Oleanane-type Saponins from Glochidion hirsutum and Their Cytotoxic Activities

Article abstract of DOI:10.1002/cbdv.201600445

Five new oleanane-type saponins, hirsutosides A?–?E, were isolated from the leaves of Glochidion hirsutum (Roxb.) Voigt. Their?structures were elucidated as 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-d-glucopyranoside (1),?21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-d-glucopyranosyl-(1?→?3)-β-d-glucopyranoside (2), 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-6-acetyl-[β-d-glucopyranosyl-(1?→?3)]-β-d-glucopyranoside (3), 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-d-glucopyranosyl-(1?→?3)-〈-l-arabinopyranoside (4), and 21β-benzoyloxy-3β,16β,23-trihydroxyolean-12-ene-28-al 3-O-β-d-glucopyranosyl-(1?→?3)-α-l-arabinopyranoside (5). All isolated compounds were evaluated for cytotoxic activities on four human cancer cell lines, HepG-2, A-549, MCF-7, and SW-626 using the SRB assay. Compounds 1, 2, 4, and 5 showed significant cytotoxic activities against all human cancer cell lines with IC₅₀ values ranging from 3.4 to 10.2?μm. Compound 3 containing acetyl group at glc C(6″) exhibited weak cytotoxic activity with IC₅₀ values ranging from 47.0 to 54.4?μm.

Full text of DOI:10.1002/cbdv.201600445

Received Date: 21-Nov-2016
Revised Date: 10-Mar-2017
Accepted Date: 14-Mar-2017
Article Type: Full Paper
Oleanane-type Saponins from Glochidion hirsutum and Their Cytotoxic Activities
Nguyen Van Thanga), Vu Kim Thub), Nguyen Xuan Nhiema), Duong Thi Dunga), Tran Hong
Quanga), Bui Huu Taia), Hoang Le Tuan Anha), Pham Hai Yena), Nguyen Thi Thanh Nganc),
Nguyen Huy Hoangc), and Phan Van Kiema,* )
aInstitute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang
Quoc Viet, Caugiay, Hanoi, Vietnam.
bFaculty of Basic Sciences, Hanoi University of Mining and Geology, Tuliem, Hanoi,
Vietnam.
cInstitute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang
Quoc Viet, Caugiay, Hanoi, Vietnam.
Corresponding Author:
* Tel.: 84-4-37560793; fax: 84-4-7564390. E-mail address: phankiem@yahoo.com
Abstract
Five new oleanane-type saponins, hirsutosides A-E, were isolated from the leaves of
Glochidion hirsutum (Roxb.) Voigt. Their structures were elucidated as 21β-benzoyloxy-
3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-D-glucopyranoside (1), 21β-benzoyloxy-
3β,16β,23,28-tetrahydroxyolean-12-ene
3-O-β-D-glucopyranosyl-(1→3)-β-D-
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201600445
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glucopyranoside (2), 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-6-acetyl-
[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside (3), 21β-benzoyloxy-3β,16β,23,28-
tetrahydroxyolean-12-ene 3-O-β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside (4), and
21β-benzoyloxy-3β,16β,23-trihydroxyolean-12-ene-28-al 3-O-β-D-glucopyranosyl-(1→3)-α-
L-arabinopyranoside (5). All isolated compounds were evaluated for cytotoxic activities on
four human cancer cell lines, HepG-2, A-549, MCF-7, and SW-626 using the SRB assay.
Compounds 1, 2, 4, and 5 showed significant cytotoxic activities against all human cancer
cell lines with IC50 values ranging from 3.4 to 10.2 μM. Compound 3 containing acetyl group
at glc C(6″) exhibited weak cytotoxic activity with IC50 values ranging from 47.0 to 54.4 μM.
Keywords: Glochidion hirsutum, Euphorbiaceae, hirsutosides A-E, oleanane-type saponin,
cytotoxic activity.
Introduction
Cancer is a group of diseases characterized by the uncontrolled growth and spread of
abnormal cells. Accordingly, the development of new and efficient anticancer drugs has been
interested from the scientists around the world. Natural products are potential sources of
novel drugs with a broad range of biological and pharmacological activities, including
anticancer activities.[1] There are more than 60% of currently anticancer drugs from natural
sources.[2] Moreover, many oleanane triterpene-type saponins from various plants such as
Bolbostemma paniculatum,[3] Platycodon grandiflorum,[4] Glochidion eriocarpum[5] exhibited
cytotoxic activities.
