Arch. Pharm. Res. (2014) 37:1515–1521 DOI 10.1007/s12272-014-0428-z
RESEARCH ARTICLE
Cytotoxic triterpenoids from Saussurea phyllocephala Jiang Hu • Yan Song • Benshou Yang • Xiang Zuo • Xiao Mao • Xiaodong Shi
Received: 30 March 2014 / Accepted: 17 June 2014 / Published online: 1 July 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Three new triterpenoids, 11a-hydroxy-lup20(29)-ene-3b-palmitate (1), 28-hydroxy-20-oxo-30-norlupane-3b-palmitate (2), and 1b,11a,28-tyrihydroxy oleana-9(11),12-dien-3b-palmitate (3), together with three known compounds were isolated from the 95 % EtOH extract of Saussurea phyllocephala. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR (COSY, HMQC, HMBC and NOESY) analyses. All the triterpenoids were in vitro evaluated for their cytotoxic activities. Compound 1–3 exhibited the significant cytotoxic activities with low IC50 values (IC50 \ 4.0 lM) against six tumor cell lines (MCF-7, HeLa, HepG2, SGC7901, NCI-H460 and BGC-823).
Jiang Hu and Yan Song have contributed equally to this work. J. Hu (&) X. Mao X. Shi (&) College of Biological Resources and Environment Science, Qujing Normal University, Sanjiang Avenue, Unicorn District, Qujing 655011, Yunnan Province, China e-mail: [email protected] X. Shi e-mail: [email protected] J. Hu Institute of Characteristic Medicinal Resource of Ethnic Minorities, Qujing Normal University, Qujing 655011, China Y. Song X. Zuo Department of Pharmacy, 455 Hospital of People’s Liberation Army, Shanghai 200052, China B. Yang Institute of Microbiology, Qujing Medical College, Qujing 655000, Yunnan Province, China
Keywords Saussurea phyllocephala Compositae Triterpenoids Cytotoxic activity
Introduction The genus Saussurea consists of about 400 species, which range widely from Asia to Europe (Li et al. 2008). About 40 species have been investigated on their chemical constituents, and sesquiterpenes (Wang et al. 2010; Robinson et al. 2008; Chhabra et al. 1998), triterpenoids (Dai et al. 2001), flavonoids (Gao et al. 2006), lignans (Duan et al. 2002), and phenolic compounds (Li et al. 2010) have been reported as the most widespread constituents. Saussurea phyllocephala Coll. et Hemsl. is distributed at the altitude of about 1,200 m above sea level in the southwestern of China. It has been used as a traditional Chinese medicine for the treatment of various disease such as hepatitis, jaundice, hyperhidrosis, lung-heat, cough, vertigo and venomous snake bite (The Editorial Board of Chinese Materia Medica 1999). The present study on the chemical constituents of the 95 % EtOH extract of S. phyllocephala from Yuanjiang, Yunnan province led to the isolation of three new lupane-type triterpenoids, 1ahydroxy-lup-20(29)-ene-3b-palmitate (1), 28-hydroxy-20oxo-30-norlupane-3b-palmitate (2), and 1b,11a,28-tyrihydroxy oleana-9(11),12-dien-3b-palmitate (3), as well as three known compounds 3b,11a-dihydroxy-lup-20(29)-ene (4), 1b,3b,11a,28-tetrahydroxy-oleana-12-ene (5), and 3b,11a-dihydroxy-ursa-12-ene (6) (Reyes et al. 2006; De la Torre et al. 1990) (Fig. 1). This paper deals with the isolation and structure elucidation of three new triterpenoids by spectral methods and all the triterpenoids were in vitro evaluated for their cytotoxic activities against six tumor cell lines (MCF-7, HeLa, HepG2, SGC-7901, NCI-H460 and BGC-823).
123
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J. Hu et al.
Fig. 1 The structures of compounds 1–6
30
O
20
19 21
29
HO
11
25 1
26
10 4
RO
H
5
18
22
13 17 14 H
28
H H O
16
15
8 7
CH3(CH2)14
O
H
24
1 R=CO(CH2)14CH3 4 R=H 29
19
HO OH 25 1
11
26
10 4
RO
5
H 23
12
9
2 3
H 6
2
30
21
20 18
13
OH
14H
8
HO
22 17
15
H
28 16
H
27
HO
7
H
24
3 R=CO(CH2)14CH3 5 R=H
Materials and methods General experimental procedures Melting points were determined on a XT-4 microscopic thermometer without correction. Optical rotations were taken on a Perkin-Elmer 341 polarimeter. IR spectra were recorded on Nicolet Magna FT-IR 750 spectrophotometer using KBr disks. NMR spectra were recorded on Bruker AM-300, AM-400, and INVOR-600 NMR spectrometers. The chemical shift (d) values are given in ppm with TMS as internal standard, and coupling constants (J) are in Hz. EIMS and HREIMS spectra were recorded on a Finnigan MAT-95 mass spectrometer. ESIMS and HRESIMS spectra were recorded on a Micromass LC–MS–MS mass spectrometer. Column chromatographic separations were carried out using silica gel (200–300 mesh and H60, QingdaoHaiyang Chemical Group Corporation, P. R. China), MCI gel CHP20P (75–150 lm, Mitsubishi Chemical Industries, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) as packing material. TLC was carried out on precoated silica gel GF254 plates (Yantai Chemical Industrials), and the TLC spots were viewed at 254 nm and visualized using 5 % sulfuric acid in
123
OH
H
27
6
H 23
12
9
2 3
H
6
alcohol containing 10 mg/mL vanillin. Analytical HPLC was performed on a Waters 2690 instrument with a 996 PAD (photodiode array detector) coupled with an Alltech ELSD 2000 detector. Semipreparative and preparative HPLC was performed on a Varian SD1 instrument with a 320 single-wave detector. Their chromatographic separations were carried out on C-18 columns (250 9 10 mm, 5 lm, Waters; 220 9 25 mm, 10 lm, Merck, respectively), using a gradient solvent system comprised of H2O and MeCN, with a flow rate of 3.0 and 15.0 mL/min, respectively. All cell lines were purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Other reagents were purchased from Shanghai Sangon Biological Engineering Technology and Services CO., Lt. Plant materials The air-dried whole plant of S. phyllocephala was collected in the Yuanjiang, Yunnan province, China, in July 2011. A specimen (SP20110701), identified by one of the authors (X. Mao), was deposited in the Herbarium of the College of Biological Resources and Environment Science, Qujing Normal University, Qujing, China.
