Original Papers
1113
Isolation and Cytotoxic Effect of Anthraquinones from Morinda umbellata
Authors
Chun-Tang Chiou 1, Ruie-Yu Hsu 2, Lie-Chwen Lin 1
Affiliations
1
Key words " Morinda umbellate l " Rubiaceae l " 1,6‑dihydroxy‑2‑methl oxymethyl‑anthraquinone " 6‑hydroxy‑7‑methl oxy‑2‑methoxymethylanthraquinone " 3,6‑dihydroxy‑7‑methl oxy‑2‑methylanthraquinone " 6‑hydroxy‑2‑methoxymethl ylanthraquinone " cytotoxicity l
received revised accepted
January 29, 2014 July 3, 2014 July 9, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382956 Published online August 19, 2014 Planta Med 2014; 80: 1113–1117 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Lie-Chwen Lin National Research Institute of Chinese Medicine Ministry of Health and Welfare Taipei 155–1, Li-Nong Street Section 2 Taipei 11221 Taiwan Phone: + 88 62 28 20 19 99 ext. 71 01 Fax: + 88 62 28 26 42 76 [email protected]
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
Abstract !
Four new anthraquinones, 1,6-dihydroxy-2-methoxymethylanthraquinone (1), 6-hydroxy-7methoxy-2-methoxymethylanthraquinone (3), 3,6-dihydroxy-7-methoxy-2-methylanthraquinone (4), and 6-hydroxy-2-methoxymethylanthraquinone (8), together with 12 known anthraquinones and 6 other known compounds, were
isolated from the EtOAc extract of Morinda umbellata. Among the isolated compounds, 1, rubiadin (14), and, 3-hydroxy-2-hydroxymethylanthraquinone (16) exhibited significant cytotoxicities against HepG2 cells, with GI50 values of 4.4, 3.6, and 4.8 µM, respectively. Supporting information available online at http://www.thieme-connect.de/products
Introduction
Results and Discussion
!
!
Morinda umbellata L. (Rubiaceae) is a scandent shrub distributed in the low elevations of Taiwanʼs mountains [1]. It is used in Chinese folk medicine as an analgesic for the treatment of rheumatism [2]. Previous chemical studies of the genus Morinda have shown the presence of a variety of anthraquinones [3–5], iridoids [5], phenylpropanoids [6], and flavonoid glycosides [7]. Some of them were reported as the active compounds responsible for antimicrobial [8], antileishmanial, antimalarial [9], antitumor [10], antiosteoporotic [11], and hypoglycemic [12] activities. In the course of our search for biologically active substances in nature, we found that the ethanolic extract from the vines of M. umbellata showed cytotoxic activities against cancer cell lines in vitro. Therefore, a series of isolation processes was conducted to study the bioactive constituents of M. umbellata. This paper describes the isolation and characterization of 22 components from the vines of M. umbellata, including 4 new compounds, 1, 3, 4, and 8. Furthermore, the cytotoxic activities of these compounds on the cancer cell lines are also presented.
The ethanolic extract of the vines of M. umbellata was fractionated by solvent partition and separated by column chromatography to afford 22 compounds, including 4 new anthraquinones " Fig. 1) and 18 known compounds, soranjidiol (l (2) [13], robustaquinone D (5) [14], 3-hydroxy-2methoxy-6-hydroxymethylanthraquinone (6) [15], pustulin (7) [13], 6-hydroxyrubiabin (9) [16], 2-hydroxy-6-methoxyanthraquinone (10) [17], 3,6-dihydroxy-2-methylanthraquinone (11), 6-hydroxy-2-methylanthraquinone (12) [18], anthragallol 1,2-dimethyl ether (13) [19], rubiadin (14) [20], 3-hydroxy-2-methyl anthraquinone (15) [21], 3-hydroxy-2-hydroxymethylanthraquinone (16) [11], alizarin 1-methyl ether (17) [11], chrysoeriol (18), scopoletin (19), gallaldehyde (20), p-oxybenzaldehyde (21), and 2-monolinolein (22). The structures of 1, 3, 4, and 8 were elucidated by extensive spectroscopic analyses. The known compounds were identified by comparison of their physical and spectral data with those reported in the literature, except for 18-22, which were identified by comparison with authentic samples. Compound 1 was obtained as yellow amorphous powder. The molecular formula of 1 was determined as C16H12O5 from HRESIMS at m/z 283.0625 [M – H]− (calcd. for C16H11O5, 283.0606). The IR spectrum of 1 showed absorp-
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
2
1114
Original Papers
Fig. 2 Key HMBC correlations of compound 1.
Chemical structures of compounds 1, 3, 4, and 8.
tion bands at 3401, 1659, 1633, 1594, and 1581 cm−1, suggesting the presence of hydroxyl, carbonyl, and aromatic groups. The 1H " Table 1) revealed the presence of a pair of orthospectrum of 1 (l coupled aromatic protons (δH 7.81/7.75), an ABX coupled aromatic proton (δH 7.61/7.32/8.20), an oxymethylene proton (δH 4.58), a methoxyl proton (δ 3.47), and a chelated hydroxyl proton (δH 13.15). Two carbon signals at δC 189.1 (C-9) and 182.7 (C-10) were attributed to two conjugated carbonyl groups, which were also associated with UV absorptions at 244, 269, 279, 291, and 410 nm. Furthermore, a chelated hydroxyl proton at δH 13.1 was observed. Thus, a 1-hydroxyanthraquinone structure was deduced. In the HMQC spectrum, the proton singlets at δH 4.58 and 3.47 correlated to carbon signals at δC 68.8 (t) and 58.9 (q), respectively, indicating the presence of a methoxymethyl group " Fig. 2), the correlations (–CH2OCH3). In the HMBC spectrum (l of H-8 (δH 8.20) to C-6 (δC 164.7), C-9 (δC 189.1), and C-10a (δC 137.0), along with H-4 (δH 7.75) and H-5 (δH 7.61) to C-10 (δ 182.7), and H-3 (δH 7.81) to C-1 (δC 160.3) and C-4a (δC 133.3) suggested that a 1,6-dihydroxyanthraquinone moiety was present. In addition, HMBC correlations of –CH2OCH3 (δH 4.58) to C-1, C-3 (δC 134.7), and –CH2OCH3 (δC 58.9) confirmed the location of the methoxymethyl group at C-2. Therefore, compound 1 was deduced as 1,6-dihydroxy-2-methoxymethylanthraquinone.
