and 6, and 4 and 6, respectively. Parentheses show the IC50 values when 1 mol + 1 mol ratio of two-sesquiterpene lactones is defined as 1 mol solution. fEtoposide was used as a positive control. g Parthenolide and costunolide were used as reference compounds.
elucidated as a highly cytotoxic substance against three tumor cell lines (HeLa, HL-60, and B16-F10 melanoma). The high cytotoxicity was partly dependent on the dimer ester structures of monomer sesquiterpene lactones, 4 and 6, or 5 and 6, enhancing the cytotoxicity against the three tumor cell lines. The sesquiterpene lactones’ compositional analyses in eight varieties of yacon leaf clarified that the important dimer sesquiterpene lactones, 1 and 2, are found only in ‘Sarada otome’ and ‘Sarada okame’ cultivars, which establishes the benefits of utilization of waste parts of some yacon leaf varieties as functional plant resources. Further research on mechanism of cytotoxicity for the dimer sesquiterpene lactones, 1 and 2, may lead to new approaches to anticancer agents.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05229. IR, NMR, and MS spectra, ORTEP diagrams, and HPLC chromatograms (PDF) 10860
DOI: 10.1021/acs.jafc.5b05229 J. Agric. Food Chem. 2015, 63, 10856−10861
Article
Journal of Agricultural and Food Chemistry
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81 87 891 3014. Fax: +81 87 891 3021. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Prof. Dr. Katsuichiro Okazaki for kindly providing the murine B16-F10 melanoma cell line for this study. We are also grateful for the Rare Sugar Research Center and instruction (Kagawa University) for providing access to a polarimeter in this study.
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DOI: 10.1021/acs.jafc.5b05229 J. Agric. Food Chem. 2015, 63, 10856−10861