Cytotoxic triterpenoids from Saussurea phyllocephala
1517
Table 1 1H-NMR spectral data of compounds 1–3 (1H: 500 MHz in CDCl3) Position
1
H-NMR (multi., J Hz)
1
2
3
H–C(1)ax
0.85 (ddd, 14.0, 13.6, 4.0)
0.82 (ddd, 13.8, 13.6, 3.6)
3.94 (dd, 13.8,3.8)
H–C(1)eq
1.70 (overlapped)
1.65 (ddd, 13.6, 4.0, 3.4)
–
H–C(2)ax
1.37 (m)
1.33 (m)
1.76 (m)
H–C(2)eq
1.62(m)
1.56 (overlapped)
1.87 (m)
H–C(3)
4.45 (dd, 13.6, 4.0)
4.40 (dd, 13.5, 4.0)
4.35 (dd, 13.6,3.8)
H–C(5)
1.32 (dd, 13.6, 3.8)
1.26 (dd, 13.8, 3.6)
0.82 (dd, 13.6,3.8)
H–C(6)ax H–C(6) eq
1.98 (overlapped) 2.14 (m)
2.00 (overlapped) 2.08 (overlapped)
1.40 (m) 1.53 (m)
H–C(7)ax
1.33 (ddd, 13.8, 13.6, 4.0)
1.26 (ddd, 13.8, 13.6, 4.0)
1.27 (overlapped)
H–C(7)eq
1.58 (overlapped)
1.50 (ddd, 13.6, 3.8, 3.6)
1.51 (ddd, 13.6, 4.0, 3.6)
H–C(9)
2.03 (d, 13.2)
1.88 (d, 13.2)
1.81 (d, 13.6)
H–C(11)ax
3.12 (m)
1.35 (m)
4.60 (dd, 13.6, 7.6)
H–C(11)eq
–
1.82 (m)
–
H–C(12)ax
1.55 (overlapped)
1.45 (m)
5.30 (d, 7.6)
H–C(12)eq
2.10 (m)
2.00 (overlapped)
–
H–C(13)
2.03 (ddd, 13.6, 13.2, 3.6)
1.92 (overlapped)
–
H–C(15)ax
1.74 (ddd, 13.8, 13.6, 4.0)
1.68 (overlapped)
1.81 (overlapped)
H–C(15)eq
1.98 (overlapped)
1.90 (ddd, 13.2, 4.0, 3.4)
2.00 (overlapped)
H–C(16)ax
1.70 (overlapped)
1.70 (ddd, 13.6, 13.0, 4.0)
1.81 (overlapped) 2.00 (overlapped)
H–C(16)eq
2.08 (overlapped)
2.12 (ddd, 13.0, 3.8, 3.4)
H–C(18)
1.92 (overlapped)
1.83 (dd, 13.6, 13.2)
1.36 (dd, 13.6,3.8)
H–C(19) H–C(21)ax
2.38 (m) 2.08 (overlapped)
2.60 (m) 2.08 (overlapped)
1.15 (m) 1.25 (overlapped)
H–C(21)eq
1.72 (overlapped)
1.68 (overlapped)
1.40 (ddd, 13.6,4.0,3.8)
H–C(22)ax
1.92 (d overlapped)
1.92 (overlapped)
1.25 (overlapped)
H–C(22)eq
1.58 (overlapped)
1.56 (overlapped)
1.44 (ddd, 13.8,4.0,3.8)
H–C(23)
0.95 (s)
0.94 (s)
0.85 (s)
H–C(24)
0.82 (s)
0.82 (s)
0.86 (s)
H–C(25)
0.83 (s)
0.88 (s)
0.93 (s)
H–C(26)
1.05 (s)
1.02 (s)
0.80 (s)
H–C(27)
0.96 (s))
0.97 (s)
0.99 (s)
H–C(28)a
0.78 (s)
3.60 (d, 12.8)
3.50 (d, 12.8)
H–C(28)b
–
3.36 (d, 12.8)
3.23 (d, 12.8)
H–C(29)
1.68 (s)
2.12 (s)
0.82 (s)
H–C(30)
4.55 (d, 2.6)
–
0.98 (s)
4.68 (d, 2.6)
–
–
H (20 )
2.26 (t, 6.6)
2.30 (t, 6.6)
2.32 (t, 6.6)
H (30 ) H(40 -150 )
1.63 (m) 1.26 (brs)
1.61 (m) 1.28 (brs)
1.69 (m) 1.27 (overlapped)
H(160 )
0.88 (t, 6.6)
0.86 (t, 6.6)
0.87 (t, 6.6)
Extraction and isolation The air-dried whole plant of S. phyllocephala (2.3 kg) was ground into powder and extracted thrice with 95 % EtOH.
After evaporation of the EtOH, the crude extract (258 g) was partitioned between water and EtOAc. The EtOAcsoluble portion (91 g) was chromatographed on a silica gel column eluted with CHCl3/MeOH (from 100:1 to 1:1) to
123
1518 Table 2 13C-NMR spectral data of compounds 1–3 (13C: 125 MHz, in CDCl 3)
J. Hu et al.
Position
Position 2
3
C-NMR
1
2
3
C(1)
39.1 (t)
38.3 (t)
75.6 (d)
C(24)
16.8 (q)
16.6 (q)
16.3 (q)
25.0 (t)
24.6 (t)
34.7 (t)
C(25)
16.1 (q)
16.0 (q)
18.5 (q)
C(3)
81.2 (d)
81.5 (d)
80.9 (d)
C(26)
16.2 (q)
15.9 (q)
20.3 (q)
C(4)
38.2 (s)
37.8 (s)
38.0 (s)
C(27)
14.8 (q)
14.6 (q)
33.7 (q)
C(5)
55.0 (d)
55.4 (d)
56.3 (d)
C(28)
18.1 (q)
28.2 (t)
27.8 (t)
C(6)
18.1 (t)
18.2 (t)
18.4 (t)
C(29)
19.4 (q)
23.6 (q)
33.2 (q)
C(7)
34.2 (t)
34.1 (t)
31.0 (t)
C(30)
109.5 (t)
–
C(8)
41.1 (s)
40.8 (s)
43.2 (s)
C(10 )
173.7 (s)
173.5 (s)
C(9)
55.6 (d)
50.2 (d)
52.3 (d)
C(20 )
34.9 (t)
34.5 (t)
C(10)
37.4 (s)
37.1 (s)
37.9 (s)
C(30 )
25.2 (t)
25.1 (t)
21.6 (q) 173.5 (s) 34.6 (t) 25.1 (t)
C(11) C(12)
70.4 (d) 27.7 (t)
21.0 (t) 25.1 (t)
67.2 (d) 126.5 (d)
C(4 ) C(50 )
29.71 (t) 29.70 (t)a
29.65 (t) 29.64 (t)b
29.65 (t)c 29.64 (t)c
C(13)
37.4 (d)
37.1 (d)
144.7 (s)
C(60 )
29.68 (t)a
29.63 (t)b
29.63 (t)c
29.62 (t)
b
29.62 (t)c
29.61 (t)
b
29.61 (t)c
29.60 (t)b
29.60 (t)c
43.5 (s)
42.3 (s)
42.9 (s)
0
0
C(7 )
a
b
29.67, t a) a
C(15)
27.3 (t)
29.5 (t)
25.8 (t)
C(80 )
29.65 (t)
C(16)
35.7 (t)
32.0 (t)
27.3 (t)
C(90 )
29.61 (t)a
32.4 (s)
0
C(18) C(19)
47.1 (s) 48.4 (d) 48.1 (d)
55.8 (s) 49.0 (d) 47.0 (d)
45.6 (d) 46.8 (t)
C(10 ) 0
C(11 ) 0
C(12 )
29.5 (t)
a
29.4 (t)
a
29.3 (t)
a a
29.5 (t)
b
29.5 (t)c
29.4 (t)
b
29.4 (t)c
29.3 (t)
b
29.3 (t)c
29.2 (t)
b
29.2 (t)b
C(20)
151.0 (s)
212.3 (s)
31.1 (s)
C(130 )
29.2 (t)
C(21)
29.8 (t)
29.6 (t)
34.7 (t)
C(140 )
31.1 (t)
31.2, t
C(22)
40.1 (t)
40.0 (t)
37.1 (t)
C(150 )
22.7 (t)
22.9, t
22.7 (t)
C(23)
28.0 (q)
28.2 (q)
28.7 (q)
C(160 )
14.1 (q)
14.2 (q)
13.2 (q)
afford fractions 1–8. Fraction 2 (7.2 g) was chromatographed on a MCI gel column eluted with MeOH/H2O (from 70 to 95 %) to yield four subfractions, 2A–2D. Subfraction 2B (1.1 g) was separated by repeated column chromatography (CC) over Sephadex LH-20 (CHCl3/ MeOH, 1:1, and MeOH) and silica gel yielding 1 (29.7 mg) and 3 (31.2 mg). Subfraction 2C (245 mg) was chromatographed on a Sephadex LH-20 column (CHCl3/MeOH, 1:1) and preparative HPLC (MeCN/H2O, from 45 to 70 %), yielding 2 (33.2 mg) and 6 (37.5 mg). Fraction 3 (6.9 g) was subjected to silica gel column chromatography eluted with CHCl3/MeOH (from 9:1 to 1:4) and then purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) CC to afford 4 (55.4 mg) and 5 (48.4 mg). 11a-Hydroxy-lup-20(29)-ene-3b-palmitate (1) White amorphous powder; [a]23.3 ? 8.7 (c = 0.023, D MeOH); UV (MeOH) kmax(log e) 216 (3.78) nm; IR (KBr) mmax 3,420, 2,937, 1,730, 1,641, 1,455, 1,255, 1,040, 860 cm-1; 1H and 13C NMR data are showed Tables 1 and 2; FAB-MS (postive): m/z 681 [M ? H]?; HR-ESI–MS: m/z 703.6001 ([M ? Na]?; C46H80O3Na; calc. 703.6005).