Table 1
1
H and 13C data of compounds 1, 3, 4, and 8a. 1b
a
Compound 3 was obtained as orange amorphous powder and its molecular formula was established as C17H14O5 from HREIMS at m/z 298.0838 [M]+ (calcd. for C17H14O5: 298.0841). It showed absorption bands at 3431, 1630, and 1600 cm−1 in the IR spectrum and UV absorptions at 245, 285, and 403 nm, suggesting an an" Table 1) thraquinone derivative. The 1H NMR spectrum of 3 (l showed an aromatic ABX system at δH 8.15/8.16/7.73 and two aromatic singlets at δH 7.68 and 7.51. In addition, a methoxymethyl group –CH2OCH3 (δH 4.61 and 3.45) and a methoxy group (δH 4.02) appeared in the aliphatic region. In addition to two carbonyl groups at δC 183.4 and 183.8, the 13C NMR spectrum showed two oxygenated quaternary carbons at δC 156.0 (C-6) and 154.8 (C-7), indicating that one of these carbons was linked to a methoxy group and the other carbon was attached to a hydroxyl group. The locations of substituents were deduced by HMBC and NOESY experiments. The HMBC correlations of H-1 (δH 8.15)/H-8 (δH 7.68) to C-9 (δC 183.4) and of H-4 (δH 8.16)/H‑5 (δH 7.51) to C10 (δC 183.8) together with the NOESY correlations of –CH2OCH3 (δH 4.61) to H-1 (δH 8.15)/H‑3 (δH 7.73) and –OCH3 (δH 4.02) to H8 (δH 7.68) confirmed the structure of 3 as 6-hydroxy-7-methoxy-2-methoxymethylanthraquinone. Compound 4 possessed the molecular formula C16H12O5 derived from the HREIMS at m/z 284.0675 [M]+ (calcd. 284.0685 for C16H12O5). The IR and UV spectra of 4 showed characteristic sig" Tanals for an anthraquinone derivative. Its 1H NMR spectrum (l ble 1) showed four aromatic singlets at δH 7.47 (H-4), 7.50 (H-5),
No.
δC
1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 2-CH2OCH3 2-CH2OCH3 -CH3 -OCH3 -OH
160.3 135.5 134.7 119.4 133.3 113.6 164.7 122.2 130.7 126.3 189.1 116.1 182.7 137.0 68.8 58.9
3c δH (J in Hz)
7.81 (d, J = 8.0) 7.75 (d, J = 8.0) 7.61 (d, J = 3.2) 7.32 (dd, J = 8.4, 3.2) 8.20 (d, J = 8.4)
4.58 (s) 3.47 (s)
4c
8b
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
126.2 146.4 133.2 128.0 134.2 114.2 154.8 156.0 109.6 130.4 183.8 135.2 183.4 128.0 74.6 58.8
8.15 (d, J = 1.2)
131.05 132.9 162.0 112.5 135.2 113.9 154.3 154.3 109.6 128.5 184.2 127.1 183.3 130.3
7.94 (s)
125.8 146.8 132.9 127.8 133.6 113.2 163.8 122.1 130.7 127.1 182.2 134.6 183.3 136.7 74.0 58.6
8.20 (d, J = 0.6)
56.6
7.73 (dd, J = 7.8, 1.2) 8.16 (d, J = 7.8) 7.51 (s)
7.68 (s)
7.47 (s) 7.50 (s)
7.67 (s)
4.61 (s) 3.45 (s) 4.02 (s)
16.5 56.7
2.31 (s) 4.02 (s)
13.15 (s)
Assignments were confirmed by DEPT, 1H-1H COSY, HMQC, and HMBC experiments. b Measured in acetone-d6; c measured in methanol-d4
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
7.81 (dd, J = 7.8, 0.6) 8.21 (d, J = 7.8) 7.64 (d, J = 2.4) 7.31 (dd, J = 8.4, 2.4) 8.19 (d, J = 8.4)
4.65 (s) 3.44 (s)
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Fig. 1
Original Papers
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 Doxorubicina a
GI50 (µM) A549
HepG2
HT29
PC3
26.3 ± 1.3 82.3 ± 2.7 25.2 ± 1.0 > 100 > 100 > 100 > 100 87.4 ± 7.5 > 100 > 100 68.4 ± 2.7 60.1 ± 1.8 22.5 ± 9.7 > 100 > 100 43.7 ± 3.7 > 100 > 100 > 100 > 100 > 100 0.07 ± 0.03
4.4 ± 0.9 45.6 ± 4.7 14.0 ± 2.2 > 100 > 100 > 100 87.3 ± 4.5 12.0 ± 1.7 59.3 ± 2.1 > 100 10.0 ± 0.8 14.7 ± 5.1 88.8 ± 4.0 3.6 ± 0.4 77.5 ± 1.1 4.8 ± 1.3 > 100 51.0 ± 4.5 > 100 > 100 77.2 ± 3.5 0.07 ± 0.01
61.0 ± 2.5 > 100 > 100 > 100 > 100 > 100 > 100 84.8 ± 5.6 > 100 > 100 90.5 ± 5.8 76.7 ± 4.1 21.7 ± 2.0 > 100 > 100 92.7 ± 4.3 > 100 > 100 > 100 > 100 > 100 0.26 ± 0.04
27.3 ± 0.3 > 100 > 100 > 100 > 100 > 100 > 100 88.0 ± 1.7 > 100 > 100 > 100 80.8 ± 2.7 53.7 ± 9.0 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 0.13 ± 0.06
Table 2 Cytotoxic effects of isolated compounds on cancer cell lines.
Doxorubicin was used as a positive control
7.67 (H-8), and 7.94 (H-1), a methyl group (δH 2.31, 2-CH3), and a methoxyl group (δH 4.02, 7-OCH3). The 13C NMR spectrum showed signals for two carbonyl groups at δC 183.3 (C-10) and 184.2 (C-9) and three oxygenated quaternary carbons at δC 162.0 (C-3) and 154.3 (C-6 and C-7), which suggested that one methoxyl and two hydroxyl groups were attached to the aromatic rings. The HMBC correlations of H-1/H-8 to C-9 and H-4/ H-5 to C-10 together with the NOESY correlations of –CH3 to H1 and –OCH3 to H-8 confirmed the structure of 4 as 3,6-dihydroxy-7-methoxy-2-methylanthraquinone. Compound 8 possessed the molecular formula C16H12O4 deduced from HRESIMS at m/z 267.0675 [M – H]− (calcd. 267.0657 for " Table 1) showed two C16H11O4). The 1H NMR spectrum of 8 (l sets of aromatic ABX spin systems at δH 8.20/8.21/7.81 and 7.31/ 7.64/8.19 and a methoxymethyl group [δH 4.65 (CH2OCH3-2)/ 3.44 (CH2OCH3-2)]. In the HMBC spectrum, the correlations of H-1 (δH 8.20)/H-8 (δH 8.19) to C-9 (δC 182.2), H-4 (δH 8.21)/H‑5 (δH 7.64) to C-10 (δC 183.3), and H-8 (δH 8.19) to C-6 (δC 163.8) suggested the presence of a 6-hydroxyanthraquinone moiety. The NOESY spectrum showed correlations of –CH2OCH3 to H-1 and H-3. Thus, the structure of 8 was determined as 6-hydroxy2-methoxymethylanthraquinone. The isolated compounds 1-16 and 18-22 were tested against a panel of cancer cell lines according to the established protocol " Table 2, compounds 1, 8, 12, and 13 displayed [22]. As shown in l cytotoxicity against A549, HepG2, HT29, and PC3 cells with GI50 values ranging from 4.4 to 88.8 µM. Compounds 1, 14, and 16 showed potent suppressions on the growth of HepG2 tumor cells with GI50 values all less than 5 µM. These results suggested that the anthraquinones from M. umbellata were cytotoxic against A549, HepG2, HT29, and PC3 cancer cells, and were especially sensitive to the HepG2 cells. In this study, compounds 1, 3, 8, 11, 12, 14, and 16 showed high cytotoxic effects against HepG2 tumor cells with GI50 values all less than 15 µM. Their structure-activity relationships revealed that no more than three substitutions in the anthraquinone skeleton, and 6-hydroxy-2-methyl or
6-hydroxy-2-methoxymethyl substitution may enhance the cytotoxic activity.
