Six new furostanol glycosides from Smilax glauco-china and their cytotoxic activity

Article abstract of DOI:10.1080/10286020.2017.1281913

Six new steroidal saponins, namely glauco-chinaosides A–F, and one known compound were isolated from the tubers of Smilax glauco-china. Their structures were elucidated by a combination of spectroscopic analysis and hydrolysis followed by spectral and chromatographic analysis. Compounds 1–7 were tested in vitro for their cytotoxic activities against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, and Lovo). Compounds 1, 2, and 5 exhibited cytotoxic activity against SGC-7901, with IC₅₀ values of 2.7, 11.5, and 6.8?μM, respectively.

Full text of DOI:10.1080/10286020.2017.1281913

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Six new furostanol glycosides from Smilax glauco-
china and their cytotoxic activity
Xing Liu, Jian Liang, Ling-Ling Pan, Jian-Yong Chen, Rong-Hua Liu, Gen-Hua
Zhu, Hui-Lian Huang, Ji-Cheng Shu, Feng Shao, Yong-Hong Liang & Jiang-Li Yu
To cite this article: Xing Liu, Jian Liang, Ling-Ling Pan, Jian-Yong Chen, Rong-Hua Liu, Gen-Hua
Zhu, Hui-Lian Huang, Ji-Cheng Shu, Feng Shao, Yong-Hong Liang & Jiang-Li Yu (2017): Six new
furostanol glycosides from Smilax glauco-china and their cytotoxic activity, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2017.1281913
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1281913
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Date: 18 February 2017, At: 06:05

Journal of asian natural Products research, 2017
http://dx.doi.org/10.1080/10286020.2017.1281913
Six new furostanol glycosides from Smilax glauco-china and
their cytotoxic activity
Xing Liua, Jian Lianga, Ling-Ling Pana, Jian-Yong Chenb, Rong-Hua Liua, Gen-Hua Zhua,
Hui-Lian Huanga, Ji-Cheng Shua, Feng Shaoa, Yong-Hong Lianga and Jiang-Li Yua
aKey laboratory of Modern Preparation oftcM, Ministry of education, Jiangxi university oftraditional chinese
Medicine, nanchang 330004, china; bGastroenterology department, Jiangxi Province People hospital,
nanchang 330006, china
ABSTRACT
ARTICLE HISTORY
received 22 september 2016
accepted 10 January 2017
Six new steroidal saponins, namely glauco-chinaosides A–F, and
one known compound were isolated from the tubers of Smilax
glauco-china. Their structures were elucidated by a combination
of spectroscopic analysis and hydrolysis followed by spectral and
chromatographic analysis. Compounds 1–7 were tested in vitro for
their cytotoxic activities against four human tumor cell lines (SH-SY5Y,
SGC-7901, HCT-116, and Lovo). Compounds 1, 2, and 5 exhibited
cytotoxic activity against SGC-7901, with IC50 values of 2.7, 11.5, and
6.8 μM, respectively.
KEYWORDS
Smilax glauco-china;
smilacaceae; furostanol
glycosides; cytotoxic activity
1. Introduction
Smilaceae comprises approximately 370 species of shrubs which grow in tropical and tem-
perate areas around the world. Smilax glauco-china, an evergreen climbing shrub mainly
distributed in the south of China (i.e. Zhejiang, Jiangxi, Hunan and Guizhou Province),
is a member of the Smilax family [1]. e tubers of S. glauco-china are commonly used as
herbal materials in traditional Chinese medicine (TCM). In TCM, the efficacy of Smilax
glauco-china was regarded as dispelling wind-evil, cleaning heat, detoxicating. and elimi-
nating damp. Based on the previous phytochemical investigation on the Smilaceae family, a
conclusion can be safely arrived at that the genus Smilax is rich in steroidal saponins [2–4].
CONTACT hui-lian huang
huilianh@163.com
the supplemental data for this article are available online at http://dx.doi.org/10.1080/10286020.2017.1281913.
© 2017 informa uK limited, trading as taylor & francis Group

