2016 Volume 64 Issue 3 Pages 276-281
Fourteen compounds were isolated from the flowers of Inula japonica THUNB. (Asteraceae), including two new compounds, (1S,2S,4S,5S,8S,10R)-2-acetoxy-4,3-dihydroxy-pseudoguai-7(11)-en-12,8-olide (1) and (1S,2S,4S,5S,8S,10R)-2,4,13-trihydroxy-pseudoguai-7(11)-en-12,8-olide (2), and twelve known compounds, budlein B (3), 6β-hydroxytomentosin (4), 6-deacetoxybritanin (5), 4-epipulchellin (6), britanin (7), tomentosin (8), (+)-dihydroquercetin (9), (−)-syringaresinol (10), quercetagetin 3,4′-dimethyl ether (11), luteolin (12), britanin G (13) and inuchinenolide C (14). Structures of 1 and 2 were determined based on one and two dimensional (1D)- and (2D)-NMR data and Mosher’s esterification method. Compounds 9 and 12 showed inhibitory activities toward DNA topoisomerase I with IC50 values of 55.7 and 37.0 µM, respectively, compared to camptothecin (CPT) with an IC50 of 24.5 µM. Compounds 7–9 and 11–14 exhibited more potent inhibitory activity against topoisomerases II with IC50 values of 6.9, 3.8, 3.0, 6.9, 10.0, 14.7 and 13.8 µM, respectively, than that of etoposide (VP-16) with an IC50 of 26.9 µM. Compounds 4–7 and 10–14 exhibited weak cytotoxicities to the selected cancer cell lines.
Inula japonica THUNB. (Asteraceae) is a traditional herbal medicine and is widely distributed in Korea, Japan and China, and the flowers of this plant have been used to relieve phlegm, peptic disorder, inflammation and detumescence.1) Some terpenes2–7) and flavonoids8,9) have previously been isolated from this medicinal plant and have been reported to possess diverse biological activities, such as anti-diabetes,10) anti-hypolipidemia,11) hepatoprotective,12) anti-inflammatory7) and anti-tumor1,2,13) effects. We have been isolating compounds that inhibited topoisomerases I and II from the source of medicinal plants.14–18) DNA topoisomerases are enzymes that control DNA topology in the cell and are targets of anticancer drugs.19,20) Topoisomerase I cleaves and reseals one DNA strand at a time and does not require ATP for the complementary strand to pass through the enzyme-linked strand break, thereby effecting DNA relaxation. Topoisomerase II cleaves both strands of DNA during catalysis. In a reaction coupled to ATP binding and hydrolysis, these proteins cleave one DNA duplex, transport a second duplex through the break, and then religate the cleaved duplex.21) Camptothecin (CPT) and etoposide (VP-16) are typical inhibitors of topoisomerases I and II, respectively.19) In this paper, we report isolation of fourteen compounds from the flowers of I. japonica and their DNA topoisomerases I and II inhibitory activities and cytotoxicities.
Two new (1, 2) and twelve known compounds (3–14) were isolated by various chromatographic separations from the EtOAc extract of the flowers of I. japonica (Fig. 1). The high resolution FAB-MS spectrum of 1 exhibited [M+H]+ peak at m/z 325.1653 (Calcd 325.1651) suggesting molecular formula of 1 as C17H24O6. The 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 showed seventeen peaks due to three methyl, four methylene, five methine and five quaternary carbons. The corresponding proton signals were determined by 1H-detected heteronuclear multiple quantum coherence (HMQC) spectrum.