123
13
C(2)
C(17)
Assignments may be interchangeable
C-NMR
1
C(14)
a,b,c
13
31.2 (t)
28-Hydroxy-20-oxo-30-norlupane-3b-palmitate (2) ? 17.3 (c = 0.090, White amorphous powder; [a]23.3 D MeOH); UVkmax(log e) 225 (3.46) nm; IR (KBr) mmax 3,425, 2,925, 1,730, 1,645, 1,460, 1,253, 1,015, 940 cm-1; 1 H and 13C NMR data are showed Tables 1 and 2; FABMS (postive): m/z 683 [M ? H]?; HR-ESI–MS: m/z 705.5795 ([M ? Na]?; C45H78O4Na; calc. 705.5798). 1b,11a,28-Tyrihydroxy oleana-9(11), 12-dien-3b-palmitate (3) White amorphous powder; [a]23.3 ? 43.7 (c = 0.036, D MeOH); UV kmax(log e) 282 (2.15) nm; IR (KBr) mmax 3,415, 2,968, 1,730, 1,630, 1,465, 1,370, 1,250, 1,080; 1H and 13C NMR data are showed Tables 1 and 2; FAB-MS (postive): m/z 713 [M ? H]?; HR-ESI–MS: m/z 735.5900 ([M ? Na]?; C46H80O5Na; calc. 735.5903). Cytotoxicity assay in vitro The revised MTT method (Chen et al. 2011) was used for in vitro evaluation of the cytotoxic potential of the isolated
Cytotoxic triterpenoids from Saussurea phyllocephala
1519
Fig. 2 The key HMBC correlations of compounds 1 and 3
H
HO
HO OH
H O CH3(CH2)14
O
H O
H
1
compounds against eight cultured human tumor cell lines. All tumor cell lines (A549, BGC-823, HCT15, HeLa, HepG2, MCF-7, SK-MEL-2, and SGC-7901 cells) were cultured on RPMI-1640 medium supplemented with 10 % foetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin in 25-cm2 culture flasks at 37 °C in humidified atmosphere with 5 % CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 9 g for 3 min, then resuspended in fresh medium at a cell density of 5 9 104 cells per mL. The cell suspension was dispensed into a 96-well microplate at 100 lL per well, and incubated in humidified atmosphere with 5 % CO2 at 37 °C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 lM). After 48 h of treatment, 50 lL of 1 mg/mL MTT solution was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 lL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested with doxorubicin as positive control in triplicate and the IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves.
Results and discussion Compound 1 was obtained as a white amorphous powder, and its molecular formula was deduced as C46H80O3 by HR-ESI–MS (m/z 703.6001 [M ? Na]?; calc. 703.6005), corresponding to seven unsaturation degrees. The IR of 1 showed the strong absorption for hydroxyl (3,420 cm-1) and ester carbonyl (1,730 and 1,255 cm-1) groups. The ester group in compound 1 could be further deduced as a palmitoyl moiety because of the signal at d 173.7 (C-10 ) in the downfield region of the 13C NMR spectrum, as well as a saturated long-chain features: a methyl signal at d 0.88 (t, J = 6.6 Hz, H-160 ), several methylene signals at d 1.26 (br s, H-150 to H-40 , 24H), 1.63 (m, H-30 ), and 2.26 (t, J = 6.6 Hz, H-20 ) in the 1H NMR spectrum (Table 1). The characteristic spectral data of palmitoyl moiety in 1 are in
CH3(CH2)14
H
OH
H O
H
3
agreement with those found in the literatures (Li et al. 2008). In addition to the long-chain ester group, the left 30 carbon signals (7 9 CH3, 10 9 CH2, 7 9 CH, 6 9 C) in the 13C NMR spectrum combined with DEPT experiment, implied the presence of a pentacyclic triterpene moiety (Table 2), that could be supported by seven singlets methyl signals (d 0.78, 0.82, 0.83, 0.95, 0.96, 1.05, 1.68) in the 1H NMR spectrum (Table 1). Taking into account the characteristic of 13C- and 1H-NMR and the molecular formula, compound 1 was ascribable to be a derivate of lupan triterpenoid (Chanita, & Pakakrong 2005). Comparing the NMR data of 1 with those of the known compound 4, the only significant difference was downfield shift of chemical shift of C-3 from d 78.7 in compound 4 to 81.2 in 1. Thus, the ester and C-10 (d 173.7) was linked at C-3 position, which was confirmed by the HMBC correlations between H-3 at d 4.45 and C-10 (d 173.7), C-1 (d 39.1), C-5 (d 55.0), C-23 (d 28.0), and C-24 (d 16.8) (Fig. 2). The spin system were identified as H-9/H-11-O-/H-12/H-13/H-18/H-19 in 1 H–1H COSY spectrum indicated that one OH was located in C-11, which was supported by the HMBC correlations of H-13 (d 2.03) with C-11 (d 70.4). The stereochemistry of 1 was determined by NOESY experiment. The NOESY correlations of H-3/H-1a, 23, H-5 and H-11/H-25, H-26 suggested that the ester group and the OH at C-3 and C-11 should be b- and a-oriented, respectively. Accordingly, the structure of 1 was established as 11a-hydroxy-lup-20(29)ene-3b-palmitate. Compound 2, a white amorphous powder, was assigned a molecular formula of C45H78O4 on the basis of its HRESI–MS (m/z 705.5795 ([M ? Na]?; calc. 705.5798). The molecular formula indicated the presence of a C29 triterpenoid and a palmitoyl moiety in 2. A typical carbonyl group signal at d 212.3 (C-20) and the downfield shift of signals for H-29 (d 2.12) relative to that (d 1.68) of 1 were observed. These spectral data revealed that 2 possessed a lupan-type triterpenoid skeleton similar to 29-nor20-oxolupeol (Zhou et al. 2014), except for the presence of the palmitoyl moiety and one more hydroxy group. The HMBC correlations of H-3 (d 4.40) with C-10 (d 173.5) of the palmitoyl moiety, C-1 (d 38.3), C-5 (d 55.4), C-23 (d
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Table 3 Cytotoxicity of compounds 1–6 against six human tumor cell lines (IC50, lM) Cell lines MCF7
HeLa
1
3.0
2.6
2
2.5
2.3
3
3.4
4 5 6 Doxorubicin
SGC7901
NCIH460
BGC823
2.8
2.6
2.8
2.3
2.7
2.9
2.9
2.1
3.8
3.6
3.5
3.8
3.7
23.1
21.3
19.5
20.5
21.0
20.6
30.2
25.1
26.5
30.1
26.4
29.1
32.0
30.2
31.0
32.5
35.5
35.0
0.02
0.05
HepG2
0.04
0.01
0.01
0.02
