Fitoterapia 93 (2014) 142–149
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Diterpenoids from Isodon sculponeatus Hua-Yi Jiang a,b, Wei-Guang Wang a, Min Zhou a,b, Hai-Yan Wu a,b, Rui Zhan a, Xiao-Nian Li a, Xue Du a, Yan Li a, Jian-Xin Pu a,⁎, Han-Dong Sun a,⁎ a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People's Republic of China b University of the Chinese Academy of Sciences, Beijing 100049, People's Republic of China
a r t i c l e
i n f o
Article history: Received 25 November 2013 Accepted in revised form 28 December 2013 Accepted 31 December 2013 Available online 10 January 2014 Chemical compounds studied in this article: Bisjaponin A (PubChem CID: 24895695) Lushanrubescensin J (PubChem CID: 11297111) Sculponeatin N (PubChem CID: 42643119) Hebeiabinin B (PubChem CID: 16112773)
a b s t r a c t Phytochemical investigation of the aerial parts of Isodon sculponeatus afforded six new 7,20-epoxy-ent-kauranoids, sculponins U–Z (1–6), and 11 known diterpenoids (7–17). The structures of these new compounds were elucidated primarily by means of extensive spectroscopic analysis, and the absolute configuration of 1 was determined by single crystal X-ray diffraction. Compound 5 exhibited weak cytotoxic activity against HL-60, SMMC-7721, MCF-7, and SW-480 cell lines, and it also inhibited NO production in LPS-stimulated RAW264.7 cells, with IC50 value of 13.8 μM. © 2014 Elsevier B.V. All rights reserved.
Keywords: Isodon sculponeatus Diterpenoids X-ray diffraction Cytotoxicity Anti-inflammatory activity
1. Introduction The genus Isodon is an abundant source of diterpenoids with diverse chemical structures, of which some have antitumor and anti-inflammatory activities [1]. Isodon sculponeatus (Vaniot) Kudo (Lamiaceae), a perennial herb, is widely distributed in southern China and has been used in folk medicines for treatment of dysentery and beriberi [2,3]. Previous phytochemical investigations of this species have resulted in the isolation of several diterpenoids exhibiting significant cytotoxicity toward tumor cell lines, especially, sculponin A displaying strong cytotoxic activity against K562, A549, and HepG2 cell lines with IC50 values equal to the positive control cisplatin [4–14]. ⁎ Corresponding authors. Tel.: +86 871 65223251; fax: +86 871 65216343. E-mail addresses: [email protected] (J.-X. Pu), [email protected] (H.-D. Sun). 0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.025
As part of our program to discover structurally interesting and bioactive diterpenoids [15], we have been interested in the chemical constituents of I. sculponeatus indigenous to Muli County of Sichuan Province, PR China. A series of 6,7-seco-entkauranoids have been obtained, and isodocarpin exhibited significant cytotoxic activity against five human tumor cell lines and intriguing inhibitory activity against NO production in LPS-stimulated RAW264.7 cells [16]. Continued investigation on this plant has now furnished an additional six new 7,20-epoxyent-kauranoids (1–6), namely sculponins U–Z, and 11 known ones, wikstroemioidin A (7) [17], 1α,6β,7β,15β-tetrahydroxy7α,20-epoxy-ent-kaur-16-ene (8) [18], bisjaponin A (9) [19], lushanrubescensin J (10) [20], ent-kaurane-7α,16β,17-triol (11) [21], sculponeatin L (12) [6], sculponeatin N (13) [14], entabienervonin C (14) [22], hebeiabinin B (15) [22], maoyecrystal G (16) [23], and maoyecrystal H (17) [23] (Fig. 1). The absolute configuration of 1 was confirmed by single crystal X-ray
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143
Fig. 1. The structures of compounds 1–17.
diffraction. Herein, we report the isolation and structure elucidation of the new compounds, as well as the cytotoxic properties and anti-inflammatory assay of the selected isolates.
Corporation, Tokyo, Japan), and Sephadex LH-20 (Pharmacia). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 5% H2SO4 in ethanol, followed by heating.
2. Experimental section 2.1. General experimental procedures X-ray data were collected using a Bruker APEX DUO instrument. Melting points were obtained on an XRC-1 apparatus and were uncorrected. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Tenor 27 spectrophotometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers with TMS as internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HREIMS was performed on an API QSTAR time-of-flight spectrometer. HSCCC was performed on a TBE-300B instrument (TAUTO). Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm) column. Column chromatography (CC) was performed on silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), MCI gel (75–150 μm, Mitsubishi Chemical
2.2. Plant material Aerial parts of I. sculponeatus were collected in August 2011 from Muli County, Sichuan Province, People's Republic of China, and identified by Prof. Xi-Wen Li, Kunming Institute of Botany. A voucher specimen (KIB 20110813) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The dried and powdered aerial parts of I. sculponeatus (8 kg) were extracted with 70% aqueous acetone (30 L) four times (2 days each time) at room temperature, then filtered. The filtrate was evaporated under reduced pressure and then partitioned between EtOAc and H2O. The EtOAc soluble portion (700 g) was subjected to column chromatography on silica gel (3 kg, 100–200 mesh), eluted with a CHCl3–Me2CO gradient system (1:0–0:1) that afforded fractions A–G. The fractions were then decolorized using MCI gel and eluted with 90% MeOH–H2O.
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Fraction B (120 g) was chromatographed via silica gel CC (1.2 kg, 200–300 mesh), eluted with CHCl3–MeOH gradient (150:1–1:1) to yield fractions B1–B5. Compound 12 (50.0 mg) was crystallized from fraction B4. Fraction B2 (10 g) was purified by medium-pressure column chromatography on RP-18 (400 g, MeOH–H2O gradient, 20%–100%), followed by repeated column chromatography over silica gel (11 g, CHCl3– MeOH gradient, 120:1–0:1), to obtain compound 13 (15.3 mg). Fraction C (80 g) was separated by medium-pressure column chromatography on RP-18 (900 g, MeOH–H2O gradient, 25%–100%) to give fractions C1–C5. Compound 9 (10.1 mg) was precipitated out from fraction C1. Fraction C2 (230 mg) was subjected to repeated CC over silica gel (7 g, 200–300 mesh), eluted with CHCl3–MeOH (gradient system: 120:1–1:1), to yield compounds 1 (2.1 mg) and 2 (2.3 mg). Compounds 3 (3.1 mg), 4 (2.3 mg), and 7 (3.2 mg) were isolated from fraction C3 by Sephadex LH-20 CC (CHCl3–MeOH 1:1) and then by repeated column chromatography over silica gel (8 g, petroleum ether–Me2CO gradient, 12:1–0:1). Fraction D (100 g) was subjected to medium-pressure column chromatography on RP-18 (900 g, MeOH–H2O gradient, 10%–100%) to give fractions D1–D6. Fraction D1 (8 g) was further purified by column chromatography on silica gel (210 g, 200–300 mesh), eluted with CHCl3–MeOH gradient system (100:1–1:2), followed by semipreparative HPLC (25% MeCN–H2O), to afford compounds 5 (5.1 mg) and 8 (4.5 mg). Fraction D4 (9 g) was further purified by column chromatography on silica gel (200 g, 200–300 mesh), eluted with CHCl3–MeOH gradient system (100:1–1:2), followed by semipreparative HPLC (23% MeCN–H2O, tR = 12.5 and 15.7 min, respectively), to afford compounds 14 (7.7 mg) and 15 (6.8 mg). Compound 10 (7.3 mg) was precipitated out from fraction D5 (3 g). Then fraction D6 (280 mg) was separated by repeated CC on silica gel (8 g, 200–300 mesh), eluted with CHCl3–MeOH (gradient system: 100:1–1:1), to yield compound 11 (13.2 mg).