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X. LIU ET AL.
Aglycone
moiety
compounds
R1
R2
R3
S1
S3
S2
S3
S1
S3
S3
CH2CH3
CH2CH3
CH2CH3
CH3
S4
S4
S4
S4
S4
S4
S4
1
3
5
7
2
4
6
I
CH2CH3
CH2CH3
CH3
Figure 1. the structures of compounds 1–7.
Steroidal saponins exhibit a range of bioactivities, such as anti-inflammatory, cytotoxic,
and anti-tumor effects [3–7]. However, to our best knowledge, there is almost no reference
regarding the constituents of S. glauco-china. As part of our continuous interests in steroidal
saponins in Smilax (Smilaceae) plants [8,9], a chemical investigation has been undertaken.
e current article reports six new steroidal saponins and one known relative from the plant.
2. Results and discussion
Compound 1 (Figure 1) was obtained as a white amorphous powder with a molecular for-
mula of C47H76O18, determined by the deprotonated molecule at m/z 927.4989 [M − H]− in
the HR-ESI-MS. e UV spectrum showed absorption maximum at 240 nm. In the IR spec-
trum, absorption bands for hydroxyl (3409 cm−1), methyl (2934 cm−1), olifin (1718 cm−1),
and ether (1038 cm−1) groups were observed. Its structure was identified by comparison
of its 1H-NMR and 13C-NMR (Table 1), HSQC, and HMBC data with the congener from
Dioscorea panthaica [10,11].

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3

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X. LIU ET AL.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 2. Key hMBc (h →c) correlations of compound 1.
Figure 3. important noe (h–h) enhancement of compound 1.
1
e H-NMR spectrum of 1 showed signals belonging to an olefinic proton at δH
5.34 (br d, J = 4.4 Hz, H-6), and six methyl protons at δH 0.95(s, H-19), 0.72 (s, H-18),
1.79(s, H-21), 1.21(t, J = 7.0, H-23-CH3), δH 1.14 (d, J = 6.6 Hz, H-27), δH 1.75(d, J = 6.2 Hz,
H-6″). e 13C-NMR spectrum displayed a total of 47 carbon signals, which were composed
of 27 signals due to aglycone moiety, 2 carbon signals due to the ethoxy group at C-23, and
18 carbon signals due to the presence of three hexoses. e DEPT spectrum of 1 showed
the presence of 6 primary carbons, 12 secondary carbons, 24 tertiary carbons, 3 quaternary
carbons, and 2 SP2 hybrid quaternary carbons. All of the spectral data showed compound
1 possessed the same partial structure in A, B, C, D, and E rings as dioscoreside C [10],
except for the different substituent of C-23. Comparison of the 13C-NMR data with those in
the reference indicated the upfield shifs of C-22 and C-23 by 0.7 and 1.4 ppm, respectively,
together with one more carbon signal at δC 15.7. Furthermore, in the HMBC spectrum, the
correlation of H-23/C-23-OCH2 was observed (Figure 2). ese data indicated a typical
ethoxy group at C-23.
e spatial configuration of compound 1 was confirmed according to the known
1
compound of dioscoreside C. e H-NMR spectrum indicated that 1 possessed one
less sugar moiety than dioscoreside C. In the NOESY spectrum of 1, the correlation of
H-23/H-25 (Figure 3) indicated that the configuration of C-23 is similar to the dioscore-
side C. us, the ethoxy group of C-23 had the α-configuration. erefore, the absolute
configuration of C-23 was assigned as S. e resonance of the protons and carbons (C-24,
C-25, C-26, and C-27) around the C-25 center and the 3JHH values (12.5, 6.2 Hz) between