The 1H-NMR spectrum exhibited two methyl singlets [δH 2.06 (3H, s, H–OCOCH3); 1.06 (3H, s, H-15)], one methyl doublet [δH 0.89 (3H, d, J=6.6 Hz, H-14)], four pair of methylene peaks [δH 4.68 (2H, s, H-13), 3.57 (1H, d, J=19.8 Hz, H-6a), 2.91 (1H, d, J=19.8 Hz, H-6b), 2.43 (1H, m, H-3a), 2.03 (1H, m, H-3b), 2.21 (1H, dt, J=13.1, 2.8 Hz, H-9a), 1.12 (1H, ddd, J=13.1, 12.5, 12.5 Hz, H-9b)], five methine protons [δH 5.33 (1H, br d, J=12.1 Hz, H-8), 5.07 (1H, m, H-2), 4.16 (1H, t, J=9.3 Hz, H-4), 1.80 (1H, m, H-10), 1.46 (1H, dd, J=11.1, 7.4 Hz, H-1)]. Analysis of the 1H–1H correlation spectroscopy (COSY) spectrum revealed coupling spin-systems among C-8/C-9/C-10(C-14)/C-1/C-2/C-3/C-4 and the heteronuclear multiple bond connectivity (HMBC) correlations of H-1/C-2, C-3, C-5, C-9 and C-15; H-6/ C-7 and C-11; H-13/ C-7, C-11 and C-12; H-14/C-1, C-9 and C-10; H-15/C-1, C-4, C-5, and C-6; H-2′/C-1′ were observed (Fig. 2). The position of the acetyl group on C-2 was determined by comparison of chemical shift values of C-1, C-2 and C-3 of 1 with those of 2, in which C-2 signal of 1 was shifted to 4.8 ppm downfield while C-1 and C-3 signals were shifted to 3.7 and 2.3 ppm upfield, respectively, compared with those of 2. In addition, protons of the acetyl group showed nuclear Overhauser effect (NOE) cross peaks with H-2, H-3 and H-4 in the nuclear Overhauser effect spectroscopy (NOESY) spectrum of 1 (Fig. 3). Coupling constant values of the axial proton, H-9b, (2JH9b-H9a=13.1 Hz, 3JH9b-H8=12.5 Hz and 3JH9b-H10=12.5 Hz), indicated the dihedral angles from H-9b to both 8-H and 10-H were about 180°. In the NOESY spectrum, the correlations of H-4/H-3b and H-1, H-1/H-6b and H-9b, H-15/H-2, H-6a, H-8 and H-10, H2/H3a and H-10, H-8/H-9a, H-10 and H-15 were identified (Fig. 3). Thus, the relative stereochemistry of compound 1 was elucidated as 2α-acetoxy-4β,13-dihydroxy-1αH,10βH-pseudoguai-7(11)-en-12,8α-olide. In order to determine the absolute stereochemistry on C-4 of 1 by Mosher’s esterification method, (S)- and (R)-α-methoxy-α-(trifluoromethyl)-phenylacetyl (MTPA) esters of 1, 1a and b, respectively, were prepared using the corresponding (R)-(−)- and (S)-(+)-MTPA chloride, respectively. The differences of proton chemical shifts between (S)-MTPA ester and (R)-MTPA esters are shown in Fig. 4. For the configuration on C-4 carbon of 1, the Δδ values (δS–δR) of protons on the β1, β2, γ, δ positions from C-4 were all positive and those on the α′, β′ positions were all negative, which indicated 4S configuration.22) Accordingly, all relevant chiral centers in 1 were determined as 1S, 2S, 4S, 5S, 8S and 10R configurations.