Fraction E (78 g) was separated by medium-pressure column chromatography on RP-18 (900 g, MeOH–H2O gradient, 10%–80%), to obtain fractions E1–E6. After CC on silica gel (330 g, 200–300 mesh), eluted with CHCl3–MeOH (gradient system: 80:1–1:1), followed by semipreparative HPLC (25% MeOH–H2O), compound 6 (2.5 mg) was obtained from fraction E3 (12 g). Then fraction E4 was separated by HSCCC (CHCl3–MeOH–H2O 4:3:2) and then by repeated CC on silica gel (30 g, 200–300 mesh), eluted with CHCl3–MeOH gradient (80:1–1:1), to yield the mixture of compounds 16 and 17 (7.8 mg). 2.4. Spectroscopic data 2.4.1. Sculponin U (1) Colorless needle crystals (MeOH); mp 247–249 °C; [α]25D –78.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (4.13) nm; IR (KBr) νmax 3468, 2952, 1750 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 371 [M + Na]+; positive-ion HREIMS [M]+ m/z 348.1943 (calculated for 348.1937). 2.4.2. Sculponin V (2) White amorphous powder; [α]26D –22.6 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 203 (3.47), 372 (1.88) nm; IR (KBr) νmax 3431, 2932, 1721, 1629 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 387 [M + Na]+; positive-ion HREIMS [M]+ m/z 364.1880 (calculated for 364.1886). 2.4.3. Sculponin W (3) White amorphous powder; [α]26D –77.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (3.61) nm; IR (KBr) νmax 3447, 1721, 1630 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 429 [M + Na]+; positive-ion HREIMS [M]+ m/z 406.2000 (calculated for 406.1992).
Table 1 13 C NMR data of compounds 1–6 (in C5D5N, δ in ppm).a Position
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc
29.8 (t) 19.3 (t) 41.0 (t) 34.3 (s) 55.4 (d) 73.0 (d) 105.7 (s) 53.0 (s) 41.9 (d) 44.8 (s) 18.1 (t) 32.6 (t) 36.1 (d) 25.8 (t) 74.9 (d) 160.8 (s) 108.4 (t) 31.6 (q) 21.2 (q) 175.9 (s)
73.9 (d) 31.2 (t) 39.3 (t) 34.1 (s) 55.7 (d) 73.2 (d) 106.3 (s) 52.9 (s) 43.7 (d) 48.5 (s) 21.7 (t) 32.9 (t) 36.3 (d) 25.6 (t) 74.9 (d) 160.9 (s) 108.0 (t) 31.2 (q) 21.0 (q) 176.5 (s)
74.6 (d) 25.2 (t) 38.6 (t) 33.7 (s) 61.1 (d) 73.8 (d) 97.8 (s) 51.7 (s) 40.9 (d) 41.3 (s) 51.6 (d) 54.6 (d) 39.0 (d) 25.8 (t) 75.0 (d) 153.3 (s) 108.6 (t) 32.0 (q) 21.3 (q) 65.3 (t) 21.3 (q) 170.6 (s)
75.6 (d) 25.1 (t) 38.4 (t) 33.6 (s) 61.3 (d) 74.0 (d) 98.7 (s) 53.3 (s) 44.8 (d) 41.5 (s) 125.5 (d) 135.4 (d) 40.2 (d) 32.1 (t) 75.7 (d) 157.6 (s) 106.0 (t) 32.1 (q) 21.1 (q) 64.1 (t) 21.4 (q) 170.3 (s)
31.4 (t) 19.2 (t) 42.1 (t) 34.0 (s) 57.9 (d) 75.3 (d) 97.8 (s) 52.1 (s) 47.2 (d) 37.3 (s) 65.1 (d) 45.3 (t) 37.3 (d) 27.1 (t) 75.8 (d) 162.8 (s) 106.6 (t) 34.5 (q) 22.9 (q) 68.8 (t)
31.3 (t) 18.9 (t) 36.1 (t) 37.9 (s) 60.0 (d) 73.9 (d) 96.6 (s) 61.7 (s) 54.9 (d) 39.0 (s) 65.6 (d) 41.6 (t) 34.9 (d) 27.4 (t) 210.9 (s) 154.4 (s) 115.7 (t) 29.1 (q) 64.7 (t) 69.8 (t)
a 1
H,
NMR data of compounds 1 and 4 were recorded at 100 MHz; data for compounds 2, 3, 5, and 6 were recorded at 125 MHz. Assignments were made based on C NMR, DEPT, HSQC, 1H–1H COSY, HMBC, and ROESY experiments.
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Table 2 1 H NMR spectroscopic data (in C5D5N, δ in ppm, J in Hz) of compounds 1–6.a Position
1
2
3
4
5
6
1a 1b 2a 2b 3a 3b 5 6 9 11a 11b 12a 12b 13 14a 14b 15 17a 17b 18 19a 19b 20a 20b OAc
2.36 (br d, 13.3) 0.95 (m) 1.95 (m)
3.55 (br s)
5.26 (dd, 11.5, 5.2)
5.13 (dd, 13.7, 6.3)
2.32 (overlapped)
2.07 1.96 1.33 1.26 1.78 4.27 2.76 2.35 1.66 2.10 1.41 2.60 2.12 1.90 5.01 5.46 5.10 1.09 0.92
1.88 (m) 1.71 (m) 1.31 (m)
1.86 1.62 1.32 1.26 1.65 4.19 3.15 5.58
1.39 (overlapped)
2.25 (br d, 13.4) 1.39 (m) 1.58 (overlapped) 1.41 (m) 2.38 (br d, 13.0) 1.11 (br t, 13.0) 1.73 (d, 7.8) 4.68 (dd, 10.8, 7.8) 1.58 (overlapped) 4.45 (d, 4.0)
1.34 1.23 1.75 4.31 2.55 1.45 1.27 1.99 1.45 2.58 2.10 1.84 5.09 5.49 5.20 1.11 0.91
(m) (m) (d, 5.6) (br t, 5.6) (m) (overlapped) (m) (m) (overlapped) (m) (dd, 12.2, 4.9) (d, 12.2) (br s) (br s) (br s) (s) (s)
(m) (m) (m) (m) (d, 6.0) (br t, 6.0) (dd, 13.3, 5.6) (m) (m) (m) (m) (m) (m) (d, 12.1) (br s) (br s) (br s) (s) (s)
1.59 4.15 2.80 3.13
(d, 3.5) (br s) (br s) (br t, 4.2)
(m) (m) (m) (m) (d, 3.1) (br s) (br s) (dd, 9.1, 2.1)
3.20 (br t, 4.2)
6.31 (br t, 9.1)
2.98 2.60 2.14 5.17 5.52 5.17 1.07 1.07
3.05 2.46 2.00 5.37 5.33 5.05 1.08 1.07
(t, 4.2) (d, 12.2) (dd, 12.2, 4.8) (overlapped) (br s) (overlapped) (overlapped) (overlapped)
4.82 (d, 9.5) 4.31 (d, 9.5) 2.18 (s)
(m) (dd, 11.4, 4.2) (d, 11.4) (s) (br s) (br s) (overlapped) (overlapped)
4.44 (d, 10.0) 4.24 (d, 10.0) 2.02 (s)
1.38 1.17 1.72 4.36 2.32 4.49
(overlapped) (m) (d, 5.7) (br t, 5.7) (overlapped) (br s)
2.53 1.96 2.88 3.24 2.18 5.28 5.45 5.19 1.24 1.13
(m) (m) (m) (d, 14.2) (dd, 14.2, 5.2) (s) (br s) (br s) (s) (s)
5.41 (d, 9.9) 4.27 (d, 9.9)
2.51 (m) 1.76 (m) 3.12 (m) 3.66 (d, 11.6) 2.59 (dd, 11.6, 4.2) 5.98 (br s) 5.30 (br s) 1.68 (s) 4.36 (d, 10.5) 4.12 (d, 10.5) 5.25 (d, 8.6) 4.38 (d, 8.6)
Assignments were made based on 1H NMR, 13C NMR, DEPT, HSQC, COSY, HMBC, and ROESY experiments. a NMR data for compounds 1 and 4 were recorded at 400 MHz, and data of compounds 2, 3, 5, and 6 were recorded at 500 MHz.
