Introduction
The genus Garcinia , belonging to the family Clusiaceae, has been widely studied for their chemical constituents
and biological activities and comprises about 20 species in Thailand
[1 ], [2 ], [3 ], [4 ], [5 ]. Garcinia
schomburgkiana Pierre, locally named “Ma dan” in Thai, is an edible plant in Southeast Asia [6 ]. In Thai folk medicine, its roots, leaves, and fruits are used
for the treatment of cough, menstrual disturbances, and diabetes as well as an expectorant
and laxative [7 ]. Previous phytochemical studies of G.
schomburgkiana showed the presence of xanthones, phloroglucinols, depsidones, biphenyls, flavonoids,
and triterpenoids, some of which exhibited cytotoxic and antimalarial activity [8 ], [9 ], [10 ], [11 ], [12 ]. Herein, we report the
isolation and structural elucidation of five undescribed polyprenylated benzoylphloroglucinol
derivatives, named garschomcinols A – E, and five known analogues from the branches
of G.
schomburgkiana . The structures of the isolated compounds were determined by spectroscopic analysis,
especially 1D and 2D NMR spectroscopy, and comparison with literature data. The
configuration of the bicyclo [3.3.1]nonane core structure of the polyprenylated benzoylphloroglucinols
was assigned by comparison of the experimental electronic circular dichroism (ECD)
data
with those of related compounds. The cytotoxicity in vitro of all isolated compounds against five cancer cell lines (KB, HeLa S3, HT-29, MCF-7,
and Hep G2) was evaluated by the MTT
colorimetric method.
Results and Discussion
Phytochemical investigation of the CH2 Cl2 crude extract from the branches of G. schomburgkiana led to the isolation of five undescribed polyprenylated
benzoylphloroglucinol derivatives, named garschomcinols A – E (1 – 5 ), and five known analogues (6 – 10 ) ([Fig. 1 ]), including
oblongifolin C (6 ) [13 ], guttiferone K (7 ) [14 ], garciyunnanin B (8 ) [15 ],
oxyguttiferone K (9 ) [16 ], and oblongifolin G (10 ) [17 ]. The chemical structures of the known compounds were confirmed
by NMR spectroscopic data and comparison with previously published data.
Fig. 1 Chemical structures of 1 –10 .
Garschomcinol A (1 ) was obtained as a yellow gum [α ]D
20 + 12.5 (c 0.20, MeOH). Its molecular formula was determined as
C43 H60 O7 from the [M + Na]+ ion peak at m /z 711.4233 (calcd. for C43 H60 O7 Na, 711.4237) in positive
HRESIMS. UV absorptions maxima at λ
max 242, 257, and 324 nm revealed aromatic and conjugated carbonyl chromophores. The
IR spectrum showed absorption bands at
3425 cm−1 (hydroxyl groups) and 1720 and 1665 cm−1 (carbonyl groups). The 1 H NMR data of 1 ([Table 1 ]) exhibited signals
for a 1,2,4-trisubstituted benzene ring at δ
H 6.69 (1H, d, J = 8.3 Hz, H-15), 6.95 (1H, dd, J = 1.4, 8.3 Hz, H-16), and 7.19 (1H, br s, H-12), a tertiary methyl
proton at δ
H 0.81 (3H, s, H-22), a methylene group at δ
H 1.44 and 2.05 (2H, H-7), a methine proton at δ
H 1.74 (1H, m, H-6), and signals
attributed to a 2,6-dimethyloct-6-en-2-ol and three 3-methylbut-2-enyl groups. The
13 C NMR data showed resonances for six aromatic carbons, a conjugated carbonyl group
at
δ
C 196.5 (C-10), an enolized 1,3-diketone at δ
C 119.2 (C-2), 191.6 (C-3), and 194.8 (C-1), a nonconjugated carbonyl at δ
C 208.9 (C-9),
three quaternary carbons at δ
C 51.4 (C-5), 64.0 (C-8), and 69.3 (C-4), a methyl at δ
C 16.2 (C-22), a methylene at δ
C 43.0 (C-7), a methine
at δ
C 42.1 (C-6), and 25 signals assignable to five isoprene units. The 1 H and 13 C NMR spectroscopic data ([Table 1 ]) of
1 were similar to those of oblongifolin C (6 ) except for the geranyl group in 6 , in which the second double bond was hydrated to a 2,6-dimethyloct-6-en-2-ol group.
