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Four new triterpenoids isolated from the resin of Garcinia hanburyi a
a
a
a
bc
a
Hong-Min Wang , Qun-Fang Liu , Yi-Wu Zhao , Shuang-Zhu Liu , bc
bc
Zhen-Hua Chen , Ru-Jun Zhang , Zhen-Zhong Wang , Wei Xiao Wei-Min Zhao
&
a
a
Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China b
Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, 222001, China c
State Key Laboratory of Pharmaceutical Process New-Tech for Chinese Medicine, Lianyungang, 222001, China Published online: 07 Jan 2014.
To cite this article: Hong-Min Wang, Qun-Fang Liu, Yi-Wu Zhao, Shuang-Zhu Liu, Zhen-Hua Chen, Ru-Jun Zhang, Zhen-Zhong Wang, Wei Xiao & Wei-Min Zhao (2014) Four new triterpenoids isolated from the resin of Garcinia hanburyi, Journal of Asian Natural Products Research, 16:1, 20-28, DOI: 10.1080/10286020.2013.875012 To link to this article: http://dx.doi.org/10.1080/10286020.2013.875012
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 1, 20–28, http://dx.doi.org/10.1080/10286020.2013.875012
Four new triterpenoids isolated from the resin of Garcinia hanburyi Hong-Min Wanga, Qun-Fang Liua, Yi-Wu Zhaob,c, Shuang-Zhu Liua, Zhen-Hua Chena, Ru-Jun Zhanga, Zhen-Zhong Wangb,c, Wei Xiaob,c* and Wei-Min Zhaoa*
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a
Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; bJiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang 222001, China; cState Key Laboratory of Pharmaceutical Process New-Tech for Chinese Medicine, Lianyungang 222001, China (Received 7 October 2013; final version received 10 December 2013) Four new triterpenoids, 2-O-acetyl-3-O-(40 -O-acetyl)-a-L -arabinopyranosylmaslinic acid (1), 2-O-acetyl-3-O-(30 -O-acetyl)-a-L -arabinopyranosylmaslinic acid (2), 2-Oacetyl-3-O-(30 ,40 -O-diacetyl)-a-L -arabinopyranosylmaslinic acid (3), and 3-O-(30 -Oacetyl)-a-L -arabinopyranosyloleanolic acid (4), together with six known triterpenoids, 3-O-(40 -O-acetyl)-a-L -arabinopyranosyloleanolic acid (5), maslinic acid (6), 2-Oacetylmaslinic acid (7), 3-O-acetylmaslinic acid (8), betulinic acid (9), and 2ahydroxy-3b-O-acetylbetulinic acid (10), were isolated from the EtOAc extract of Garcinia hanburyi resin. Their structures were elucidated by analysis of the spectroscopic data and chemical methods. Keywords: Garcinia hanburyi; Guttiferae; triterpenoid; saponin
1.
Introduction
Gamboge, the pulverized golden color resin collected primarily from Garcinia hanburyi (Guttiferae), and to a lesser extent from G. Morella, has a particularly long and rich history in the arts and sciences [1–4]. It has been used as dye and folk medicine for the treatment of infected wound and tumors [1–4]. Previous phytochemical investigations of gamboge have mainly resulted in the identification of caged polyprenylated xanthones [1–8]. In this study, 10 triterpenoids including four new triterpenoids (1–4) and six known ones (5–10) were isolated from the resin of G. hanburyi (Figure 1). The structures of these compounds were elucidated by spectroscopic analysis and chemical methods. 2. Results and discussion Compound 1 was obtained as white amorphous powder. Its molecular formula
C39H60O10 was established by HR-TOFMS (m/z 711.4089 [M þ Na]þ), implying 10 degrees of unsaturation. Its 1H (Table 1) spectrum indicated the presence of one triterpene aglycone [dH 1.10 (s, 3H), 1.08 (s, 3H); 1.03 (s, 3H); 0.92 (s, 3H); 0.91 (s, 3H); 0.90 (s, 3H); 0.73 (s, 3H)], one sugar moiety (dH 3.51 –4.33), and two acetyl methyl groups [dH 2.11 (s, 3H), 1.99 (s, 3H)]. The 13C NMR (Table 1) spectrum of 1 exhibited 39 carbon signals, of which nine carbon signals were consistent with the presence of an arabinopyranosyl moiety [dC 104.5 (d), 72.6 (d), 71.5 (d), 70.4 (d), 63.5 (t)] [9 – 12] and two acetyl methyl groups [dC 171.1 (s), 171.1 (s), 21.6 (q), 21.2 (q)]. The remaining 30 carbon signals strongly resembled those of maslinic acid, except for the downfield shift of C-3 (from dC 83.5 to 89.2), due to the glycosylation of 3-hydroxy group in 1. Moreover, the HMBC correlations from H-10 (dH 4.33) to C-3 confirmed the
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
Journal of Asian Natural Products Research
21
29 20 12
R1 R2 O O 3
4'
1'
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R3
OH
1 1a 2 3 4 4a 5
H
1 10
COOH
H
R1 H
COOH
H
27
R2
H 23
H
6 R1 = R2 = OH 7 R1 = OAc, R2 = OH 8 R1 = OH, R2 = OAc
R1 = R2 = OAc, R3 = OH R1 = R2 = R3 = OH R1 = R3 = OAc, R2 = OH R1 = R2 = R3 = OAc R1 = H, R2 = OH, R3 = OAc R1 = H, R2 = R3 = OH R1 = H, R2 = OAc, R3 = OH
H H
R1
COOH
H R2
H 9 R1 = H, R2 = OH 10 R1 = OH, R2 = OAc
Figure 1. Structures of compounds 1 –10, 1a, and 4a.
connection between the maslinic acid moiety and the arabinopyranosyl moiety (Figure 2). Furthermore, the locations of two acetyl groups at 2- and 40 -hydroxy groups were established based on the HMBC correlations from H-2 (dH 5.14) to an acetyl carbonyl carbon (dC 171.1), and from H-40 (dH 5.08) to the other acetyl carbonyl carbon (dC 171.1) (Figure 2). The arabinopyranosyl unit in 1 was determined to connect to the aglycone in a-configuration in 4C1 form on the basis of the 3 0 0 JH-1,H-2 value (6.4 Hz) and the NOE correlations between H-10 and H-30 and between H-10 and H-50 in the NOESY experiment (Figure 2) [9 – 12]. The absolute configuration of the arabinopyranosyl unit in 1 was found to belong to L series by GC-MS analysis of its peracetylated thiazolidine derivative [13,14]. Hydrolysis of 1 with 10% KOH yielded 1a, which was further hydrolyzed with acid to give maslinic acid and arabinose identified by
comparison of their spectral data with those in the literature and co-TLC [15,16]. Derivation of the arabinose hydrolysate from 1 with L -cysteine methyl ester gave a GC-MS peak at 16.09 min, same as that of a standard L -arabinose derivative. Accordingly, the structure of 1 was established as 2-O-acetyl-3-O-(40 -O-acetyl)-a-L -arabinopyranosylmaslinic acid. Compound 2, a white amorphous powder, possessed the same molecular formula as 1, based on HR-TOF-MS (m/z 711.4090 [M þ Na]þ). Its 1H and 13C NMR data (Table 1) were very similar to those of 1 and displayed signals belonging to one maslinic acid moiety, one arabinopyranosyl unit, and two acetyl groups. The HMBC correlations from H-10 (dH 4.89) to C-3 (dC 89.9) established the connection between the maslinic acid moiety and the arabinopyranosyl moiety (Figure 2). Assignment of two O-acetyl groups at C2 and C-30 was evidenced from the HMBC
20 21
17 18 19
16
12 13 14 15
8 9 10 11
7
2 3 4 5 6
30.8 (s) 33.8 (t)
46.6 (s) 41.0 (d) 45.9 (t)
22.8 (t)
122.4 (d) 144.8 (s) 41.6 (s) 27.7 (t)
39.3 (s) 47.7 (d) 38.4 (s) 23.5 (t)
32.5 (t)
69.7 (d) 89.2 (d) 41.6 (s) 55.0 (d) 18.1 (t)
2.04 (m) 1.19 (m) 5.58 (m) 3.53 (d, 9.7) – 0.95 (m) 1.52 (m) 1.29 (m) 1.49 (m) 0.98 (m) – 1.75 (m) – 2.00 (m) 1.02 (m) 5.49 (br s) – – 2.18 (m) 1.21 (m) 2.16 (m) 1.31 (m) – 3.33 (dd, 12.3, 3.0) 1.83 (m) 1.33 (m) – 1.48 (m) 1.27 (m)
1.90 (m) 1.03 (m) 5.14 (m) 3.25 (d, 9.5) – 0.83 (m) 1.57 (m) 1.38 (m) 1.76 (m) 1.31 (m) – 1.58 (m) 1.88 (m) 1.07 (m) 5.26 (br s) – – 1.71 (m) 1.06 (m) 2.01 (m) 1.61 (m) – 2.82 (dd, 12.3, 3.0) 1.59 (m) 1.13 (m) – 1.20 (m) 1.32 (m)
1
44.4 (t)
dH (mult, J in Hz)
dH (mult, J in Hz)
No.
dC
2b
1a
Table 1. NMR data of compounds 1 – 5 and 1a.