Glochidion is a large genus of the Euphorbiaceae family, comprising more than 250 species
in the world. Glochidion hirsutum (Roxb.) Voigt is a shrub or small tree distributed
throughout Southeast Asia. The leaves of G. hirsutum have been used in folk medicine to
treat toothaches; the roots are used as medicine for rheumatism and pneumonia.[6]
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Phytochemical studies of G. hirsutum have shown the presence of flavonols.[7, 8] Previous our
investigation program on cytotoxic constituents of Glochidion genus identified cytotoxic
oleanane saponins from G. eriocarpum[5] and G. glomerulatum.[9, 10] Herein, we reported the
isolation, structural elucidation of oleanane-type saponins from the leaves of G. hirsutum, and
their cytotoxic activity against four human cancer cell lines, HepG-2, A-549, MCF-7, and
SW-626.
Results and Discussion
Structure Elucidation
The methanol extract of the G. hirsutum leaves was suspended in water and then partitioned
with CH2Cl2 and EtOAc to obtain three layers. The EtOAc layer was separated using a
combination of silica gel and RP-18 column chromatographic steps to afford five new
oleanane-type saponins (Fig. 1). Their structures were elucidated by extensive spectroscopic
methods including 1D and 2D NMR experiments, as well as by HR ESI MS analysis.
Compound 1 was obtained as a white amorphous powder and its molecular formula was
determined as C43H64O11 by HR ESI MS ion at m/z 779.4370 [M + Na]+ (calcd for
1
C43H64O11Na, 779.4346). The H NMR spectrum of 1 showed the signals of six methyl
groups at δ(H) 0.75, 0.96, 1.04, 1.06, 1.17, and 1.34 (each, 3H, s) and one olefinic proton at
δ(H) 5.37 (1H, t, J = 3.0 Hz), which indicated an oleanane aglycone. In addition to these,
protons of a benzoyloxy were observed at δ(H) 7.51 (2H, t, J = 8.0 Hz), 7.62 (1H, t, J = 8.0
Hz), and 8.04 (2H, d, J = 8.0 Hz). One anomeric proton at δ(H) 4.43 (1H, d, J = 8.0 Hz)
showed the presence of a sugar moiety. The 13C NMR and DEPT spectra of 1 showed the
presence of 43 carbons, including 1 carbonyl, 8 quaternary carbons, 17 methines,11
methylenes, and 6 methyl carbons (Table 1). The analysis of 1H and 13C NMR spectroscopic
data indicated that the aglycone of 1 was similar to those of 21β-(benzoyloxy)olean-12-ene-
3β,16β,23,28-tetraol, an oleanane-type triterpene isolated from Glochidion assamicum.[11]
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The HMBC correlations between H-C(18) (δ(H) 2.51) and C(13) (δ(C) 143.0)/C(16) (δ(C)
67.9)/C(17) (δ(C) 44.7)/C(28) (δ(C) 66.6) as well as the COSY correlations between H-C(15)
(δ(H) 1.40-1.46 and 1.80-1.84) and H-C(16) (δ(H) 4.36) confirmed the positions of two
hydroxyl groups at C(16) and C(28) (Fig. 2). The β configuration (equatorial orientation) of
the hydroxyl group at C(16) was confirmed by NOESY correlations between H-C(16) (δ(H)
4.36) and H-C(27) (δ(H) 1.34)/Hα- C(19) (δ(H) 2.10) as well as by the coupling constant of
H-C(15) and H-C(16), Jeq-ax = 5.0 Hz and Jax-ax = 12.0 Hz. The location of a oxygenated group
at C(21) was assigned based on the HMBC correlations between H-C(29) (δ(H) 0.96)/H-C(30)
(δ(H) 1.17) and C(19) (δ(C) 48.0)/C(20) (δ(C) 36.6)/C(21) (δ(C) 78.2). Furthermore, the
esterification location of benzoic acid at C-21 was confirmed by HMBC correlation between
H-C(21) (δ(H) 5.16) and Bz C(7ʹ) (δ(C) 167.9). The configuration of the benzoyloxy group
was determined as β by the NOESY observations between H-C(21) (δ(H) 5.16) and Hα-C(19)
(δ(H) 2.08-2.12)/H-C(29) (δ(H) 0.96). The HMBC correlations from H-C(3) (δ(H) 3.67) to
C(4) (δ(C) 43.9)/C(5) (δ(C) 48.1)/C(23) (δ(C) 64.8)/C(24) (δ(C) 13.4), from H-C(23) (δ(H)
3.31 and 3.67)/H-C(24) (δ(H) 0.75) to C(3) (δ(C) 83.3)/C(4) (δ(C) 43.9)/C(5) (δ(C) 48.1)
suggested the location of the oxygenated and hydroxyl groups at C(3) and C(23), respectively.
The α-orientations of H-C(3) and the hydroxylmethylene group at C(4) were determined by
the NOESY observation of H-C(3) (δ(H) 3.67) and H-C(5) (δ(H) 1.63-1.69)/H-C(23) (δ(H)
3.31 and 3.67) and of H-C(24) (δ(H) 0.75) and H-C(25) (δ(H) 1.04). Acid hydrolysis of 1
afforded D-glucose as sugar component (identified as TMS derivatives by GC). Also, the
HMBC correlation from glc H-C(1″) (δ(H) 4.43) to C(3) (δ(C) 83.3) confirmed the location
of glucopyranosyl moiety at C(3). Consequently, the structure of 1 was elucidated to be 21β-
benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-D-glucopyranoside and named
hirsutoside A.