The
structure of the side chain was confirmed by the presence of an oxygenated carbon
in the 13 C NMR spectrum at δ
C 71.4 (C-41), the COSY correlations of H-24/H-25,
H-27/H-39 and H-39/H-40, and the HMBC correlations of H-25 with C-6, C-26, C-27, and
C-28, H-42 and H-43 with C-40 and C-41, and H-39 with C-26, C-27, C-40, and C-41 ([Fig. 2 ]). The relative configuration of 1 was determined using 1 H-1 H coupling constants and NOESY correlations. The coupling constant J = 12.8 Hz of
H-6/H-7ax and the NOESY interactions between H-7ax /H-24, H-7ax /H-29, H-22/H-17, H-22/H-24, and H-25/H-27 suggested an axial orientation of H-6 and
H-22, an equatorial
orientation of H-17, H-24, and H-29, and an E -configuration of the Δ
25,26 double bond ([Fig. 3 ]). The 13 C NMR data at
δ
C 42.1 supported an equatorial orientation of the C-6 substituent since the C-6 resonance
with an axial substituent is reportedly observed at δ
C 46 – 48
[16 ]. Thus, the structure of 1 was assigned as shown in [Fig. 1 ].
Table 1 1 H (400 MHz) and 13 C (100 MHz) NMR data of compounds 1 – 3 in CD3 OD.
Position
1
2
3
δ
H
(J in Hz)
δ
C
δ
H (J in Hz)
δ
C
δ
H (J in Hz)
δ
C
1
194.8
194.5
194.7
2
119.2
119.3
119.3
3
191.6
191.4
191.4
4
69.3
69.4
69.4
5
51.4
51.5
51.5
6
1.74, m
42.1
1.78, m
42.0
1.79, m
42.0
7eq
2.05, m
43.0
2.07, m
43.1
2.09, m
43.1
7ax
1.44, t (12.8)
1.47, t (12.6)
1.47, t (13.0)
8
64.0
63.9
63.9
9
208.9
208.9
208.9
10
196.5
196.5
196.5
11
130.0
130.0
130.0
12
7.19, br s
117.4
7.21, d (1.8)
117.4
7.21, d (1.5)
117.4
13
146.1
146.1
146.2
14
152.3
152.3
152.3
15
6.69, d (8.3)
115.0
6.70, d (8.3)
115.1
6.71, d (8.3)
115.1
16
6.95, dd (1.4, 8.3)
124.9
6.97, dd (1.9, 8.3)
124.9
6.97, dd (1.5, 8.3)
124.9
17
2.70, m
26.6
2.71, m
26.6
2.72, m
26.6
18
4.87, br s
121.3
4.86, br s
121.3
4.87, br s
121.3
19
134.8
134.9
134.9
20
1.62, s
26.3
1.64, s
26.3
1.65, s
26.3
21
1.69, s
18.4
1.71, s
18.4
1.72, s
18.4
22
0.81, s
16.2
0.83, m
16.2
0.84, m
16.2
23
1.67, m
37.4
1.68, m
37.4
1.69, m
37.4
24
1.76, m, 2.07, m
29.9
1.78, m, 2.09, m
29.9
1.78, m, 2.10, m
29.9
25
5.01, br s
123.7
5.04, m
123.9
5.04, m
123.9
26
138.1
138.0
138.0
27
1.95, m
41.1
1.99, m
40.9
2.00, m
40.9
28
1.56, s
16.4
1.57, s
16.4
1.58, s
16.4
29
2.49, m
31.6
2.51, m
31.6
2.53, m
31.6
30
5.13, br s
120.9
5.13, br s
120.9
5.14, br s
120.9
31
135.4
135.4
135.4
32
1.71, s
26.3
1.72, s
26.3
1.73, s
26.3
33
1.66, s
18.3
1.68, s
18.3
1.69, s
18.3
34
1.97, m
25.2
1.99, m
25.2
1.99, m
25.2
35
5.06, br s
125.5
5.07, m
125.5
5.08, m
125.5
36
132.3
132.4
132.4
37
1.66, s
26.0
1.68, s
25.9
1.69, s
25.9
38
1.59, s
18.0
1.61, s
18.0
1.62, s
18.0
39
1.44, m
23.3
1.39, m
22.7
1.40, m
22.7
40
1.34, m
44.1
1.39, m
39.6
1.40, m
40.0
41
71.4
76.3
76.0
42
1.12, s
29.2
1.11, s
25.5
1.13, s
26.2
43
1.14, s
29.2
1.12, s
25.5
1.13, s
26.2
44
3.13, s
49.4
3.36, m
57.6
45
1.11, m
16.4
Fig. 2 Key HMBC (arrow curves) and COSY (bold lines) correlations of 1 –5 .