(d) (d) (s) (d) (t)
(s) (d) (s) (t)
31.3 (s) 34.6 (t)
46.8 (s) 42.3 (d) 46.8 (t)
24.0 (t)
122.6 (d) 145.3 (s) 42.5 (s) 28.6 (t)
41.6 48.3 38.5 24.2
33.5 (t)
70.2 89.9 40.0 55.4 18.7
45.2 (t)
dC
31.5 (s) 34.7 (t)
47.1 (s) 42.4 (d) 46.9 (t)
24.1 (t)
122.7 (d) 145.5 (s) 42.7 (s) 28.7 (t)
41.7 (s) 48.4 (d) 38.6 (s) 24.4 (t)
33.7 (t)
69.6 (d) 89.7 (d) 40.2 (s) 55.6 (d) 18.8 (t)
45.3 (t)
dC
3c,d
30.3 (s) 34.5 (t)
46.8 (s) 42.3 (d) 47.0 (t)
24.0 (t)
122.8 (d) 145.1 (s) 42.4 (s) 26.9 (t)
40.0 (s) 48.3 (d) 37.3 (s) 24.1 (t)
33.5 (t)
28.6 (t) 89.1 (d) 39.9 (s) 56.1 (d) 18.8 (t)
39.0 (t)
dC
4b,d
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30.3 (s) 34.5 (t)
46.8 (s) 42.3 (d) 47.0 (t)
24.0 (t)
122.8 (d) 145.1 (s) 42.5 (s) 27.0 (t)
40.0 (s) 48.4 (d) 37.3 (s) 24.2 (t)
33.5 (t)
28.6 (t) 89.3 (d) 39.9 (s) 56.2 (d) 18.8 (t)
39.0 (t)
dC
5b
(Continued)
31.0 (s) 34.2 (t)
46.7 (s) 42.0 (d) 47.2 (t)
23.7 (t)
122.5 (d) 144.8 (s) 42.2 (s) 28.3 (t)
40.8 (s) 48.0 (d) 37.9 (s) 23.9 (t)
33.2e (t)
66.5 (t) 95.2 (d) 40.8 (s) 55.6 (d) 18.5 (t)
46.4 (t)
dC
1ab,d
22 H.-M. Wang et al.
171.1 (s) 171.1 (s) 21.6 (q) 21.2 (q)
– 1.99 (s)
2.11 (s)
28.8 (q) 17.9 (q) 16.6 (q) 17.3 (q) 26.0 (q) 184.1 (s) 33.5 (q) 23.7 (q) 104.5 (d) 72.6 (d) 71.5 (d) 70.4 (d) 63.5 (d)
H (300 MHz) and 13C NMR (100 MHz) tested in CDCl3. H (300 MHz) and 13C NMR (100 MHz) tested in C5D5N. c 13 C NMR (125 MHz) tested in C5D5N. d The assignments are based on literature data [9,17– 20]. e The chemical shifts may interchange.
b1
a1
23 24 25 26 27 28 29 30 10 20 30 40 50 a 50 b 2-O-COCH3 30 -O-COCH3 40 -O-COCH3 2-O-COC H3 30 -O-COC H3 40 -O-COC H3 1.95 (s) 2.28 (s)
2.04 (m) 1.82 (m) 1.32 (s) 0.97 (s) 0.97 (s) 1.00 (s) 1.32 (s) – 0.97 (s) 1.02 (s) 4.89 (d, 6.4) 4.61 (m) 5.49 (m) 4.61 (m) 4.32 (dd, 12.2, 3.7) 3.88 (d, 12.2) – –
1.58 (m) 1.41 (m) 1.08 (s) 0.91 (s) 1.03 (s) 0.73 (s) 1.10 (s) – 0.90 (s) 0.92 (s) 4.33 (d, 6.4) 3.68 (dd, 7.8, 6.4) 3.77 (d, 7.8, 3.0) 5.08 (br s) 3.98 (dd, 12.6, 3.7) 3.51 (d, 12.6) –
22
32.5 (t)
dH (mult, J in Hz)
dH (mult, J in Hz)
No.
dC
2b
1a
Table 1 – continued
22.2 (q) 21.4 (q)
171.2 (s) 171.3 (s)
28.9 (q) 17.6 (q) 16.8 (q) 18.4 (q) 26.5 (q) 180.5 (s) 33.6 (q) 24.1 (q) 106.9 (d) 70.2 (d) 77.4 (d) 67.0 (d) 67.0 (d)
33.3 (t)
dC
170.3e (s) 171.0e (s) 171.1e (s) 22.2e (q) 21.2e (q) 21.3e (q)
29.0 (q) 17.8 (q) 17.0 (q) 18.5 (q) 26.6 (q) 180.6 (s) 33.8 (q) 24.2 (q) 106.6 (d) 70.2e (d) 74.4 (d) 70.6e (d) 64.2 (t)
33.5 (t)
dC
3c,d
21.5 (q)
171.2 (s)
28.5 (q) 17.2 (q) 15.8 (q) 17.7 (q) 26.5 (q) 180.5 (s) 33.6 (q) 24.1 (q) 107.5 (d) 70.0 (d) 77.4 (d) 67.2 (d) 66.8 (t)
33.5 (t)
dC
4b,d
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21.5 (q)
171.2 (s)
28.5 (q) 17.2 (q) 15.8 (q) 17.7 (q) 26.5 (q) 180.6 (s) 33.6 (q) 24.1 (q) 107.5 (d) 70.0 (d) 77.4 (d) 67.2 (d) 66.8 (t)
33.5 (t)
dC
5b
28.5 (q) 17.4 (q) 16.7 (q) 18.1 (q) 26.1 (q) 180.2 (s) 33.3 (q) 23.8 (q) 107.4 (d) 73.0 (d) 74.9 (d) 69.8 (d) 67.9 (t)
33.1e (d)
dC
1ab,d
Journal of Asian Natural Products Research 23
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H.-M. Wang et al. 29
29 12
O O
10
O
3
O
HO
H
1
O
4'
20 18
O
O
H
1
H
27
OH 4'
23
O
O
H
3
O
O 1'
OH
27
H 23
1
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COOH
10
H
1'
OH
12
O COOH
20 18
2 29
COOH
4'
O
H
3
O 1' O OH
COSY HMBC
10
O O
H
1
O
O
20 18
12
O
27
H
NOE
23 3
Figure 2. Selected 1H– 1H COSY, HMBC, and NOE correlations of compounds 1 – 3.