Materials and Methods !
General experimental procedures IR spectra were obtained as KBr pellets on a Nicolet Avatar 320 IR spectrometer. UV spectra were obtained in MeOH on a Hitachi U2001 spectrophotometer. 1D and 2D NMR spectra were measured on a Varian VNMRS-600 spectrometer with deuterated solvent as internal standards. EIMS, HREIMS, ESIMS, and HRESIMS were recorded on Finnigan MAT95S and Shimadzu LCMS‑IT‑TOF mass spectrometers. Column chromatography was performed on Sephadex LH-20 (Pharmacia) or silica gel 60 (70–230 mesh or 230–400 mesh, Merck). The semipreparative HPLC system consisted of a chromatographic pump (LC-10AD, Shimadzu) and a UV-Visible detector (SPD-10A vp, Shimadzu). A Lichrosorb® Si60 column (10 × 250 mm; particle size 7 µm; Merck) or Purospher® STAR RP-18e column (10 × 250 mm; particle size 5 µm; Merck) was used for separation.
Plant material The vines of M. umbellata were collected in New Taipei City, Taiwan. M. umbellata was identified by Dr. Cheng-Jen Chiou, retired research fellow of the National Research Institute of Chinese Medicine and reconfirmed by comparison with a voucher specimen (No. 298 775), which has been deposited in the herbarium of the Taiwan Forestry Research Institute.
Extraction and isolation The vines of M. umbellata (4.7 kg) were sliced and extracted with 95 % EtOH (30 L × 3). The ethanolic extract was concentrated to a volume of 200 mL. The concentrated ethanolic extract was then partitioned successively between H2O and n-hexane, followed by EtOAc and n-BuOH (each 200 mL × 3) to give n-hexane, EtOAc,
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Compound
1115
Original Papers
n-BuOH, and H2O extracts. The EtOAc extract displayed cytotoxicity against A549 and HepG2 cells with GI50 values of 54.8 and 48.6 µg/mL, respectively. Therefore, the EtOAc extract (126.6 g) was further separated using silica gel column chromatography (10 × 90 cm, 70–230 mesh) with a solvent system of 10%, 30 %, 50 %, 70% EtOAc in n-hexane and finally EtOAc (3 L for each portion), monitored by TLC. Six fractions (Frs. 1–6) were collected. Fr. 3 (5.7 g) was repeatedly chromatographed over medium pressure silica gel column (4.8 × 45 cm, 230–400 mesh, ~ 10 bar, 10–50 % EtOAc/n-hexane over 90 min) and semipreparative HPLC (40– 50 % CHCl3/n-hexane over 40 min, Lichrosorb® Si60 column, 7 µm, 10 × 250 mm, flow rate = 3.7 mL/min, UV= 254 nm) to give 1 (4.0 mg, rt = 26 min), 2 (8.6 mg, rt = 18 min), 5 (2.3 mg, rt = 15 min), 8 (2.2 mg, rt = 30.5 min), 12 (3.8 mg, rt = 24 min), 13 (8.6 mg, rt = 25 min), 14 (5.8 mg, rt = 14 min), 15 (21.1 mg, rt = 16.5 min), and 21 (1.4 mg, rt = 32 min). Fr. 4 (1.4 g) was purified by Sephadex LH-20 (90% MeOH/CHCl3) and semipreparative HPLC (50–90% MeOH/H2O over 30 min, Purospher® STAR RP18e column, 5 µm, 10 × 250 mm, flow rate = 3.7 mL/min, UV = 254 nm) to afford 3 (1.3 mg, rt = 11 min), 4 (2.1 mg, rt = 9 min), 6 (10.9 mg, rt = 11 min), 7 (6.2 mg, rt = 13 min), 9 (6.9 mg, rt = 10 min), 11 (1.2 mg; rt = 8.5 min), 16 (61.7 mg, rt = 7 min), 17 (2.9 mg, rt = 10 min), 18 (3.0 mg, rt = 6.5 min), and 22 (17.0 mg, rt = 12.5 min). Fr. 5 (2.87 g) was purified by Sephadex LH-20 (90% MeOH/CHCl3) and semipreparative HPLC (80 % MeOH/H2O, Purospher® STAR RP-18e column, 5 µm, 10 × 250 mm, flow rate = 3.7 mL/min, UV= 254 nm) to yield 6 (1.1 mg, rt = 11 min), 10 (3.8 mg, rt = 6 min), 19 (16 mg, rt = 13 min), and 20 (5.4 mg, rt = 4.5 min).
285 (4.71), 403 (3.32) nm; IR (KBr) υmax 3431 (OH), 1630 (C=O), 1600 (C=C), 1383, 1326, 1082, 1028 cm−1; 1H and 13C NMR data, " Table 1; EIMS m/z 298 [M]+, HREIMS m/z 298.0838 [M]+ see l (calcd. 298.0841 for C17H14O5). 2,7-Dihydroxy-3-methoxy-6-methylanthraquinone (4): yellow powder (> 95%, purity); UV (MeOH) λmax (log ε) 285 (4.31), 311 sh. (3.80), 349 sh. (3.47) nm; IR (KBr) υmax 3399 (OH), 1663, 1630 (C=O), 1602, 1383, 1350, 1060, 1030 cm−1; 1H and 13C NMR " Table 1; EIMS m/z 284 [M]+, HREIMS m/z 284.0675 data, see l [M]+ (calcd. 284.0685 for C16H12O5). 6-Hydroxy-2-methoxymethylanthraquinone (8): yellow powder (> 95%, purity); UV (MeOH) λmax (log ε) 246 sh. (4.21), 270 (4.37), 283 sh. (4.29), 333 (3.54), 383 (3.32) nm; IR (KBr) υmax 3305 (OH), 1671(C=O), 1658 (C=O), 1582, 1566, 1317, 1108, " Table 1; ESIMS m/z 267 1086 cm−1; 1H and 13C NMR data, see l [M – H]−, HRESIMS m/z 267.0675 [M – H]− (calcd. 267.0657 for C16H11O4).