Fig. 3 Key NOESY correlations of 1 .
Garschomcinol B (2 ) was obtained as a yellow gum with [α ]D
20 + 13.5 (c 0.28, MeOH). Its molecular formula of
C44 H62 O7 was established by the positive HRESIMS [M + Na]+ ion peak at m /z 725.4382 (calcd. for
C44 H62 O7 Na, 725.4393). The NMR data ([Table 1 ]) of 2 and 1 were nearly identical except for the side chain of
2 , which showed a signal for a methoxy group at C-41. The presence of the methoxy group
was confirmed by the 13 C NMR resonance at δ
C 49.4 (C-44) and the
HMBC correlation of the methoxy protons at δ
H 3.13 (3H, s, H-44) with C-41 (δ
C 76.3) ([Fig. 2 ]). Finally, the structure of
2 was determined as shown in [Fig. 1 ].
Garschomcinol C (3 ) was obtained as a yellow gum with [α ]D
20 + 14.7 (c 0.34, MeOH). Its molecular formula was determined to be
C45 H64 O7 by HRESIMS ([M + Na]+
m /z 739.4524, calcd. for C45 H64 O7 Na, 739.4550). According to the NMR
analysis ([Table 1 ]), compound 3 possessed the same structure as 2 , except that the methoxy group of the side chain was replaced by an ethoxy group.
The 1 H and 13 C NMR spectra showed protons at δ
H 1.11 (3H, m, H-45) and 3.36 (2H, m, H-44), which were correlated in the HSQC spectrum
with carbons at
δ
C 16.4 and 57.6, respectively. The HMBC spectrum showed cross-peaks between H-44 and
C-41 (δ
C 76.0) and C-45 ([Fig. 2 ]),
indicating that the ethoxy group was attached at C-41 in the side chain. Thus, the
structure of 3 was characterized as shown in [Fig. 1 ].
Garschomcinol D (4 ) was obtained as a yellow gum with [α ]D
20 + 13.1 (c 0.30, MeOH). Its molecular formula of
C36 H54 O3 was suggested by the positive HRESIMS [M + K]+ ion peak at m /z 573.3786 (calcd. for
C36 H54 O3 K, 573.3710). The IR spectrum displayed bands at 1719, 1653, and 1642 cm−1 for carbonyl groups. The 1 H NMR data of 4
([Table 2 ]) exhibited signals for two methine protons at δ
H 2.02 (1H, m, H-6) and 5.99 (1H, s, H-2), a methylene group at δ
H
1.35 and 1.93 (2H, H-7), the tertiary methyl protons at δ
H 0.68 (3H, s, H-15), and signals attributed to a 2.2-dimethylpyran ring, a 3,7-dimethylocta-2,6-dienyl,
and two
3-methylbut-2-enyl groups. The 13 C NMR data showed resonances for an enolized 1,3-diketone at δ
C 120.2 (C-2), 175.2 (C-3), and 198.7 (C-1), a non-conjugated
carbonyl at δ
C 208.1 (C-9), three quaternary carbons at δ
C 47.4 (C-5), 62.6 (C-4), and 63.2 (C-8), a methyl at δ
C 17.1 (C-15), a methylene
at δ
C 39.6 (C-7), a methine at δ
C 36.3 (C-6), and 25 signals assignable to five isoprene units. The NMR data of 4 showed close similarity to those of
6 except for the absence of a benzoyl group at C-2 in 4 , which was confirmed by the HMBC correlations ([Fig. 2 ]) from H-2 to C-1, C-3, C-4, and C-8.