correlations between H-2 (dH 5.58) and one acetyl carbonyl carbon at dC 171.2 and between H-30 (dH 5.49) and the other acetyl carbonyl carbon at dC 171.3 (Figure 2). In addition, hydrolysis of 2 with 10% KOH yielded 1a, which confirmed that the absolute configuration of arabinopyranosyl moiety in 2 was L. Thus, the structure of 2 was characterized as 2-O-acetyl-3-O-(30 -O-acetyl)-a-L arabinopyranosylmaslinic acid. Compound 3, a white amorphous powder, gave the molecular formula as C41H62O11 according to its HR-TOF-MS (m/z 753.4197 [M þ Na]þ), 42 Dalton higher than that of 2. Comparison of its 1H and 13C NMR spectra with those of 2 revealed that the only difference was the presence of an additional acetyl group. The downfield shift of H-40 (dH 5.66) assigned by 1H – 1H COSY spectrum of 3 (Figure 2) indicated that the 40 -hydroxy group was acetylated in 3. In addition, hydrolysis of 3 with 10% KOH yielded 1a, suggesting that the arabinopyranosyl moiety in 3 belongs to L series. Therefore, compound 3 was characterized as 2-O-acetyl-3-O-(30 ,40 -O-
diacetyl)-a- L -arabinopyranosylmaslinic acid. Compound 4 was obtained as white amorphous powder and exhibited a quasimolecular ion peak at m/z 653.4065 [M þ Na]þ, corresponding to the molecular formula C37H58O8. The 1H and 13C NMR spectra of 4 were very close to those of 5 [17] and showed one oleanolic acid moiety, one arabinopyranosyl moiety, and one acetyl group. Hydrolysis of 4 with 10% KOH yielded 4a [17], suggesting that the structural difference between 4 and 5 laid in the location of the acetyl group. The connectivity of the acetyl group at 30 hydroxy of the sugar residue was determined by the downfield chemical shift of C-30 and upfield chemical shift of C-20 and C-40 compared with those of 4a. Accordingly, the structure of 4 was determined as 3-O-(30 -O-acetyl)-a-L -arabinopyranosyloleanolic acid. Six known compounds, namely 3-O(40 -O-acetyl)-a-L -arabinopyranosyloleanolic acid (5) [17], maslinic acid (6) [15], 2-O-acetylmaslinic acid (7) [18], 3-Oacetylmaslinic acid (8) [19], betulinic acid
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Journal of Asian Natural Products Research (9) [20], and 2a-hydroxy-3b-O-acetylbetulinic acid (10) [17], were also isolated from the EtOAc extract of G. hanburyi resin. Their 1H and 13C NMR data were identical to those reported in the literature. To evaluate the biological activities of the four new compounds, compounds 1– 4 were tested in an array of bioassay (see Bioassay section in the Supporting information). Unfortunately, compounds 1– 4 showed no cytotoxicity against NCI-H460 cell lines at 1 mM and no inhibitory effect against KDR protein tyrosine kinase at 10 mM. They were also inactive in the T-cell immunomodulatory bioassay at 1, 10, and 100 mM, respectively. 3. 3.1
25
Shimadu GC-MS-QP2010E (Shimadzu, Kyoto, Japan) using a RXIw-5 ms capillary column (30 m £ 0.25 mm, 0.25 mm) (RESTEK, Bellefonte, PA, USA). 3.2
Plant material
The resin of G. hanburyi (Guttiferae) collected in Vietnam was purchased from the Bozhou Herbal Medicine Market, Anhui Province, in December 2010, and was authenticated by Professor Qinan Wu of Nanjing University of Traditional Chinese Medicine. A specimen (No. SIMM20101228) is deposited at Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
Experimental General experimental procedures
Optical rotations were measured with a Perkin-Elmer 341 polarimeter (PerkinElmer, Waltham, MA, USA). IR spectra were recorded using a Perkin-Elmer 577 spectrometer (PerkinElmer, Waltham, MA, USA). NMR spectra were recorded on a Varian-MERCURY Plus-300 (Varian, Palo Alto, CA, USA), Bruker AM 400 (Bruker, Ettlingen, Germany), Varian-MERCURY Plus-400 (Varian, Palo Alto, CA, USA), Bruker Advance III 500 (Bruker, Ettlingen, Germany), or Bruker Advance III 600 (Bruker, Ettlingen, Germany) spectrometer with TMS as internal standard. ESI-MS and HR-ESIMS data were measured using a Shimadzu LC-MS-2020 (Shimadzu, Kyoto, Japan) and a Bruker Daltonics microTOF QII mass spectrometer (Bruker, Ettlingen, Germany), respectively. Column chromatographic separations were conducted by using silica gel H60 (3002400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) as packing materials. HSGF254 silica gel TLC plates (Yantai Chemical Industrial Institute, Yantai, China) were used for analysis. GC analysis was carried out on a
3.3
Extraction and isolation
Dried and ground resin (1.5 kg) was successively extracted with EtOAc (9 l) three times at room temperature. After combination and removal of the solvent under vacuum, the residue (392 g) was subjected to column chromatography over silica gel (200 –300 mesh) and eluted with a petroleum ether – EtOAc gradient (from 50:1 to 0:1, v/v) to yield 11 fractions (Fr. A – Fr.K). Test with vanillin –sulfuric acid solution on TLC plates suggested the presence of triterpenoids in Fr.G, Fr.I, and Fr.K. The three fractions were analyzed by LC-PDA-MS, revealing the existence of four compounds with molecular weights 688, 688, 730, and 630 not reported in the genus Garcinia in the Reaxys database. Fr.G (7 g) was subjected to RP-18 column chromatography eluted with a 70 – 100% aqueous MeOH gradient to give six subfractions (Fr.G1 – Fr.G6). Fr.G2 (0.9 g) was further subjected to repeated chromatography over silica gel (300 – 400 mesh) to afford compounds 7 (123 mg), 8 (243 mg), 9 (58 mg), and 10 (31 mg). Fr.I (12 g) was separated by column chromatography over silica gel (300 – 400 mesh) eluted with a CHCl3 – MeOH gradient (from 50:1 to 10:1, v/v), affording four
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26
H.-M. Wang et al.
subfractions (Fr.I1 – Fr.I4). Fr.I2 (1.5 g) was chromatographed over silica gel (300 – 400 mesh) eluted with CHCl3 – MeOH (40:1, v/v), followed by RP-18 column chromatography eluted with a 50– 75% aqueous CH3CN gradient to give compounds 3 (198 mg), 4 (43 mg), and 5 (135 mg). Fr.I4 (1.5 g) was subjected to column chromatography over silica gel (300 – 400 mesh) and Sephadex LH-20 gel (95% EtOH), affording compound 6 (35 mg). Fr.K (2.5 g) was subjected repeatedly to chromatography over silica gel (300 –400 mesh) eluted with CHCl3 – MeOH (30:1, v/v) to give compounds 1 (98 mg) and 2 (18 mg). 3.3.1 2-O-Acetyl-3-O-(3 0 -O-acetyl)-a-L arabinopyranosylmaslinic acid (1) White amorphous powder; ½a22 D þ 35.0 (c ¼ 0.20, MeOH); IR(KBr): nmax 3373, 2945, 2908, 1738, 1720, 1695, 1641, 1454, 1367, 1252, 1095, 1034 cm21; for 1H NMR (300 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Table 1. ESI-MS: m/z 711 [M þ Na]þ; HR-TOF-MS m/z 711.4089 [M þ Na]þ (calcd for C39H60O10Na, 711.4084). 3.3.2 2-O-Acetyl-3-O-(4 0 -O-acetyl)-a-L arabinopyranosylmaslinic acid (2) White amorphous powder; ½a22 D þ 31.0 (c ¼ 0.21, MeOH); IR(KBr): nmax 3429, 2947, 1720, 1695, 1642, 1365, 1252, 1142, 1080, 1047 cm21; for 1H NMR (300 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) spectroscopic data, see Table 1. ESI-MS: m/z 711 [M þ Na]þ; HR-TOF-MS m/z 711.4090 [M þ Na] þ (calcd for C39H60O10Na, 711.4084). 3.3.3 2-O-Acetyl-3-O-(3 0 ,4 0 -O-diacetyl)a-L -arabinopyranosylmaslinic acid (3) White amorphous powder; ½a26 D þ 16.4 (c ¼ 0.20, MeOH); IR(KBr): nmax 3435, 2945, 2864, 1732, 1693, 1641, 1464, 1369,