6
X. LIU ET AL.
H-25 and H-26 provided the evidence for the C-25 R configuration of 1, as described in
1
the previous report [11,12]. Based on the inspection on the H and 13C NMR spectral
data of compound 1, the structure of the aglycone moiety was found to be identical to
3β,26-dihydroxy-23(S)-ethoxy-25(R)-furosta-5,20(22)-diene.
1
e H-NMR spectrum of 1 displayed signals for three anomeric protons at δH 4.98
(d, J = 7.6 Hz), δH 5.94 (br s), and δH 4.88 (d, J = 7.7 Hz), which gave correlations in the HSQC
spectrum with 13C NMR signals at δC 102.5, δC 102.7, and δC 104.9, respectively. e identity
of the monosaccharides and the sequence of the oligosaccharide chain were determined by
the analysis of HMQC, HMBC, COSY, and NOESY spectra. All of the data showed that com-
pound 1 possessed two glucoses and one rhamnose. Acid hydrolysis of 1 yielded d-glucose
and l-rhamnose, as revealed by HPLC analysis and comparison with authentic standards.
e α-anomeric configuration for the rhamnose was determined by its C-5 data (δC 70.4)
[13], and the β-anomeric configurations for the two glucoses were determined from their
large 3J1,2 coupling constants (7.6, 7.7 Hz). e attachments of the rhamnose moiety to C-4′
of the glucose moiety were established by the correlation of H-1″/C-4′, and the glucose moi-
ety to C-3 of the aglycone was based on a correlation of H-1′/C-3. In addition, the remaining
glucose moiety to C-26 of the aglycone was based on a correlation of H-1″′/C-26. Based on
all the data mentioned above, compound 1 was determined to be 26-O-β-d-glucopyranosyl-
3β,26-dihydroxy-23(S)–ethoxy -25(R)-furosta-5,20(22)-diene-3-O-α-l-rhamnopyranosyl
(1→4)-β-d-glucopyranoside, and given the trivial name glauco-chinaoside A.
Compound 2 (Figure 1) was isolated as a white amorphous powder with a molecular
formula of C47H76O18, determined by the deprotonated molecular ion at m/z 927.4965
[M − H]− in the HR-ESI-MS. e UV spectrum showed absorption maximum at 260 nm.
In the IR spectrum, absorption bands of hydroxyl (3406 cm−1), methyl (2934 cm−1), olif-
1
ine (1664 cm−1), and ether (1074 cm−1) groups can be found. H-NMR and 13C-NMR
spectral data (Table 1) indicated that compound 2 had the same aglycone moiety and
sugar arrangement as that of 1. e HMQC, HMBC, and NOESY spectra indicated that
the aglycone of 2 possessed the same partial structure of A, B, C, D, and E rings, together
with the ethoxy group at C-23, of which absolute configuration of C-23 was assigned as S.
Indeed, the NMR spectroscopic data of compound 2 were very similar to that of 1, with the
main difference at the downfield shifs of C-27 (δC 18.1) and C-25 (δC 31.0) and the upfield
shif of C-24 (δC 37.5). According to the reference, the C-25 S configuration of 2 could be
deduced [10]. Based on all the data mentioned above, compound 2 was determined to be
26-O-β-d-glucopyranosyl-3β,26-dihydroxy-23(S)-ethoxy-25(S)-furosta- 5,20(22)-diene-3-
O-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranoside, and was named glauco-chinaoside B.
Compound 3 (Figure 1) was obtained as a white amorphous powder. e molecular
formula of compound 3 was C53H86O22, determined by the deprotonated molecule at m/z
1073.5599 [M − H]− in the HR-ESI-MS. e UV spectrum showed absorption maximum at
210 nm. In the IR spectrum, absorption bands of hydroxyl (3426 cm−1), methyl (2924 cm−1),
olifine (1742 cm−1), and ether (1041 cm−1) groups can be found. According to the NMR
data (Table 1), it had the same aglycone moiety as compound 1, but as its molecular mass
was 146 Da higher, an additional 6-deoxyhexose moiety could be presumed. e spatial
configuration of compound 3 was confirmed according to the resonance data, which were
very similar to that of compound 1. e 1H-NMR spectrum of 3 displayed signals for four
anomeric protons of δH 4.98, 6.44, 5.89, and 4.86, which gave correlations in the HSQC
spectrum with 13C NMR signals at δC 100.3, 102.0, 102.9, and 104.9, respectively. According