2.4.4. Sculponin X (4) White amorphous powder; [α]25D –271.0 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 204 (4.16), 245 (3.34) nm; IR (KBr) νmax 3429, 3253, 2949, 1723 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 413 [M + Na]+; positive-ion HREIMS [M]+ m/z 390.2038 (calculated for 390.2042). 2.4.5. Sculponin Y (5) White amorphous powder; [α]21D –40.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (3.68), 254 (2.85) nm; IR (KBr) νmax 3420, 2927, 1640 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 373 [M + Na]+; positive-ion HREIMS [M]+ m/z 350.2102 (calculated for 350.2093). 2.4.6. Sculponin Z (6) White amorphous powder; [α]24D –111.0 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 236 (3.87), 202 (3.79) nm; IR (KBr) νmax 3416, 2930, 1705, 1639 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 387 [M + Na]+; positive-ion HREIMS [M]+ m/z 364.1879 (calculated for 364.1886). 2.5. The cytotoxicity assay The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI-1640 or DMEM (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan
formed in living cells based on the reduction of 3-(4,5dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4sulfopheny)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) [24]. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS–50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench's method [25]. 2.6. Nitric oxide production in RAW264.7 macrophages Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI-1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with serial dilutions of the compounds, with the maximum concentration of 25 μM, in the presence of 1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (Reagent A & Reagent B, respectively, Sigma) to 100 μL of each supernatant from LPS (Sigma)-treated or LPS- and compound-treated cells in triplicate. After 5 min incubation, the
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absorbance was measured at 570 nm with a 2104 Envision Multilabel Plate Reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [26]. 2.7. X-ray crystal structure analysis Colorless crystals of 1 were obtained from CH3OH. Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97 [27]. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms [27]. The H-atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 972948 for 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033); e-mail: [email protected]]. Crystallographic data for sculponin U (1): C20H28O5, M = 348.42, orthorhombic, space group P212121, Z = 4, a = 6.59370 (10) Å, b = 9.90360 (10) Å, c = 26.0159 (3) Å; α = β = γ = 90.00°, V = 1698.87 (4) Å3, T = 100 (2) K, μ (Cu Kα) = 0.786 mm−1; 7116 reflections measured, 2708 independent reflections (Rint = 0.0480); the final R1 values were 0.0949 [I N 2σ(I)]; the final wR(F2) values were 0.2180 [I N 2σ (I)]; the final R1 values were 0.0952 (all data); the final wR(F2) values were 0.2191 (all data); the goodness of fit on F2 was 1.132; Flack parameter = 0.1(3); the Hooft parameter is 0.03 (7) for 1052 Bijvoet pairs. 3. Results and discussion Compound 1 had molecular formula C20H28O5, as deduced from HREIMS ([M]+ m/z 348.1943, calculated 348.1937), indicating seven degrees of unsaturation. Its IR absorption bands at 3468 and 1750 cm−1 suggested the presence of hydroxy and carbonyl groups. The 13C NMR and DEPT spectra exhibited 20 carbon resonances attributed to two methyls,
1H-1H
COSY: H
H
HMBC : H
seven methylenes (one olefinic), five methines (two oxygenated), and six quaternary carbons (one carbonyl, one olefinic, and one oxygenated) (Table 1). The 1H–1H COSY and HSQC spectra established the spin systems for the molecular fragments of C-1–C-2–C-3, C-5–C-6, and C-9–C-11–C-12– C-13–C-14, and their connectivity was deduced from the HMBC experiment (Fig. 2). The HMBC correlations from Me-18 (δH 1.11, s) and Me-19 (δH 0.91, s) to C-3 (δC 41.0, t), C-4 (δC 34.3, s), and C-5 (δC 55.4, d), and from H-5 (δH 1.75, d, J = 5.6 Hz) to C-4, C-18 (δC 31.6, q), and C-19 (δC 21.2, q) were consistent with the C-4 quaternary carbon being connected with Me-18 and Me-19, and C-3 being connected to C-5 through C-4. The correlations from H-1b (δH 0.95, m), H-5, and H-9 (δH 2.55, m) to both of C-10 (δC 44.8, s) and C-20 (δC 175.9, s) suggested that C-10 was connected to C-1, C-5, C-9, and C-20. The HMBC relationships from H-11 to C-10, from H-14 to C-7 (δC 105.7, s), C-8 (δC 53.0, s), C-9 (δC 41.9, d), C-15 (δC 74.9, d) and C-16 (δC 160.8, s), and from H-13 (δH 2.58, m) to C-15, C-16 and C-17 suggested that C-8, C-9, and C-11–C-14 constituted the six-membered ring C, and allowed the connections of C-13 and C-16, and of C-8 and C-15 to form a five-membered ring D. Thus, the basic skeleton of 1 was characteristic of an ent-kauranoid, similar to rabdoternin A [28]. The location of a hydroxy group at C-6 was determined on the basis of the 1H–1H COSY correlation between H-5 and H-6 (δH 4.31), as well as HMBC correlations from H-6 to C-4, C-7, and C-8. A hydroxy group was connected to C-15 as evidenced by HMBC correlations from H-9, H2-14, and H2-17 to C-15. Additionally, HMBC correlations from H-1b, H-5, and H-9 to the lactonic carbonyl group (δC 175.9) inferred the location of a carbonyl group at C-20. Hence, the planar structure of 1 was elucidated. In the ROESY spectrum, H-9 correlated to H-5, signifying that they were located on the same side of the molecule (Fig. 2). ROESY correlation between H-15 and H-14β (δH 2.10) disclosed the β-orientation of HO-15. To determine the absolute configuration, a single crystal X-ray diffraction analysis of 1 was performed. The final refinement on the Cu Kα data resulted in a Hoof parameter of 0.03 (7) for 1052 Bijvoet pairs, allowing unambiguous assignment of the absolute configuration of 1 as shown in Fig. 3 [29]. All chiral centers, C-5, C-6, C-7, C-8, C-9, C-10, C-13, and C-15, were determined as R, S, S, S, S, R, R, and R, respectively. Thus, the structure of 1
C
ROESY : H
Fig. 2. 1H–1H COSY, selected HMBC and key ROESY correlations of 1.
H
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Fig. 3. X-ray crystal structure of 1.
was determined as 6β,7β,15β-trihydroxy-7α,20-olide-ent-kaur16-ene, and it was named sculponin U. Compound 2 had the molecular formula C20H28O6 by HREIMS, indicating that it had one more oxygen atom than 1. The 1H and 13C NMR spectra of 2 (Tables 1 and 2) resembled those of 1 except that the C-1 methylene (δC 29.8) in 1 was replaced by an oxygenated methine (δC 73.9) in 2, which was supported by the following observations: (i) the HSQC and 1 H–1H COSY analysis revealed the spin system of C(1)H–C(2) H2–C(3)H2; (ii) the HMBC correlations from H-3b (δH 1.26, m) and H-9 (δH 2.76, dd, J = 13.3, 5.6 Hz) to C-1 (δC 73.9) implied the location of a hydroxy group at C-1. The observed ROESY correlations of H-1 with H-5β and H-9β, and of H-6 with Me-19α established the respective β- and α-orientation of H-1 and H-6. The HO-15 of 2 was assigned to be β-oriented for the ROESY correlation between H-15 and H-14β (δH 2.12), and further supported by the upfield shift of C-9 (δC 43.7) compared with that of xerophilusin N (δC 50.4) [30], which was caused by the γ-steric compression effect between HO-15
1H-1H
COSY: H
H
HMBC : H
147
and H-9β. Hence, compound 2 was established as 1α,6β,7β,15βtetrahydroxy-7α,20-olide-ent-kaur-16-ene, and it was named sculponin V. Compound 3 possessed the molecular formula C22H30O7 by HREIMS, indicating eight degrees of unsaturation. Its IR spectrum revealed the presence of OH, C =O, and C = C groups from absorptions at 3447, 1721, and 1630 cm−1, respectively. The 13C NMR and DEPT spectra (Tables 1 and 2) suggested that 3 was a diterpenoid derivative with 22 carbons, consisting of three methyls, five methylenes (one oxygenated and one olefinic), eight methines (five oxygenated), and six quaternary carbons (one oxygenated, one olefinic, and one carbonyl carbon). These data revealed that 3 was an ent-kaurane diterpenoid, similar to wikstroemioidin A (7) [17]. The location of one acetoxy group at C-1 was determined on the basis of the HMBC correlations from H-1 (δH 5.26, dd, J = 11.5, 5.2 Hz) and a methyl (δH 2.18, s) to the acetyl carbonyl carbon (δC 170.6) (Fig. 4). The presence of a three-membered ring between C-11 and C-12 was evident from the chemical shift of C-11 (δC 51.6, d) and C-12 (δC 54.6, d), along with the HMBC correlations between H-11 (δH 3.13, br t, J = 4.2 Hz) and C-12, and between H-12 (δH 3.20, br t, J = 4.2 Hz) and C-14 (δC 25.8, t). The connectivity of C-7 and C-20 through an oxygen atom was deduced by the HMBC correlations from H-6 (δH 4.15, br s) and H-20 to C-7 (δC 97.8, s), and by the requirement for a degree of unsaturation. In the ROESY spectrum (Fig. 4), H-1 showed correlations to H-5β and H-9β, and H-11 correlated to H-1 and H-9β, revealing H-1, H-11, and H-12 to be β-oriented. In addition, the HO-15 of 3 was assigned to be β-oriented for the upfield shift of C-9 (δC 40.9) compared with that of longikaurin D (δC 54.7) [31,32], which was caused by the γ-steric compression effect between HO-15 and H-9β. Consequently, compound 3 was elucidated as 1α-acetoxy-6β,7β,15β-trihydroxy-7α,20:11α,12-diepoxy-entkaur-16-ene and given the name sculponin W. The molecular formula of compound 4 was determined to be C22H30O6, indicating that 4 had one less oxygen atom than 3. The similarities of the NMR data between 3 and 4 denoted that they were based on the same carbon skeleton (Tables 1 and 2). The differences between them were that in the 13C NMR spectrum of 4 two additional olefinic methines (δC 125.5, d; δC 135.4, d) were present instead of two oxygenated
C
ROESY : H
Fig. 4. 1H–1H COSY, selected HMBC and key ROESY correlations of 3.