1250, 1084, 1049 cm21; 1H NMR (300 MHz, CDCl3): dH 5.25 (br s, 1H), 5.19 (br s, 1H), 5.13 (m, 1H, H-2), 4.93 (dd, J ¼ 9.9, 3.4 Hz, 1H, H-30 ), 4.36 (d, J ¼ 7.2 Hz, 1H, H-10 ), 3.93 (d, J ¼ 12.5 Hz, 1H, H-5 0 a), 3.80 (dd, J ¼ 9.9, 7.2 Hz, 1H, H-2 0 ), 3.57 (d, J ¼ 12.5 Hz, 1H, H-5 0 b), 3.27 (d, J ¼ 9.8 Hz, 1H, H-3), 2.80 (dd, J ¼ 13.0, 3.5 Hz, 1H, H-18), 2.09 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.10 (s, 3H), 1.07 (s, 3H), 1.02 (s, 3H), 0.91 (s, 3H), 0.89 (s, 6H), 0.72 (s, 3H). 1H NMR (500 MHz, C5D5N): dH 5.66 (br s, 1H, H40 ), 5.58 (m, 1H, H-2), 5.55 (br s, 1H, H30 ), 5.51 (s, 1H, H-12), 4.85 (d, J ¼ 6.8 Hz, 1H, H-10 ), 4.36 (dd, J ¼ 8.0, 6.8 Hz, 1H, H-20 ), 4.17 (d, J ¼ 12.6 Hz, 1H, H-50 a), 3.86 (d, J ¼ 12.6 Hz, 1H, H-50 b), 3.52 (d, J ¼ 9.8 Hz, 1H, H-3), 3.32 (d, J ¼ 13.0, 2.9 Hz, 1H, H-18), 2.27 (s, 3H, OAc), 2.01 (s, 6H, OAc), 1.31 (m, 6H), 1.02 (s, 3H), 0.99 (s, 3H), 0.97 (s, 9H). For 13C NMR (125 MHz, C5D5N) spectroscopic data, see Table 1; ESI-MS: m/z 753 [M þ Na]þ; HR-TOF-MS: m/z 753.4197 [M þ Na]þ (calcd for C41H62O11Na, 753.4190). 3.3.4 3-O-(3 0 -O-Acetyl)-a-L arabinopyranosyloleanolic acid (4) White amorphous powder; ½a22 D þ 43.1 (c ¼ 0.20, MeOH); IR(KBr): nmax 3340, 2941, 2877, 2858, 1724, 1705, 1649, 1464, 1377, 1267, 1086, 1022 cm21; 1H NMR (300 MHz, CDCl3): dH 5.27 (br s, 1H, H12), 4.87 (d, J ¼ 9.7 Hz, 1H, H-30 ), 4.32 (d, J ¼ 7.2 Hz, 1H, H-10 ), 4.02 (br s, 1H, H-40 ), 3.97 (d, J ¼ 13.2 Hz, 1H, H-50 a), 3.85 (dd, J ¼ 9.7, 7.2 Hz, 1H, H-20 ), 3.58 (d, J ¼ 13.2 Hz, 1H, H-50 b), 3.17 (dd, J ¼ 10.9, 4.2 Hz, 1H, H-3), 2.81 (d, J ¼ 12.3 Hz, 1H, H-18), 2.16 (s, 3H, OAc), 1.12 (s, 3H), 0.99 (s, 3H), 0.92 (s, 6H), 0.90 (s, 3H), 0.81 (s, 3H), 0.74 (s, 3H). 1H NMR (300 MHz, C5D5N): dH 5.51 (m, 2H, H-12 and H-3 0 ), 4.88 (d, J ¼ 6.6 Hz, 1H, H-1 0 ), 4.64 (t, J ¼ 7.8 Hz, 1H, H-20 ), 4.60 (br s, 1H, H-
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40 ), 4.34 (dd, J ¼ 12.2, 3.4 Hz, 1H, H-50 a), 3.91 (d, J ¼ 11.4 Hz, 1H, H-50 b), 3.35 (m, 2H, H-3 and H-18), 1.97 (s, 3H, OAc), 1.32 (s, 6H), 1.03 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H), 0.87 (s, 3H). For 13 C NMR (100 MHz, C5D5N) spectroscopic data, see Table 1; ESI-MS: m/z 653 [M þ Na]þ; HR-TOF-MS m/z 653.4065 [M þ Na] þ (calcd for C 37H 58O 8Na, 653.4029). 3.4
27
NMR (300 MHz, C5D5N): dH 5.41 (br s, 1H, H-12), 4.71 (d, J ¼ 7.6 Hz, 1H, H-10 ), 4.48 (dd, J ¼ 8.5, 7.6 Hz, 1H, H-20 ), 4.26 (m, 2H, H-30 and H-40 ), 4.12 (dd, J ¼ 11.4, 3.3 Hz, 1H, H-50 a), 3.99 (dt, J ¼ 11.4, 3.3 Hz, 1H, H-2), 3.84 (d, J ¼ 11.4 Hz, 1H, H-50 b), 3.24 (m, 2H, H-3 and H-18), 2.21 (dd, J ¼ 12.5, 4.4 Hz, 1H), 1.36 (s, 3H), 1.24 (s, 3H), 0.97 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H), 0.86 (s, 3H); for 13C NMR (100 MHz, C5D5N) spectroscopic data, see Table 1.
Hydrolysis and derivatization
Compounds 1 –4 (10 mg) were hydrolyzed with 10% KOH (2 ml) at room temperature for 2 h. The reaction products of 1– 3 were diluted with water and extracted with nBuOH (3 £ 5 ml) to yield compound 1a (6 mg). The reaction product of 4 was treated in the same way to give 4a (5 mg). The structure of 1a was elucidated by detailed spectroscopic analysis and chemical methods, while 4a was identified by comparison of NMR data with those in the literature [17]. Compound 1a (5 mg) was refluxed with 1 M HCl (2 ml) for 2 h under constant stirring. The reaction product was diluted with water and extracted with EtOAc (3 £ 5 ml). The aqueous layer and EtOAc layer were lyophilized to dryness, respectively, to give arabinose and maslinic acid which was identified by comparison with NMR data in the literature and co-TLC with authentic sample [15,16]. The arabinose hydrolyzed from 1a was dissolved in dry pyridine (1 ml), and L -cysteine methyl ester hydrochloride (3 mg) was added. The mixture was stirred at 608C for 2 h, 150 ml of Ac2O was added, and stirred at 908C for another 1 h. The reaction product was evaporated to dryness to give methyl 3acetyl-2R-(L -arabino-10 ,20 ,30 ,40 -tetraacetoxybutyl)-thiazolidine-4R-carbonylate [13]. Compound 1a: white amorphous powder; ½a26 D þ 16.7 (c ¼ 0.10, MeOH); IR (KBr): nmax 3431, 2925, 2854, 1714, 1645, 1462, 1365, 1253, 1088, 644 cm21; 1H
3.5 GC analysis The peracetylated derivative was analyzed by GC under the following conditions: Column, RXIw-5 ms, 30 m £ 25 mm, 0.25 mm; carrier gas: He (17.8 ml/min), injection temperature at 2808C, detection temperature at 2008C, column temperature at 1008C, initial temperature was maintained at 1008C for 5 min, and then raised to 2808C at the rate of 208C/min and maintained at 2808C for 10 min; injection volume: 1.0 ml, split ratio: 1/10. The absolute configuration of sugar moiety was determined by comparing their retention times with the derivatives of authentic D and L -arabinose prepared in the same manner [13,14]. The derivative of arabinose hydrolyzed from compound 1a gave a peak with retention time of 16.09 min, while the derivatives of standard D - and L -arabinose had GC retention time of 16.36 and 16.10 min, respectively. References [1] Q.B. Han and H.X. Xu, Curr. Med. Chem. 16, 3775 (2009). [2] O. Chantarasriwong, A. Batova, W. Chavasiri, and E.A. Theodorakis, Chemistry 16, 9944 (2010). [3] M. Hemshekhar, K. Sunitha, M.S. Santhosh, S. Devaraja, K. Kemparaju, B.S. Vishwanath, S.R. Niranjana, and K.S. Girish, Phytochem. Rev. 10, 325 (2011). [4] N. Anantachoke, P. Tuchinda, C. Kuhakarn, M. Pohmakotr, and V. Reutrakul, Pharm. Biol. 50, 78 (2012).
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[5] J. Asano, K. Chiba, M. Tada, and T. Yoshii, Phytochemistry. 41, 815 (1996). [6] Q.B. Han, Y.L. Wang, L. Yang, T.F. Tso, C.F. Qiao, J.Z. Song, L.J. Xu, S.L. Chen, D.J. Yang, and H.X. Xu, Chem. Pharm. Bull. 54, 265 (2006). [7] S.J. Tao, S.H. Guan, W. Wang, Z.Q. Lu, G.T. Chen, N. Sha, Q.X. Yue, X. Liu, and D.A. Guo, J. Nat. Prod. 72, 117 (2009). [8] Y.X. Deng, S.L. Pan, S.Y. Zhao, M.Q. Wu, Z.Q. Sun, X.H. Chen, and Z.Y. Shao, Fitoterapia 83, 1548 (2012). [9] N. De Tommasi, S. Piacente, F. De Simone, C. Pizza, and Z.L. Zhou, J. Nat. Prod. 56, 1669 (1993). [10] N.P. Sahu, K. Koike, Z. Jia, and T. Nikaido, Tetrahedron 51, 13435 (1995). [11] A. Braca, G. Autore, F. De Simone, S. Marzocco, I. Morelli, F. Venturella, and N. De Tommasi, Planta Med. 70, 960 (2004). [12] X. Chang, W. Li, Z. Jia, T. Satou, S. Fushiya, and K. Koike, J. Nat. Prod. 70, 179 (2007).
[13] S. Hara, H. Okabe, and K. Mihashi, Chem. Pharm. Bull. 35, 501 (1987). [14] K.Y. Niu, L.Y. Wang, S.Z. Liu, and W.M. Zhao, J. Asian Nat. Prod. Res. 15, 1 (2013). [15] Q.S. Zhao, J. Tian, J.M. Yue, S.N. Chen, Z.W. Lin, and H.D. Sun, Phytochemistry 48, 1025 (1998). [16] A.J. Benesi, C.J. Falzone, S. Banerjee, and G.K. Farber, Carbohydr. Res. 258, 27 (1994). [17] L.L. Wang, Z.L. Li, D.D. Song, L. Sun, Y. H. Pei, Y.K. Jing, and H.M. Hua, Planta Med. 74, 1735 (2008). [18] O. Thoison, T. Sevenet, H.M. Niemeyer, and G.B. Russell, Phytochemistry 65, 2173 (2004). [19] A.S.R. Anjaneyulu, A.V.R. Reddy, G.R. Mallavarapu, and R.S. Chandrasekhara, Phytochemistry 25, 2670 (1986). [20] P. Ghosh, A. Mandal, J. Ghosh, C. Pal, and A.K. Nanda, J. Asian Nat. Prod. Res. 14, 141 (2012).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Four new triterpenoids isolated from the resin of Garcinia hanburyi a
a
a
a
bc
a
Hong-Min Wang , Qun-Fang Liu , Yi-Wu Zhao , Shuang-Zhu Liu , bc
bc
Zhen-Hua Chen , Ru-Jun Zhang , Zhen-Zhong Wang , Wei Xiao Wei-Min Zhao
&
a
a
Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China b
Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, 222001, China c
State Key Laboratory of Pharmaceutical Process New-Tech for Chinese Medicine, Lianyungang, 222001, China Published online: 07 Jan 2014.
