2012

NPC Natural Product Communications Vol. 7

No. 1
Xanthones with Antiproliferative Effects on Prostate Cancer Cells 53 - 56
from the Stem Bark of Garcinia xanthochymus
Feng Jia,b, Zhanlin Lia,b, Gaofeng Liuc, Shengli Niua,b, Nan Zhaoa,b, Xiaoqiu Liub and Huiming Huaa,b*
a
Key Laborarory of Structure-Based Drug Design & Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, P. R. China
b
School of Traditional Chinese Materia Media, Shenyang Pharmaceutical University, 110016, Shenyang,
P. R. China
c
Department of Pharmacy, the Second Affiliated Hospital of Harbin Medical University, 150086,
Harbin, P. R. China

huimhua@163.com

Received: September 21st, 2011; Accepted: November 21st, 2011

Investigations of the constituents of the stem barks of Garcinia xanthochymus have yielded two new compounds, garcinenones X (1) and Y (2), along with five
known xanthones, 1,4,5,6-tetrahydroxy-7-(3-methylbut-2-enyl)xanthone (3), 1,4,6-trihydroxy-5-methoxy-7-(3-methylbut-2-enyl)xanthone (4), 1,4,5,6-
tetrahydroxy-7,8-di(3-methylbut-2-enyl)xanthone (5), 1,3,5,6-tetrahydroxy-4,7,8-tri(3-methylbut-2-enyl)xanthone (6), and 1,5,6-trihydroxy-7,8-di(3-
methylbut-2-enyl)-6',6'dimethylpyrano(2',3':3,4)xanthone (7). The structures of the compounds were determined by spectroscopic methods. The cell growth
inhibitory activity of the isolated compounds against the PC-3 cell line was measured. Among them, compounds 2, 3, 5, and 6 exhibited significant inhibitory
effects with IG50 values of 14.3, 15.5, 11.1, and 6.8 µM, respectively.

Keywords: Garcinia xanthochymus, Guttiferae, Xanthone, Cell growth inhibition, PC-3 cell line.

5'' 4''
Garcinia xanthochymus (Guttiferae) is a traditional Dai medicine 3''

native to the south and southwest of Yunnan Province of China, 2''

4''' 1''
which grows up to 10-20 m. Garcinia species are well-known as 1'''
O OH

3'''
rich sources of xanthones [1], biflavones, and benzophenones [2,3].
5''' 2'''
These phenolic constituents have been reported to possess a wide
range of biological and pharmacological properties, such as HO O
O

5'
antibacterial [4,5], antimalarial [6], and cytotoxic activities [7,8]. In OH
1'
2'

the course of our search for anticancer agents from natural sources, 3'
OH

we launched a systematic investigation of the chemical constituents 4'