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
to the HMBC data, the attachments of the rhamnoses moiety to C-2′ and C-4′ of the glu-
cose moiety were established by the correlation of H-1″/C-2′, H-1″′/C-4′. In conclusion,
compound 3 was elucidated as 26-O-β-d-glucopyranosyl-3β,26-dihydroxy-23(S)-ethoxy-
25(R)-furosta-5,20(22)-diene-3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyrano-
syl(1→4)]-β-d-glucopyranoside, and was named glauco-chinaoside C.
Compound 4 was isolated as a white amorphous powder with a molecular formula of
C53H86O22, determined by the pseudo-molecular ion at m/z 1097.546 [M + Na]+ in the
HR-ESI-MS. e UV spectrum showed absorption maximum at 265 nm. In the IR spec-
trum, absorption bands for hydroxyl (3431 cm−1), methyl (2925 cm−1), olifin (1737 cm−1),
and ether (1164 cm−1) groups can be found. 1H-NMR and 13C-NMR spectral data (Table 1)
indicated that compound 4 had the same aglycone moiety and spatial configuration as
that of compound 2. 1H-NMR and 13C-NMR spectral data indicated the configuration of
C-25 in compounds 2 and 3 was different. So, compounds 4 and 3 were different in the
configuration of C-25. Similar sugar part and molecular weight of compounds 4 and 3
all indicated that they are structural isomers. us, the structure of 4 was established as
26-O-β-d-glucopyranosyl-3β,26-dihydroxy-23(S)-ethoxy-25(S)-furosta-5,20(22) -diene-3-
O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl(1→4)]-β-d-glucopyranoside, and
was named glauco-chinaoside D.
Compound 5 was obtained as a white amorphous powder with a molecular formula
of C47H76O18, determined by the deprotonated molecular ion at m/z 927.5032 [M − H]−
in the HR-ESI-MS, similar molecular weight to compounds 1 and 2. e UV spectrum
showed absorption maximum at 260 nm. In the IR spectrum, absorption bands for hydroxyl
(3432 cm−1), methyl (2924 cm−1), olifin (1736 cm−1), and ether (1163 cm−1) groups can be
found. From the NMR data (Table 1), the same aglycone moiety and spatial configuration
as that of compound 1 could be identified. e resonance of the protons and carbons pro-
vided the evidence for the C-23 S and C-25 R configurations of 5. Compound 5 possessed
two glucoses and one rhamnose based on the 1H-NMR spectrum that displayed signals for
three anomeric protons at δH 5.08, 6.43, and 4.87 and three anomeric carbons at δC 100.4,
102.7, and 105.0, respectively. Indeed, the NMR spectroscopic data of compound 5 were
very similar to that of 1, whereas the main difference was the attachment of rhamnose. e
attachment of the rhamnose moiety to C-2′ of the glucose moiety was established by the
HMBC correlation of H-1″/C-2′. ese data allowed the structure of 5 to be assigned as
26-O-β-d-glucopyranosyl-3β,26-dihydroxy-23(S)-ethoxy-25(R)- furosta-5,20(22)-diene-3-
O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranoside, and was named glauco-chinaoside E.
e molecular formula of compound 6 was determined as C52H84O22 by the ion peak at
m/z 1083.5369 [M + Na]+ in HR-ESI-MS, which is 14 Da less than compound 4. e UV
(MeOH) spectrum showed absorption maximum at 210 nm. In the IR (KBr)spectrum,
absorption bands for hydroxyl (3425 cm−1), methyl (2922 cm−1), olifin (1731 cm−1), and
ether (1040 cm−1) groups can be found. From the NMR data (Table 1), the same sugar
chain could be identified. Further interpretation of the NMR data revealed the differ-
ence in C-23, and the ethoxy group (δC 63.8 and δC 15.7) was displaced by the meth-
oxy group (δC 56.3). In addition, the absolute configuration of C-23 was assigned as S
according to the reference [10]. e resonance of the protons and carbons (C-24, C-25,
C-26, and C-27) around the C-25 center provided the evidence for the C-25 S configura-
tion of compound 6 as described in compound 2. us, compound 6 was elucidated as

8
X. LIU ET AL.
26-O-β-d-glucopyranosyl-3β,26-dihydroxy-23(S)-methoxy-25(S)-furosta-5,20(22)-diene-
3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl(1→4)]-β-d-glucopyranoside, and
was named glauco-chinaoside F.
Additionally, one known compound 7 was isolated. Based on the NMR spectroscopic
data and comparison with the literature [10], its structure was determined to be 26-O-β-
d-glucopyranosyl-3β,26-dihydroxy-23(S)-methoxy-25(R)-furosta-5,20(22)-diene-3-O-α-
l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl(1→4)]-β-d-glucopyranoside, and was
named dioscoreside C.
Compounds 1–7 were evaluated for cytotoxic activities against four human cell lines
(SH-SY5Y, SGC-7901, HCT-116, and Lovo), with Vero as a positive control. e purity
of compounds 1–7 was 94, 93.2, 92.1, 91.0, 91.7, 94.2, and 91.2%, respectively. Cisplatin
(≥99.9%; Sigma–Aldrich, St. Louis, MO, USA), a well-known anticancer drug, was used
in this experiment as a positive control. Compounds 1–7 were inactive (IC50 > 100 μM) to
SH-SY5Y, Lovo, and HCT-116 cell lines. Compounds 1, 2, and 5 exhibited cytotoxic activity
against SGC-7901, with IC50 values of 2.7, 11.5, and 6.8 μM, respectively.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured with a Perkin–Elmer 241 MC polarimeter (Co. Perkin
Elmer, Liantrisant, UK). IR spectra were recorded on L1600401 Spectrum TWO DTGS
(Co. Perkin Elmer, Liantrisant, UK). NMR spectra were obtained on a Bruker AM-400
spectrometer (400-MHz) in pyridine-d5 at room temperature (25 °C) (Bruker, Fällanden,
Switzerland); Electrospray ionization (ESI) mass spectra were employed in the positive ion
mode on a LCQ DECAXP instrument (ermo Finnigan, San Jose, CA, USA) equipped
with an ion-trap mass analyzer. HR-ESI-MS were obtained in the positive ion mode using
a Waters UPLC Premier Q-TOF system. Preparative HPLC was operated using a Prevail
C18 column (5 μm, 10.0 mm I.D × 250 mm) at rate of 3.0 ml/min and detection wave-
length of 210 nm. UV spectra and HPLC analysis was carried out on an Agilent 1200
system equipped with a quaternary solvent delivery system, an autosampler, and a DAD
detector. e column was a ermo C18 (5 μm, 4.6 mm I.D × 250 mm). Column chro-
matography was performed using MCI GEL (Mitsubishi Chemical Corporation, Japan),
Sephadex LH-20 (Amersham Bio-sciences AB, Uppsala, Sweden) and C18 SPE by Bulk
Sorbent (Grace Davison Discovery Sciences), and silica gel (SiO2; 200–300 mesh; Qingdao
Haiyang Chemical Co., Ltd., Qingdao, China). TLC plates were HSGF254 SiO2 from Yantai
Jiangyou Silica Gel Development Co., Ltd. (Yantai, China).
3.2. Plant material
Rhizomes of Smilax glauco-china (Smilacaceae) were collected in Wuyunjie Nature Reserve,
Taoyuan County, Hunan Province, China, in May 2013. All plants were identified by Daigui
Zhang, a lecturer at Jishou University. A voucher specimen (No. 20130513) was deposited
at the Key Laboratory of Modern Preparation of TCM, Jiangxi University of TCM, China.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9