To cite this article: Hong-Min Wang, Qun-Fang Liu, Yi-Wu Zhao, Shuang-Zhu Liu, Zhen-Hua Chen, Ru-Jun Zhang, Zhen-Zhong Wang, Wei Xiao & Wei-Min Zhao (2014) Four new triterpenoids isolated from the resin of Garcinia hanburyi, Journal of Asian Natural Products Research, 16:1, 20-28, DOI: 10.1080/10286020.2013.875012 To link to this article: http://dx.doi.org/10.1080/10286020.2013.875012
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 1, 20–28, http://dx.doi.org/10.1080/10286020.2013.875012
Four new triterpenoids isolated from the resin of Garcinia hanburyi Hong-Min Wanga, Qun-Fang Liua, Yi-Wu Zhaob,c, Shuang-Zhu Liua, Zhen-Hua Chena, Ru-Jun Zhanga, Zhen-Zhong Wangb,c, Wei Xiaob,c* and Wei-Min Zhaoa*
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a
Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; bJiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang 222001, China; cState Key Laboratory of Pharmaceutical Process New-Tech for Chinese Medicine, Lianyungang 222001, China (Received 7 October 2013; final version received 10 December 2013) Four new triterpenoids, 2-O-acetyl-3-O-(40 -O-acetyl)-a-L -arabinopyranosylmaslinic acid (1), 2-O-acetyl-3-O-(30 -O-acetyl)-a-L -arabinopyranosylmaslinic acid (2), 2-Oacetyl-3-O-(30 ,40 -O-diacetyl)-a-L -arabinopyranosylmaslinic acid (3), and 3-O-(30 -Oacetyl)-a-L -arabinopyranosyloleanolic acid (4), together with six known triterpenoids, 3-O-(40 -O-acetyl)-a-L -arabinopyranosyloleanolic acid (5), maslinic acid (6), 2-Oacetylmaslinic acid (7), 3-O-acetylmaslinic acid (8), betulinic acid (9), and 2ahydroxy-3b-O-acetylbetulinic acid (10), were isolated from the EtOAc extract of Garcinia hanburyi resin. Their structures were elucidated by analysis of the spectroscopic data and chemical methods. Keywords: Garcinia hanburyi; Guttiferae; triterpenoid; saponin
1.
Introduction
Gamboge, the pulverized golden color resin collected primarily from Garcinia hanburyi (Guttiferae), and to a lesser extent from G. Morella, has a particularly long and rich history in the arts and sciences [1–4]. It has been used as dye and folk medicine for the treatment of infected wound and tumors [1–4]. Previous phytochemical investigations of gamboge have mainly resulted in the identification of caged polyprenylated xanthones [1–8]. In this study, 10 triterpenoids including four new triterpenoids (1–4) and six known ones (5–10) were isolated from the resin of G. hanburyi (Figure 1). The structures of these compounds were elucidated by spectroscopic analysis and chemical methods. 2. Results and discussion Compound 1 was obtained as white amorphous powder. Its molecular formula
C39H60O10 was established by HR-TOFMS (m/z 711.4089 [M þ Na]þ), implying 10 degrees of unsaturation. Its 1H (Table 1) spectrum indicated the presence of one triterpene aglycone [dH 1.10 (s, 3H), 1.08 (s, 3H); 1.03 (s, 3H); 0.92 (s, 3H); 0.91 (s, 3H); 0.90 (s, 3H); 0.73 (s, 3H)], one sugar moiety (dH 3.51 –4.33), and two acetyl methyl groups [dH 2.11 (s, 3H), 1.99 (s, 3H)]. The 13C NMR (Table 1) spectrum of 1 exhibited 39 carbon signals, of which nine carbon signals were consistent with the presence of an arabinopyranosyl moiety [dC 104.5 (d), 72.6 (d), 71.5 (d), 70.4 (d), 63.5 (t)] [9 – 12] and two acetyl methyl groups [dC 171.1 (s), 171.1 (s), 21.6 (q), 21.2 (q)]. The remaining 30 carbon signals strongly resembled those of maslinic acid, except for the downfield shift of C-3 (from dC 83.5 to 89.2), due to the glycosylation of 3-hydroxy group in 1. Moreover, the HMBC correlations from H-10 (dH 4.33) to C-3 confirmed the
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
Journal of Asian Natural Products Research
21
29 20 12
R1 R2 O O 3
4'
1'
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R3
OH
1 1a 2 3 4 4a 5
H
1 10
COOH
H
R1 H
COOH
H
27
R2
H 23
H
6 R1 = R2 = OH 7 R1 = OAc, R2 = OH 8 R1 = OH, R2 = OAc
R1 = R2 = OAc, R3 = OH R1 = R2 = R3 = OH R1 = R3 = OAc, R2 = OH R1 = R2 = R3 = OAc R1 = H, R2 = OH, R3 = OAc R1 = H, R2 = R3 = OH R1 = H, R2 = OAc, R3 = OH
H H
R1
COOH
H R2
H 9 R1 = H, R2 = OH 10 R1 = OH, R2 = OAc
Figure 1. Structures of compounds 1 –10, 1a, and 4a.
connection between the maslinic acid moiety and the arabinopyranosyl moiety (Figure 2). Furthermore, the locations of two acetyl groups at 2- and 40 -hydroxy groups were established based on the HMBC correlations from H-2 (dH 5.14) to an acetyl carbonyl carbon (dC 171.1), and from H-40 (dH 5.08) to the other acetyl carbonyl carbon (dC 171.1) (Figure 2). The arabinopyranosyl unit in 1 was determined to connect to the aglycone in a-configuration in 4C1 form on the basis of the 3 0 0 JH-1,H-2 value (6.4 Hz) and the NOE correlations between H-10 and H-30 and between H-10 and H-50 in the NOESY experiment (Figure 2) [9 – 12]. The absolute configuration of the arabinopyranosyl unit in 1 was found to belong to L series by GC-MS analysis of its peracetylated thiazolidine derivative [13,14]. Hydrolysis of 1 with 10% KOH yielded 1a, which was further hydrolyzed with acid to give maslinic acid and arabinose identified by
comparison of their spectral data with those in the literature and co-TLC [15,16]. Derivation of the arabinose hydrolysate from 1 with L -cysteine methyl ester gave a GC-MS peak at 16.09 min, same as that of a standard L -arabinose derivative. Accordingly, the structure of 1 was established as 2-O-acetyl-3-O-(40 -O-acetyl)-a-L -arabinopyranosylmaslinic acid. Compound 2, a white amorphous powder, possessed the same molecular formula as 1, based on HR-TOF-MS (m/z 711.4090 [M þ Na]þ). Its 1H and 13C NMR data (Table 1) were very similar to those of 1 and displayed signals belonging to one maslinic acid moiety, one arabinopyranosyl unit, and two acetyl groups. The HMBC correlations from H-10 (dH 4.89) to C-3 (dC 89.9) established the connection between the maslinic acid moiety and the arabinopyranosyl moiety (Figure 2). Assignment of two O-acetyl groups at C2 and C-30 was evidenced from the HMBC
20 21
17 18 19
16
12 13 14 15
8 9 10 11
7
2 3 4 5 6
30.8 (s) 33.8 (t)
46.6 (s) 41.0 (d) 45.9 (t)
22.8 (t)
122.4 (d) 144.8 (s) 41.6 (s) 27.7 (t)
39.3 (s) 47.7 (d) 38.4 (s) 23.5 (t)
32.5 (t)
69.7 (d) 89.2 (d) 41.6 (s) 55.0 (d) 18.1 (t)
2.04 (m) 1.19 (m) 5.58 (m) 3.53 (d, 9.7) – 0.95 (m) 1.52 (m) 1.29 (m) 1.49 (m) 0.98 (m) – 1.75 (m) – 2.00 (m) 1.02 (m) 5.49 (br s) – – 2.18 (m) 1.21 (m) 2.16 (m) 1.31 (m) – 3.33 (dd, 12.3, 3.0) 1.83 (m) 1.33 (m) – 1.48 (m) 1.27 (m)
1.90 (m) 1.03 (m) 5.14 (m) 3.25 (d, 9.5) – 0.83 (m) 1.57 (m) 1.38 (m) 1.76 (m) 1.31 (m) – 1.58 (m) 1.88 (m) 1.07 (m) 5.26 (br s) – – 1.71 (m) 1.06 (m) 2.01 (m) 1.61 (m) – 2.82 (dd, 12.3, 3.0) 1.59 (m) 1.13 (m) – 1.20 (m) 1.32 (m)
1
44.4 (t)
dH (mult, J in Hz)
dH (mult, J in Hz)
No.
dC
2b
1a
Table 1. NMR data of compounds 1 – 5 and 1a.