H
148
H.-Y. Jiang et al. / Fitoterapia 93 (2014) 142–149
methines (δC 51.6, d; δC 54.6, d) in 3, suggesting a double bond in 4 rather than the three-membered epoxy ring in 3. The corresponding protons of the olefinic methines (δH 5.58, dd, J = 9.1, 2.1 Hz; δH 6.31, br t, J = 9.1 Hz) showed 1H–1H COSY correlations with H-9 (δH 3.15, br s) and H-13 (δH 3.05, m), respectively, indicating a double bond between C-11 and C-12, which was further confirmed by the HMBC correlations of H-11 with C-8 (δC 53.3, s), C-9 (δC 44.8, d), and C-13 (δC 40.2, d), and of H-12 with C-9 and C-13. Comparison of its ROESY spectrum with that of 3 indicated that the relative configurations of the chiral carbons in 4 were identical to those in 3. Thus, the structure of 4 was deduced as 1α-acetoxy6β,7β,15β-trihydroxy-7α,20-epoxy-ent-kaur-11(12),16(17)diene, and it was named sculponin X. The molecular formula of compound 5 was determined as C20H30O5, indicating that 5 possessed the same molecular formula as 1α,6β,7β,15β-tetrahydroxy-7α,20-epoxy-ent-kaur16-ene (8) [18]. Analysis of the IR spectrum revealed that compounds 5 and 8 had the same functional groups. The NMR spectra of both compounds were quite similar, except that the hydroxy group connected to C-1 in 8 was attached to C-11 in 5. This was established by the upfield chemical shift of an oxygenated methine in 5 (δC 65.1) compared with that of 8 (δC 73.9). In addition, the oxygenated methine proton H-11 (δH 4.49) coupled to H-9 (δH 2.32) and H2-12 (δH 2.53, 1.96) in the 1 H–1H COSY spectrum and H-11 correlated to C-8 (δC 52.1, s) and C-13 (δC 37.3, d) in the HMBC spectrum, further identifying the substitution of a hydroxy group at C-11 of 5. The ROESY correlations from H-11 to H-9β and H-12β (δH 1.96) indicated the β-orientation of the H-11 in 5. The β-orientation for H-5 and HO-6 was evident from the ROESY correlations from H-5 to H-9β, and from H-6 to Me-19α. In addition, the β-orientation of HO-15 was determined from the upfield shift of C-9 (δC 47.2) in 5 compared with that of C-9 in longikaurin D (δC 54.7) [31,32]. Thus, the structure of compound 5 was defined as 6β,7β,11α,15β-tetrahydroxy-7α,20-epoxy-ent-kaur-16-ene, and it was named sculponin Y. Compound 6 had the molecular formula C20H28O6 by HREIMS. Analysis of its 1H and 13C NMR spectra (Tables 1 and 2) indicated that 6 possessed the same carbon skeleton as that of 5. However, a tertiary methyl (Me-19) and an oxygenated methine group (C-15) in 5 were changed to an oxymethylene group (δC 64.7, t) and a carbonyl group (δC 210.9, s) in 6, respectively. In the HMBC spectrum, protons of the oxymethylene group (δH 4.36, 4.12) correlated to C-18 (δC 29.1, q), and H-5 (δH 1.73) and Me-18 (δH 1.68, s) correlated to the oxymethylene group, indicating that a hydroxy group was located at C-19. In addition, the C-15 was replaced by a carbonyl group on the basis of HMBC correlations from H-9 (δH 1.58), H-13 (δH 3.12, m), H-14b (δH 2.59, dd, J = 11.6, 4.2 Hz), and
H2-17 (δH 5.98, br s; 5.30, br s) to C-15. In the ROESY spectrum, H2-19 correlated to H-6α, and H-11 correlated to H-9β, suggesting the α-orientation of C-19 and HO-11. Therefore, the structure of 6 was determined to be 6β,7β,11α,19tetrahydroxy-7α,20-epoxy-ent-kaur-16-en-15-one, and it was named sculponin Z. Compounds 7–17 were identified by comparison of their physical constant data with data in the literature [6,14,17–23]. Compound 9, a dimeric ent-kaurane diterpenoid connected with a rare four-membered carbon ring, is reported from the species I. sculponeatus for the first time. Considering the cytotoxicity of diterpenoids previously isolated from the plants of the genus Isodon [1,4–8], compounds 5 and 8–15 were evaluated for their in vitro cytotoxicity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) according to a previously described procedure [24]. Other compounds were not tested for their activity due to the sample limitation. Compound 5 showed weak cytotoxic activity against HL-60, SMMC-7721, MCF-7, and SW-480 cell lines with respective IC50 value of 19.2, 22.3, 16.7, and 19.0 μM (Table 3). Furthermore, due to the folk use of I. sculponeatus [2,3], and since NO is an essential component of the host innate immune and inflammatory response to a variety of pathogens [33], the anti-inflammatory assay in LPS-stimulated RAW264.7 cells was carried out on compound 5 by MTT assay. As a result, it exhibited moderate inhibitory activity against LPS-induced NO production with IC50 value of 13.8 μM. At the highest concentration used, the tested compound 5 did not exhibit inhibitory activities, which suggests that the inhibitory activities against NO production in LPS-stimulated RAW264.7 cells were not induced by the cytotoxicity of the compounds tested.
Table 3 IC50 values (μM) of diterpenoids from I. sculponeatus for human tumor cell lines.
[1] (a) Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their biological activities. Nat Prod Rep 2006;23:673–98. (b) Zhan R, Li XN, Wang WG, Pu JX, Sun HD, et al. Bioative ent-kaurane diterpenoids from Isodon rosthornii. J Nat Prod 2006;76:1267–77. (c) Liu X, Wang WG, Pu JX, Wu JZ, Sun HD, et al. Enmein-type diterpenoids from the aerial parts of Isodon rubescens and their cytotoxicity. Fitoterapia 2006;83:1451–5. [2] Compiling groups of compilation of countrywide herbal medicine of ChinaCompilation of countrywide herbal medicine of China. Beijing: People's Medical Publishing House; 1996 853. [3] Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae EditaFlora Reipublicae Popularis Sinica. Beijing: Science Press; 1977 504.
Compounda
HL-60
SMMC-7721
A-549
MCF-7
SW480
5 DDPb Paclitaxelb
19.2 1.8 b0.008
22.3 4.5 b0.008
N40 7.6 b0.008
16.7 15.7 b0.008
19.0 15.0 b0.008
a Other selected ones not listed in the table were inactive (IC50 N 40 μM) for all cell lines. b DDP (cisplatin) and paclitaxel were used as positive controls.