Supporting information 1D and 2D NMR spectra of anthraquinones 1, 3, 4, and 8 are available as Supporting Information.
Acknowledgements !
This study was supported by a research grant (NSC 102–2320B‑077-002-MY3) from the National Science Council, Taiwan.
Conflict of Interest Cell lines
!
A549, HepG2, HT29, and PC3 cell lines were utilized as target cells in the cytotoxic assay. A549 is a human lung adenocarcinoma epithelial cell line, HepG2 is a human hepatocellular liver carcinoma cell lines, HT29 is a human colon adenocarcinoma grade II cell line, and PC3 is a human prostate cancer cell line. These four cell lines were obtained from the Bioresources Collection and Research Center (BCRC, Hsin-Chu, Taiwan) and cultured in RPMI1640 medium (Hyclone) containing 10% FBS (Gibco), 100 u/mL penicillin (Gibco), 100 µg/mL streptomycin, and 1 × non-essential amino acid (Gibco), at 37 °C in an atmosphere of humidified 5 % CO2.
The authors report no conflicts of interest in this work.
Sulforhodamine B (SRB) assay Growth inhibition was assessed as described previously [22]. Each tumor cell line was cultured with or without various concentrations of test compounds and doxorubicin (positive control, purchased from Sigma-Aldrich, purity > 98 %) for 3 days, treated with 4% SRB reagent for 10 min and then 10 mM TrisBase were added. OD values in the wavelength 515 nm were determined by an ELISA reader, and inhibitory activity was calculated as GI50 (concentration of inhibition of 50% cell growth).
Isolates 1,6-Dihydroxy-2-methoxymethylanthraquinone (1): yellow powder (90.5 %, purity); UV (MeOH) λmax (log ε) 220 (4.16), 268 (4.15), 408 (3.64) nm; IR (KBr) υmax 3401 (OH), 1659 (C=O), 1633(C=O), 1594 (C=C), 1580 (C=C), 1428, 1277, 1247 cm−1; 1H " Table 1; ESIMS m/z 283 [M – H]−, HREand 13C NMR data, see l SIMS m/z 283.0625 [M – H]− (calcd. 283.0606 for C16H11O5). 7-Hydroxy-6-methoxy-2-methoxymethylanthraquinone (3): orange powder (> 95%, purity); UV (MeOH) λmax (log ε) 245 (4.21), Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
References 1 Yang TYA. Morinda L. In: Editorial Committee of the Flora of Taiwan, editor. Flora of Taiwan, 2nd edition, Volume IV. Taipei: Department of Botany, National Taiwan University; 1998: 299–301 2 Juangsu New Medical College. Zhong Yao Da Ci Dian (Dictionary of Chinese Materia Medica). Shanghai: Shanghai Scientific and Technological Publishers; 1977: 807 3 Ismail NH, Lajis NH, Ali AM, Aimi N, Kitajima M, Takayama H. Anthraquinones from Morinda elliptica. Phytochemistry 1997; 45: 1723–1725 4 Ee GC, Wen YP, Sukari MA, Go R, Lee HL. A new anthraquinone from Morinda citrifolia roots. Nat Prod Res 2009; 23: 1322–1329 5 Kamiya K, Satake T, Tanaka Y, Kamiya K, Satake T, Endang H, Umar M. New anthraquinone and iridoid from the fruits of Morinda citrifolia. Chem Pharm Bull 2005; 53: 1597–1599 6 He QW, Wang G, Feng T, He QW, Liu JK. Two new phenylpropanoids and one propanoate from Morinda citrifolia. J Asian Nat Prod Res 2011; 13: 238–241 7 Cimanga K, Bruyne TD, Lasure A, Li Q, Pieters L, Claeys M, Berghe DV, Kambu K, Tona L, Vlietinck A. Flavonoid O-glycosides from the leaves of Morinda morindoides. Phytochemistry 1995; 38: 1301–1303 8 Guo SP, Song QS, Xiang W, Xiang W, Zhang HJ. Antimicrobial anthraquinones from Morinda angustifolia. Fitoterapia 2008; 79: 501–504 9 Sittie AA, Lemmich E, Olsen CE, Hviid L, Kharazmi A, Nkrumah FK, Christensen SB. Structure-activity studies: in vitro antileishmanial and antimalarial activities of anthraquinones from Morinda lucida. Planta Med 1999; 65: 259–261 10 Chang P, Chen CF. Isolation and characterization of antitumor anthraquinones from Morinda umbellata. Chin Pharm J 1995; 47: 347–353 11 Wu YB, Han T, Jiao L, Qin LP, Sun LN, Wu YB, Zhang QY, Zheng CJ, Wu JZ. Antiosteoporotic activity of anthraquinones from Morinda officinalis on osteoblasts and osteoclasts. Molecules 2009; 14: 573–583 12 Hamabe W, Harada S, Kamiya K, Murakami R, Satake T, Tokuyama S. Chemical constituents of Morinda citrifolia roots exhibit hypoglycemic effects in streptozotocin-induced diabetic mice. Biol Pharm Bull 2008; 31: 935–938
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
1116
Original Papers
19 Wijnsma R, Verpoorte R, Mulder-Krieger T, Svendsen AB. Anthraquinones in callus cultures of Cinchona ledgeriana. Phytochemistry 1984; 23: 2307–2312 20 Jang DS, Jeong IH, Kim JS, Lee YM, Yoo NH, Cho JH, Kim JH. Anthraquinones from the roots of Knoxia valerianoides inhibit the formation of advanced glycation end products and rat lens aldose reductase in vitro. Arch Pharm Res 2010; 33: 209–214 21 Fox BW, Hadfield JA, Zhang X. Preparation of naturally occurring anthraquinones. Synthetic Commun 1996; 26: 49–62 22 Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 2007; 42: 377–387
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
13 Núñez Montoya SC, Agnese AM, Cabrera JL. Anthraquinone derivatives from Heterophyllaea pustulata. J Nat Prod 2006; 69: 801–803 14 Schripsema J, Ramos-Valdivia A, Verpoorte R. Robustaquinones, novel anthraquinones from an elicited Cinchona robusta suspension culture. Phytochemistry 1999; 51: 55–60 15 Huang WH, Yu SH, Li YB, Jiang JQ. Two new anthraquinones from Hedyotis diffusa. J Asian Nat Prod Res 2008; 10: 467–471 16 Itokawa H, Mihara K, Takeya K. Studies on a novel anthraquinone and its glycosides isolated from Rubia cordifolia and R. akane. Chem Pharm Bull 1983; 31: 2353–2358 17 Cameron DW, Schütz PE. Oxidation and reduction of some methoxy-anthracenes and their derivatives. J Chem Soc C 1967; 2121–2125 18 Ali M, Akhtar MSM, Mir SR, Singh O. New anthraquinones from Rubia cordifolia roots. Indian J Chem B 2006; 45: 1945–1950
1117
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
1113
Isolation and Cytotoxic Effect of Anthraquinones from Morinda umbellata
Authors
Chun-Tang Chiou 1, Ruie-Yu Hsu 2, Lie-Chwen Lin 1
Affiliations
1
Key words " Morinda umbellate l " Rubiaceae l " 1,6‑dihydroxy‑2‑methl oxymethyl‑anthraquinone " 6‑hydroxy‑7‑methl oxy‑2‑methoxymethylanthraquinone " 3,6‑dihydroxy‑7‑methl oxy‑2‑methylanthraquinone " 6‑hydroxy‑2‑methoxymethl ylanthraquinone " cytotoxicity l
received revised accepted
January 29, 2014 July 3, 2014 July 9, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382956 Published online August 19, 2014 Planta Med 2014; 80: 1113–1117 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Lie-Chwen Lin National Research Institute of Chinese Medicine Ministry of Health and Welfare Taipei 155–1, Li-Nong Street Section 2 Taipei 11221 Taiwan Phone: + 88 62 28 20 19 99 ext. 71 01 Fax: + 88 62 28 26 42 76 [email protected]
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
Abstract !