In addition, the prenyl group at C-4 in 6 was cyclized to build a pyran ring, which was determined by the HMBC correlations
from H-10 [δ
H 1.71, 2.52 (2H, m)] to C-3,
C-4, and C-12 (δ
C 83.5), and from H-11 [δ
H 1.27, 1.71 (2H, m)] to C-4, C-12, C-13 (δ
C 29.5), and C-14 (δ
C 26.2). The
relative configuration of 4 was assigned using 1 H-1 H coupling constants and NOESY correlations as in 1 . The coupling constant J = 13.1 Hz of
H-6/H-7ax and the interactions in the NOESY spectrum between H-7ax /H-17, H-7ax /H-22, H-15/H-10, H-15/H-17, and H-18/H-20 suggested an axial orientation of H-6 and
H-15, an
equatorial orientation of H-10, H-17, and H-22, and the E -configuration of the Δ
18,19 double bond. Thus, the structure of 4 was assigned as shown in [Fig. 1 ].
Table 2 1 H (400 MHz) and 13 C (100 MHz) NMR data of compounds 4 and 5 in CDCl3 .
Position
4
5
δ
H
(J in Hz)
δ
C
δ
H (J in Hz)
δ
C
1
198.7
198.9
2
5.99, s
120.2
5.95, s
119.9
3
175.2
175.4
4
62.6
62.5
5
47.4
47.6
6
2.02, m
36.3
1.87, m
36.0
7eq
1.93, m
39.6
1.92, m
39.5
7ax
1.35, t (13.1)
1.34, t (13.0)
8
63.2
62.8
9
208.1
207.7
10
1.71, m, 2.52, m
18.1
2.47, m
18.0
11
1.27, m, 1.71, m
33.7
1.21, m, 1.67, m
33.5
12
83.5
83.5
13
1.23, s
29.5
1.20, s
29.5
14
1.42, s
26.2
1.39, s
26.0
15
0.68, s
17.1
0.61, s
17.1
16
1.39, m, 1.63, m
37.0
1.31, m, 1.56, m
36.8
17
1.69, m, 1.99, m
29.1
0.89, m, 1.36, m
28.1
18
5.05, m
124.2
1.26, m, 1.73, m
32.2
19
137.1
2.33, m
39.9
20
1.96, m
39.9
181.5
21
1.54, s
16.4
1.13, d (7.0)
17.3
22
2.42, d (6.7)
30.1
2.40, m
30.1
23
4.93, m
120.2
4.86, t (6.6)
119.7
24
133.6
133.7
25
1.69, s
26.2
1.62, s
25.8
26
1.66, s
18.1
1.62, s
18.0
27
1.85, m, 2.04, m
23.0
1.73, m, 1.95, m
23.0
28
4.96, m
124.4
4.92, t (6.1)
124.2
29
131.6
131.5
30
1.66, s
25.9
1.59, s
25.9
31
1.57, s
18.0
1.53, s
17.9
32
2.04, m
26.8
33
5.00, m
122.5
34
133.4
35
1.63, s
26.0
36
1.59, s
18.1
Garschomcinol E (5 ) was obtained as a yellow gum with [α ]D
20 + 12.5 (c 0.20, MeOH). Its molecular formula was determined as
C31 H46 O5 from the positive HRESIMS [M + Na]+ ion peak at m /z 521.3233 (calcd. for C31 H46 O5 Na,
521.3243). The NMR data ([Table 2 ]) of 5 were similar with those of 4 except for the geranyl group at C-6, which was replaced by a 2-methylbutanoic
acid group in 5 . The structure of the side chain was confirmed by the presence of a carboxylic acid
carbon at δ
C 181.5 (C-20) in the 13 C NMR spectrum, the
COSY correlations of H-17/H-18, H-18/H-19 and H-19/H-21, and the HMBC correlations
([Fig. 2 ]) of H-17 [δ
H 0.89, 1.36 (2H, m)] with C-5
(δ
C 47.6), C-7 (δ
C 39.5), and C-19 (δ
C 39.9), H-18 [δ
H 1.26, 1.73 (2H, m)] with C-6 (δ
C 36.0), C-20,
and C-21 (δ
C 17.3), H-19 [δ
H 2.33 (1H, m)] with C-17 (δ
C 28.1), C-20, and C-21, and H-21 [δ
H 1.13 (3H, d,
J = 7.0 Hz)] with C-18 (δ
C 32.2), C-19, and C-20. Compound 5 had the same relative configuration as 4 based on the J value and NOESY analysis,
while the configuration at C-19 remains undetermined. Finally, the structure of 5 was determined as shown in [Fig. 1 ].