28.2), and C-24 (d 16.6), and of H-28 (d 3.60 and 3.36) with C-16 (d 32.0) and C-22 (d 40.0) indicted that the longchain ester group and one OH were located in C-3 and C-28, respectively. The NOESY correlations of H-3/H-1a, 23, H-5 suggested that the long-chain ester group at C-3 should be b-oriented. Accordingly, the structure of 2 was established as 28-hydroxy-20-oxo-30-norlupane-3bpalmitate. Compound 3, obtained as a white amorphous solid, exhibited a quasimolecular ion peak at m/z 735.5900 [M ? Na]? calc. (735.5903) in the high-resolution mass spectrometry, which corresponded to the molecular formula C46H80O5. The 1H and 13C NMR spectra of 3 showed characteristic resonances of a triterpenoid and a palmitoyl moiety (Tables 1, 2). The 13C NMR and DEPT spectra showed the triterpenoid contained seven quaternary C-atoms, seven CH (including three oxygen-bearing carbons), nine CH2 (including one oxygenated CH2, and eight sp3 ones) and seven Me (Table 2), and seven singlets methyl signals (d 0.80, 0.82, 0.85, 0.86, 0.93, 0.98, 0.99) were observed in the 1H NMR spectrum (Table 1). These results and typical resonances at d 126.5 (C-12) and 144.7 (C-13) further indicated a oleanane-type triterpenoid skeleton. The HMBC correlations between H-3 at d 4.35 and C-10 (d 173.5), C-1 (d 75.6), C-23 (d 28.7), and C-24 (d 16.3) suggested that the ester group (palmitoyl) linked at C-3 position. In 1H-1H COSY spectrum of 3, the correlations between H-1 at d 3.94 with H-2 (d 1.76 and 1.87) suggested the presence a hydroxy group at C-1, which was supported by the HMBC experiments. 1H–1H COSY spectrum of 3 implied the connectivity for H-12 to the oxygenated methane proton at d 4.60 suggested that a hydroxy group was linked to C-11, which was further confirmed by the HMBC correlations from H-11 to C-8 (d 43.2), C-10 (d 37.9), and C-13 (d 144.7). In the HMBC plot (Fig. 2), the correlations of H-28 (d 3.50 and 3.23) with C-16 (d 27.3) and C-22 (d 37.1) indicated that a OH group was located at C-28. Furthermore, In the NOESY
123
spectrum, the correlations of H-3/H-1, 23, H-5 suggested that the long-chain ester group and the OH should be boriented, and the H-11/H-25, 26 correlations indicated that the OH at C-11 should be a-oriented. Accordingly, the structure of 3 was established as 1b,11a,28-tyrihydroxy oleana-9(11), 12-dien-3b-palmitate. The cytotoxic activities of the isolated compounds were determined using the revised MTT method (Chen, Hung, Sung, Chen, and Kuo) against MCF-7 cells (human breast cancer), HeLa cells (human cervical cancer), HepG2 cells (Human hepatocellular carcinoma), SGC-7901 cells (human gastric adenocarcinoma), NCI-H460 (large cell lung carcinoma), and BGC-823 cells (human gastric carcinoma) with doxorubicin as positive control. The IC50 value was defined as the concentration of the test compound necessary to inhibit the growth to 50 % of the control in the MTT assay. The IC50 value (Table 3) showed that compound 1–3 with a palmitoyl moiety at C-3 exhibited the potent cytotoxic activities with low IC50 values (IC50 \ 4.0 lM) for all tested tumor cells while compound 4 displayed weak cytotoxic activities with higher IC50 values (19.5–23.1 lM). The other compounds had no cytotoxicities although they possess the similar triterpenoid skeleton. These results indicated that the palmitoyl moiety at C-3 which could assist in influx of excellular compounds into cell maybe enhance the cytotoxic activities. The lupane- and oleanane-type triterpenoid with a long chain fatty acid at C-3 show strong cytotoxic and antioxidant activities against various types of cancer cell lines (Chaturvedula et al. 2004; Faizi and Aneela 2004), which were agreed well with our results. Acknowledgments The above research was made possible by the grant from the Scientific Planning Project of the Applied Basic Research of Yunnan Province (S2012FZ0005), the Key Projects of Scientific Research Foundation of the Department of Education of Yunnan Province (2013Z095), and the Developing Key Subject of Ecology of Qujing Normal University.
References Chanita, P., and T. Pakakrong. 2005. Lupane-triterpene esters from the leaves of Ceriops decandra (Griff.) Ding Hou. Australian Journal of Chemistry 58: 615–618. Chaturvedula, V.S., B.N. Zhou, Z. Gao, S.J. Thomas, S.M. Hecht, and D.G. Kingston. 2004. New lupane triterpenoids from Solidago canadensis that inhibit the lyase activity of DNA polymerase beta. Bioorganic and Medicinal Chemistry 12: 6271–6275. Chhabra, B.R., S. Gupta, M. Jain, and P.S. Kalsi. 1998. Sesquiterpene lactones from Saussurea lappa. Phytochemistry 49: 801–804. Chen, J.J., H.C. Hung, P.J. Sung, I.S. Chen, and W.L. Kuo. 2011. Aporphine alkaloids and cytotoxic lignans from the roots of Illigera luzonensis. Phytochemistry 72: 523–532. Dai, J., C.Y. Zhao, Q. Zhang, Z.L. Liu, R.L. Zheng, and L. Yang. 2001. Taraxastane-type triterpenoids from Saussurea petrovii. Phytochemistry 58: 1107–1111.
Cytotoxic triterpenoids from Saussurea phyllocephala De la Torre, M.C., M. Bruno, F. Piozzi, G. Savona, B. Rodriguez, and N. Apostolides Arnold. 1990. Terpenoids from Salvia willeana and S. virgata. Phytochemistry 29: 668–670. Duan, H.Q., Y. Takaishi, H. Momota, Y. Ohmoto, and T. Taki. 2002. Immunosuppressive constituents from Saussurea medusa. Phytochemistry 59: 85–90. Faizi, S., and Naz Aneela. 2004. Palmitoleate (_(9Z)-hexadeca-9enoate) esters of oleanane triterpenoids from the golden flowers of Tagetes erecta: isolation and autoxidation products. Helvetica Chimica Acta 87: 46–56. Gao, M., B.Z. Song, and C.Z. Liu. 2006. Dynamic microwaveassisted extraction of flavonoids from Saussurea medusa Maxim cultured cells. Biochemical Engineering Journal 32: 79–83. Li, X.H., J.T. Feng, and Y.P. Shi. 2008. Triterpenoids from Saussurea ussuriensis. Canadian Journal of Chemistry 86: 281–284. Li, X.W., Z.T. Guo, Y. Zhao, Z. Zhao, and J.F. Hu. 2010. Chemical constituents from Saussurea cordifolia. Phytochemistry 71: 682–687.