of the chloroform extract of the stem bark of G. xanthochymus. In 1 2

the present study, seven xanthones, including two new and five
known compounds (see Fig. 2), have been isolated. Herein, we
report the isolation, structural elucidation, and antitumor assay of
these compounds.
Garcinenone X (1) was obtained as yellow powder from methanol.
Its molecular formula was determined to be C19H18O6 from a quasi-
molecular ion peak at m/z = 365.1003 [M+Na]+ in its HR-ESI-MS.
The IR spectrum of 1 showed absorption bands at 3382 cm-1 and
1641 cm-1, indicative of a hydroxyl group and a conjugated
carbonyl group. The UV absorption at 213, 243, and 319 nm,
combined with NMR spectroscopic data indicated that 1 was a
polyhydroxylated xanthone [9]. The 1H NMR spectrum of 1
revealed the presence of three aromatic protons in an ABC spin
system at δ 7.32 (1H, d, J = 7.8 Hz, H-6), 7.24 (1H, t, J = 7.8 Hz,
H-7), and 7.55 (1H, d, J = 7.8 Hz, H-8), one chelated hydroxyl
group at δ 12.92 (1H, s), two phenolic hydroxyl groups at δ 10.45
(2H, s), one methoxy group at δ 3.87 (3H, s) and a prenyl moiety at
δ 5.18 (1H, t, J =7.2 Hz, H-2'), 3.26 (2H, d, J = 7.1 Hz, H-1'), 1.73
(3H, s, H-4'), and 1.62 (3H, s, H-5'). The 13C NMR spectrum of 1
showed nineteen carbon signals, including one methoxy, four sp2
methines, ten sp2 quaternary carbons, one carbonyl carbon and three
saturated carbons. The above data suggested that 1 was a xanthone
Figure 1: The structures and key HMBC correlations of garcinenones X (1) and
Y (2)
derivative possessing three hydroxyl groups, a methoxy group and a
prenyl moiety.
HMBC correlations established the structures of two aromatic rings.
The hydroxyl group at δ 12.92 was located at C-1, based on its
downfield chemical shift. In the HMBC spectrum, the hydroxyl
group at δ 12.92 showed long-rang correlations with the carbon
signals at δ 102.0 (C-9a), 110.5 (C-2), and 155.4 (C-1), and a signal
at δ 3.26 (H-1') showed long-rang correlations with δ 110.5 (C-2),
155.4 (C-1), 131.2 (C-3'), and 122.5 (C-2'), which indicated that a
prenyl group was placed at C-2. Because a proton at δ 7.55 (H-8)
showed long-rang correlations with δ 180.8 (C-9), 145.3 (C-5a),
and 121.3 (C-6), combined with the downfield chemical shift, the
aromatic proton at δ 7.55 was assigned to H-8. Based on the
coupling constants, H-8 was clearly one of three methines (δ 7.55,
7.32, and 7.24) in an ABC spin system. So, two aromatic protons at
δ 7.32 and 7.24 were assigned to H-6 and H-7, which were
supported by the long-range correlations of H-6 at δ 7.32 with
δ 115.0 (C-8), 145.3 (C-5a), and 146.9 (C-5), and of H-7 at δ 7.24
with δ 120.9 (C-8a) and 146.9 (C-5). The significant downfield shift
54 Natural Product Communications Vol. 7 (1) 2012 Ji et al.