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The molecular formula of 2 was determined as C49H74O16 by the HR ESI MS ion at m/z
1
941.4896 [M + Na]+ (calcd for C49H74O16Na, 941.4875). The H and 13C NMR spectra
exhibited the presence of one oleanane aglycone, one benzoyloxy, and two sugar moieties
(Table 1). The NMR data of 2 were similar to those of hirsutoside A (1), except for the
addition of a sugar moiety at glc C(3ʺ). The aglycone was recognized to be 21β-(benzoyloxy)
olean-12-ene-3β,16β,23,28-tetraol.[11] The configurations of functional groups for aglycone 2
were similar to those of 1, confirmed by NOESY experiments. Acid hydrolysis of 2 gave D-
glucose (identified as TMS derivatives by GC). The HMBC correlations between glc H-C(1‴)
(δ(H) 4.57) and glc C(3″) (δ(C) 88.3); glc H-C(3″) (δ(H) 3.55) and glc C(1‴) (δ(C) 105.3)
confirmed the sequence of sugar linkages as 3-O-β-D-glucopyranosyl (1→3)-β-D-
glucopyranoside. The sequence of sugar linkages at C(3) of aglycone was proved by HMBC
correlations between glc H-C(1″) (δ(H) 4.49) and C(3) (δ(C) 83.4). Consequently, the
structure of 2 was determined as 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-
O-β-D-glucopyranosyl-(1→3)-β-D-glucopyranoside and named hirsutoside B.
The molecular formula of 3 was deduced as C51H76O17 by the HR ESI MS ion at m/z
983.4965 [M + Na]+ (calcd for C51H76O17Na, 983.4980). Analysis of the NMR data of 3
indicated that the structure of aglycone was similar to those of 1 and 2. The HMBC
correlations between glc H-C(1‴) (δ(H) 4.53) and glc C(3″) (δ(C) 83.4); between glc H-C(6″)
(δ(H) 4.18 and 4.36) and acetyl group (δ(C) 172.8) confirmed the sugar linkages to be 3-O-6-
acetyl-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside. Moreover, the sequence of sugar
linkages was located at C(3) of aglycone by HMBC correlation between glc H-C(1″) (δ(H)
4.68) and C(3) (δ(C) 83.3). Based on the above evidence, compound 3 was defined as 21β-
benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene
(1→3)]-β-D-glucopyranoside and named hirsutoside C.
3-O-6-acetyl-[β-D-glucopyranosyl-
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Compound 4 was also obtained as a white amorphous powder. The molecular formula was
determined as C48H72O15 by the HR ESI MS ion peak at m/z 911.4779 [M + Na]+ (calcd for
1
C48H72O15Na, 911.4769). The H, 13C NMR, and DEPT spectra of compound 4 showed one
olean-12-ene triterpene aglycone, one benzoyloxy, and two sugar moieties (Table 2). The
aglycone of 4 was found to be similar to those of hirsutoside A (1). Acid hydrolysis and GC
analysis of 4 confirmed the presence of D-glucose and L-arabinose. In addition, the coupling
constants of ara H-C(1″) and ara H-C(2″), J = 7.5 Hz; glc H-C(1‴) and glc H-C(2‴), J = 8.0
Hz, confirmed the configurations of the O-glycoside bonds as β-D-glucopyranosyl and α-L-
arabinopyranosyl. The sequence of sugar linkages was as 3-O-β-D-glucopyranosyl-(1→3)-α-
L-arabinopyranoside confirming by the HMBC correlation from glc H-C(1‴) (δ(H) 4.57) to
ara C(3″) (δ(C) 84.2). Furthermore, the location of sugar at C(3) of aglycone was confirmed
by the HMBC correlation between ara H-C(1″) (δ(H) 4.38) and C(3) (δ(C) 83.3).
Consequently, compound 4 was defined as 21β-benzoyloxy-3β,16β,23,28-tetrahydroxyolean-
12-ene 3-O-β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside and named hirsutoside D.