1521 Reyes, C.P., M.J. Nunez, I.A. Jimenez, J. Busserolles, M.J. Alcaraz, and I.L. Bazzocchi. 2006. Activity of lupane triterpenoids from Maytenus species as inhibitors of nitric oxide and prostaglandin E2. Bioorganic and Medicinal Chemistry 14: 1573–1579. Robinson, A., T.V. Kumar, E. Sreedhar, V.G. Naidu, S.R. Krishna, K.S. Babu, P.V. Srinivas, and J.M. Rao. 2008. A new sesquiterpene lactone from the roots of Saussurea lappa: structure– anticancer activity study. Bioorganic & Medicinal Chemistry Letters 18: 4015–4017. The Editorial Board of Chinese Materia Medica. 1999. Chinese materia medica, vol. 7. Shanghai: Shanghai Science and Technology Publish House. Wang, H.B., J.P. Zuo, and G.W. Qin. 2010. One new sesquiterpene from Saussurea laniceps. Fitoterapia 81: 937–939. Zhou, J., C.J. Li, J.Z. Yang, J. Ma, Y. Li, X.Q. Bao, X.G. Chen, D. Zhang, and D.M. Zhang. 2014. Lupane triterpenoids from the Stems of Euonymus carnosus. Journal of Natural Products 77: 276–284.
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RESEARCH ARTICLE
Cytotoxic triterpenoids from Saussurea phyllocephala Jiang Hu • Yan Song • Benshou Yang • Xiang Zuo • Xiao Mao • Xiaodong Shi
Received: 30 March 2014 / Accepted: 17 June 2014 / Published online: 1 July 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Three new triterpenoids, 11a-hydroxy-lup20(29)-ene-3b-palmitate (1), 28-hydroxy-20-oxo-30-norlupane-3b-palmitate (2), and 1b,11a,28-tyrihydroxy oleana-9(11),12-dien-3b-palmitate (3), together with three known compounds were isolated from the 95 % EtOH extract of Saussurea phyllocephala. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR (COSY, HMQC, HMBC and NOESY) analyses. All the triterpenoids were in vitro evaluated for their cytotoxic activities. Compound 1–3 exhibited the significant cytotoxic activities with low IC50 values (IC50 \ 4.0 lM) against six tumor cell lines (MCF-7, HeLa, HepG2, SGC7901, NCI-H460 and BGC-823).
Jiang Hu and Yan Song have contributed equally to this work. J. Hu (&) X. Mao X. Shi (&) College of Biological Resources and Environment Science, Qujing Normal University, Sanjiang Avenue, Unicorn District, Qujing 655011, Yunnan Province, China e-mail: [email protected] X. Shi e-mail: [email protected] J. Hu Institute of Characteristic Medicinal Resource of Ethnic Minorities, Qujing Normal University, Qujing 655011, China Y. Song X. Zuo Department of Pharmacy, 455 Hospital of People’s Liberation Army, Shanghai 200052, China B. Yang Institute of Microbiology, Qujing Medical College, Qujing 655000, Yunnan Province, China
Keywords Saussurea phyllocephala Compositae Triterpenoids Cytotoxic activity
Introduction The genus Saussurea consists of about 400 species, which range widely from Asia to Europe (Li et al. 2008). About 40 species have been investigated on their chemical constituents, and sesquiterpenes (Wang et al. 2010; Robinson et al. 2008; Chhabra et al. 1998), triterpenoids (Dai et al. 2001), flavonoids (Gao et al. 2006), lignans (Duan et al. 2002), and phenolic compounds (Li et al. 2010) have been reported as the most widespread constituents. Saussurea phyllocephala Coll. et Hemsl. is distributed at the altitude of about 1,200 m above sea level in the southwestern of China. It has been used as a traditional Chinese medicine for the treatment of various disease such as hepatitis, jaundice, hyperhidrosis, lung-heat, cough, vertigo and venomous snake bite (The Editorial Board of Chinese Materia Medica 1999). The present study on the chemical constituents of the 95 % EtOH extract of S. phyllocephala from Yuanjiang, Yunnan province led to the isolation of three new lupane-type triterpenoids, 1ahydroxy-lup-20(29)-ene-3b-palmitate (1), 28-hydroxy-20oxo-30-norlupane-3b-palmitate (2), and 1b,11a,28-tyrihydroxy oleana-9(11),12-dien-3b-palmitate (3), as well as three known compounds 3b,11a-dihydroxy-lup-20(29)-ene (4), 1b,3b,11a,28-tetrahydroxy-oleana-12-ene (5), and 3b,11a-dihydroxy-ursa-12-ene (6) (Reyes et al. 2006; De la Torre et al. 1990) (Fig. 1). This paper deals with the isolation and structure elucidation of three new triterpenoids by spectral methods and all the triterpenoids were in vitro evaluated for their cytotoxic activities against six tumor cell lines (MCF-7, HeLa, HepG2, SGC-7901, NCI-H460 and BGC-823).
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1516
J. Hu et al.
Fig. 1 The structures of compounds 1–6
30
O
20
19 21
29
HO
11
25 1
26
10 4
RO
H
5
18
22
13 17 14 H
28
H H O
16
15
8 7
CH3(CH2)14
O
H
24
1 R=CO(CH2)14CH3 4 R=H 29
19
HO OH 25 1
11
26
10 4
RO
5
H 23
12
9
2 3
H 6
2
30
21
20 18
13
OH
14H
8
HO
22 17
15
H
28 16
H
27
HO
7
H
24
3 R=CO(CH2)14CH3 5 R=H
Materials and methods General experimental procedures Melting points were determined on a XT-4 microscopic thermometer without correction. Optical rotations were taken on a Perkin-Elmer 341 polarimeter. IR spectra were recorded on Nicolet Magna FT-IR 750 spectrophotometer using KBr disks. NMR spectra were recorded on Bruker AM-300, AM-400, and INVOR-600 NMR spectrometers. The chemical shift (d) values are given in ppm with TMS as internal standard, and coupling constants (J) are in Hz. EIMS and HREIMS spectra were recorded on a Finnigan MAT-95 mass spectrometer. ESIMS and HRESIMS spectra were recorded on a Micromass LC–MS–MS mass spectrometer. Column chromatographic separations were carried out using silica gel (200–300 mesh and H60, QingdaoHaiyang Chemical Group Corporation, P. R. China), MCI gel CHP20P (75–150 lm, Mitsubishi Chemical Industries, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) as packing material. TLC was carried out on precoated silica gel GF254 plates (Yantai Chemical Industrials), and the TLC spots were viewed at 254 nm and visualized using 5 % sulfuric acid in
123
OH
H
27
6
H 23
12
9
2 3
H
6
alcohol containing 10 mg/mL vanillin. Analytical HPLC was performed on a Waters 2690 instrument with a 996 PAD (photodiode array detector) coupled with an Alltech ELSD 2000 detector. Semipreparative and preparative HPLC was performed on a Varian SD1 instrument with a 320 single-wave detector. Their chromatographic separations were carried out on C-18 columns (250 9 10 mm, 5 lm, Waters; 220 9 25 mm, 10 lm, Merck, respectively), using a gradient solvent system comprised of H2O and MeCN, with a flow rate of 3.0 and 15.0 mL/min, respectively. All cell lines were purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Other reagents were purchased from Shanghai Sangon Biological Engineering Technology and Services CO., Lt. Plant materials The air-dried whole plant of S. phyllocephala was collected in the Yuanjiang, Yunnan province, China, in July 2011. A specimen (SP20110701), identified by one of the authors (X. Mao), was deposited in the Herbarium of the College of Biological Resources and Environment Science, Qujing Normal University, Qujing, China.