Four new anthraquinones, 1,6-dihydroxy-2-methoxymethylanthraquinone (1), 6-hydroxy-7methoxy-2-methoxymethylanthraquinone (3), 3,6-dihydroxy-7-methoxy-2-methylanthraquinone (4), and 6-hydroxy-2-methoxymethylanthraquinone (8), together with 12 known anthraquinones and 6 other known compounds, were
isolated from the EtOAc extract of Morinda umbellata. Among the isolated compounds, 1, rubiadin (14), and, 3-hydroxy-2-hydroxymethylanthraquinone (16) exhibited significant cytotoxicities against HepG2 cells, with GI50 values of 4.4, 3.6, and 4.8 µM, respectively. Supporting information available online at http://www.thieme-connect.de/products
Introduction
Results and Discussion
!
!
Morinda umbellata L. (Rubiaceae) is a scandent shrub distributed in the low elevations of Taiwanʼs mountains [1]. It is used in Chinese folk medicine as an analgesic for the treatment of rheumatism [2]. Previous chemical studies of the genus Morinda have shown the presence of a variety of anthraquinones [3–5], iridoids [5], phenylpropanoids [6], and flavonoid glycosides [7]. Some of them were reported as the active compounds responsible for antimicrobial [8], antileishmanial, antimalarial [9], antitumor [10], antiosteoporotic [11], and hypoglycemic [12] activities. In the course of our search for biologically active substances in nature, we found that the ethanolic extract from the vines of M. umbellata showed cytotoxic activities against cancer cell lines in vitro. Therefore, a series of isolation processes was conducted to study the bioactive constituents of M. umbellata. This paper describes the isolation and characterization of 22 components from the vines of M. umbellata, including 4 new compounds, 1, 3, 4, and 8. Furthermore, the cytotoxic activities of these compounds on the cancer cell lines are also presented.
The ethanolic extract of the vines of M. umbellata was fractionated by solvent partition and separated by column chromatography to afford 22 compounds, including 4 new anthraquinones " Fig. 1) and 18 known compounds, soranjidiol (l (2) [13], robustaquinone D (5) [14], 3-hydroxy-2methoxy-6-hydroxymethylanthraquinone (6) [15], pustulin (7) [13], 6-hydroxyrubiabin (9) [16], 2-hydroxy-6-methoxyanthraquinone (10) [17], 3,6-dihydroxy-2-methylanthraquinone (11), 6-hydroxy-2-methylanthraquinone (12) [18], anthragallol 1,2-dimethyl ether (13) [19], rubiadin (14) [20], 3-hydroxy-2-methyl anthraquinone (15) [21], 3-hydroxy-2-hydroxymethylanthraquinone (16) [11], alizarin 1-methyl ether (17) [11], chrysoeriol (18), scopoletin (19), gallaldehyde (20), p-oxybenzaldehyde (21), and 2-monolinolein (22). The structures of 1, 3, 4, and 8 were elucidated by extensive spectroscopic analyses. The known compounds were identified by comparison of their physical and spectral data with those reported in the literature, except for 18-22, which were identified by comparison with authentic samples. Compound 1 was obtained as yellow amorphous powder. The molecular formula of 1 was determined as C16H12O5 from HRESIMS at m/z 283.0625 [M – H]− (calcd. for C16H11O5, 283.0606). The IR spectrum of 1 showed absorp-
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
2
1114
Original Papers
Fig. 2 Key HMBC correlations of compound 1.
Chemical structures of compounds 1, 3, 4, and 8.
tion bands at 3401, 1659, 1633, 1594, and 1581 cm−1, suggesting the presence of hydroxyl, carbonyl, and aromatic groups. The 1H " Table 1) revealed the presence of a pair of orthospectrum of 1 (l coupled aromatic protons (δH 7.81/7.75), an ABX coupled aromatic proton (δH 7.61/7.32/8.20), an oxymethylene proton (δH 4.58), a methoxyl proton (δ 3.47), and a chelated hydroxyl proton (δH 13.15). Two carbon signals at δC 189.1 (C-9) and 182.7 (C-10) were attributed to two conjugated carbonyl groups, which were also associated with UV absorptions at 244, 269, 279, 291, and 410 nm. Furthermore, a chelated hydroxyl proton at δH 13.1 was observed. Thus, a 1-hydroxyanthraquinone structure was deduced. In the HMQC spectrum, the proton singlets at δH 4.58 and 3.47 correlated to carbon signals at δC 68.8 (t) and 58.9 (q), respectively, indicating the presence of a methoxymethyl group " Fig. 2), the correlations (–CH2OCH3). In the HMBC spectrum (l of H-8 (δH 8.20) to C-6 (δC 164.7), C-9 (δC 189.1), and C-10a (δC 137.0), along with H-4 (δH 7.75) and H-5 (δH 7.61) to C-10 (δ 182.7), and H-3 (δH 7.81) to C-1 (δC 160.3) and C-4a (δC 133.3) suggested that a 1,6-dihydroxyanthraquinone moiety was present. In addition, HMBC correlations of –CH2OCH3 (δH 4.58) to C-1, C-3 (δC 134.7), and –CH2OCH3 (δC 58.9) confirmed the location of the methoxymethyl group at C-2. Therefore, compound 1 was deduced as 1,6-dihydroxy-2-methoxymethylanthraquinone.