The absolute configurations of the bicyclo [3.3.1]nonane core in 1 – 5 were assigned by comparison of the experimental ECD data with data reported for related
compounds. The
experimental ECD curves of 1 – 3 (Fig. 6S , Supporting Information) showed similarities to those of 6 and 7
[9 ] (positive Cotton
effect at 200 – 240 nm, negative Cotton effect at 240 – 310 nm, and positive Cotton
effect at 310 – 400 nm), thereby suggesting the 4S , 5S , 6R , 8S absolute
configuration of 1 – 3 . The experimental ECD curves of 4 and 5 (Fig. 6S , Supporting Information) showed a similar pattern to those of
32-hydroxy-ent -guttiferone M [18 ] (two negative high-amplitude Cotton effects at 254 and 320 nm, along with a positive
Cotton effect at 215 nm), which
revealed the 4R , 5S , 6R , 8S absolute configuration of 4 and 5 .
Most isolated compounds showed potent cytotoxicity against cancer cell lines, while
no cytotoxicity was found for 4 and 5 (IC50 > 100 µM) ([Table 3 ]). It is worth noting that compound 6 showed cytotoxicity against all five cancer cell lines including KB, HeLa S3, HT-29,
MCF-7, and Hep G2 with IC50 values
in the range of 5.05 – 7.03 µM. Compounds 1 and 7 exhibited cytotoxicity against four cell lines (KB, HeLa S3, HT-29, and MCF-7) with
IC50 values in the range of
4.06 – 7.89 µM. Compounds 2 and 3 were cytotoxic against three cell lines (KB, HeLa S3, and MCF-7) with IC50 values in the range of 6.24 – 9.37 µM. Compound 10
was cytotoxic against KB and HeLa S3 cells with IC50 values of 2.52 and 6.39 µM, respectively. In addition, compounds 8 and 9 showed significant cytotoxic effects
against KB cells with IC50 values of 5.70 and 5.03 µM, respectively. These data suggest that the presence of
a 3,4-dihydroxybenzoyl group at C-2 might improve the cytotoxicity of
phloroglucinols.
Table 3 In vitro cytotoxicity of compounds 1 – 10 against five human cancer cell lines.
Compounds
IC50 (µM); 95% CI
KB
HeLa S3
HT-29
MCF-7
Hep G2
NT = not tested; a doxorubicin was used as the positive control
1
5.93; 5.36 – 6.49
5.33; 5.15 – 5.50
7.89; 7.28 – 8.50
5.42; 5.00 – 5.84
12.03; 11.39 – 12.66
2
7.13; 6.06 – 8.20
6.24; 4.6#7 – 7.81
12.73; 6.58 – 18.87
6.72; 6.03 – 7.41
14.39; 13.98 – 14.79
3
9.37; 7.33 – 11.41
6.65; 5.80 – 7.49
14.13; 10.64 – 17.63
6.50; 6.30 – 6.70
14.12; 11.96 – 11.28
4
> 100
> 100
NT
NT
NT
5
> 100
> 100
NT
NT
NT
6
5.38; 5.10 – 5.65
5.22; 3.85 – 6.60
5.05; 4.77 – 5.33
5.90; 4.33 – 7.47
7.03; 6.20 – 7.86
7
6.39; 5.91 – 6.88
5.48; 5.45 – 5.51
6.29; 4.63 – 7.95
4.06; 2.90 – 5.22
11.46; 9.34 – 13.58
8
5.70; 4.79 – 6.61
16.09; 15.95 – 16.24
20.80; 20.18 – 21.42
18.13; 14.47 – 21.49
> 100
9
5.03; 4.92 – 5.15
11.05; 10.35 – 11.74
19.17; 18.19 – 20.14
18.29; 9.13 – 27.44
> 100
10
2.52; 2.42 – 2.63
6.39; 5.44 – 7.34
13.48; 9.45 – 17.51
12.12; 2.57 – 21.67
11.20; 9.40 – 13.01
Doxorubicina
0.02; 0.00 – 0.02
0.15; 0.11 – 0.19
0.59; 0.51 – 0.67
1.29; 1.25 – 1.34
1.00; 0.57 – 1.43
Material and Methods
General experimental procedures
UV-visible absorption spectra were recorded on a UV-2550 UV-vis spectrometer. IR spectra
were measured on a Nicolet 6700 FT-IR spectrometer using KBr discs. Optical rotations
were measured
with a Jasco P-1010 polarimeter. NMR spectra were recorded on a Bruker 400 AVANCE
spectrometer (400 MHz for 1 H and 100 MHz for 13 C). The HRESIMS were obtained using a
Bruker MICROTOF model mass spectrometer. ECD data were recorded on a JASCO J-815 spectropolarimeter.