(d) (d) (s) (d) (t)
(s) (d) (s) (t)
31.3 (s) 34.6 (t)
46.8 (s) 42.3 (d) 46.8 (t)
24.0 (t)
122.6 (d) 145.3 (s) 42.5 (s) 28.6 (t)
41.6 48.3 38.5 24.2
33.5 (t)
70.2 89.9 40.0 55.4 18.7
45.2 (t)
dC
31.5 (s) 34.7 (t)
47.1 (s) 42.4 (d) 46.9 (t)
24.1 (t)
122.7 (d) 145.5 (s) 42.7 (s) 28.7 (t)
41.7 (s) 48.4 (d) 38.6 (s) 24.4 (t)
33.7 (t)
69.6 (d) 89.7 (d) 40.2 (s) 55.6 (d) 18.8 (t)
45.3 (t)
dC
3c,d
30.3 (s) 34.5 (t)
46.8 (s) 42.3 (d) 47.0 (t)
24.0 (t)
122.8 (d) 145.1 (s) 42.4 (s) 26.9 (t)
40.0 (s) 48.3 (d) 37.3 (s) 24.1 (t)
33.5 (t)
28.6 (t) 89.1 (d) 39.9 (s) 56.1 (d) 18.8 (t)
39.0 (t)
dC
4b,d
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30.3 (s) 34.5 (t)
46.8 (s) 42.3 (d) 47.0 (t)
24.0 (t)
122.8 (d) 145.1 (s) 42.5 (s) 27.0 (t)
40.0 (s) 48.4 (d) 37.3 (s) 24.2 (t)
33.5 (t)
28.6 (t) 89.3 (d) 39.9 (s) 56.2 (d) 18.8 (t)
39.0 (t)
dC
5b
(Continued)
31.0 (s) 34.2 (t)
46.7 (s) 42.0 (d) 47.2 (t)
23.7 (t)
122.5 (d) 144.8 (s) 42.2 (s) 28.3 (t)
40.8 (s) 48.0 (d) 37.9 (s) 23.9 (t)
33.2e (t)
66.5 (t) 95.2 (d) 40.8 (s) 55.6 (d) 18.5 (t)
46.4 (t)
dC
1ab,d
22 H.-M. Wang et al.
171.1 (s) 171.1 (s) 21.6 (q) 21.2 (q)
– 1.99 (s)
2.11 (s)
28.8 (q) 17.9 (q) 16.6 (q) 17.3 (q) 26.0 (q) 184.1 (s) 33.5 (q) 23.7 (q) 104.5 (d) 72.6 (d) 71.5 (d) 70.4 (d) 63.5 (d)
H (300 MHz) and 13C NMR (100 MHz) tested in CDCl3. H (300 MHz) and 13C NMR (100 MHz) tested in C5D5N. c 13 C NMR (125 MHz) tested in C5D5N. d The assignments are based on literature data [9,17– 20]. e The chemical shifts may interchange.
b1
a1
23 24 25 26 27 28 29 30 10 20 30 40 50 a 50 b 2-O-COCH3 30 -O-COCH3 40 -O-COCH3 2-O-COC H3 30 -O-COC H3 40 -O-COC H3 1.95 (s) 2.28 (s)
2.04 (m) 1.82 (m) 1.32 (s) 0.97 (s) 0.97 (s) 1.00 (s) 1.32 (s) – 0.97 (s) 1.02 (s) 4.89 (d, 6.4) 4.61 (m) 5.49 (m) 4.61 (m) 4.32 (dd, 12.2, 3.7) 3.88 (d, 12.2) – –
1.58 (m) 1.41 (m) 1.08 (s) 0.91 (s) 1.03 (s) 0.73 (s) 1.10 (s) – 0.90 (s) 0.92 (s) 4.33 (d, 6.4) 3.68 (dd, 7.8, 6.4) 3.77 (d, 7.8, 3.0) 5.08 (br s) 3.98 (dd, 12.6, 3.7) 3.51 (d, 12.6) –
22
32.5 (t)
dH (mult, J in Hz)
dH (mult, J in Hz)
No.
dC
2b
1a
Table 1 – continued
22.2 (q) 21.4 (q)
171.2 (s) 171.3 (s)
28.9 (q) 17.6 (q) 16.8 (q) 18.4 (q) 26.5 (q) 180.5 (s) 33.6 (q) 24.1 (q) 106.9 (d) 70.2 (d) 77.4 (d) 67.0 (d) 67.0 (d)
33.3 (t)
dC
170.3e (s) 171.0e (s) 171.1e (s) 22.2e (q) 21.2e (q) 21.3e (q)
29.0 (q) 17.8 (q) 17.0 (q) 18.5 (q) 26.6 (q) 180.6 (s) 33.8 (q) 24.2 (q) 106.6 (d) 70.2e (d) 74.4 (d) 70.6e (d) 64.2 (t)
33.5 (t)
dC
3c,d
21.5 (q)
171.2 (s)
28.5 (q) 17.2 (q) 15.8 (q) 17.7 (q) 26.5 (q) 180.5 (s) 33.6 (q) 24.1 (q) 107.5 (d) 70.0 (d) 77.4 (d) 67.2 (d) 66.8 (t)
33.5 (t)
dC
4b,d
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21.5 (q)
171.2 (s)
28.5 (q) 17.2 (q) 15.8 (q) 17.7 (q) 26.5 (q) 180.6 (s) 33.6 (q) 24.1 (q) 107.5 (d) 70.0 (d) 77.4 (d) 67.2 (d) 66.8 (t)
33.5 (t)
dC
5b
28.5 (q) 17.4 (q) 16.7 (q) 18.1 (q) 26.1 (q) 180.2 (s) 33.3 (q) 23.8 (q) 107.4 (d) 73.0 (d) 74.9 (d) 69.8 (d) 67.9 (t)
33.1e (d)
dC
1ab,d
Journal of Asian Natural Products Research 23
24
H.-M. Wang et al. 29
29 12
O O
10
O
3
O
HO
H
1
O
4'
20 18
O
O
H
1
H
27
OH 4'
23
O
O
H
3
O
O 1'
OH
27
H 23
1
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COOH
10
H
1'
OH
12
O COOH
20 18
2 29
COOH
4'
O
H
3
O 1' O OH
COSY HMBC
10
O O
H
1
O
O
20 18
12
O
27
H
NOE
23 3
Figure 2. Selected 1H– 1H COSY, HMBC, and NOE correlations of compounds 1 – 3.