Table 1
1
H and 13C data of compounds 1, 3, 4, and 8a. 1b
a
Compound 3 was obtained as orange amorphous powder and its molecular formula was established as C17H14O5 from HREIMS at m/z 298.0838 [M]+ (calcd. for C17H14O5: 298.0841). It showed absorption bands at 3431, 1630, and 1600 cm−1 in the IR spectrum and UV absorptions at 245, 285, and 403 nm, suggesting an an" Table 1) thraquinone derivative. The 1H NMR spectrum of 3 (l showed an aromatic ABX system at δH 8.15/8.16/7.73 and two aromatic singlets at δH 7.68 and 7.51. In addition, a methoxymethyl group –CH2OCH3 (δH 4.61 and 3.45) and a methoxy group (δH 4.02) appeared in the aliphatic region. In addition to two carbonyl groups at δC 183.4 and 183.8, the 13C NMR spectrum showed two oxygenated quaternary carbons at δC 156.0 (C-6) and 154.8 (C-7), indicating that one of these carbons was linked to a methoxy group and the other carbon was attached to a hydroxyl group. The locations of substituents were deduced by HMBC and NOESY experiments. The HMBC correlations of H-1 (δH 8.15)/H-8 (δH 7.68) to C-9 (δC 183.4) and of H-4 (δH 8.16)/H‑5 (δH 7.51) to C10 (δC 183.8) together with the NOESY correlations of –CH2OCH3 (δH 4.61) to H-1 (δH 8.15)/H‑3 (δH 7.73) and –OCH3 (δH 4.02) to H8 (δH 7.68) confirmed the structure of 3 as 6-hydroxy-7-methoxy-2-methoxymethylanthraquinone. Compound 4 possessed the molecular formula C16H12O5 derived from the HREIMS at m/z 284.0675 [M]+ (calcd. 284.0685 for C16H12O5). The IR and UV spectra of 4 showed characteristic sig" Tanals for an anthraquinone derivative. Its 1H NMR spectrum (l ble 1) showed four aromatic singlets at δH 7.47 (H-4), 7.50 (H-5),
No.
δC
1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 2-CH2OCH3 2-CH2OCH3 -CH3 -OCH3 -OH
160.3 135.5 134.7 119.4 133.3 113.6 164.7 122.2 130.7 126.3 189.1 116.1 182.7 137.0 68.8 58.9
3c δH (J in Hz)
7.81 (d, J = 8.0) 7.75 (d, J = 8.0) 7.61 (d, J = 3.2) 7.32 (dd, J = 8.4, 3.2) 8.20 (d, J = 8.4)
4.58 (s) 3.47 (s)
4c
8b
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
126.2 146.4 133.2 128.0 134.2 114.2 154.8 156.0 109.6 130.4 183.8 135.2 183.4 128.0 74.6 58.8
8.15 (d, J = 1.2)
131.05 132.9 162.0 112.5 135.2 113.9 154.3 154.3 109.6 128.5 184.2 127.1 183.3 130.3
7.94 (s)
125.8 146.8 132.9 127.8 133.6 113.2 163.8 122.1 130.7 127.1 182.2 134.6 183.3 136.7 74.0 58.6
8.20 (d, J = 0.6)
56.6
7.73 (dd, J = 7.8, 1.2) 8.16 (d, J = 7.8) 7.51 (s)
7.68 (s)
7.47 (s) 7.50 (s)
7.67 (s)
4.61 (s) 3.45 (s) 4.02 (s)
16.5 56.7
2.31 (s) 4.02 (s)
13.15 (s)
Assignments were confirmed by DEPT, 1H-1H COSY, HMQC, and HMBC experiments. b Measured in acetone-d6; c measured in methanol-d4
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
7.81 (dd, J = 7.8, 0.6) 8.21 (d, J = 7.8) 7.64 (d, J = 2.4) 7.31 (dd, J = 8.4, 2.4) 8.19 (d, J = 8.4)
4.65 (s) 3.44 (s)
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Fig. 1
Original Papers
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 Doxorubicina a
GI50 (µM) A549
HepG2
HT29
PC3
26.3 ± 1.3 82.3 ± 2.7 25.2 ± 1.0 > 100 > 100 > 100 > 100 87.4 ± 7.5 > 100 > 100 68.4 ± 2.7 60.1 ± 1.8 22.5 ± 9.7 > 100 > 100 43.7 ± 3.7 > 100 > 100 > 100 > 100 > 100 0.07 ± 0.03
4.4 ± 0.9 45.6 ± 4.7 14.0 ± 2.2 > 100 > 100 > 100 87.3 ± 4.5 12.0 ± 1.7 59.3 ± 2.1 > 100 10.0 ± 0.8 14.7 ± 5.1 88.8 ± 4.0 3.6 ± 0.4 77.5 ± 1.1 4.8 ± 1.3 > 100 51.0 ± 4.5 > 100 > 100 77.2 ± 3.5 0.07 ± 0.01
61.0 ± 2.5 > 100 > 100 > 100 > 100 > 100 > 100 84.8 ± 5.6 > 100 > 100 90.5 ± 5.8 76.7 ± 4.1 21.7 ± 2.0 > 100 > 100 92.7 ± 4.3 > 100 > 100 > 100 > 100 > 100 0.26 ± 0.04
27.3 ± 0.3 > 100 > 100 > 100 > 100 > 100 > 100 88.0 ± 1.7 > 100 > 100 > 100 80.8 ± 2.7 53.7 ± 9.0 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 0.13 ± 0.06
Table 2 Cytotoxic effects of isolated compounds on cancer cell lines.
Doxorubicin was used as a positive control
7.67 (H-8), and 7.94 (H-1), a methyl group (δH 2.31, 2-CH3), and a methoxyl group (δH 4.02, 7-OCH3). The 13C NMR spectrum showed signals for two carbonyl groups at δC 183.3 (C-10) and 184.2 (C-9) and three oxygenated quaternary carbons at δC 162.0 (C-3) and 154.3 (C-6 and C-7), which suggested that one methoxyl and two hydroxyl groups were attached to the aromatic rings. The HMBC correlations of H-1/H-8 to C-9 and H-4/ H-5 to C-10 together with the NOESY correlations of –CH3 to H1 and –OCH3 to H-8 confirmed the structure of 4 as 3,6-dihydroxy-7-methoxy-2-methylanthraquinone. Compound 8 possessed the molecular formula C16H12O4 deduced from HRESIMS at m/z 267.0675 [M – H]− (calcd. 267.0657 for " Table 1) showed two C16H11O4). The 1H NMR spectrum of 8 (l sets of aromatic ABX spin systems at δH 8.20/8.21/7.81 and 7.31/ 7.64/8.19 and a methoxymethyl group [δH 4.65 (CH2OCH3-2)/ 3.44 (CH2OCH3-2)]. In the HMBC spectrum, the correlations of H-1 (δH 8.20)/H-8 (δH 8.19) to C-9 (δC 182.2), H-4 (δH 8.21)/H‑5 (δH 7.64) to C-10 (δC 183.3), and H-8 (δH 8.19) to C-6 (δC 163.8) suggested the presence of a 6-hydroxyanthraquinone moiety. The NOESY spectrum showed correlations of –CH2OCH3 to H-1 and H-3. Thus, the structure of 8 was determined as 6-hydroxy2-methoxymethylanthraquinone. The isolated compounds 1-16 and 18-22 were tested against a panel of cancer cell lines according to the established protocol " Table 2, compounds 1, 8, 12, and 13 displayed [22]. As shown in l cytotoxicity against A549, HepG2, HT29, and PC3 cells with GI50 values ranging from 4.4 to 88.8 µM. Compounds 1, 14, and 16 showed potent suppressions on the growth of HepG2 tumor cells with GI50 values all less than 5 µM. These results suggested that the anthraquinones from M. umbellata were cytotoxic against A549, HepG2, HT29, and PC3 cancer cells, and were especially sensitive to the HepG2 cells. In this study, compounds 1, 3, 8, 11, 12, 14, and 16 showed high cytotoxic effects against HepG2 tumor cells with GI50 values all less than 15 µM. Their structure-activity relationships revealed that no more than three substitutions in the anthraquinone skeleton, and 6-hydroxy-2-methyl or
6-hydroxy-2-methoxymethyl substitution may enhance the cytotoxic activity.