Cytotoxic triterpenoids from Saussurea phyllocephala
1517
Table 1 1H-NMR spectral data of compounds 1–3 (1H: 500 MHz in CDCl3) Position
1
H-NMR (multi., J Hz)
1
2
3
H–C(1)ax
0.85 (ddd, 14.0, 13.6, 4.0)
0.82 (ddd, 13.8, 13.6, 3.6)
3.94 (dd, 13.8,3.8)
H–C(1)eq
1.70 (overlapped)
1.65 (ddd, 13.6, 4.0, 3.4)
–
H–C(2)ax
1.37 (m)
1.33 (m)
1.76 (m)
H–C(2)eq
1.62(m)
1.56 (overlapped)
1.87 (m)
H–C(3)
4.45 (dd, 13.6, 4.0)
4.40 (dd, 13.5, 4.0)
4.35 (dd, 13.6,3.8)
H–C(5)
1.32 (dd, 13.6, 3.8)
1.26 (dd, 13.8, 3.6)
0.82 (dd, 13.6,3.8)
H–C(6)ax H–C(6) eq
1.98 (overlapped) 2.14 (m)
2.00 (overlapped) 2.08 (overlapped)
1.40 (m) 1.53 (m)
H–C(7)ax
1.33 (ddd, 13.8, 13.6, 4.0)
1.26 (ddd, 13.8, 13.6, 4.0)
1.27 (overlapped)
H–C(7)eq
1.58 (overlapped)
1.50 (ddd, 13.6, 3.8, 3.6)
1.51 (ddd, 13.6, 4.0, 3.6)
H–C(9)
2.03 (d, 13.2)
1.88 (d, 13.2)
1.81 (d, 13.6)
H–C(11)ax
3.12 (m)
1.35 (m)
4.60 (dd, 13.6, 7.6)
H–C(11)eq
–
1.82 (m)
–
H–C(12)ax
1.55 (overlapped)
1.45 (m)
5.30 (d, 7.6)
H–C(12)eq
2.10 (m)
2.00 (overlapped)
–
H–C(13)
2.03 (ddd, 13.6, 13.2, 3.6)
1.92 (overlapped)
–
H–C(15)ax
1.74 (ddd, 13.8, 13.6, 4.0)
1.68 (overlapped)
1.81 (overlapped)
H–C(15)eq
1.98 (overlapped)
1.90 (ddd, 13.2, 4.0, 3.4)
2.00 (overlapped)
H–C(16)ax
1.70 (overlapped)
1.70 (ddd, 13.6, 13.0, 4.0)
1.81 (overlapped) 2.00 (overlapped)
H–C(16)eq
2.08 (overlapped)
2.12 (ddd, 13.0, 3.8, 3.4)
H–C(18)
1.92 (overlapped)
1.83 (dd, 13.6, 13.2)
1.36 (dd, 13.6,3.8)
H–C(19) H–C(21)ax
2.38 (m) 2.08 (overlapped)
2.60 (m) 2.08 (overlapped)
1.15 (m) 1.25 (overlapped)
H–C(21)eq
1.72 (overlapped)
1.68 (overlapped)
1.40 (ddd, 13.6,4.0,3.8)
H–C(22)ax
1.92 (d overlapped)
1.92 (overlapped)
1.25 (overlapped)
H–C(22)eq
1.58 (overlapped)
1.56 (overlapped)
1.44 (ddd, 13.8,4.0,3.8)
H–C(23)
0.95 (s)
0.94 (s)
0.85 (s)
H–C(24)
0.82 (s)
0.82 (s)
0.86 (s)
H–C(25)
0.83 (s)
0.88 (s)
0.93 (s)
H–C(26)
1.05 (s)
1.02 (s)
0.80 (s)
H–C(27)
0.96 (s))
0.97 (s)
0.99 (s)
H–C(28)a
0.78 (s)
3.60 (d, 12.8)
3.50 (d, 12.8)
H–C(28)b
–
3.36 (d, 12.8)
3.23 (d, 12.8)
H–C(29)
1.68 (s)
2.12 (s)
0.82 (s)
H–C(30)
4.55 (d, 2.6)
–
0.98 (s)
4.68 (d, 2.6)
–
–
H (20 )
2.26 (t, 6.6)
2.30 (t, 6.6)
2.32 (t, 6.6)
H (30 ) H(40 -150 )
1.63 (m) 1.26 (brs)
1.61 (m) 1.28 (brs)
1.69 (m) 1.27 (overlapped)
H(160 )
0.88 (t, 6.6)
0.86 (t, 6.6)
0.87 (t, 6.6)
Extraction and isolation The air-dried whole plant of S. phyllocephala (2.3 kg) was ground into powder and extracted thrice with 95 % EtOH.
After evaporation of the EtOH, the crude extract (258 g) was partitioned between water and EtOAc. The EtOAcsoluble portion (91 g) was chromatographed on a silica gel column eluted with CHCl3/MeOH (from 100:1 to 1:1) to