Table 1: 1H (600 MHz) and 13C (150 MHz) NMR data for 1 in DMSO-d6. Table 2: 1H (600 MHz) and 13C (150 MHz) NMR data for 2 in acetone-d6.
1 13
1
H (Hz) 13
C HMBC (H→C) H (Hz) C HMBC (H→C)
1 164.8
1 155.4
2 6.11(1H, s) 92.64 C(1),C(3),C(4)
2 110.5
3 167.3
3 156.7
4 103.4
4 127.7
4a 151.3
4a 147.5
5 130.0
5 146.9
5a 145.3 5a 145.6
6 7.32(1H, d, J = 7.8) 121.3 C(5),C(5a),C(8) 6 150.0
7 7.24(1H, t, J = 7.8) 124.4 C(5),C(8a) 7 125.3
8 7.55(1H, d, J = 7.8) 115.0 C(5a),C(6),C(9) 8 134.4
8a 120.9 8a 111.1
9 180.8 9 182.6
9a 102.0 9a 103.2
1' 3.32(2H, m) 26.9 C(3),C(4),C(4a),C(2'),C(3')
1' 3.26(2H, d, J = 7.2) 21.7 C(1),C(2),C(2'),C(3')
2' 4.82(1H, t-like, J = 8.4) 91.9
2' 5.18(1H, t, J = 7.2) 122.5 C(1'),C(4'),C(5')
3' 70.7
3' 131.2
4' 1.28(3H, s) 24.6 C(2'),C(3'),C(5')
4' 1.73(3H, s) 18.2 C(2'),C(3'),C(5')
5' 1.25(3H, s) 25.4 C(2'),C(3'),C(4')
5' 1.62(3H, s) 25.9 C(2'),C(3'),C(4')
1'' 4.11(2H, d, J = 5.3) 28.3 C(7),C(8),C(8a),C(2''),C(3'')
4-OMe 3.87(3H, s) 61.6 C(4)
1-OH 12.92(1H, s) C(1),C(2),C(9a) 2'' 5.09(1H, m) 124.7
3-OH 10.45(1H, s) 3'' 129.9
5-OH 10.45(1H, s) 4'' 1.67(3H, s) 25.1 C(5'')
5'' 1.78(3H, s) 17.4 C(4'')
1''' 3.46(2H, d, J = 6.2) 24.5 C(6),C(7),C(8),C(2'''),C(3''')
of the methoxy carbon at δ 61.6 suggested that both of the ortho- 2''' 5.09(1H, m) 123.1
3''' 131.2
positions of this methoxy group are substituted [10,11]. Therefore, 4''' 1.66(3H, s) 25.1 C(5''')
the methoxy group was located at C-4, which was supported by 5''' 1.78(3H, s) 17.5 C(4''')
long-rang correlation between the methoxy group at δ 3.87 (3H, s) 1-OH 13.88(1H, s) C(1),C(2),C(9a)
5-OH 8.85 (1H, br s)
and C-4 (δ 127.7). Thus, two hydroxyl groups were located at C-3 6-OH 8.64 (1H, br s)
and C-5, respectively. Consequently, compound 1 was established
as 1,3,5-trihydroxy-4-methoxy-2-prenylxanthone and was named 13
garcinenone X. C NMR data of 2 with those of 1,5,6-trihydroxy-7-(3-methyl-2-
butenyl)-8-(3-hydroxy-3-methylbutyl)-5′-(1-hydroxy-1-methyl-
Garcinenone Y (2) was obtained as yellow amorphous powder and ethyl) -4′,5′-dihydro-furano(2′,3′:3,4)xanthone [14]. In the HMBC
its molecular formula was determined to be C28H32O7 based on the spectrum, the long rang correlations between the chelated hydroxyl
[M+Na]+ ion at m/z = 503.2011 in its HR-ESI-MS. The IR spectrum proton at δ 13.88 and the carbon at δ 164.8 (C-1), 92.6 (C-2), and
of 2 indicated the presence of a hydroxyl group at 3445 cm-1 and a 103.2 (C-9a), and between δ 6.11 (H-2) and δ 167.3 (C-3), 164.8
conjugated carbonyl group at 1660 cm-1. The UV at 257, 285, 331 (C-1), and 103.4 (C-4) were observed. Furthermore, the presence of
nm indicated that 2 was a polyhydroxylated xanthone [12]. The 1H a 4-(1-hydroxy-1-methylethyl)-3, 4-dihydrofuran unit was proposed
NMR spectrum of 2 showed two prenyl moieties at δ 4.11 (2H, d, by the proton to carbon connectivities of δ 3.32 (H-1') to δ 167.3
J = 5.3 Hz, H-1''), 5.09 (1H, m, H-2''), 1.67 (3H, s, H-4'') and 1.78 (C-3), 151.3 (C-4a), 103.4 (C-4), 91.9 (C-2'), and 70.7 (C-3'). The
(3H, s, H-5''), and at δ 3.46 (2H, d, J = 6.2 Hz, H-1'''), 5.09 (1H, m, above data allowed us to establish an unequivocal assignment of
H-2'''), 1.66 (3H, s, H-4''') and 1.78 (3H, s, H-5'''). Also it gave one the A ring of 2 as that of 1,5,6-trihydroxy-7-(3-methyl-2-butenyl)-
chelated hydroxyl group at δ 13.88 (1H, s), two free hydroxyl 8-(3-hydroxy-3-methylbutyl)-5′-(1-hydroxy-1-methylethyl)-4′,5′-
groups at δ 8.85 (1H, br s) and 8.64 (1H, br s), and one aromatic dihydrofurano(2′,3′:3,4)xanthone. Therefore, the structure of 2 was
proton at δ 6.11 (1H, s, H-2). Furthermore, the 1H NMR spectrum determined as 1,5,6-trihydroxy-7,8-di(3-methylbut-2-enyl)-5′-(1-
indicated the presence of a 4-(1-hydroxy-1-methylethyl)-3,4- hydroxy-1-methylethyl)-4′,5′-dihydro-furano(2′,3′:3,4)xanthone and
dihydrofuran ring at δ 4.82 (1H, t-like, J = 8.4 Hz, H-2'), 3.32 (2H, was named garcinenone Y.
m, H-1'), 1.28 (3H, s, H-4') and 1.25 (3H, s, H-5') [12]. The 13C
NMR spectrum displayed 28 signals consisting of 4 methines, 14
quaternary carbons, a carbonyl, 3 methylenes and 6 methyls. These
data suggested a trihydroxyxanthone with two 3-methylbut-2-enyl
groups and a 4-(1-hydroxy-1-methylethyl)-3, 4-dihydrofuran ring.