The molecular formula of compound 5 was determined as C48H70O15 by the HR ESI MS ion
at m/z 909.4602 [M + Na]+ (calcd for C48H70O15Na, 909.4612). Analysis of the NMR data of
5 indicated that the structure of 5 was similar to those of 4 except for the presence of
aldehydic group instead of hydroxylmethylene at C(17). The HMBC correlations from H-
C(16) (δ(H) 4.45)/H-C(22) (δ(H) 1.45 and 2.46) to C(28) (δ(C) 207.4); from H-C(18) (δ(H)
2.92) to C(12) (δ(C) 125.2)/C(13) (δ(C) 142.1)/C(16) (δ(C) 66.1)/C(17) (δ(C) 42.7)
confirmed the positions of hydroxyl and aldehyde groups at C(16) and C(17), respectively. In
addition, the β configuration of the hydroxyl group at C(16) was confirmed by NOESY
correlations between H-C(16) (δ(H) 4.45) and H-C(27) (δ(H) 1.32). The location of a
benzoyloxy group at C(21) was proved by the HMBC correlations between H-C(29) (δ(H)
0.99)/H-C(30) (δ(H) 1.17) and C(19) (δ(C) 47.8)/C(20) (δ(C) 36.5)/C(21) (δ(C) 77.1);
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between H-C(21) (δ(H) 5.15) and Bz C(7ʹ) (δ(C) 167.7). The configuration of this
benzoyloxy group was determined as β by the NOESY observations between H-C(21) (δ(H)
5.15) and H-C(29) (δ(H) 0.99). The orientation of remaining functional groups of aglycone
were based on NOESY experiments and coupling constant analysis. The position and
sequence of sugar linkages were similar to those of compound 4. Consequently, the structure
of 5 was elucidated to be 21β-benzoyloxy-3β,16β,23-trihydroxyolean-12-ene-28-al 3-O-β-D-
glucopyranosyl-(1→3)-α-L-arabinopyranoside, and named hirsutoside E.
Biological Studies
All compounds were evaluated against four human cancer cell lines, HepG-2 (human liver
hepatocellular carcinoma), A-549 (human lung carcinoma), MCF-7 (human breast carcinoma)
and SW-626 (human ovarian carcinoma) using the SRB assay. Ellipticine, an anticancer
agent, was used as a positive control with IC50 values ranging from 1.4 to 2.1 μM for all the
human cancer cell lines (Table 3).
Comparing to ellipticine, compounds 1, 2, 4, and 5 showed significant cytotoxic activities
against all human cancer cells with IC50 values ranging from 3.4 to 10.2 μM. Compound 3
containing acetyl group at glc C(6″) exhibited weak cytotoxic activity with IC50 values
ranging from 47.0 to 54.4 μM. In the structure-activity relationship of isolated compounds 1-
3, when additional sugar moiety at glc C(3″) (2), the cytotoxic activity exhibited stronger,
however, when acetyl group at glc C(6″) (3) the cytotoxic activity decreased. The current
study demonstrated that the cytotoxic activity of 2 on all tested human cancer cell lines
comparable to those of ellipticine. This work has thus provided a further example of the
importance of oleanane-type saponins contain a benzoyloxy group at C(21) as potential
anticancer agents.
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Experimental Section
General
Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. The NMR
spectra were recorded using a Bruker DRX 500 spectrometer (1H, 500 MHz; 13C, 125 MHz).
The HR-ESI-MS were obtained using an Agilent 6550 iFunnel Q-TOF LC/MS system.
Column chromatography was performed using silica-gel (Kieselgel 60, 70-230 mesh and
230-400 mesh, Merck) or RP-18 resins (30 - 50 μm, Fujisilisa Chemical Ltd.), and thin layer
chromatography (TLC) was performed using a pre-coated silica-gel 60 F254 (0.25 mm, Merck)
and RP-18 F254S plates (0.25 mm, Merck).
Plant Material
The leaves of Glochidion hirsutum (Roxb.) Voigt were collected in Sondong, Bacgiang,
Vietnam in December 2012 and identified by Dr. Nguyen The Cuong, Institute of Ecology
and Biological Resources, VAST. A voucher specimen (GH1212) was deposited at the
Herbarium of the Institute of Marine Biochemistry, Hanoi, Vietnam.
Extraction and Isolation
Dried leaves of G. hirsutum (4.0 kg) were sonicated in MeOH (7 L × 3 times) for 15 h to
yield a MeOH extract (355 g) after evaporating under reduced pressure. The MeOH extract
was suspended in H2O and successively partitioned with CH2Cl2 and EtOAc to obtain CH2Cl2
(GH1, 120.0 g), EtOAc (GH2, 50.0 g), and H2O (GH3, 180.0 g) layers after removal of the
solvents in vacuo. The GH2 layer was applied to a silica gel CC eluted with a gradient elution
of CH2Cl2 – MeOH (100:1, 30:1, 10:1, 5:1, 1:1, 0:1, v/v) to give six smaller fractions, GH2A
(4.0 g), GH2B (3.1 g), GH2C (3.6 g), GH2D (8.5 g), GH2E (4.3 g), and GH2F (7.0 g). The
GH2C fraction was applied to a silica gel CC eluting with CH2Cl2 – MeOH (8:1, v/v) to give
two fractions, GH2C1 and GH2C2. The GH2C1 fraction was further purified by silica gel CC
eluting with EtOAc – MeOH – H2O (12:1:0.01, v/v/v) to yield 1 (12.0 mg). The GH2D
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fraction was subjected to a silica gel CC eluting with CH2Cl2 –MeOH – H2O (5:1:0.1, v/v/v)
to give three smaller fractions, GH2D1-GH2D3. The GH2D1 fraction was applied to a silica
gel CC eluting with EtOAc – MeOH – H2O (6:1:0.01, v/v/v) to yield compound 4 (18.0 mg).