) and HMBC (
) Correlations of Compound 1
) Correlations of Compound 1
Compound 2 had the molecular formula of C15H22O5 as established from the high resolution (HR)-FAB-MS at m/z 283.1544 [M+H]+ (Calcd 283.1545). The 1H- and 13C-NMR spectra of 2 were similar to those of 1, except that no characteristic peaks of acetyl group appeared in the spectra of 2. All other chemical shift values of the 13C-NMR spectra of 1 and 2 were consistent within difference of 1 ppm except those of C-1, C-2 and C-3. Analyses of the 1H–1H COSY and NOESY spectrum of 2 showed same patterns of coupling and NOE correlations as those of 1 (see the Supplementary materials). Based on these results, the relative stereochemistry of 2 was assigned as 2α,4β-dihydroxy-13-acetoxy-1αH,10βH-pseudoguai-7(11)-en-12,8α-olide. The (S)- and (R)-MTPA esters of 2 (2a and b, respectively) were prepared using the corresponding (R)-(−)- and (S)-(+)-MTPA chloride, respectively. For the configuration on C-2 carbon of 2, the Δδ values (δS–δR) of three protons on the α and β positions from C-2 were all negative and those on the β′, γ1′, γ2′ and δ′ positions were all positive. The negative value for the α′ position was probably due to the anisotropic effect of the other α-methoxy-α-(trifluoromethyl)phenyl-acetyl functions on the C-4 positions of 2a and b. Therefore, S configuration on C-2 of 2 was suggested and all relevant chiral centers in 2 were assigned as 1S, 2S, 4S, 5S, 8S and 10R configurations. Structures of 3–14 were identified by comparison of 1H- and C13-NMR, FAB-MS spectral data and optical rotation values with those previously reported to be budlein B (3),23) 6β-hydroxytomentosin (4),24) 6-deacetoxybritanin (5),25) 4-epipulchellin (6),26) britanin (7),27) tomentosin (8),28) (+)-dihydroquercetin (9),29) (−)-syringaresinol (10),30) quercetagetin 3,4′-dimethy ether (11),31) luteolin (12),32) britanin G (13)33) and inuchinenolide C (14)34) (see the Supplementary materials).
These compounds (1–14) were tested for inhibitory activities of DNA topoisomerases I and II (Fig. 5, Table 1). Compounds 9 and 12 showed inhibitory activities toward DNA topoisomerase I with IC50 values of 55.7 and 37.0 µM, respectively, compared with IC50 values of 24.5 µM for the positive control, CPT. Compounds 7–9 and 11–14 exhibited more potent inhibitory activity against topoisomerase II (IC50; 6.9, 3.8, 3.0, 6.9, 9.9, 14.7 and 13.8 µM, respectively) than that of the positive control, Vp-16 (IC50 ; 26.9 µM) (Fig. 6, Table 1). These are the first report of topoisomerases I and II inhibitory activities of compounds 1–14 except that topoisomerase II inhibitory activity of 9, 10 and 12 were reported previously.35–37)
D: supercoiled DNA only. T: supercoiled DNA+topoisomerase I. C1, C2, C3, C4: supercoiled DNA+topoisomerase I+camptothecin (10, 20, 40, 80 µM). a, b, c, d: supercoiled DNA+topoisomerase I+compound (10, 20, 40, 80 µM).
| Compounds | Topoisomerase I IC50 (µM) | Topoisomerase II IC50 (µM) | Cytotoxicity IC50 (µM) | |||
|---|---|---|---|---|---|---|
| A549a) | SK-OV-3b) | HepG-2c) | HT-29d) | |||
| 1 | >80 | >80 | >80 | >80 | >80 | >80 |
| 2 | >80 | >80 | >80 | >80 | >80 | >80 |
| 3 | >80 | >80 | >80 | >80 | >80 | >80 |
| 4 | >80 | >80 | >80 | >80 | 16.8 | 18.2 |
| 5 | >80 | >80 | 24.8 | >80 | 50.3 | 1.1 |
| 6 | >80 | >80 | >80 | >80 | >80 | 47.3 |
| 7 | >80 | 6.9 | 44.1 | >80 | 35.5 | 3.9 |
| 8 | >80 | 3.8 | >80 | >80 | >80 | 80 |
| 9 | 55.7 | 3.0 | >80 | >80 | >80 | >80 |
| 10 | >80 | 28.9 | 65.6 | >80 | 30.0 | 57.5 |
| 11 | >80 | 6.9 | 59.3 | >80 | >80 | 30.9 |
| 12 | 37.0 | 9.9 | >80 | >80 | >80 | 29.6 |
| 13 | >80 | 14.7 | 32.8 | >80 | 80 | 7.8 |
| 14 | >80 | 13.8 | 35.6 | >80 | >80 | 21.3 |
| CPTe) | 24.5 | — | 0.8 | 10.0 | 0.7 | 0.4 |
| VP-16f) | — | 26.9 | — | — | — | — |
a) A549: lung carcinoma. b) SK-OV-3: ovary adenocarcinoma. c) HepG-2: live hepatoblastoma. d) HT-29: colon adenocarcinoma. e) CPT: positive control. f) Vp-16: positive control. All data was the arithmetic mean of triplicate determinations.