123
1518 Table 2 13C-NMR spectral data of compounds 1–3 (13C: 125 MHz, in CDCl 3)
J. Hu et al.
Position
Position 2
3
C-NMR
1
2
3
C(1)
39.1 (t)
38.3 (t)
75.6 (d)
C(24)
16.8 (q)
16.6 (q)
16.3 (q)
25.0 (t)
24.6 (t)
34.7 (t)
C(25)
16.1 (q)
16.0 (q)
18.5 (q)
C(3)
81.2 (d)
81.5 (d)
80.9 (d)
C(26)
16.2 (q)
15.9 (q)
20.3 (q)
C(4)
38.2 (s)
37.8 (s)
38.0 (s)
C(27)
14.8 (q)
14.6 (q)
33.7 (q)
C(5)
55.0 (d)
55.4 (d)
56.3 (d)
C(28)
18.1 (q)
28.2 (t)
27.8 (t)
C(6)
18.1 (t)
18.2 (t)
18.4 (t)
C(29)
19.4 (q)
23.6 (q)
33.2 (q)
C(7)
34.2 (t)
34.1 (t)
31.0 (t)
C(30)
109.5 (t)
–
C(8)
41.1 (s)
40.8 (s)
43.2 (s)
C(10 )
173.7 (s)
173.5 (s)
C(9)
55.6 (d)
50.2 (d)
52.3 (d)
C(20 )
34.9 (t)
34.5 (t)
C(10)
37.4 (s)
37.1 (s)
37.9 (s)
C(30 )
25.2 (t)
25.1 (t)
21.6 (q) 173.5 (s) 34.6 (t) 25.1 (t)
C(11) C(12)
70.4 (d) 27.7 (t)
21.0 (t) 25.1 (t)
67.2 (d) 126.5 (d)
C(4 ) C(50 )
29.71 (t) 29.70 (t)a
29.65 (t) 29.64 (t)b
29.65 (t)c 29.64 (t)c
C(13)
37.4 (d)
37.1 (d)
144.7 (s)
C(60 )
29.68 (t)a
29.63 (t)b
29.63 (t)c
29.62 (t)
b
29.62 (t)c
29.61 (t)
b
29.61 (t)c
29.60 (t)b
29.60 (t)c
43.5 (s)
42.3 (s)
42.9 (s)
0
0
C(7 )
a
b
29.67, t a) a
C(15)
27.3 (t)
29.5 (t)
25.8 (t)
C(80 )
29.65 (t)
C(16)
35.7 (t)
32.0 (t)
27.3 (t)
C(90 )
29.61 (t)a
32.4 (s)
0
C(18) C(19)
47.1 (s) 48.4 (d) 48.1 (d)
55.8 (s) 49.0 (d) 47.0 (d)
45.6 (d) 46.8 (t)
C(10 ) 0
C(11 ) 0
C(12 )
29.5 (t)
a
29.4 (t)
a
29.3 (t)
a a
29.5 (t)
b
29.5 (t)c
29.4 (t)
b
29.4 (t)c
29.3 (t)
b
29.3 (t)c
29.2 (t)
b
29.2 (t)b
C(20)
151.0 (s)
212.3 (s)
31.1 (s)
C(130 )
29.2 (t)
C(21)
29.8 (t)
29.6 (t)
34.7 (t)
C(140 )
31.1 (t)
31.2, t
C(22)
40.1 (t)
40.0 (t)
37.1 (t)
C(150 )
22.7 (t)
22.9, t
22.7 (t)
C(23)
28.0 (q)
28.2 (q)
28.7 (q)
C(160 )
14.1 (q)
14.2 (q)
13.2 (q)
afford fractions 1–8. Fraction 2 (7.2 g) was chromatographed on a MCI gel column eluted with MeOH/H2O (from 70 to 95 %) to yield four subfractions, 2A–2D. Subfraction 2B (1.1 g) was separated by repeated column chromatography (CC) over Sephadex LH-20 (CHCl3/ MeOH, 1:1, and MeOH) and silica gel yielding 1 (29.7 mg) and 3 (31.2 mg). Subfraction 2C (245 mg) was chromatographed on a Sephadex LH-20 column (CHCl3/MeOH, 1:1) and preparative HPLC (MeCN/H2O, from 45 to 70 %), yielding 2 (33.2 mg) and 6 (37.5 mg). Fraction 3 (6.9 g) was subjected to silica gel column chromatography eluted with CHCl3/MeOH (from 9:1 to 1:4) and then purified by Sephadex LH-20 (CHCl3/MeOH, 1:1) CC to afford 4 (55.4 mg) and 5 (48.4 mg). 11a-Hydroxy-lup-20(29)-ene-3b-palmitate (1) White amorphous powder; [a]23.3 ? 8.7 (c = 0.023, D MeOH); UV (MeOH) kmax(log e) 216 (3.78) nm; IR (KBr) mmax 3,420, 2,937, 1,730, 1,641, 1,455, 1,255, 1,040, 860 cm-1; 1H and 13C NMR data are showed Tables 1 and 2; FAB-MS (postive): m/z 681 [M ? H]?; HR-ESI–MS: m/z 703.6001 ([M ? Na]?; C46H80O3Na; calc. 703.6005).
123
13
C(2)
C(17)
Assignments may be interchangeable
C-NMR
1
C(14)
a,b,c
13
31.2 (t)
28-Hydroxy-20-oxo-30-norlupane-3b-palmitate (2) ? 17.3 (c = 0.090, White amorphous powder; [a]23.3 D MeOH); UVkmax(log e) 225 (3.46) nm; IR (KBr) mmax 3,425, 2,925, 1,730, 1,645, 1,460, 1,253, 1,015, 940 cm-1; 1 H and 13C NMR data are showed Tables 1 and 2; FABMS (postive): m/z 683 [M ? H]?; HR-ESI–MS: m/z 705.5795 ([M ? Na]?; C45H78O4Na; calc. 705.5798). 1b,11a,28-Tyrihydroxy oleana-9(11), 12-dien-3b-palmitate (3) White amorphous powder; [a]23.3 ? 43.7 (c = 0.036, D MeOH); UV kmax(log e) 282 (2.15) nm; IR (KBr) mmax 3,415, 2,968, 1,730, 1,630, 1,465, 1,370, 1,250, 1,080; 1H and 13C NMR data are showed Tables 1 and 2; FAB-MS (postive): m/z 713 [M ? H]?; HR-ESI–MS: m/z 735.5900 ([M ? Na]?; C46H80O5Na; calc. 735.5903). Cytotoxicity assay in vitro The revised MTT method (Chen et al. 2011) was used for in vitro evaluation of the cytotoxic potential of the isolated
Cytotoxic triterpenoids from Saussurea phyllocephala
1519
Fig. 2 The key HMBC correlations of compounds 1 and 3
H
HO
HO OH
H O CH3(CH2)14
O
H O
H
1
compounds against eight cultured human tumor cell lines. All tumor cell lines (A549, BGC-823, HCT15, HeLa, HepG2, MCF-7, SK-MEL-2, and SGC-7901 cells) were cultured on RPMI-1640 medium supplemented with 10 % foetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin in 25-cm2 culture flasks at 37 °C in humidified atmosphere with 5 % CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 9 g for 3 min, then resuspended in fresh medium at a cell density of 5 9 104 cells per mL. The cell suspension was dispensed into a 96-well microplate at 100 lL per well, and incubated in humidified atmosphere with 5 % CO2 at 37 °C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 lM). After 48 h of treatment, 50 lL of 1 mg/mL MTT solution was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 lL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested with doxorubicin as positive control in triplicate and the IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves.