The GH2D2 fraction was subjected to an RP-18 CC eluting with MeOH – H2O (3.5:1, v/v) to
yield compound 5 (32.0 mg). The GH2D3 fraction was applied to a silica gel CC, eluted with
CH2Cl2 – MeOH – H2O (6:1:0.05, v/v/v) to yield compound 3 (21.0 mg). Compound 2 (9.0
mg) was obtained from GH2E fraction, using a silica gel CC eluting with CH2Cl2 – MeOH –
H2O (6:1:0.05, v/v/v) then an RP-18 CC eluent of MeOH – H2O (3:1, v/v). The purity of the
compounds was assessed by HPLC-DAD at 210 nm as > 95%.
21β-Benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-D-glucopyranoside (1):
1
White amorphous powder; [α]25 = +30.0 (c 0.1, MeOH); H (CD3OD, 500 MHz) and 13C
D
NMR (CD3OD, 125 MHz) data, see Table 1; C43H64O11, HR ESI MS m/z: 779.4370 [M +
Na]+ (calcd for C43H64O11Na, 779.4346).
21β-Benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-D-glucopyranosyl-(1→3)-
1
β-D-glucopyranoside (2): White amorphous powder; [α]2D5 = + 50.0 (c 0.1, MeOH); H
(CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1; C49H74O16, HR ESI
MS m/z: 941.4896 [M + Na]+ (calcd for C49H74O16Na, 941.4875).
21β-Benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene
3-O-6-acetyl-[β-D-
glucopyranosyl-(1→3)]-β-D-glucopyranoside (3): White amorphous powder; [α]2D5 = ‒20.0
(c 0.1, MeOH); 1H (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1;
C51H76O17, HR ESI MS m/z: 983.4965 [M + Na]+ (calcd for C51H76O17Na, 983.4980).
21β-Benzoyloxy-3β,16β,23,28-tetrahydroxyolean-12-ene 3-O-β-D-glucopyranosyl-(1→3)-
1
α-L-arabinopyranoside (4): White amorphous powder; [α]2D5 = + 41.0 (c 0.1, MeOH); H
(CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 2; C48H72O15, HR ESI
MS m/z: 911.4779 [M + Na]+ (calcd for C48H72O15Na, 911.4769).
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21β-Benzoyloxy-3β,16β,23-trihydroxyolean-12-ene-28-al 3-O-β-D-glucopyranosyl-
(1→3)-α-L-arabinopyranoside (5): White amorphous powder; [α]2D5 = ‒9.0 (c 0.1, MeOH);
1H (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 2; C48H70O15, HR
ESI MS m/z: 909.4602 [M + Na]+ (calcd for C48H70O15Na, 909.4612).
Acid Hydrolysis
Each compound (1-5, 2.0 mg) was separately dissolved in 1.0 N HCl (dioxane - H2O, 1:1, v/v,
1.0 mL) and heated to 80 °C in a water bath for 3 h. The solvent in acidic solution was
removed under an N2 stream. After extraction with CHCl3, the aqueous layer was
concentrated to dryness using N2. The residue was dissolved in dry pyridine (0.1 mL),
followed by addition of L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL).
The reaction mixture was heated at 6 °C for 2 h. Trimethylsilylimidazole solution (0.1 mL)
was then added, followed by heating at 60 °C for 1.5 h. The dried product was partitioned
with n-hexane and H2O (0.1 mL each), and the organic layer was analyzed by gas
chromatography (GC): column DB-5 (0.32 mm ID × 30 m length), detector FID, column
temp 210 °C, injector temp 270 °C, detector temp 300 °C, carrier gas He (2 mL/min). Under
these conditions, the standard sugars gave peaks at tR (min) 14.11 and 14.26 for D- and L-
glucose, 9.82 and 15.24 for D- and L-arabinose, respectively. Peaks at tR (min) 14.11 of D-
glucose for 1 ‒ 3; 14.11 and 15.24 of D-glucose and L-arabinose for 4 and 5, were observed.
Cytotoxic Assay
Tumour cells were cultivated in a humidified atmosphere of 5% CO2 at 37 °C for 48 h. Cell
viability was examined by SRB method for the determination of cell density, based on the
measurement of cellular protein content. Viable cells were seeded in the growth medium (180
μL) into 96-well microwell plates (4 × 104 cells per well) and allowed to attach overnight.