D: supercoiled DNA only. T: supercoiled DNA+topoisomerase II. V1, V2, V3, V4: supercoiled DNA+topoisomerase II+etoposide (1, 5, 10, 20 µM). a, b, c, d: supercoiled DNA+topoisomerase II+compound (1, 5, 10, 20 µM).
The cytotoxicity of all the compounds isolated were tested on selected human cancer cell lines, lung carcinoma (A549), ovary adenocarcinoma (SK-OV-3), liver hepatoblastoma (HepG-2) and colon adenocarcinoma (HT-29) cell lines (Table 1). Compounds 5, 7, 10, 11, 13 and 14 exhibited weak cytotoxicities on the A549 in the range of IC50 values, 24.8–65.6 µM (IC50 of CPT, 0.8 µM). Compounds 4, 5, 7 and 10 showed weak cytotoxicities against the HepG-2 with IC50 values of 16.8–50.3 µM (IC50 of CPT, 0.7 µM). In the HT-29 cell line, compounds 4–7 and 10–14 showed cytotoxic activities with IC50 values of 1.1–57.3 µM (IC50 of CPT, 0.4 µM). None of the compounds exhibited cytotoxicity against SK-OV-3 cell line.
In conclusion, fourteen compounds including two new guaiane type sesquiterpenoids were isolated from the flowers of I. japonica. Structures of 1 and 2 were determined based on spectroscopic data. Compounds 9 and 12 showed inhibitory activities on DNA topoisomerases I, and 7–9 and 11–14 exhibited more potent inhibitory activity against topoisomerase II than that of VP-16. Inhibitory activities toward DNA topoisomerases I and II of these compounds except those of 9, 10 and 12 are firstly reported. Compounds 4, 5, 7, 10, 11, 13 and 14 showed weak cytotoxicity to the selected cancer cell lines.
The NMR spectra were recorded on Bruker 250 MHz (DMX 250, Germany) and Varian 600 MHz (VNS 600, Australia) spectrometers using their standard pulse program. FAB-MS spectra were recorded on a JEOL JMS-700 (Tokyo, Japan) mass spectrometer at the Daegu center of KBSI, Korea. Stationary phases for column chromatography (silica gel 60, 70–230 and 230–400 mesh, LiChroprep RP-18 gel, 40–63 µm, Sephadex LH-20) and TLC plates (silica-gel 60 F254 and RP-18 F254, 0.25 mm) were purchased from Merck KGaA (Darmstadt, Germany). Spots were detected under UV radiation and by spraying with 10% H2SO4, followed by heating. The NMR solvents, chloroform-d, pyridine-d5 and methanol-d4, were purchased from Aldrich (St. Louis, MO, U.S.A.). For preparative HPLC, the LC-10AD pump, SPD-10A detector and Shim-Pack prep-ODS (20×250 mm) column (Shimadzu, Kyoto, Japan) were used.
Plant MaterialThe flowers of I. japonica was purchased in May 2008 from Dongkyung Pharm Co., Ltd. in Seoul, South Korea. These materials were confirmed taxonomically by Professor Gi-Hwan Bae, Chungnam National University, Daejeon, South Korea. A voucher specimen (YNIJ-2008) has been deposited at the College of Pharmacy, Yeungnam University, South Korea.