Acknowledgment This project was supported financially by the National Natural Science Foundation of China (Nos. 21322204 and 81172939), the reservation-talent project of Yunnan Province (2011CI043), the Major Direction Projection Foundation of CAS Intellectual Innovation Project (No. KSCX2-EW-J-24), and West Light Foundation of the Chinese Academy of Sciences (J.-X. Pu). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.12.025. References
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Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Diterpenoids from Isodon sculponeatus Hua-Yi Jiang a,b, Wei-Guang Wang a, Min Zhou a,b, Hai-Yan Wu a,b, Rui Zhan a, Xiao-Nian Li a, Xue Du a, Yan Li a, Jian-Xin Pu a,⁎, Han-Dong Sun a,⁎ a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People's Republic of China b University of the Chinese Academy of Sciences, Beijing 100049, People's Republic of China
a r t i c l e
i n f o
Article history: Received 25 November 2013 Accepted in revised form 28 December 2013 Accepted 31 December 2013 Available online 10 January 2014 Chemical compounds studied in this article: Bisjaponin A (PubChem CID: 24895695) Lushanrubescensin J (PubChem CID: 11297111) Sculponeatin N (PubChem CID: 42643119) Hebeiabinin B (PubChem CID: 16112773)
a b s t r a c t Phytochemical investigation of the aerial parts of Isodon sculponeatus afforded six new 7,20-epoxy-ent-kauranoids, sculponins U–Z (1–6), and 11 known diterpenoids (7–17). The structures of these new compounds were elucidated primarily by means of extensive spectroscopic analysis, and the absolute configuration of 1 was determined by single crystal X-ray diffraction. Compound 5 exhibited weak cytotoxic activity against HL-60, SMMC-7721, MCF-7, and SW-480 cell lines, and it also inhibited NO production in LPS-stimulated RAW264.7 cells, with IC50 value of 13.8 μM. © 2014 Elsevier B.V. All rights reserved.
Keywords: Isodon sculponeatus Diterpenoids X-ray diffraction Cytotoxicity Anti-inflammatory activity
1. Introduction The genus Isodon is an abundant source of diterpenoids with diverse chemical structures, of which some have antitumor and anti-inflammatory activities [1]. Isodon sculponeatus (Vaniot) Kudo (Lamiaceae), a perennial herb, is widely distributed in southern China and has been used in folk medicines for treatment of dysentery and beriberi [2,3]. Previous phytochemical investigations of this species have resulted in the isolation of several diterpenoids exhibiting significant cytotoxicity toward tumor cell lines, especially, sculponin A displaying strong cytotoxic activity against K562, A549, and HepG2 cell lines with IC50 values equal to the positive control cisplatin [4–14]. ⁎ Corresponding authors. Tel.: +86 871 65223251; fax: +86 871 65216343. E-mail addresses: [email protected] (J.-X. Pu), [email protected] (H.-D. Sun). 0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.025
As part of our program to discover structurally interesting and bioactive diterpenoids [15], we have been interested in the chemical constituents of I. sculponeatus indigenous to Muli County of Sichuan Province, PR China. A series of 6,7-seco-entkauranoids have been obtained, and isodocarpin exhibited significant cytotoxic activity against five human tumor cell lines and intriguing inhibitory activity against NO production in LPS-stimulated RAW264.7 cells [16]. Continued investigation on this plant has now furnished an additional six new 7,20-epoxyent-kauranoids (1–6), namely sculponins U–Z, and 11 known ones, wikstroemioidin A (7) [17], 1α,6β,7β,15β-tetrahydroxy7α,20-epoxy-ent-kaur-16-ene (8) [18], bisjaponin A (9) [19], lushanrubescensin J (10) [20], ent-kaurane-7α,16β,17-triol (11) [21], sculponeatin L (12) [6], sculponeatin N (13) [14], entabienervonin C (14) [22], hebeiabinin B (15) [22], maoyecrystal G (16) [23], and maoyecrystal H (17) [23] (Fig. 1). The absolute configuration of 1 was confirmed by single crystal X-ray
H.-Y. Jiang et al. / Fitoterapia 93 (2014) 142–149
143
Fig. 1. The structures of compounds 1–17.
diffraction. Herein, we report the isolation and structure elucidation of the new compounds, as well as the cytotoxic properties and anti-inflammatory assay of the selected isolates.
Corporation, Tokyo, Japan), and Sephadex LH-20 (Pharmacia). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 5% H2SO4 in ethanol, followed by heating.
2. Experimental section 2.1. General experimental procedures X-ray data were collected using a Bruker APEX DUO instrument. Melting points were obtained on an XRC-1 apparatus and were uncorrected. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Tenor 27 spectrophotometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers with TMS as internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HREIMS was performed on an API QSTAR time-of-flight spectrometer. HSCCC was performed on a TBE-300B instrument (TAUTO). Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm) column. Column chromatography (CC) was performed on silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), MCI gel (75–150 μm, Mitsubishi Chemical
2.2. Plant material Aerial parts of I. sculponeatus were collected in August 2011 from Muli County, Sichuan Province, People's Republic of China, and identified by Prof. Xi-Wen Li, Kunming Institute of Botany. A voucher specimen (KIB 20110813) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The dried and powdered aerial parts of I. sculponeatus (8 kg) were extracted with 70% aqueous acetone (30 L) four times (2 days each time) at room temperature, then filtered. The filtrate was evaporated under reduced pressure and then partitioned between EtOAc and H2O. The EtOAc soluble portion (700 g) was subjected to column chromatography on silica gel (3 kg, 100–200 mesh), eluted with a CHCl3–Me2CO gradient system (1:0–0:1) that afforded fractions A–G. The fractions were then decolorized using MCI gel and eluted with 90% MeOH–H2O.
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Fraction B (120 g) was chromatographed via silica gel CC (1.2 kg, 200–300 mesh), eluted with CHCl3–MeOH gradient (150:1–1:1) to yield fractions B1–B5. Compound 12 (50.0 mg) was crystallized from fraction B4. Fraction B2 (10 g) was purified by medium-pressure column chromatography on RP-18 (400 g, MeOH–H2O gradient, 20%–100%), followed by repeated column chromatography over silica gel (11 g, CHCl3– MeOH gradient, 120:1–0:1), to obtain compound 13 (15.3 mg). Fraction C (80 g) was separated by medium-pressure column chromatography on RP-18 (900 g, MeOH–H2O gradient, 25%–100%) to give fractions C1–C5. Compound 9 (10.1 mg) was precipitated out from fraction C1. Fraction C2 (230 mg) was subjected to repeated CC over silica gel (7 g, 200–300 mesh), eluted with CHCl3–MeOH (gradient system: 120:1–1:1), to yield compounds 1 (2.1 mg) and 2 (2.3 mg). Compounds 3 (3.1 mg), 4 (2.3 mg), and 7 (3.2 mg) were isolated from fraction C3 by Sephadex LH-20 CC (CHCl3–MeOH 1:1) and then by repeated column chromatography over silica gel (8 g, petroleum ether–Me2CO gradient, 12:1–0:1). Fraction D (100 g) was subjected to medium-pressure column chromatography on RP-18 (900 g, MeOH–H2O gradient, 10%–100%) to give fractions D1–D6. Fraction D1 (8 g) was further purified by column chromatography on silica gel (210 g, 200–300 mesh), eluted with CHCl3–MeOH gradient system (100:1–1:2), followed by semipreparative HPLC (25% MeCN–H2O), to afford compounds 5 (5.1 mg) and 8 (4.5 mg). Fraction D4 (9 g) was further purified by column chromatography on silica gel (200 g, 200–300 mesh), eluted with CHCl3–MeOH gradient system (100:1–1:2), followed by semipreparative HPLC (23% MeCN–H2O, tR = 12.5 and 15.7 min, respectively), to afford compounds 14 (7.7 mg) and 15 (6.8 mg). Compound 10 (7.3 mg) was precipitated out from fraction D5 (3 g). Then fraction D6 (280 mg) was separated by repeated CC on silica gel (8 g, 200–300 mesh), eluted with CHCl3–MeOH (gradient system: 100:1–1:1), to yield compound 11 (13.2 mg).