Comparison of 13C NMR spectroscopic data of 2 with those of

1,5,6-trihydroxy-7,8-di(3-methylbut-2-enyl)-6',6'-dimethylpyrano
(2',3':3,4)xanthone (7) [13] indicated that the substitution pattern of
ring B was the same as that of 7, which was confirmed by HMBC
experiment. The downfield shift of a methene at δ 4.11 (H-1'') in a
prenyl group suggested a prenyl group located at C-8. In the HMBC
spectrum, the proton at δ 4.11 (H-1'') showed long-range
correlations with δ 111.1 (C-8a), 124.7 (C-2''), 125.3 (C-7), 134.4
(C-8), and 129.9 (C-3''), which confirmed the location of a prenyl
moiety was placed at the C-8. The other signal at δ 3.46 (H-1''')
showed long-range correlations with δ 123.1 (C-2'''), 125.3 (C-7),
131.2 (C-3'''), 134.4 (C-8), and 150.0 (C-6), which indicated that
another prenyl group was located at C-7. The substitution pattern of
ring A was elucidated by HMBC experiment, and by comparing the
7
Figure 2: Structures of the isolated compounds 3-7.
The known compounds were identified as 1,4,5,6-tetrahydroxy-
7-(3-methylbut-2-enyl)xanthone (3) [15], 1,4,6-trihydroxy-5-
methoxy-7-(3-methylbut-2-enyl)xanthone (4) [15], 1,4,5,6-
tetrahydroxy-7,8-di(3-methylbut-2-enyl) xanthone (5) [9], 1,3,5,6-
tetrahydroxy-4,7,8-tri(3-methyl-but-2-enyl)xanthone (6) [16], and
1,5,6-trihydroxy-7,8-di(3-methylbut-2-enyl)-6',6'dimethylpyrano
(2',3':3,4)xanthone (7) [13] by comparison of the NMR data with
those reported.
Xanthones from Garcinia xanthochymus Natural Product Communications Vol. 7 (1) 2012 55