Extraction and IsolationThe flowers of I. japonica (10 kg) were extracted with 70% EtOH at 40°C for 12 h and the EtOH solution was evaporated to dryness at 40°C (1.5 kg). The dried EtOH extract was suspended with H2O, and the resulting H2O layer was partitioned three times with n-hexane and EtOAc, successively. The EtOAc extract (130 g) was loaded on a silica gel column (11×22 cm, silica-gel 70–230 mesh), and the column was eluted with n-hexane–EtOAc (gradient from n-hexane to EtOAc) and then EtOAc–MeOH (gradient from EtOAc to MeOH). The eluent was combined on the basis of TLC analyses, giving 27 fractions (E1–E27). Fraction E-18 (4 g) was chromatographed on a reverse-phase column (4×25 cm, LiChroprep Rp-18) with MeOH–H2O (gradient from 30 : 70 to 100 : 0) to give compound 3 (62 mg). Fraction E15 (10 g) was chromatographed on a silica gel column (4×25 cm) with n-hexane–EtOAc (gradient from 90 : 10 to 0 : 100) giving 20 fractions (E15-1–E15-20). Fraction E15-16 (1.1 g) was chromatographed on a reverse-phase column (3×22 cm) with MeOH–H2O (gradient from 25 : 75 to 100 : 0) to give compound 4 (96 mg) and compound 5 (210 mg). Fraction E-21 (3 g) was chromatographed on a reverse-phase column (4×25 cm) with MeOH–H2O (gradient from 20 : 80 to 100 : 0), giving 32 fractions (E21-1–E21-32). Fraction E21-5 (800 mg) was eluted through a reverse-phase column (4×25 cm) with MeOH–H2O (gradient from 20 : 80 to 100 : 0), and the main fraction from the column was subjected to preparative HPLC (gradient from 30 to 100%), resulting in purification of compounds 1 (8 mg), 2 (5 mg), and 6 (7 mg). Fraction E10 (3.5 g) was chromatographed on a silica-gel column (5×25 cm) with ethyl acetate–methanol (gradient from 60 : 40 to 0 : 100), affording compound 8 (2 g). Fraction E12 (3 g) was chromatographed on a silica-gel column (5×25 cm) with hexane–ethyl acetate (gradient from 90 : 10 to 0 : 100) and the mixture was subjected to preparative HPLC, resulting in the purification of compound 13 (10 mg) and compound 14 (10 mg). Fraction E13 (10 g) was recrystallized with chloroform and methanol, standing for 2 h at room temperature to give compound 7 (5 g). Fraction E18 (4 g) was chromatographed on a reverse-phase column (4×25 cm) with methanol–H2O (gradient from 30 : 70 to 100 : 0) to give 35 fractions (E18-1–E18-35). Fraction E18-6 (100 mg) was passed through a Sephadex LH-20 column (3×80 cm) with MeOH to give compound 9 (4 mg). Fraction E18-14 (120 mg) was purified on a Sephadex LH-20 column (3×80 cm) with MeOH as a mobile phase to give compound 10 (13 mg). Fraction E18-21 (93 mg) was chromatographed on a Sephadex LH-20 column (3×80 cm) eluted with MeOH to give compound 11 (6 mg). Fraction E18-26 (246 mg) was separated on a Sephadex LH-20 column (3×80 cm) with MeOH to give compound 12 (10 mg).
Determination of Configuration of Hydroxyl Group by Mosher’s Esterification(S)- and (R)-MTPA [α-methoxy-α-(trifluoromethyl)phenyl-acetyl]esters of 1 and 2 were prepared using Mosher’s esterification method in NMR tubes.38) Compounds (1 and 2, 1 mg each) and 4-(dimethylamino)-pyridine (0.2 mg) were transferred into each vial, and the mixtures were dried under vacuum. S-(+) and R-(−)-α-methoxy-α-(trifluoromethyl)phenyl-acetyl chloride (6.0 µL) were immediately added to each vial in pyridine-d5 (0.5 mL), then the vial was sealed and shaken to mix the sample and MTPA chloride evenly. The reaction was permitted to stand at room temperature for 1 h. The solution of derivatives was moved to an NMR tube then monitored by 1H-NMR.