Fraction E (78 g) was separated by medium-pressure column chromatography on RP-18 (900 g, MeOH–H2O gradient, 10%–80%), to obtain fractions E1–E6. After CC on silica gel (330 g, 200–300 mesh), eluted with CHCl3–MeOH (gradient system: 80:1–1:1), followed by semipreparative HPLC (25% MeOH–H2O), compound 6 (2.5 mg) was obtained from fraction E3 (12 g). Then fraction E4 was separated by HSCCC (CHCl3–MeOH–H2O 4:3:2) and then by repeated CC on silica gel (30 g, 200–300 mesh), eluted with CHCl3–MeOH gradient (80:1–1:1), to yield the mixture of compounds 16 and 17 (7.8 mg). 2.4. Spectroscopic data 2.4.1. Sculponin U (1) Colorless needle crystals (MeOH); mp 247–249 °C; [α]25D –78.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (4.13) nm; IR (KBr) νmax 3468, 2952, 1750 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 371 [M + Na]+; positive-ion HREIMS [M]+ m/z 348.1943 (calculated for 348.1937). 2.4.2. Sculponin V (2) White amorphous powder; [α]26D –22.6 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 203 (3.47), 372 (1.88) nm; IR (KBr) νmax 3431, 2932, 1721, 1629 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 387 [M + Na]+; positive-ion HREIMS [M]+ m/z 364.1880 (calculated for 364.1886). 2.4.3. Sculponin W (3) White amorphous powder; [α]26D –77.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (3.61) nm; IR (KBr) νmax 3447, 1721, 1630 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 429 [M + Na]+; positive-ion HREIMS [M]+ m/z 406.2000 (calculated for 406.1992).
Table 1 13 C NMR data of compounds 1–6 (in C5D5N, δ in ppm).a Position
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc
29.8 (t) 19.3 (t) 41.0 (t) 34.3 (s) 55.4 (d) 73.0 (d) 105.7 (s) 53.0 (s) 41.9 (d) 44.8 (s) 18.1 (t) 32.6 (t) 36.1 (d) 25.8 (t) 74.9 (d) 160.8 (s) 108.4 (t) 31.6 (q) 21.2 (q) 175.9 (s)
73.9 (d) 31.2 (t) 39.3 (t) 34.1 (s) 55.7 (d) 73.2 (d) 106.3 (s) 52.9 (s) 43.7 (d) 48.5 (s) 21.7 (t) 32.9 (t) 36.3 (d) 25.6 (t) 74.9 (d) 160.9 (s) 108.0 (t) 31.2 (q) 21.0 (q) 176.5 (s)
74.6 (d) 25.2 (t) 38.6 (t) 33.7 (s) 61.1 (d) 73.8 (d) 97.8 (s) 51.7 (s) 40.9 (d) 41.3 (s) 51.6 (d) 54.6 (d) 39.0 (d) 25.8 (t) 75.0 (d) 153.3 (s) 108.6 (t) 32.0 (q) 21.3 (q) 65.3 (t) 21.3 (q) 170.6 (s)
75.6 (d) 25.1 (t) 38.4 (t) 33.6 (s) 61.3 (d) 74.0 (d) 98.7 (s) 53.3 (s) 44.8 (d) 41.5 (s) 125.5 (d) 135.4 (d) 40.2 (d) 32.1 (t) 75.7 (d) 157.6 (s) 106.0 (t) 32.1 (q) 21.1 (q) 64.1 (t) 21.4 (q) 170.3 (s)
31.4 (t) 19.2 (t) 42.1 (t) 34.0 (s) 57.9 (d) 75.3 (d) 97.8 (s) 52.1 (s) 47.2 (d) 37.3 (s) 65.1 (d) 45.3 (t) 37.3 (d) 27.1 (t) 75.8 (d) 162.8 (s) 106.6 (t) 34.5 (q) 22.9 (q) 68.8 (t)
31.3 (t) 18.9 (t) 36.1 (t) 37.9 (s) 60.0 (d) 73.9 (d) 96.6 (s) 61.7 (s) 54.9 (d) 39.0 (s) 65.6 (d) 41.6 (t) 34.9 (d) 27.4 (t) 210.9 (s) 154.4 (s) 115.7 (t) 29.1 (q) 64.7 (t) 69.8 (t)
a 1
H,
NMR data of compounds 1 and 4 were recorded at 100 MHz; data for compounds 2, 3, 5, and 6 were recorded at 125 MHz. Assignments were made based on C NMR, DEPT, HSQC, 1H–1H COSY, HMBC, and ROESY experiments.
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145
Table 2 1 H NMR spectroscopic data (in C5D5N, δ in ppm, J in Hz) of compounds 1–6.a Position
1
2
3
4
5
6
1a 1b 2a 2b 3a 3b 5 6 9 11a 11b 12a 12b 13 14a 14b 15 17a 17b 18 19a 19b 20a 20b OAc
2.36 (br d, 13.3) 0.95 (m) 1.95 (m)
3.55 (br s)
5.26 (dd, 11.5, 5.2)
5.13 (dd, 13.7, 6.3)
2.32 (overlapped)
2.07 1.96 1.33 1.26 1.78 4.27 2.76 2.35 1.66 2.10 1.41 2.60 2.12 1.90 5.01 5.46 5.10 1.09 0.92
1.88 (m) 1.71 (m) 1.31 (m)
1.86 1.62 1.32 1.26 1.65 4.19 3.15 5.58
1.39 (overlapped)
2.25 (br d, 13.4) 1.39 (m) 1.58 (overlapped) 1.41 (m) 2.38 (br d, 13.0) 1.11 (br t, 13.0) 1.73 (d, 7.8) 4.68 (dd, 10.8, 7.8) 1.58 (overlapped) 4.45 (d, 4.0)
1.34 1.23 1.75 4.31 2.55 1.45 1.27 1.99 1.45 2.58 2.10 1.84 5.09 5.49 5.20 1.11 0.91
(m) (m) (d, 5.6) (br t, 5.6) (m) (overlapped) (m) (m) (overlapped) (m) (dd, 12.2, 4.9) (d, 12.2) (br s) (br s) (br s) (s) (s)
(m) (m) (m) (m) (d, 6.0) (br t, 6.0) (dd, 13.3, 5.6) (m) (m) (m) (m) (m) (m) (d, 12.1) (br s) (br s) (br s) (s) (s)
1.59 4.15 2.80 3.13
(d, 3.5) (br s) (br s) (br t, 4.2)
(m) (m) (m) (m) (d, 3.1) (br s) (br s) (dd, 9.1, 2.1)
3.20 (br t, 4.2)
6.31 (br t, 9.1)
2.98 2.60 2.14 5.17 5.52 5.17 1.07 1.07
3.05 2.46 2.00 5.37 5.33 5.05 1.08 1.07
(t, 4.2) (d, 12.2) (dd, 12.2, 4.8) (overlapped) (br s) (overlapped) (overlapped) (overlapped)
4.82 (d, 9.5) 4.31 (d, 9.5) 2.18 (s)
(m) (dd, 11.4, 4.2) (d, 11.4) (s) (br s) (br s) (overlapped) (overlapped)
4.44 (d, 10.0) 4.24 (d, 10.0) 2.02 (s)
1.38 1.17 1.72 4.36 2.32 4.49
(overlapped) (m) (d, 5.7) (br t, 5.7) (overlapped) (br s)
2.53 1.96 2.88 3.24 2.18 5.28 5.45 5.19 1.24 1.13
(m) (m) (m) (d, 14.2) (dd, 14.2, 5.2) (s) (br s) (br s) (s) (s)
5.41 (d, 9.9) 4.27 (d, 9.9)
2.51 (m) 1.76 (m) 3.12 (m) 3.66 (d, 11.6) 2.59 (dd, 11.6, 4.2) 5.98 (br s) 5.30 (br s) 1.68 (s) 4.36 (d, 10.5) 4.12 (d, 10.5) 5.25 (d, 8.6) 4.38 (d, 8.6)
Assignments were made based on 1H NMR, 13C NMR, DEPT, HSQC, COSY, HMBC, and ROESY experiments. a NMR data for compounds 1 and 4 were recorded at 400 MHz, and data of compounds 2, 3, 5, and 6 were recorded at 500 MHz.