Silica gel 60 G, silica gel 70 – 230 mesh, silica gel RP-C18 40 – 63 µm, and Sephadex
LH-20 (all Merck) were used for column chromatography.
Plant material
Branches of G. schomburgkiana were collected in January 2020 from Pho Si Suwan district, Sisaket province, Thailand
(15°16′55″ N 104°01′40″ E). The plant material was identified by
Dr. Suttira Sedlak, botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham
University. A voucher specimen (Khumkratok no. 92 – 08) was deposited at Mahasarakham
University.
Extraction and isolation
The air-dried branches of G. schomburgkiana (10.0 kg) were ground and then macerated with CH2 Cl2 over a period of 5 days at room temperature with 2 × 15 L.
Removal of the solvent under reduced pressure provided the CH2 Cl2 crude extract (120.0 g), which was further separated by column chromatography (45 × 10 cm,
i. d.) over
silica gel with a gradient of n -hexane-EtOAc (1 : 0, 8 : 2, 6 : 4, 4 : 6, 2 : 8, each 5 L) to give 12 fractions (A – L).
Fraction C (1.5 g) was purified by a silica gel RP-C18 column
(55 × 3 cm, i. d.) with H2 O-MeOH (2 : 8, 1 L) to afford compound 4 (6.5 mg). Fraction D (6.2 g) was applied to a Sephadex LH-20 column (75 × 5 cm, i. d.)
with
CH2 Cl2 -MeOH (8 : 2, 2 L) and further purified by a silica gel RP-C18 column (55 × 3 cm,
i. d.) with H2 O-MeOH (2 : 8, 1 L) to obtain compounds 8
(8.5 mg), 9 (4.2 mg), and 10 (6.0 mg). Compounds 3 (10.8 mg), 6 (15.5 mg), and 7 (16.2 mg) were obtained from fraction E (10.5 g) by chromatography on a
Sephadex LH-20 column (75 × 5 cm, i. d.) with CH2 Cl2 -MeOH (8 : 2, 2 L) followed by a silica gel RP-C18 column (55 × 3 cm, i. d.) with
H2 O-MeOH (2 : 8, 1 L).
Compound 2 (12.5 mg) was isolated from fraction F (3.0 g) using a silica gel RP-C18 column (55 × 3 cm,
i. d.) with H2 O-MeOH (2 : 8, 1 L). Finally, Fraction G (3.5 g) was
subjected to a Sephadex LH-20 column (75 × 5 cm, i. d.) using CH2 Cl2 -MeOH (8 : 2, 2 L) to provide compounds 1 (15.5 mg) and 5 (7.5 mg).