correlations between H-2 (dH 5.58) and one acetyl carbonyl carbon at dC 171.2 and between H-30 (dH 5.49) and the other acetyl carbonyl carbon at dC 171.3 (Figure 2). In addition, hydrolysis of 2 with 10% KOH yielded 1a, which confirmed that the absolute configuration of arabinopyranosyl moiety in 2 was L. Thus, the structure of 2 was characterized as 2-O-acetyl-3-O-(30 -O-acetyl)-a-L arabinopyranosylmaslinic acid. Compound 3, a white amorphous powder, gave the molecular formula as C41H62O11 according to its HR-TOF-MS (m/z 753.4197 [M þ Na]þ), 42 Dalton higher than that of 2. Comparison of its 1H and 13C NMR spectra with those of 2 revealed that the only difference was the presence of an additional acetyl group. The downfield shift of H-40 (dH 5.66) assigned by 1H – 1H COSY spectrum of 3 (Figure 2) indicated that the 40 -hydroxy group was acetylated in 3. In addition, hydrolysis of 3 with 10% KOH yielded 1a, suggesting that the arabinopyranosyl moiety in 3 belongs to L series. Therefore, compound 3 was characterized as 2-O-acetyl-3-O-(30 ,40 -O-
diacetyl)-a- L -arabinopyranosylmaslinic acid. Compound 4 was obtained as white amorphous powder and exhibited a quasimolecular ion peak at m/z 653.4065 [M þ Na]þ, corresponding to the molecular formula C37H58O8. The 1H and 13C NMR spectra of 4 were very close to those of 5 [17] and showed one oleanolic acid moiety, one arabinopyranosyl moiety, and one acetyl group. Hydrolysis of 4 with 10% KOH yielded 4a [17], suggesting that the structural difference between 4 and 5 laid in the location of the acetyl group. The connectivity of the acetyl group at 30 hydroxy of the sugar residue was determined by the downfield chemical shift of C-30 and upfield chemical shift of C-20 and C-40 compared with those of 4a. Accordingly, the structure of 4 was determined as 3-O-(30 -O-acetyl)-a-L -arabinopyranosyloleanolic acid. Six known compounds, namely 3-O(40 -O-acetyl)-a-L -arabinopyranosyloleanolic acid (5) [17], maslinic acid (6) [15], 2-O-acetylmaslinic acid (7) [18], 3-Oacetylmaslinic acid (8) [19], betulinic acid
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Journal of Asian Natural Products Research (9) [20], and 2a-hydroxy-3b-O-acetylbetulinic acid (10) [17], were also isolated from the EtOAc extract of G. hanburyi resin. Their 1H and 13C NMR data were identical to those reported in the literature. To evaluate the biological activities of the four new compounds, compounds 1– 4 were tested in an array of bioassay (see Bioassay section in the Supporting information). Unfortunately, compounds 1– 4 showed no cytotoxicity against NCI-H460 cell lines at 1 mM and no inhibitory effect against KDR protein tyrosine kinase at 10 mM. They were also inactive in the T-cell immunomodulatory bioassay at 1, 10, and 100 mM, respectively. 3. 3.1
25
Shimadu GC-MS-QP2010E (Shimadzu, Kyoto, Japan) using a RXIw-5 ms capillary column (30 m £ 0.25 mm, 0.25 mm) (RESTEK, Bellefonte, PA, USA). 3.2
Plant material
The resin of G. hanburyi (Guttiferae) collected in Vietnam was purchased from the Bozhou Herbal Medicine Market, Anhui Province, in December 2010, and was authenticated by Professor Qinan Wu of Nanjing University of Traditional Chinese Medicine. A specimen (No. SIMM20101228) is deposited at Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
Experimental General experimental procedures
Optical rotations were measured with a Perkin-Elmer 341 polarimeter (PerkinElmer, Waltham, MA, USA). IR spectra were recorded using a Perkin-Elmer 577 spectrometer (PerkinElmer, Waltham, MA, USA). NMR spectra were recorded on a Varian-MERCURY Plus-300 (Varian, Palo Alto, CA, USA), Bruker AM 400 (Bruker, Ettlingen, Germany), Varian-MERCURY Plus-400 (Varian, Palo Alto, CA, USA), Bruker Advance III 500 (Bruker, Ettlingen, Germany), or Bruker Advance III 600 (Bruker, Ettlingen, Germany) spectrometer with TMS as internal standard. ESI-MS and HR-ESIMS data were measured using a Shimadzu LC-MS-2020 (Shimadzu, Kyoto, Japan) and a Bruker Daltonics microTOF QII mass spectrometer (Bruker, Ettlingen, Germany), respectively. Column chromatographic separations were conducted by using silica gel H60 (3002400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) as packing materials. HSGF254 silica gel TLC plates (Yantai Chemical Industrial Institute, Yantai, China) were used for analysis. GC analysis was carried out on a
3.3
Extraction and isolation
Dried and ground resin (1.5 kg) was successively extracted with EtOAc (9 l) three times at room temperature. After combination and removal of the solvent under vacuum, the residue (392 g) was subjected to column chromatography over silica gel (200 –300 mesh) and eluted with a petroleum ether – EtOAc gradient (from 50:1 to 0:1, v/v) to yield 11 fractions (Fr. A – Fr.K). Test with vanillin –sulfuric acid solution on TLC plates suggested the presence of triterpenoids in Fr.G, Fr.I, and Fr.K. The three fractions were analyzed by LC-PDA-MS, revealing the existence of four compounds with molecular weights 688, 688, 730, and 630 not reported in the genus Garcinia in the Reaxys database. Fr.G (7 g) was subjected to RP-18 column chromatography eluted with a 70 – 100% aqueous MeOH gradient to give six subfractions (Fr.G1 – Fr.G6). Fr.G2 (0.9 g) was further subjected to repeated chromatography over silica gel (300 – 400 mesh) to afford compounds 7 (123 mg), 8 (243 mg), 9 (58 mg), and 10 (31 mg). Fr.I (12 g) was separated by column chromatography over silica gel (300 – 400 mesh) eluted with a CHCl3 – MeOH gradient (from 50:1 to 10:1, v/v), affording four
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26
H.-M. Wang et al.
subfractions (Fr.I1 – Fr.I4). Fr.I2 (1.5 g) was chromatographed over silica gel (300 – 400 mesh) eluted with CHCl3 – MeOH (40:1, v/v), followed by RP-18 column chromatography eluted with a 50– 75% aqueous CH3CN gradient to give compounds 3 (198 mg), 4 (43 mg), and 5 (135 mg). Fr.I4 (1.5 g) was subjected to column chromatography over silica gel (300 – 400 mesh) and Sephadex LH-20 gel (95% EtOH), affording compound 6 (35 mg). Fr.K (2.5 g) was subjected repeatedly to chromatography over silica gel (300 –400 mesh) eluted with CHCl3 – MeOH (30:1, v/v) to give compounds 1 (98 mg) and 2 (18 mg). 3.3.1 2-O-Acetyl-3-O-(3 0 -O-acetyl)-a-L arabinopyranosylmaslinic acid (1) White amorphous powder; ½a22 D þ 35.0 (c ¼ 0.20, MeOH); IR(KBr): nmax 3373, 2945, 2908, 1738, 1720, 1695, 1641, 1454, 1367, 1252, 1095, 1034 cm21; for 1H NMR (300 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Table 1. ESI-MS: m/z 711 [M þ Na]þ; HR-TOF-MS m/z 711.4089 [M þ Na]þ (calcd for C39H60O10Na, 711.4084). 3.3.2 2-O-Acetyl-3-O-(4 0 -O-acetyl)-a-L arabinopyranosylmaslinic acid (2) White amorphous powder; ½a22 D þ 31.0 (c ¼ 0.21, MeOH); IR(KBr): nmax 3429, 2947, 1720, 1695, 1642, 1365, 1252, 1142, 1080, 1047 cm21; for 1H NMR (300 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) spectroscopic data, see Table 1. ESI-MS: m/z 711 [M þ Na]þ; HR-TOF-MS m/z 711.4090 [M þ Na] þ (calcd for C39H60O10Na, 711.4084). 3.3.3 2-O-Acetyl-3-O-(3 0 ,4 0 -O-diacetyl)a-L -arabinopyranosylmaslinic acid (3) White amorphous powder; ½a26 D þ 16.4 (c ¼ 0.20, MeOH); IR(KBr): nmax 3435, 2945, 2864, 1732, 1693, 1641, 1464, 1369,