(1S,2S,4S,5S,8S,10R)-2-Acetoxy-4,13-dihydroxy-pseudoguai-7(11)-en-12,8-olide (1)White powder; 1H-NMR (pyridine-d5, 250 MHz) δ: 5.33 (1H, br d, J=12.1 Hz, H-8), 5.07 (1H, m, H-2), 4.68 (2H, s, H-13), 4.16 (1H, t, J=9.3 Hz, H-4), 3.57 (1H, d, J=19.8 Hz, H-6a), 2.91 (1H, d, J=19.8 Hz, H-6b), 2.43 (1H, m, H-3a), 2.21 (1H, dt, J=13.1, 2.8 Hz, H-9a), 2.06 (3H, s, H-2′), 2.03 (1H, m, H-3b), 1.80 (1H, m, H-10), 1.46 (1H, dd, J=11.1, 7.4 Hz, H-1), 1.12 (1H, ddd, J=13.1, 12.5, 12.5 Hz, H-9b), 1.06 (3H, s, H-15), 0.89 (3H, d, J=6.6 Hz, H-14); 13C-NMR (pyridine-d5, 62.5 MHz) δ: 173.5 (C-12), 170.8 (C-1ʹ), 167.6 (C-7), 127.7 (C-11), 82.1 (C-8), 79.4 (C-4), 76.0 (C-2), 57.8 (C-1), 54.3 (C-13), 46.4 (C-5), 43.1 (C-9), 39.3 (C-3), 38.8 (C-6), 31.7 (C-10), 21.5 (C-2′), 21.0 (C-14), 14.9 (C-15). Positive HR-FAB-MS m/z 325.1653 [M+H]+ (Calcd for C17H25O6: 325.1651). [α]D20: +11.9 (c=0.30, MeOH). UV (MeOH) λmax nm (log ε): 215 (3.85). IR (KBr) νmax cm−1: 3288, 2994, 2932, 2881, 2855, 1736, 1678 1457, 1381, 1253.
(1S,2S,4S,5S,8S,10R)-2,4,13-Trihydroxy-pseudoguai-7(11)-en-12,8-olide (2)Yellow crystal; 1H-NMR (pyridine-d5, 250 MHz) δ: 5.35 (1H, br d, J=12.3 Hz, H-8), 4.65 (2H, s, H-13), 4.34 (1H, dd, J=9.9, 8.7 Hz, H-4), 4.19 (1H, m, H-2), 3.62 (1H, d, J=20.0 Hz, H-6a), 2.88 (1H, d, J=20.0 Hz, H-6b), 2.36 (2H, m, H-3), 2.26 (1H, dt, J=13.2, 2.5 Hz, H-9a), 1.87 (1H, m, H-10), 1.46 (1H, dd, J=12.4, 6.9 Hz, H-1), 1.30 (3H, d, J=6.5 Hz, H-14), 1.17 (1H, m, H-9b), 1.10 (3H, s, H-15); 13C-NMR (pyridine-d5, 62.5 MHz) δ: 172.6 (C-12), 167.2 (C-7), 126.5 (C-11), 81.3 (C-8), 78.7 (C-4), 71.6 (C-2), 61.5 (C-1), 53.3 (C-13), 46.0 (C-5), 42.7 (C-9), 41.7 (C-3), 38.1 (C-6), 31.4 (C-10), 21.1 (C-14), 14.2 (C-15). Positive HR-FAB-MS m/z 283.1544 [M+H]+ (Calcd for C15H23O5: 283.1545). [α]D20: +24.8 (c=0.10, MeOH). UV (MeOH) λmax nm (log ε): 215 (3.88). IR (KBr) νmax cm−1: 3445, 2961, 2926, 2856, 1745, 1632, 1456, 1384.
Assay for DNA Topoisomerases I and II Inhibition in VitroDNA topoisomerase I inhibition assay was carried out according to the reported method.18,39) Each sample was tested triplicate.
Assay for CytotoxicityThe tetrazolium-based colorimetric assay (MTT assay) was used for the in vitro assay of cytotoxicity in selected cancer cell lines, including lung carcinoma (A549), ovary adenocarcinoma (SK-OV-3), liver hepatoblastoma, (HepG-2) and colon adenocarcinoma (HT-29) cells.18) Each sample was tested triplicate.
This research was supported by a Yeungnam University research Grant in 2013.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