Test samples were added carefully into wells of 96-well plates and the cultivation was
continued under the same conditions for another 48 h. Thereafter, the medium was removed
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and the remaining cell monolayers are fixed with the cold 20% (w/v) trichloroacetic acid for
1 h at 4 °C and stained by 1X SRB staining solution at room temperature for 30 min, after
which the unbound dye was removed by washing repeatedly with 1% (v/v) acetic acid. The
proteinbound dye is dissolved in 10 mM Tris base solution for OD determination at 515 nm
on an ELISA Plate Reader (Bio-Rad). DMSO 10% was used as blank sample while
ellipticine was used as positive control. The cytotoxicity was measured at doses of 100.0,
20.0, 4.0, and 0.8 μM and estimated as a half maximal inhibitory concentration (IC50), which
was calculated by the program TableCurve Version 4.0. All experiments were prepared in
triplicates. The inhibition rate (IR) of cells was calculated by the following formula IR% =
[100 – (absorbancet – absorbance0)/(absorbancec – absorbance0)] × 100%, where IR indicates
inhibition rate of cell growth, absorbancet indicates average optical density value at day 2;
absorbance0 indicates average optical density value at time-zero and absorbancec indicates
average optical density value of the blank DMSO control sample.
Supplementary Material
1D and 2D NMR, and HR ESI MS are included for all compounds. Supplementary data
associated with this article can be found, in the online version, at
http://dx.doi.org//10.1002/cbdv.xxx
Notes
The authors declare no competing financial interest.
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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1 – 3
1
2
3
C
Aglycone
1
δ(C) δ(H) (mult., J, in Hz)
δ(C) δ(H) (mult., J, in Hz)
δ(C) δ(H) (mult., J, in Hz)
39.6 0.98 – 1.02 (m)
1.64 – 1.68 (m)
26.3 1.70 – 1.76(m)
1.95 – 2.01 (m)
83.3 3.67 (dd, 3.5, 13.0)
43.9 -
48.1 1.63 – 1.69 (m)
18.8 1.42 – 1.46 (m)
1.55 – 1.59 (m)
33.3 1.33 – 1.39 (m)
1.72 – 1.76 (m)
41.1 -
39.6 1.01 – 1.05 (m)
1.64 – 1.70 (m)
26.3 1.75 – 1.79 (m)
1.95 – 2.01 (m)
83.4 3.68 (m)
39.7 0.99 – 1.03 (m)
1.64 – 1.68(m)
26.4 1.74 – 1.80 (m)
1.97 – 2.03 (m)
83.3 3.65 (dd, 4.5, 12.0)
44.1 -
47.8 1.63 – 1.70 (m)
18.9 1.42 – 1.46 (m)
1.52 – 1.56 (m)
33.3 1.35 – 1.41 (m)
1.72 – 1.79 (m)
41.1 -
2
3
4
5
43.9 -
48.2 1.64 – 1.70 (m)
18.9 1.45 – 1.49 (m)
1.55 – 1.59(m)
33.3 1.35 – 1.41 (m)
1.73 – 1.79 (m)
41.1 -
6
7
8
9
48.1 1.25 – 1.30 (m)
48.5 1.25 – 1.30 (m)
48.2 1.25 – 1.30 (m)
10
11
12
13
14
37.5 -
37.5 -
37.5 -