2.4.4. Sculponin X (4) White amorphous powder; [α]25D –271.0 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 204 (4.16), 245 (3.34) nm; IR (KBr) νmax 3429, 3253, 2949, 1723 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 413 [M + Na]+; positive-ion HREIMS [M]+ m/z 390.2038 (calculated for 390.2042). 2.4.5. Sculponin Y (5) White amorphous powder; [α]21D –40.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (3.68), 254 (2.85) nm; IR (KBr) νmax 3420, 2927, 1640 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 373 [M + Na]+; positive-ion HREIMS [M]+ m/z 350.2102 (calculated for 350.2093). 2.4.6. Sculponin Z (6) White amorphous powder; [α]24D –111.0 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 236 (3.87), 202 (3.79) nm; IR (KBr) νmax 3416, 2930, 1705, 1639 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 387 [M + Na]+; positive-ion HREIMS [M]+ m/z 364.1879 (calculated for 364.1886). 2.5. The cytotoxicity assay The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI-1640 or DMEM (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan
formed in living cells based on the reduction of 3-(4,5dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4sulfopheny)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) [24]. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS–50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench's method [25]. 2.6. Nitric oxide production in RAW264.7 macrophages Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI-1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with serial dilutions of the compounds, with the maximum concentration of 25 μM, in the presence of 1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (Reagent A & Reagent B, respectively, Sigma) to 100 μL of each supernatant from LPS (Sigma)-treated or LPS- and compound-treated cells in triplicate. After 5 min incubation, the
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absorbance was measured at 570 nm with a 2104 Envision Multilabel Plate Reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [26]. 2.7. X-ray crystal structure analysis Colorless crystals of 1 were obtained from CH3OH. Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97 [27]. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms [27]. The H-atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 972948 for 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033); e-mail: [email protected]]. Crystallographic data for sculponin U (1): C20H28O5, M = 348.42, orthorhombic, space group P212121, Z = 4, a = 6.59370 (10) Å, b = 9.90360 (10) Å, c = 26.0159 (3) Å; α = β = γ = 90.00°, V = 1698.87 (4) Å3, T = 100 (2) K, μ (Cu Kα) = 0.786 mm−1; 7116 reflections measured, 2708 independent reflections (Rint = 0.0480); the final R1 values were 0.0949 [I N 2σ(I)]; the final wR(F2) values were 0.2180 [I N 2σ (I)]; the final R1 values were 0.0952 (all data); the final wR(F2) values were 0.2191 (all data); the goodness of fit on F2 was 1.132; Flack parameter = 0.1(3); the Hooft parameter is 0.03 (7) for 1052 Bijvoet pairs. 3. Results and discussion Compound 1 had molecular formula C20H28O5, as deduced from HREIMS ([M]+ m/z 348.1943, calculated 348.1937), indicating seven degrees of unsaturation. Its IR absorption bands at 3468 and 1750 cm−1 suggested the presence of hydroxy and carbonyl groups. The 13C NMR and DEPT spectra exhibited 20 carbon resonances attributed to two methyls,
1H-1H
COSY: H
H
HMBC : H
seven methylenes (one olefinic), five methines (two oxygenated), and six quaternary carbons (one carbonyl, one olefinic, and one oxygenated) (Table 1). The 1H–1H COSY and HSQC spectra established the spin systems for the molecular fragments of C-1–C-2–C-3, C-5–C-6, and C-9–C-11–C-12– C-13–C-14, and their connectivity was deduced from the HMBC experiment (Fig. 2). The HMBC correlations from Me-18 (δH 1.11, s) and Me-19 (δH 0.91, s) to C-3 (δC 41.0, t), C-4 (δC 34.3, s), and C-5 (δC 55.4, d), and from H-5 (δH 1.75, d, J = 5.6 Hz) to C-4, C-18 (δC 31.6, q), and C-19 (δC 21.2, q) were consistent with the C-4 quaternary carbon being connected with Me-18 and Me-19, and C-3 being connected to C-5 through C-4. The correlations from H-1b (δH 0.95, m), H-5, and H-9 (δH 2.55, m) to both of C-10 (δC 44.8, s) and C-20 (δC 175.9, s) suggested that C-10 was connected to C-1, C-5, C-9, and C-20. The HMBC relationships from H-11 to C-10, from H-14 to C-7 (δC 105.7, s), C-8 (δC 53.0, s), C-9 (δC 41.9, d), C-15 (δC 74.9, d) and C-16 (δC 160.8, s), and from H-13 (δH 2.58, m) to C-15, C-16 and C-17 suggested that C-8, C-9, and C-11–C-14 constituted the six-membered ring C, and allowed the connections of C-13 and C-16, and of C-8 and C-15 to form a five-membered ring D. Thus, the basic skeleton of 1 was characteristic of an ent-kauranoid, similar to rabdoternin A [28]. The location of a hydroxy group at C-6 was determined on the basis of the 1H–1H COSY correlation between H-5 and H-6 (δH 4.31), as well as HMBC correlations from H-6 to C-4, C-7, and C-8. A hydroxy group was connected to C-15 as evidenced by HMBC correlations from H-9, H2-14, and H2-17 to C-15. Additionally, HMBC correlations from H-1b, H-5, and H-9 to the lactonic carbonyl group (δC 175.9) inferred the location of a carbonyl group at C-20. Hence, the planar structure of 1 was elucidated. In the ROESY spectrum, H-9 correlated to H-5, signifying that they were located on the same side of the molecule (Fig. 2). ROESY correlation between H-15 and H-14β (δH 2.10) disclosed the β-orientation of HO-15. To determine the absolute configuration, a single crystal X-ray diffraction analysis of 1 was performed. The final refinement on the Cu Kα data resulted in a Hoof parameter of 0.03 (7) for 1052 Bijvoet pairs, allowing unambiguous assignment of the absolute configuration of 1 as shown in Fig. 3 [29]. All chiral centers, C-5, C-6, C-7, C-8, C-9, C-10, C-13, and C-15, were determined as R, S, S, S, S, R, R, and R, respectively. Thus, the structure of 1
C
ROESY : H
Fig. 2. 1H–1H COSY, selected HMBC and key ROESY correlations of 1.
H
H.-Y. Jiang et al. / Fitoterapia 93 (2014) 142–149
Fig. 3. X-ray crystal structure of 1.
was determined as 6β,7β,15β-trihydroxy-7α,20-olide-ent-kaur16-ene, and it was named sculponin U. Compound 2 had the molecular formula C20H28O6 by HREIMS, indicating that it had one more oxygen atom than 1. The 1H and 13C NMR spectra of 2 (Tables 1 and 2) resembled those of 1 except that the C-1 methylene (δC 29.8) in 1 was replaced by an oxygenated methine (δC 73.9) in 2, which was supported by the following observations: (i) the HSQC and 1 H–1H COSY analysis revealed the spin system of C(1)H–C(2) H2–C(3)H2; (ii) the HMBC correlations from H-3b (δH 1.26, m) and H-9 (δH 2.76, dd, J = 13.3, 5.6 Hz) to C-1 (δC 73.9) implied the location of a hydroxy group at C-1. The observed ROESY correlations of H-1 with H-5β and H-9β, and of H-6 with Me-19α established the respective β- and α-orientation of H-1 and H-6. The HO-15 of 2 was assigned to be β-oriented for the ROESY correlation between H-15 and H-14β (δH 2.12), and further supported by the upfield shift of C-9 (δC 43.7) compared with that of xerophilusin N (δC 50.4) [30], which was caused by the γ-steric compression effect between HO-15
1H-1H
COSY: H
H
HMBC : H
147
and H-9β. Hence, compound 2 was established as 1α,6β,7β,15βtetrahydroxy-7α,20-olide-ent-kaur-16-ene, and it was named sculponin V. Compound 3 possessed the molecular formula C22H30O7 by HREIMS, indicating eight degrees of unsaturation. Its IR spectrum revealed the presence of OH, C =O, and C = C groups from absorptions at 3447, 1721, and 1630 cm−1, respectively. The 13C NMR and DEPT spectra (Tables 1 and 2) suggested that 3 was a diterpenoid derivative with 22 carbons, consisting of three methyls, five methylenes (one oxygenated and one olefinic), eight methines (five oxygenated), and six quaternary carbons (one oxygenated, one olefinic, and one carbonyl carbon). These data revealed that 3 was an ent-kaurane diterpenoid, similar to wikstroemioidin A (7) [17]. The location of one acetoxy group at C-1 was determined on the basis of the HMBC correlations from H-1 (δH 5.26, dd, J = 11.5, 5.2 Hz) and a methyl (δH 2.18, s) to the acetyl carbonyl carbon (δC 170.6) (Fig. 4). The presence of a three-membered ring between C-11 and C-12 was evident from the chemical shift of C-11 (δC 51.6, d) and C-12 (δC 54.6, d), along with the HMBC correlations between H-11 (δH 3.13, br t, J = 4.2 Hz) and C-12, and between H-12 (δH 3.20, br t, J = 4.2 Hz) and C-14 (δC 25.8, t). The connectivity of C-7 and C-20 through an oxygen atom was deduced by the HMBC correlations from H-6 (δH 4.15, br s) and H-20 to C-7 (δC 97.8, s), and by the requirement for a degree of unsaturation. In the ROESY spectrum (Fig. 4), H-1 showed correlations to H-5β and H-9β, and H-11 correlated to H-1 and H-9β, revealing H-1, H-11, and H-12 to be β-oriented. In addition, the HO-15 of 3 was assigned to be β-oriented for the upfield shift of C-9 (δC 40.9) compared with that of longikaurin D (δC 54.7) [31,32], which was caused by the γ-steric compression effect between HO-15 and H-9β. Consequently, compound 3 was elucidated as 1α-acetoxy-6β,7β,15β-trihydroxy-7α,20:11α,12-diepoxy-entkaur-16-ene and given the name sculponin W. The molecular formula of compound 4 was determined to be C22H30O6, indicating that 4 had one less oxygen atom than 3. The similarities of the NMR data between 3 and 4 denoted that they were based on the same carbon skeleton (Tables 1 and 2). The differences between them were that in the 13C NMR spectrum of 4 two additional olefinic methines (δC 125.5, d; δC 135.4, d) were present instead of two oxygenated
C
ROESY : H
Fig. 4. 1H–1H COSY, selected HMBC and key ROESY correlations of 3.