Garschomcinol A (1 ): yellow gum; [α ]D
20 + 12.5 (c 0.20, MeOH); UV (MeOH) λ
max (log ε ) 324 (0.2), 257 (0.5), and
242 (0.5) nm.; IR (KBr) ν
max 3425, 1720, 1665 cm−1 ; ECD (c 0.05, MeOH) λ
max (Δε ) 330 (+ 4.9), 250 (− 21.0), 220 (+ 25.2) nm.;
1 H and 13 C NMR data, see [Table 1 ]; HRESIMS (positive ion mode) m/z 711.4233 [M + Na]+ (calcd. for
C43 H60 O7 Na, 711.4237).
Garschomcinol B (2 ): yellow gum; [α ]D
20 + 13.5 (c 0.28, MeOH); UV (MeOH) λ
max (log ε ) 326 (0.3), 253 (0.4), and
240 (0.5) nm.; IR (KBr) ν
max 3428, 1728, 1645 cm−1 ; ECD (c 0.05, MeOH) λ
max (Δε ) 332 (+ 5.7), 252 (− 32.2), 221 (+ 38.0) nm.;
1 H and 13 C NMR data, see [Table 1 ]; HRESIMS (positive ion mode) m/z 725.4382 [M + Na]+ (calcd. for
C44 H62 O7 Na, 725.4393).
Garschomcinol C (3 ): yellow gum; [α ]D
20 + 14.7 (c 0.34, MeOH); UV (MeOH) λ
max (log ε ) 318 (0.1), 260 (0.3), and
238 (0.2) nm.; IR (KBr) ν
max 3423, 1727, 1647 cm−1 ; ECD (c 0.05, MeOH) λ
max (Δε ) 331 (+ 5.2), 250 (− 25.8), 220 (+ 30.3) nm.;
1 H and 13 C NMR data, see [Table 1 ]; HRESIMS (positive ion mode) m/z 739.4524 [M + Na]+ (calcd. for
C45 H64 O7 Na, 739.4550).
Garschomcinol D (4 ): yellow gum; [α ]D
20 + 13.1 (c 0.30, MeOH); UV (MeOH) λ
max (log ε ) 328 (0.3), 259 (0.4), and
246 (0.2) nm.; IR (KBr) ν
max 1719, 1653, 1642 cm−1 ; ECD (c 0.05, MeOH) λ
max (Δε ) 316 (− 18.7), 254 (− 39.7), 212 (+ 49.9) nm.;
1 H and 13 C NMR data, see [Table 2 ]; HRESIMS (positive ion mode) m/z 573.3786 [M + K]+ (calcd. for
C36 H54 O3 K, 573.3710).
Garschomcinol E (5 ): yellow gum; [α ]D
20 + 12.5 (c 0.20, MeOH); UV (MeOH) λ
max (log ε ) 318 (0.2), 245 (0.2), and
238 (0.1) nm.; IR (KBr) ν
max 1725, 1648, 1635 cm−1 ; ECD (c 0.05, MeOH) λ
max (Δε ) 316 (− 22.7), 253 (− 52.5), 208 (+ 72.2) nm.;
1 H and 13 C NMR data, see [Table 2 ]; HRESIMS (positive ion mode) m/z 521.3233 [M + Na]+ (calcd. for
C31 H46 O5 Na, 521.3243).
Cytotoxicity assay
The cytotoxicity of compounds 1 – 10 was evaluated using the MTT colorimetric method against KB, HeLa S3, HT-29, MCF-7,
and Hep G2 cell lines as previously reported [19 ], with doxorubicin as the positive control. The cancer cells were cultured in 100 µL/well
of MEM containing 10% fetal bovine serum and 1% streptomycin-penicillin,
seeded in a 96-well plate (3000 cells/well), and preincubated in a 5% CO2 incubator at 37 °C for 24 h. Various concentrations of the sample, DMSO as the negative
control, and the
positive control (10 µL/well) were added, and then incubated for 72 h under the above
conditions. The supernatant was removed and 100 µL of MTT solution (0.5 mg/mL) were
added into each well
and further incubated for 3 h. The supernatant was decanted and DMSO (100 µL/well)
was added to dissolve Formosan, which was measured at 550 nm by a microplate reader.
The tests were
performed in triplicate. The IC50 value was calculated by curve fitting with SigmaPlot 10 (Systat Software Inc.) and
the 95% confidence interval for the mean values was identified
by using IBM SPSS Amos 19 (SPSS Inc.).