24.7 1.94 – 2.00 (m)
124.9 5.37 (t, 3.0)
143.0 -
24.7 1.94 – 2.00 (m)
124.9 5.37 (br s)
143.0 -
24.7 1.94 – 2.00 (m)
125.0 5.37 (t, 3.0)
143.0 -
44.6 -
44.6 -
44.6 -
36.5 1.40 – 1.46 (m)
1.80 – 1.84 (m)
67.9 4.36 (dd, 5.0, 12.0)
44.7 -
43.6 2.51 (dd, 4.5, 14.0)
48.0 1.30 – 1.36 (m)
2.08 – 2.12 (m)
36.6 -
78.2 5.16 (dd, 5.0, 12.0)
30.2 1.73 (dd, 12.0, 13.5)
2.39 (dd, 5.0, 13.5)
64.8 3.31 (d, 13.0)
3.67 (d, 13.0)
36.5 1.40 – 1.46 (m)
1.81 – 1.85 (m)
67.9 4.36 (dd, 5.0, 12.0)
44.8 -
43.6 2.52 (dd, 4.5, 13.5)
48.0 1.30 – 1.35 (m)
2.00 – 2.06 (m)
36.6 -
78.2 5.16 (dd, 4.5, 12.0)
30.2 1.73 (dd, 12.0, 13.5)
2.38 (dd, 4.5, 13.5)
65.0 3.32 (d, 13.0)
3.68 (d, 13.0)
36.5 1.40 – 1.46 (m)
1.80 – 1.84 (m)
67.9 4.36 (dd, 5.0, 12.0)
44.8 -
43.7 2.52 (dd, 4.5, 13.0)
48.0 1.30 – 1.35 (m)
2.00 – 2.06 (m)
36.6 -
78.2 5.16 (dd, 5.0, 12.5)
30.2 1.73 (dd, 12.5, 13.5)
2.38 (dd, 4.5, 13.5)
64.3 3.23 (d, 11.0)
3.83 (d, 11.0)
15
16
17
18
19
20
21
22
23
24
25
26
27
28
13.4 0.75 (s)
16.6 1.04 (s)
17.5 1.06 (s)
27.4 1.34 (s)
66.6 3.42 (d, 11.0)
3.73 (d, 11.0)
13.4 0.75 (s)
16.6 1.05 (s)
17.5 1.07 (s)
27.4 1.32 (s)
66.6 3.42 (d, 10.5)
3.73 (d, 10.5)
13.5 0.71 (s)
16.6 1.04 (s)
17.5 1.06 (s)
27.4 1.32 (s)
66.6 3.41 (d, 11.0)
3.72 (d, 11.0)
29
30
29.4 0.96 (s)
18.9 1.17 (s)
29.4 0.96 (s)
18.9 1.17 (s)
29.4 0.96 (s)
18.9 1.17 (s)
21-O-Bz
134.2 -
134.2 -
134.2 -
1′
130.4 8.04 (d, 8.0)
129.6 7.51 (t, 8.0)
131.9 7.62 (t, 8.0)
167.9 -
130.4 8.05 (d, 8.0)
129.6 7.51 (t, 8.0)
131.9 7.63 (t, 8.0)
167.8 -
130.4 8.05 (d, 8.0)
129.6 7.51 (t, 8.0)
131.9 7.63 (t, 8.0)
167.8 -
172.8 -
20.9 2.08 (s)
2′, 6′
3′, 5′
4′
7′
CH3CO
CH3CO
3-O-Glc
1″
105.7 4.43 (d, 8.0)
105.3 4.49 (d, 8.0)
105.3 4.68 (d, 7.5)
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2″
3″
4″
5″
75.6 3.20 (t, 8.0)
74.9 3.38 – 3.42 (m)
88.3 3.53 – 3.57 (m)
70.0 3.40 – 3.46 (m)
77.4 3.30 – 3.34 (m)
62.4 3.71 (dd, 6.0, 11.5)
3.90 (dd, 2.0, 11.5)
76.2 3.26 – 3.30 (m)
83.4 3.43 – 3.49 (m)
71.3 3.30 – 3.36 (m)
77.6 3.26 – 3.32 (m)
64.9 4.18 (dd, 5.0, 12.0)
4.36 (dd, 2.0, 12.0)
77.7 3.34 – 3.38 (m)
71.6 3.29 – 3.33 (m)
78.3 3.28 – 3.32 (m)
62.7 3.69 (dd, 4.5, 12.0)
3.86 (dd, 2.0, 12.0)
6″
3″-O-Glc
1‴
2‴
3‴
4‴
5‴
105.3 4.57 (d, 8.0)
75.5 3.29 – 3.33(m)
77.8 3.38 – 3.42 (m)
71.6 3.37 – 3.36(m)
78.2 3.32 – 3.38(m)
62.4 3.64 (dd, 6.0, 11.5)
3.88 (dd, 2.0, 11.5)
104.4 4.53 (d, 8.0)
75.4 3.42 – 3.48 (m)
77.8 3.37 – 3.41(m)
71.3 3.31 – 3.35(m)
78.6 3.56 – 3.60(m)
62.7 3.67 (dd, 5.0, 12.0)
3.86 (dd, 2.0, 12.0)
6‴
Assignments were done by HSQC, HMBC, COSY, and NOESY experiments. Ara, arabinopyranosyl; Bz,
benzoyl; Glc, glucopyranosyl.
Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 4 and 5
C
4
5
δ(C) δ(H) (mult., J, in Hz)
δ(C) δ(H) (mult., J, in Hz)
Aglycone
1
39.6 0.98 – 1.02 (m)
1.64 – 1.68 (m)
26.3 1.70 – 1.76 (m)
1.96 – 2.00 (m)
83.3 3.67 (dd, 3.5, 13.0)
43.9 -
48.1 1.63 – 1.66 (m)
18.8 1.44 – 1.48 (m)
1.52 – 1.56 (m)
33.3 1.35 – 1.41 (m)
1.72 – 1.78 (m)
41.1 -
39.5 0.98 – 1.02 (m)
1.64 – 1.68 (m)
26.3 1.73 – 2.00 (m)
1.88 – 1.92 (m)
83.4 3.61 – 3.67(m)
43.9 -
48.1 1.62 – 1.68 (m)
18.8 1.42 – 1.46 (m)
1.52 – 1.56(m)
2
3
4
5
6
7
33.6 1.35 – 1.41 (m)
1.69 – 1.75 (m)
40.9 -
8
9
48.1 1.25 – 1.29 (m)
37.5 -
24.7 1.94 – 1.98 (m)
124.9 5.37 (br s)
143.0 -
48.3 1.25 – 1.29 (m)
37.6 -
24.6 1.95 – 1.99 (m)
125.2 5.42 (t, 3.0)
142.1 -
10
11
12
13
14
15
44.6 -
44.9 -
36.5 1.41 – 1.45 (m)
1.80 – 1.84 (m)
67.9 4.36 (dd, 5.0, 12.0)
43.9 -
43.6 2.51 (dd, 4.5, 14.0)
48.0 1.30 – 1.36 (m)
2.09 – 2.11 (m)
36.6 -
78.2 5.16 (dd, 5.0, 12.0)
30.2 1.73 (dd, 12.0, 13.5)
2.39 (dd, 4.5, 13.5)
64.8 3.31 (d, 13.0)
3.67 (d, 13.0)
38.1 1.53 – 1.60 (m)
1.81 – 1.85 (m)
66.1 4.45 (dd, 5.0, 12.0)
42.7 -
43.9 2.92 (dd, 4.5, 14.0)
47.8 1.37 – 1.43 (m)
2.09 – 2.11 (m)
36.5 -
77.1 5.15 (dd, 4.5, 12.0)
28.3 1.45 (dd, 12.0, 13.0)
2.46 (dd, 4.5, 13.0)
65.1 3.33 (d, 13.0)
3.67 (d, 13.0)
16
17
18
19
20
21
22
23
24
13.4 0.75 (s)
13.4 0.75 (s)
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25
26
27
28
16.6 1.04 (s)
17.5 1.06 (s)
27.4 1.34 (s)
66.6 3.42 (d, 11.0)
3.73 (d, 11.0)
29.4 0.96 (s)
18.9 1.17 (s)
16.5 1.03 (s)
17.7 0.87 (s)
26.9 1.32 (s)
207.4 9.78 (s)
29
30
29.2 0.99 (s)
18.8 1.17 (s)
21-O-Bz
134.2 -
134.4 -
1′
130.4 8.04 (d, 8.0)
129.6 7.51 (t, 8.0)
131.8 7.63 (t, 8.0)
167.8 -
130.5 8.04 (d, 8.0)
129.6 7.50 (t, 8.0)
131.6 7.64 (t, 8.0)
167.7 -
2′, 6′
3′, 5′
4′
7′
3-O-Ara
1″
106.1 4.38 (d, 7.5)
72.1 3.70 – 3.74 (m)
84.2 3.65 – 3.71 (m)
69.5 4.06 (br s)
66.9 3.60 (br d, 12.0)
3.88 (br d, 12.0)
106.1 4.37 (d, 7.5)
72.1 3.69 – 3.73 (m)
84.3 3.63 – 3.69 (m)
69.6 4.06 (br s)
66.9 3.60 (br d, 14.0)
3.88 (dd, 2.0, 14.0)
2″
3″
4″
5″
3″-O-Glc
1‴
2‴
3‴
4‴
5‴
6‴
105.5 4.57 (d, 8.0)
75.3 3.30 – 3.34 (m)
77.6 3.37 – 3.43 (m)
71.1 3.34 – 3.40 (m)
77.9 3.29 – 3.33 (m)
62.3 3.71 (dd, 5.0, 12.0)
3.87 (br d, 12.0)
105.5 4.56 (d, 8.0)
75.3 3.30 – 3.34 (m)
77.7 3.37 – 3.43 (m)
71.2 3.31 – 3.37 (m)
78.0 3.29 – 3.33 (m)
62.4 3.71 (dd, 5.0, 12.0)
3.85 (dd, 2.0, 12.0)
Assignments were done by HSQC, HMBC, COSY, and NOESY experiments. Ara, arabinopyranosyl, Bz,
benzoyl, Glc, glucopyranosyl.
Table 3. Effects of 1−5 from G. hirsutum on the Growth of Human Cancer Cells
IC50 (µM)
Compound
HepG-2
8.2 1.3
3.4 0.3
47.0 5.6
7.6 0.8
9.9 3.1
1.4 0.2
A-549
9.3 0.3
4.4 0.7
49.3 4.1
8.0 2.2
8.6 1.3
1.8 0.3
MCF-7
9.2 0.5
4.7 0.6
51.9 3.7
8.8 1.3
10.2 2.4
2.0 0.3
SW-626
8.5 1.3
6.6 1.0
54.4 1.5
9.1 1.1
10.1 1.9
2.1 0.3
1
2
3
4
5
Ellipticine
Ellipticine was used as a positive control.
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Figure 1. Chemical Structures of Compounds 1 − 5 from G. hirsutum
Figure 2. The Key HMBC, 1H−1H COSY, and NOESY Correlations of 1, 3, and 5
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