H
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methines (δC 51.6, d; δC 54.6, d) in 3, suggesting a double bond in 4 rather than the three-membered epoxy ring in 3. The corresponding protons of the olefinic methines (δH 5.58, dd, J = 9.1, 2.1 Hz; δH 6.31, br t, J = 9.1 Hz) showed 1H–1H COSY correlations with H-9 (δH 3.15, br s) and H-13 (δH 3.05, m), respectively, indicating a double bond between C-11 and C-12, which was further confirmed by the HMBC correlations of H-11 with C-8 (δC 53.3, s), C-9 (δC 44.8, d), and C-13 (δC 40.2, d), and of H-12 with C-9 and C-13. Comparison of its ROESY spectrum with that of 3 indicated that the relative configurations of the chiral carbons in 4 were identical to those in 3. Thus, the structure of 4 was deduced as 1α-acetoxy6β,7β,15β-trihydroxy-7α,20-epoxy-ent-kaur-11(12),16(17)diene, and it was named sculponin X. The molecular formula of compound 5 was determined as C20H30O5, indicating that 5 possessed the same molecular formula as 1α,6β,7β,15β-tetrahydroxy-7α,20-epoxy-ent-kaur16-ene (8) [18]. Analysis of the IR spectrum revealed that compounds 5 and 8 had the same functional groups. The NMR spectra of both compounds were quite similar, except that the hydroxy group connected to C-1 in 8 was attached to C-11 in 5. This was established by the upfield chemical shift of an oxygenated methine in 5 (δC 65.1) compared with that of 8 (δC 73.9). In addition, the oxygenated methine proton H-11 (δH 4.49) coupled to H-9 (δH 2.32) and H2-12 (δH 2.53, 1.96) in the 1 H–1H COSY spectrum and H-11 correlated to C-8 (δC 52.1, s) and C-13 (δC 37.3, d) in the HMBC spectrum, further identifying the substitution of a hydroxy group at C-11 of 5. The ROESY correlations from H-11 to H-9β and H-12β (δH 1.96) indicated the β-orientation of the H-11 in 5. The β-orientation for H-5 and HO-6 was evident from the ROESY correlations from H-5 to H-9β, and from H-6 to Me-19α. In addition, the β-orientation of HO-15 was determined from the upfield shift of C-9 (δC 47.2) in 5 compared with that of C-9 in longikaurin D (δC 54.7) [31,32]. Thus, the structure of compound 5 was defined as 6β,7β,11α,15β-tetrahydroxy-7α,20-epoxy-ent-kaur-16-ene, and it was named sculponin Y. Compound 6 had the molecular formula C20H28O6 by HREIMS. Analysis of its 1H and 13C NMR spectra (Tables 1 and 2) indicated that 6 possessed the same carbon skeleton as that of 5. However, a tertiary methyl (Me-19) and an oxygenated methine group (C-15) in 5 were changed to an oxymethylene group (δC 64.7, t) and a carbonyl group (δC 210.9, s) in 6, respectively. In the HMBC spectrum, protons of the oxymethylene group (δH 4.36, 4.12) correlated to C-18 (δC 29.1, q), and H-5 (δH 1.73) and Me-18 (δH 1.68, s) correlated to the oxymethylene group, indicating that a hydroxy group was located at C-19. In addition, the C-15 was replaced by a carbonyl group on the basis of HMBC correlations from H-9 (δH 1.58), H-13 (δH 3.12, m), H-14b (δH 2.59, dd, J = 11.6, 4.2 Hz), and
H2-17 (δH 5.98, br s; 5.30, br s) to C-15. In the ROESY spectrum, H2-19 correlated to H-6α, and H-11 correlated to H-9β, suggesting the α-orientation of C-19 and HO-11. Therefore, the structure of 6 was determined to be 6β,7β,11α,19tetrahydroxy-7α,20-epoxy-ent-kaur-16-en-15-one, and it was named sculponin Z. Compounds 7–17 were identified by comparison of their physical constant data with data in the literature [6,14,17–23]. Compound 9, a dimeric ent-kaurane diterpenoid connected with a rare four-membered carbon ring, is reported from the species I. sculponeatus for the first time. Considering the cytotoxicity of diterpenoids previously isolated from the plants of the genus Isodon [1,4–8], compounds 5 and 8–15 were evaluated for their in vitro cytotoxicity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) according to a previously described procedure [24]. Other compounds were not tested for their activity due to the sample limitation. Compound 5 showed weak cytotoxic activity against HL-60, SMMC-7721, MCF-7, and SW-480 cell lines with respective IC50 value of 19.2, 22.3, 16.7, and 19.0 μM (Table 3). Furthermore, due to the folk use of I. sculponeatus [2,3], and since NO is an essential component of the host innate immune and inflammatory response to a variety of pathogens [33], the anti-inflammatory assay in LPS-stimulated RAW264.7 cells was carried out on compound 5 by MTT assay. As a result, it exhibited moderate inhibitory activity against LPS-induced NO production with IC50 value of 13.8 μM. At the highest concentration used, the tested compound 5 did not exhibit inhibitory activities, which suggests that the inhibitory activities against NO production in LPS-stimulated RAW264.7 cells were not induced by the cytotoxicity of the compounds tested.
Table 3 IC50 values (μM) of diterpenoids from I. sculponeatus for human tumor cell lines.
[1] (a) Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their biological activities. Nat Prod Rep 2006;23:673–98. (b) Zhan R, Li XN, Wang WG, Pu JX, Sun HD, et al. Bioative ent-kaurane diterpenoids from Isodon rosthornii. J Nat Prod 2006;76:1267–77. (c) Liu X, Wang WG, Pu JX, Wu JZ, Sun HD, et al. Enmein-type diterpenoids from the aerial parts of Isodon rubescens and their cytotoxicity. Fitoterapia 2006;83:1451–5. [2] Compiling groups of compilation of countrywide herbal medicine of ChinaCompilation of countrywide herbal medicine of China. Beijing: People's Medical Publishing House; 1996 853. [3] Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae EditaFlora Reipublicae Popularis Sinica. Beijing: Science Press; 1977 504.
Compounda
HL-60
SMMC-7721
A-549
MCF-7
SW480
5 DDPb Paclitaxelb
19.2 1.8 b0.008
22.3 4.5 b0.008
N40 7.6 b0.008
16.7 15.7 b0.008
19.0 15.0 b0.008
a Other selected ones not listed in the table were inactive (IC50 N 40 μM) for all cell lines. b DDP (cisplatin) and paclitaxel were used as positive controls.
Acknowledgment This project was supported financially by the National Natural Science Foundation of China (Nos. 21322204 and 81172939), the reservation-talent project of Yunnan Province (2011CI043), the Major Direction Projection Foundation of CAS Intellectual Innovation Project (No. KSCX2-EW-J-24), and West Light Foundation of the Chinese Academy of Sciences (J.-X. Pu). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.12.025. References
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