Results and discussion Compound 1 was obtained as a white amorphous powder, and its molecular formula was deduced as C46H80O3 by HR-ESI–MS (m/z 703.6001 [M ? Na]?; calc. 703.6005), corresponding to seven unsaturation degrees. The IR of 1 showed the strong absorption for hydroxyl (3,420 cm-1) and ester carbonyl (1,730 and 1,255 cm-1) groups. The ester group in compound 1 could be further deduced as a palmitoyl moiety because of the signal at d 173.7 (C-10 ) in the downfield region of the 13C NMR spectrum, as well as a saturated long-chain features: a methyl signal at d 0.88 (t, J = 6.6 Hz, H-160 ), several methylene signals at d 1.26 (br s, H-150 to H-40 , 24H), 1.63 (m, H-30 ), and 2.26 (t, J = 6.6 Hz, H-20 ) in the 1H NMR spectrum (Table 1). The characteristic spectral data of palmitoyl moiety in 1 are in
CH3(CH2)14
H
OH
H O
H
3
agreement with those found in the literatures (Li et al. 2008). In addition to the long-chain ester group, the left 30 carbon signals (7 9 CH3, 10 9 CH2, 7 9 CH, 6 9 C) in the 13C NMR spectrum combined with DEPT experiment, implied the presence of a pentacyclic triterpene moiety (Table 2), that could be supported by seven singlets methyl signals (d 0.78, 0.82, 0.83, 0.95, 0.96, 1.05, 1.68) in the 1H NMR spectrum (Table 1). Taking into account the characteristic of 13C- and 1H-NMR and the molecular formula, compound 1 was ascribable to be a derivate of lupan triterpenoid (Chanita, & Pakakrong 2005). Comparing the NMR data of 1 with those of the known compound 4, the only significant difference was downfield shift of chemical shift of C-3 from d 78.7 in compound 4 to 81.2 in 1. Thus, the ester and C-10 (d 173.7) was linked at C-3 position, which was confirmed by the HMBC correlations between H-3 at d 4.45 and C-10 (d 173.7), C-1 (d 39.1), C-5 (d 55.0), C-23 (d 28.0), and C-24 (d 16.8) (Fig. 2). The spin system were identified as H-9/H-11-O-/H-12/H-13/H-18/H-19 in 1 H–1H COSY spectrum indicated that one OH was located in C-11, which was supported by the HMBC correlations of H-13 (d 2.03) with C-11 (d 70.4). The stereochemistry of 1 was determined by NOESY experiment. The NOESY correlations of H-3/H-1a, 23, H-5 and H-11/H-25, H-26 suggested that the ester group and the OH at C-3 and C-11 should be b- and a-oriented, respectively. Accordingly, the structure of 1 was established as 11a-hydroxy-lup-20(29)ene-3b-palmitate. Compound 2, a white amorphous powder, was assigned a molecular formula of C45H78O4 on the basis of its HRESI–MS (m/z 705.5795 ([M ? Na]?; calc. 705.5798). The molecular formula indicated the presence of a C29 triterpenoid and a palmitoyl moiety in 2. A typical carbonyl group signal at d 212.3 (C-20) and the downfield shift of signals for H-29 (d 2.12) relative to that (d 1.68) of 1 were observed. These spectral data revealed that 2 possessed a lupan-type triterpenoid skeleton similar to 29-nor20-oxolupeol (Zhou et al. 2014), except for the presence of the palmitoyl moiety and one more hydroxy group. The HMBC correlations of H-3 (d 4.40) with C-10 (d 173.5) of the palmitoyl moiety, C-1 (d 38.3), C-5 (d 55.4), C-23 (d
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J. Hu et al.
Table 3 Cytotoxicity of compounds 1–6 against six human tumor cell lines (IC50, lM) Cell lines MCF7
HeLa
1
3.0
2.6
2
2.5
2.3
3
3.4
4 5 6 Doxorubicin
SGC7901
NCIH460
BGC823
2.8
2.6
2.8
2.3
2.7
2.9
2.9
2.1
3.8
3.6
3.5
3.8
3.7
23.1
21.3
19.5
20.5
21.0
20.6
30.2
25.1
26.5
30.1
26.4
29.1
32.0
30.2
31.0
32.5
35.5
35.0
0.02
0.05
HepG2
0.04
0.01
0.01
0.02
28.2), and C-24 (d 16.6), and of H-28 (d 3.60 and 3.36) with C-16 (d 32.0) and C-22 (d 40.0) indicted that the longchain ester group and one OH were located in C-3 and C-28, respectively. The NOESY correlations of H-3/H-1a, 23, H-5 suggested that the long-chain ester group at C-3 should be b-oriented. Accordingly, the structure of 2 was established as 28-hydroxy-20-oxo-30-norlupane-3bpalmitate. Compound 3, obtained as a white amorphous solid, exhibited a quasimolecular ion peak at m/z 735.5900 [M ? Na]? calc. (735.5903) in the high-resolution mass spectrometry, which corresponded to the molecular formula C46H80O5. The 1H and 13C NMR spectra of 3 showed characteristic resonances of a triterpenoid and a palmitoyl moiety (Tables 1, 2). The 13C NMR and DEPT spectra showed the triterpenoid contained seven quaternary C-atoms, seven CH (including three oxygen-bearing carbons), nine CH2 (including one oxygenated CH2, and eight sp3 ones) and seven Me (Table 2), and seven singlets methyl signals (d 0.80, 0.82, 0.85, 0.86, 0.93, 0.98, 0.99) were observed in the 1H NMR spectrum (Table 1). These results and typical resonances at d 126.5 (C-12) and 144.7 (C-13) further indicated a oleanane-type triterpenoid skeleton. The HMBC correlations between H-3 at d 4.35 and C-10 (d 173.5), C-1 (d 75.6), C-23 (d 28.7), and C-24 (d 16.3) suggested that the ester group (palmitoyl) linked at C-3 position. In 1H-1H COSY spectrum of 3, the correlations between H-1 at d 3.94 with H-2 (d 1.76 and 1.87) suggested the presence a hydroxy group at C-1, which was supported by the HMBC experiments. 1H–1H COSY spectrum of 3 implied the connectivity for H-12 to the oxygenated methane proton at d 4.60 suggested that a hydroxy group was linked to C-11, which was further confirmed by the HMBC correlations from H-11 to C-8 (d 43.2), C-10 (d 37.9), and C-13 (d 144.7). In the HMBC plot (Fig. 2), the correlations of H-28 (d 3.50 and 3.23) with C-16 (d 27.3) and C-22 (d 37.1) indicated that a OH group was located at C-28. Furthermore, In the NOESY
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spectrum, the correlations of H-3/H-1, 23, H-5 suggested that the long-chain ester group and the OH should be boriented, and the H-11/H-25, 26 correlations indicated that the OH at C-11 should be a-oriented. Accordingly, the structure of 3 was established as 1b,11a,28-tyrihydroxy oleana-9(11), 12-dien-3b-palmitate. The cytotoxic activities of the isolated compounds were determined using the revised MTT method (Chen, Hung, Sung, Chen, and Kuo) against MCF-7 cells (human breast cancer), HeLa cells (human cervical cancer), HepG2 cells (Human hepatocellular carcinoma), SGC-7901 cells (human gastric adenocarcinoma), NCI-H460 (large cell lung carcinoma), and BGC-823 cells (human gastric carcinoma) with doxorubicin as positive control. The IC50 value was defined as the concentration of the test compound necessary to inhibit the growth to 50 % of the control in the MTT assay. The IC50 value (Table 3) showed that compound 1–3 with a palmitoyl moiety at C-3 exhibited the potent cytotoxic activities with low IC50 values (IC50 \ 4.0 lM) for all tested tumor cells while compound 4 displayed weak cytotoxic activities with higher IC50 values (19.5–23.1 lM). The other compounds had no cytotoxicities although they possess the similar triterpenoid skeleton. These results indicated that the palmitoyl moiety at C-3 which could assist in influx of excellular compounds into cell maybe enhance the cytotoxic activities. The lupane- and oleanane-type triterpenoid with a long chain fatty acid at C-3 show strong cytotoxic and antioxidant activities against various types of cancer cell lines (Chaturvedula et al. 2004; Faizi and Aneela 2004), which were agreed well with our results. Acknowledgments The above research was made possible by the grant from the Scientific Planning Project of the Applied Basic Research of Yunnan Province (S2012FZ0005), the Key Projects of Scientific Research Foundation of the Department of Education of Yunnan Province (2013Z095), and the Developing Key Subject of Ecology of Qujing Normal University.
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