1250, 1084, 1049 cm21; 1H NMR (300 MHz, CDCl3): dH 5.25 (br s, 1H), 5.19 (br s, 1H), 5.13 (m, 1H, H-2), 4.93 (dd, J ¼ 9.9, 3.4 Hz, 1H, H-30 ), 4.36 (d, J ¼ 7.2 Hz, 1H, H-10 ), 3.93 (d, J ¼ 12.5 Hz, 1H, H-5 0 a), 3.80 (dd, J ¼ 9.9, 7.2 Hz, 1H, H-2 0 ), 3.57 (d, J ¼ 12.5 Hz, 1H, H-5 0 b), 3.27 (d, J ¼ 9.8 Hz, 1H, H-3), 2.80 (dd, J ¼ 13.0, 3.5 Hz, 1H, H-18), 2.09 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.10 (s, 3H), 1.07 (s, 3H), 1.02 (s, 3H), 0.91 (s, 3H), 0.89 (s, 6H), 0.72 (s, 3H). 1H NMR (500 MHz, C5D5N): dH 5.66 (br s, 1H, H40 ), 5.58 (m, 1H, H-2), 5.55 (br s, 1H, H30 ), 5.51 (s, 1H, H-12), 4.85 (d, J ¼ 6.8 Hz, 1H, H-10 ), 4.36 (dd, J ¼ 8.0, 6.8 Hz, 1H, H-20 ), 4.17 (d, J ¼ 12.6 Hz, 1H, H-50 a), 3.86 (d, J ¼ 12.6 Hz, 1H, H-50 b), 3.52 (d, J ¼ 9.8 Hz, 1H, H-3), 3.32 (d, J ¼ 13.0, 2.9 Hz, 1H, H-18), 2.27 (s, 3H, OAc), 2.01 (s, 6H, OAc), 1.31 (m, 6H), 1.02 (s, 3H), 0.99 (s, 3H), 0.97 (s, 9H). For 13C NMR (125 MHz, C5D5N) spectroscopic data, see Table 1; ESI-MS: m/z 753 [M þ Na]þ; HR-TOF-MS: m/z 753.4197 [M þ Na]þ (calcd for C41H62O11Na, 753.4190). 3.3.4 3-O-(3 0 -O-Acetyl)-a-L arabinopyranosyloleanolic acid (4) White amorphous powder; ½a22 D þ 43.1 (c ¼ 0.20, MeOH); IR(KBr): nmax 3340, 2941, 2877, 2858, 1724, 1705, 1649, 1464, 1377, 1267, 1086, 1022 cm21; 1H NMR (300 MHz, CDCl3): dH 5.27 (br s, 1H, H12), 4.87 (d, J ¼ 9.7 Hz, 1H, H-30 ), 4.32 (d, J ¼ 7.2 Hz, 1H, H-10 ), 4.02 (br s, 1H, H-40 ), 3.97 (d, J ¼ 13.2 Hz, 1H, H-50 a), 3.85 (dd, J ¼ 9.7, 7.2 Hz, 1H, H-20 ), 3.58 (d, J ¼ 13.2 Hz, 1H, H-50 b), 3.17 (dd, J ¼ 10.9, 4.2 Hz, 1H, H-3), 2.81 (d, J ¼ 12.3 Hz, 1H, H-18), 2.16 (s, 3H, OAc), 1.12 (s, 3H), 0.99 (s, 3H), 0.92 (s, 6H), 0.90 (s, 3H), 0.81 (s, 3H), 0.74 (s, 3H). 1H NMR (300 MHz, C5D5N): dH 5.51 (m, 2H, H-12 and H-3 0 ), 4.88 (d, J ¼ 6.6 Hz, 1H, H-1 0 ), 4.64 (t, J ¼ 7.8 Hz, 1H, H-20 ), 4.60 (br s, 1H, H-
Journal of Asian Natural Products Research
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40 ), 4.34 (dd, J ¼ 12.2, 3.4 Hz, 1H, H-50 a), 3.91 (d, J ¼ 11.4 Hz, 1H, H-50 b), 3.35 (m, 2H, H-3 and H-18), 1.97 (s, 3H, OAc), 1.32 (s, 6H), 1.03 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H), 0.87 (s, 3H). For 13 C NMR (100 MHz, C5D5N) spectroscopic data, see Table 1; ESI-MS: m/z 653 [M þ Na]þ; HR-TOF-MS m/z 653.4065 [M þ Na] þ (calcd for C 37H 58O 8Na, 653.4029). 3.4
27
NMR (300 MHz, C5D5N): dH 5.41 (br s, 1H, H-12), 4.71 (d, J ¼ 7.6 Hz, 1H, H-10 ), 4.48 (dd, J ¼ 8.5, 7.6 Hz, 1H, H-20 ), 4.26 (m, 2H, H-30 and H-40 ), 4.12 (dd, J ¼ 11.4, 3.3 Hz, 1H, H-50 a), 3.99 (dt, J ¼ 11.4, 3.3 Hz, 1H, H-2), 3.84 (d, J ¼ 11.4 Hz, 1H, H-50 b), 3.24 (m, 2H, H-3 and H-18), 2.21 (dd, J ¼ 12.5, 4.4 Hz, 1H), 1.36 (s, 3H), 1.24 (s, 3H), 0.97 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H), 0.86 (s, 3H); for 13C NMR (100 MHz, C5D5N) spectroscopic data, see Table 1.
Hydrolysis and derivatization
Compounds 1 –4 (10 mg) were hydrolyzed with 10% KOH (2 ml) at room temperature for 2 h. The reaction products of 1– 3 were diluted with water and extracted with nBuOH (3 £ 5 ml) to yield compound 1a (6 mg). The reaction product of 4 was treated in the same way to give 4a (5 mg). The structure of 1a was elucidated by detailed spectroscopic analysis and chemical methods, while 4a was identified by comparison of NMR data with those in the literature [17]. Compound 1a (5 mg) was refluxed with 1 M HCl (2 ml) for 2 h under constant stirring. The reaction product was diluted with water and extracted with EtOAc (3 £ 5 ml). The aqueous layer and EtOAc layer were lyophilized to dryness, respectively, to give arabinose and maslinic acid which was identified by comparison with NMR data in the literature and co-TLC with authentic sample [15,16]. The arabinose hydrolyzed from 1a was dissolved in dry pyridine (1 ml), and L -cysteine methyl ester hydrochloride (3 mg) was added. The mixture was stirred at 608C for 2 h, 150 ml of Ac2O was added, and stirred at 908C for another 1 h. The reaction product was evaporated to dryness to give methyl 3acetyl-2R-(L -arabino-10 ,20 ,30 ,40 -tetraacetoxybutyl)-thiazolidine-4R-carbonylate [13]. Compound 1a: white amorphous powder; ½a26 D þ 16.7 (c ¼ 0.10, MeOH); IR (KBr): nmax 3431, 2925, 2854, 1714, 1645, 1462, 1365, 1253, 1088, 644 cm21; 1H
3.5 GC analysis The peracetylated derivative was analyzed by GC under the following conditions: Column, RXIw-5 ms, 30 m £ 25 mm, 0.25 mm; carrier gas: He (17.8 ml/min), injection temperature at 2808C, detection temperature at 2008C, column temperature at 1008C, initial temperature was maintained at 1008C for 5 min, and then raised to 2808C at the rate of 208C/min and maintained at 2808C for 10 min; injection volume: 1.0 ml, split ratio: 1/10. The absolute configuration of sugar moiety was determined by comparing their retention times with the derivatives of authentic D and L -arabinose prepared in the same manner [13,14]. The derivative of arabinose hydrolyzed from compound 1a gave a peak with retention time of 16.09 min, while the derivatives of standard D - and L -arabinose had GC retention time of 16.36 and 16.10 min, respectively. References [1] Q.B. Han and H.X. Xu, Curr. Med. Chem. 16, 3775 (2009). [2] O. Chantarasriwong, A. Batova, W. Chavasiri, and E.A. Theodorakis, Chemistry 16, 9944 (2010). [3] M. Hemshekhar, K. Sunitha, M.S. Santhosh, S. Devaraja, K. Kemparaju, B.S. Vishwanath, S.R. Niranjana, and K.S. Girish, Phytochem. Rev. 10, 325 (2011). [4] N. Anantachoke, P. Tuchinda, C. Kuhakarn, M. Pohmakotr, and V. Reutrakul, Pharm. Biol. 50, 78 (2012).
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[5] J. Asano, K. Chiba, M. Tada, and T. Yoshii, Phytochemistry. 41, 815 (1996). [6] Q.B. Han, Y.L. Wang, L. Yang, T.F. Tso, C.F. Qiao, J.Z. Song, L.J. Xu, S.L. Chen, D.J. Yang, and H.X. Xu, Chem. Pharm. Bull. 54, 265 (2006). [7] S.J. Tao, S.H. Guan, W. Wang, Z.Q. Lu, G.T. Chen, N. Sha, Q.X. Yue, X. Liu, and D.A. Guo, J. Nat. Prod. 72, 117 (2009). [8] Y.X. Deng, S.L. Pan, S.Y. Zhao, M.Q. Wu, Z.Q. Sun, X.H. Chen, and Z.Y. Shao, Fitoterapia 83, 1548 (2012). [9] N. De Tommasi, S. Piacente, F. De Simone, C. Pizza, and Z.L. Zhou, J. Nat. Prod. 56, 1669 (1993). [10] N.P. Sahu, K. Koike, Z. Jia, and T. Nikaido, Tetrahedron 51, 13435 (1995). [11] A. Braca, G. Autore, F. De Simone, S. Marzocco, I. Morelli, F. Venturella, and N. De Tommasi, Planta Med. 70, 960 (2004). [12] X. Chang, W. Li, Z. Jia, T. Satou, S. Fushiya, and K. Koike, J. Nat. Prod. 70, 179 (2007).
[13] S. Hara, H. Okabe, and K. Mihashi, Chem. Pharm. Bull. 35, 501 (1987). [14] K.Y. Niu, L.Y. Wang, S.Z. Liu, and W.M. Zhao, J. Asian Nat. Prod. Res. 15, 1 (2013). [15] Q.S. Zhao, J. Tian, J.M. Yue, S.N. Chen, Z.W. Lin, and H.D. Sun, Phytochemistry 48, 1025 (1998). [16] A.J. Benesi, C.J. Falzone, S. Banerjee, and G.K. Farber, Carbohydr. Res. 258, 27 (1994). [17] L.L. Wang, Z.L. Li, D.D. Song, L. Sun, Y. H. Pei, Y.K. Jing, and H.M. Hua, Planta Med. 74, 1735 (2008). [18] O. Thoison, T. Sevenet, H.M. Niemeyer, and G.B. Russell, Phytochemistry 65, 2173 (2004). [19] A.S.R. Anjaneyulu, A.V.R. Reddy, G.R. Mallavarapu, and R.S. Chandrasekhara, Phytochemistry 25, 2670 (1986). [20] P. Ghosh, A. Mandal, J. Ghosh, C. Pal, and A.K. Nanda, J. Asian Nat. Prod. Res. 14, 141 (2012).