Materials and Methods !
General experimental procedures IR spectra were obtained as KBr pellets on a Nicolet Avatar 320 IR spectrometer. UV spectra were obtained in MeOH on a Hitachi U2001 spectrophotometer. 1D and 2D NMR spectra were measured on a Varian VNMRS-600 spectrometer with deuterated solvent as internal standards. EIMS, HREIMS, ESIMS, and HRESIMS were recorded on Finnigan MAT95S and Shimadzu LCMS‑IT‑TOF mass spectrometers. Column chromatography was performed on Sephadex LH-20 (Pharmacia) or silica gel 60 (70–230 mesh or 230–400 mesh, Merck). The semipreparative HPLC system consisted of a chromatographic pump (LC-10AD, Shimadzu) and a UV-Visible detector (SPD-10A vp, Shimadzu). A Lichrosorb® Si60 column (10 × 250 mm; particle size 7 µm; Merck) or Purospher® STAR RP-18e column (10 × 250 mm; particle size 5 µm; Merck) was used for separation.
Plant material The vines of M. umbellata were collected in New Taipei City, Taiwan. M. umbellata was identified by Dr. Cheng-Jen Chiou, retired research fellow of the National Research Institute of Chinese Medicine and reconfirmed by comparison with a voucher specimen (No. 298 775), which has been deposited in the herbarium of the Taiwan Forestry Research Institute.
Extraction and isolation The vines of M. umbellata (4.7 kg) were sliced and extracted with 95 % EtOH (30 L × 3). The ethanolic extract was concentrated to a volume of 200 mL. The concentrated ethanolic extract was then partitioned successively between H2O and n-hexane, followed by EtOAc and n-BuOH (each 200 mL × 3) to give n-hexane, EtOAc,
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Compound
1115
Original Papers
n-BuOH, and H2O extracts. The EtOAc extract displayed cytotoxicity against A549 and HepG2 cells with GI50 values of 54.8 and 48.6 µg/mL, respectively. Therefore, the EtOAc extract (126.6 g) was further separated using silica gel column chromatography (10 × 90 cm, 70–230 mesh) with a solvent system of 10%, 30 %, 50 %, 70% EtOAc in n-hexane and finally EtOAc (3 L for each portion), monitored by TLC. Six fractions (Frs. 1–6) were collected. Fr. 3 (5.7 g) was repeatedly chromatographed over medium pressure silica gel column (4.8 × 45 cm, 230–400 mesh, ~ 10 bar, 10–50 % EtOAc/n-hexane over 90 min) and semipreparative HPLC (40– 50 % CHCl3/n-hexane over 40 min, Lichrosorb® Si60 column, 7 µm, 10 × 250 mm, flow rate = 3.7 mL/min, UV= 254 nm) to give 1 (4.0 mg, rt = 26 min), 2 (8.6 mg, rt = 18 min), 5 (2.3 mg, rt = 15 min), 8 (2.2 mg, rt = 30.5 min), 12 (3.8 mg, rt = 24 min), 13 (8.6 mg, rt = 25 min), 14 (5.8 mg, rt = 14 min), 15 (21.1 mg, rt = 16.5 min), and 21 (1.4 mg, rt = 32 min). Fr. 4 (1.4 g) was purified by Sephadex LH-20 (90% MeOH/CHCl3) and semipreparative HPLC (50–90% MeOH/H2O over 30 min, Purospher® STAR RP18e column, 5 µm, 10 × 250 mm, flow rate = 3.7 mL/min, UV = 254 nm) to afford 3 (1.3 mg, rt = 11 min), 4 (2.1 mg, rt = 9 min), 6 (10.9 mg, rt = 11 min), 7 (6.2 mg, rt = 13 min), 9 (6.9 mg, rt = 10 min), 11 (1.2 mg; rt = 8.5 min), 16 (61.7 mg, rt = 7 min), 17 (2.9 mg, rt = 10 min), 18 (3.0 mg, rt = 6.5 min), and 22 (17.0 mg, rt = 12.5 min). Fr. 5 (2.87 g) was purified by Sephadex LH-20 (90% MeOH/CHCl3) and semipreparative HPLC (80 % MeOH/H2O, Purospher® STAR RP-18e column, 5 µm, 10 × 250 mm, flow rate = 3.7 mL/min, UV= 254 nm) to yield 6 (1.1 mg, rt = 11 min), 10 (3.8 mg, rt = 6 min), 19 (16 mg, rt = 13 min), and 20 (5.4 mg, rt = 4.5 min).
285 (4.71), 403 (3.32) nm; IR (KBr) υmax 3431 (OH), 1630 (C=O), 1600 (C=C), 1383, 1326, 1082, 1028 cm−1; 1H and 13C NMR data, " Table 1; EIMS m/z 298 [M]+, HREIMS m/z 298.0838 [M]+ see l (calcd. 298.0841 for C17H14O5). 2,7-Dihydroxy-3-methoxy-6-methylanthraquinone (4): yellow powder (> 95%, purity); UV (MeOH) λmax (log ε) 285 (4.31), 311 sh. (3.80), 349 sh. (3.47) nm; IR (KBr) υmax 3399 (OH), 1663, 1630 (C=O), 1602, 1383, 1350, 1060, 1030 cm−1; 1H and 13C NMR " Table 1; EIMS m/z 284 [M]+, HREIMS m/z 284.0675 data, see l [M]+ (calcd. 284.0685 for C16H12O5). 6-Hydroxy-2-methoxymethylanthraquinone (8): yellow powder (> 95%, purity); UV (MeOH) λmax (log ε) 246 sh. (4.21), 270 (4.37), 283 sh. (4.29), 333 (3.54), 383 (3.32) nm; IR (KBr) υmax 3305 (OH), 1671(C=O), 1658 (C=O), 1582, 1566, 1317, 1108, " Table 1; ESIMS m/z 267 1086 cm−1; 1H and 13C NMR data, see l [M – H]−, HRESIMS m/z 267.0675 [M – H]− (calcd. 267.0657 for C16H11O4).
Supporting information 1D and 2D NMR spectra of anthraquinones 1, 3, 4, and 8 are available as Supporting Information.
Acknowledgements !