Table 3: The antiproliferative effects of compounds 1-7 on PC-3 cell line
(IG50 M)a.
compounds IG50(M) prostate cancer cells
1 87.8 ± 3.1
2 14.3 ± 2.6
3 15.5 ± 4.4
4 52.8 ± 4.0
5 11.1 ± 3.1
6 6.8 ± 2.5
7 36.1 ± 8.1
Doxorubicin hydrochlorideb 0.70 ± 0.04
a
IG50 is the concentration that inhibited 50% of cell growth.
b
Positive control.
All compounds were evaluated for their cell growth inhibitory
activity against the PC-3 cell line. Almost all the compounds
exhibited inhibitory effects, with IG50 values ranging from 6.8 to
87.8M (IG50 values are shown in Table 3). Among them,
compound 6 was most effective with an IG50 value of 6.8 M.
Experimental
General: The UV spectra were conducted on a Shimadzu UV-2201
spectrometer. The FT-IR spectra were obtained on a Bruker IFS-55
spectrometer (KBr disk). HRESI mass spectra were recorded on
Varian QFT-ESI and Bruker micrOTOF-Q mass spectrometers.
NMR spectra were recorded on Bruker ARX-300 and AV-600
NMR spectrometers using TMS as an internal standard.
The chromatographic silica gel (200-300 mesh) was purchased from
Qingdao Ocean Chemical Factory (Qingdao, China), and ODS (50
m) was purchased from YMC Co. Ltd., Kyoto, Japan. Sephadex
LH-20 was purchased from GE Healthcare.
Plant material: The plant material was collected from Kunming,
Yunnan Province, China, in April, 2010 and identified as the stem
bark of Garcinia xanthyochmus C. Y. Wu by Mr Jingyun Cui
(Xishuangbanna Tropical Botanic Garden of the Chinese Academy
of Sciences, People’s Republic of China). The voucher sample
(DYTH-20100401) was deposited in the Department of Natural
Products Chemistry, Shenyang Pharmaceutical University,
Shenyang, China.
Extraction and isolation: The dried stem bark of Garcinia
xanthyochmus (2.0 kg) were extracted 3 times (each for 2 h) with
95% EtOH (3 × 3 L) under reflux. The EtOH extract was
concentrated in vacuo to yield a brown-yellow gum (400 g). The
residue was then suspended in water (2 L) and partitioned
successively with CHCl3 (3 × 2 L) and n-BuOH (3 × 2 L). The
CHCl3 extract (80 g) was chromatographed over silica gel using a
gradient system of light petroleum (P. E.) (60～90°C)-acetone (100:
0-0: 100). The collected fractions were combined on the basis of
their TLC characteristics to yield 4 fractions (Fr. A-Fr. D). Fr. A
(3.1 g) was subjected to Sephadex LH-20 column chromatography
(CC) eluted with MeOH to yield 10 fractions (Fr. A1-Fr. A10). Fr.
A4 was separated by reversed-phase ODS CC eluted with MeOH-
H2O (80:20) to afford compound 5 (3.3 mg). Fr. B (3.6 g) was
submitted to Sephadex LH-20 CC eluted with CHCl3-MeOH (1:1)
to yield 9 fractions (Fr. B1-Fr. B9). Fr. B6 was repeatedly
recrystallized in P. E.-acetone (5:1) to give 1 (3.4 mg) and 6
(3.7 mg). Fr. C (3.2 g) was purified by Sephadex LH-20 CC eluted
with CHCl3-MeOH (1:1) to yield 7 fractions (Fr. C1-Fr. C7). Fr. C5
was further purified by ODS CC eluted with a MeOH-H2O gradient
system from 60:40 to 90:10 to yield 3 subfractions (Fr. C2A- Fr.
C2C). Fr. C2B was separated by preparative TLC with a developing
solvent system of P. E.-acetone (4:1) to provide 3 (5.1 mg) and 4
(3.2 mg), which were further purified on Sephadex LH-20 with
MeOH. Fr. D (5.2 g) was loaded onto a Sephadex LH-20 C column
and eluted with CHCl3-MeOH (1:1) to yield 5 fractions (Fr. D1-Fr.
D5). Compounds 2 (3.6 mg) and 7 (4.1mg) were obtained from Fr.
D2 by ODS CC eluted with a MeOH-H2O gradient system from
30:70 to 90:10, and were further purified on Sephadex LH-20 with
MeOH.
Garcinenone X
Rf: 0.4 (CHCl3-MeOH, 10:1).
IR (KBr): 3382, 2924, 1641, 1615, 1563, 1477, 1222, 1037,
778 cm-1.
UV/Vis λmax (CH3CN)max: 213, 243, 319 nm.
1
H NMR: Table 1
13
C NMR: Table 1
HR-ESI-MS: m/z [M+Na]+ calcd for C19H18NaO6: 365.1001; found:
365.1003.
Garcinenone Y
Rf: 0.4 (CHCl3-MeOH, 8:1).
IR (KBr): 3445, 2976, 2927, 1660, 1601, 1579, 1255, 1143, 999,
818 cm-1.
UV/Vis λmax (CH3CN)max: 257, 285, 331 nm.
1
H NMR: Table 2
13
C NMR: Table 2
HR-ESI-MS: m/z [M+Na]+ calcd for C28H32NaO7: 503.2046; found:
503.2011.
Acknowledgments - The authors wish to thank Mr Yi Sha and Mrs
Wen Li (Shenyang Pharmaceutical University, Shenyang, People’s
Republic of China) for the measurements of NMR data.

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