This study was supported by a research grant (NSC 102–2320B‑077-002-MY3) from the National Science Council, Taiwan.
Conflict of Interest Cell lines
!
A549, HepG2, HT29, and PC3 cell lines were utilized as target cells in the cytotoxic assay. A549 is a human lung adenocarcinoma epithelial cell line, HepG2 is a human hepatocellular liver carcinoma cell lines, HT29 is a human colon adenocarcinoma grade II cell line, and PC3 is a human prostate cancer cell line. These four cell lines were obtained from the Bioresources Collection and Research Center (BCRC, Hsin-Chu, Taiwan) and cultured in RPMI1640 medium (Hyclone) containing 10% FBS (Gibco), 100 u/mL penicillin (Gibco), 100 µg/mL streptomycin, and 1 × non-essential amino acid (Gibco), at 37 °C in an atmosphere of humidified 5 % CO2.
The authors report no conflicts of interest in this work.
Sulforhodamine B (SRB) assay Growth inhibition was assessed as described previously [22]. Each tumor cell line was cultured with or without various concentrations of test compounds and doxorubicin (positive control, purchased from Sigma-Aldrich, purity > 98 %) for 3 days, treated with 4% SRB reagent for 10 min and then 10 mM TrisBase were added. OD values in the wavelength 515 nm were determined by an ELISA reader, and inhibitory activity was calculated as GI50 (concentration of inhibition of 50% cell growth).
Isolates 1,6-Dihydroxy-2-methoxymethylanthraquinone (1): yellow powder (90.5 %, purity); UV (MeOH) λmax (log ε) 220 (4.16), 268 (4.15), 408 (3.64) nm; IR (KBr) υmax 3401 (OH), 1659 (C=O), 1633(C=O), 1594 (C=C), 1580 (C=C), 1428, 1277, 1247 cm−1; 1H " Table 1; ESIMS m/z 283 [M – H]−, HREand 13C NMR data, see l SIMS m/z 283.0625 [M – H]− (calcd. 283.0606 for C16H11O5). 7-Hydroxy-6-methoxy-2-methoxymethylanthraquinone (3): orange powder (> 95%, purity); UV (MeOH) λmax (log ε) 245 (4.21), Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
References 1 Yang TYA. Morinda L. In: Editorial Committee of the Flora of Taiwan, editor. Flora of Taiwan, 2nd edition, Volume IV. Taipei: Department of Botany, National Taiwan University; 1998: 299–301 2 Juangsu New Medical College. Zhong Yao Da Ci Dian (Dictionary of Chinese Materia Medica). Shanghai: Shanghai Scientific and Technological Publishers; 1977: 807 3 Ismail NH, Lajis NH, Ali AM, Aimi N, Kitajima M, Takayama H. Anthraquinones from Morinda elliptica. Phytochemistry 1997; 45: 1723–1725 4 Ee GC, Wen YP, Sukari MA, Go R, Lee HL. A new anthraquinone from Morinda citrifolia roots. Nat Prod Res 2009; 23: 1322–1329 5 Kamiya K, Satake T, Tanaka Y, Kamiya K, Satake T, Endang H, Umar M. New anthraquinone and iridoid from the fruits of Morinda citrifolia. Chem Pharm Bull 2005; 53: 1597–1599 6 He QW, Wang G, Feng T, He QW, Liu JK. Two new phenylpropanoids and one propanoate from Morinda citrifolia. J Asian Nat Prod Res 2011; 13: 238–241 7 Cimanga K, Bruyne TD, Lasure A, Li Q, Pieters L, Claeys M, Berghe DV, Kambu K, Tona L, Vlietinck A. Flavonoid O-glycosides from the leaves of Morinda morindoides. Phytochemistry 1995; 38: 1301–1303 8 Guo SP, Song QS, Xiang W, Xiang W, Zhang HJ. Antimicrobial anthraquinones from Morinda angustifolia. Fitoterapia 2008; 79: 501–504 9 Sittie AA, Lemmich E, Olsen CE, Hviid L, Kharazmi A, Nkrumah FK, Christensen SB. Structure-activity studies: in vitro antileishmanial and antimalarial activities of anthraquinones from Morinda lucida. Planta Med 1999; 65: 259–261 10 Chang P, Chen CF. Isolation and characterization of antitumor anthraquinones from Morinda umbellata. Chin Pharm J 1995; 47: 347–353 11 Wu YB, Han T, Jiao L, Qin LP, Sun LN, Wu YB, Zhang QY, Zheng CJ, Wu JZ. Antiosteoporotic activity of anthraquinones from Morinda officinalis on osteoblasts and osteoclasts. Molecules 2009; 14: 573–583 12 Hamabe W, Harada S, Kamiya K, Murakami R, Satake T, Tokuyama S. Chemical constituents of Morinda citrifolia roots exhibit hypoglycemic effects in streptozotocin-induced diabetic mice. Biol Pharm Bull 2008; 31: 935–938
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
1116
Original Papers
19 Wijnsma R, Verpoorte R, Mulder-Krieger T, Svendsen AB. Anthraquinones in callus cultures of Cinchona ledgeriana. Phytochemistry 1984; 23: 2307–2312 20 Jang DS, Jeong IH, Kim JS, Lee YM, Yoo NH, Cho JH, Kim JH. Anthraquinones from the roots of Knoxia valerianoides inhibit the formation of advanced glycation end products and rat lens aldose reductase in vitro. Arch Pharm Res 2010; 33: 209–214 21 Fox BW, Hadfield JA, Zhang X. Preparation of naturally occurring anthraquinones. Synthetic Commun 1996; 26: 49–62 22 Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 2007; 42: 377–387
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
13 Núñez Montoya SC, Agnese AM, Cabrera JL. Anthraquinone derivatives from Heterophyllaea pustulata. J Nat Prod 2006; 69: 801–803 14 Schripsema J, Ramos-Valdivia A, Verpoorte R. Robustaquinones, novel anthraquinones from an elicited Cinchona robusta suspension culture. Phytochemistry 1999; 51: 55–60 15 Huang WH, Yu SH, Li YB, Jiang JQ. Two new anthraquinones from Hedyotis diffusa. J Asian Nat Prod Res 2008; 10: 467–471 16 Itokawa H, Mihara K, Takeya K. Studies on a novel anthraquinone and its glycosides isolated from Rubia cordifolia and R. akane. Chem Pharm Bull 1983; 31: 2353–2358 17 Cameron DW, Schütz PE. Oxidation and reduction of some methoxy-anthracenes and their derivatives. J Chem Soc C 1967; 2121–2125 18 Ali M, Akhtar MSM, Mir SR, Singh O. New anthraquinones from Rubia cordifolia roots. Indian J Chem B 2006; 45: 1945–1950
1117
Chiou C-T et al. Isolation and Cytotoxic …
Planta Med 2014; 80: 1113–1117
Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
![[Isolation, identification and biosynthesis of anthraquinones in cell suspension cultures of Morinda citrifolia (author's transl)].](/assets/img/hold.png)