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X. LIU ET AL.
3.3. Extraction and isolation
Air-dried and powdered rhizomes and roots of S. glauco-china (5 kg) were extracted twice
with 70% EtOH and concentrated in vacuum to give crude extract, which was suspended in
H2O and partitioned successively with EtOAc and n-BuOH. e n-BuOH soluble portion
(201 g) was first chromatographed on a HP-20 macroporous adsorption resin and eluted
with 30, 50, 70, and 95% EtOH, successively, to obtain four fractions (Fr. A, B, C, and D).
Fr. A (31 g) was subjected to a silica gel column using a stepwise gradient of CHCl3: MeOH
(from 7:1 to 0:1), then purified by a Sephadex LH-20 column, and eluted with MeOH. A
further purification was made by a preparative HPLC with solvent of CH3CN:H2O (22:78)
to obtain 3 (15 mg, tR = 15.3 min), 4 (22.8 mg tR = 15.2 min), 6 (8.6 mg, tR = 12.2 min), and
7 (12.9 mg, tR = 20.5 min). Fr. B (26 g) was subjected to a MCI column using a stepwise
gradient of MeOH: H2O (20:80, 40:60, 60:40, 80:20) to obtain four sub-fractions. Fr.B.3
(5.2 g) was subjected to a silica gel column using a stepwise gradient of CHCl3: MeOH
(from 8:1 to 0:1); it was further purified by preparative HPLC with solvent of CH3CN:H2O
(18:82) to obtain compounds 1 (23 mg, tR 17.3 min) and 2 (33.8 mg, tR 19.1 min). Likewise,
Fr.B.4 (3.9 g) was further purified by preparative HPLC with CH3CN:H2O (16:84) to obtain
compound 5 (5.5 mg, tR 22.5 min). All the detection was carried out using wavelength of
210 nm and a flow rate of 3.0 ml/min.
3.3.1. Glauco-chinaoside A (1)
White amorphous powder (MeOH);[ꢀ]2D5− 53.19 (c 0.0188, MeOH); UV (MeOH) λmax (log ε)
240 (0.59) nm; IR (KBr) vmax cm−1: 3409, 2934, 1718, 1038; 1H NMR and 13C NMR spactral
data see Tables 1 and 2; HR-ESI-MS: m/z 927.4989 [M − H]− (calcd for C47H75O18, 927.4953).
3.3.2. Glauco-chinaoside B (2)
White amorphous powder (MeOH);[ꢀ]2D5− 35.02 (c 0.0256, MeOH); UV (MeOH) λmax (log ε)
260 (0.27) nm; IR (KBr) vmax cm−1: 3406, 2934, 1664, 1074; 1H NMR and 13C NMR spactral
data see Tables 1 and 2; HR-ESI-MS: m/z 927.4965 [M − H]− (calcd for C47H75O18, 927.4953).
3.3.3. Glauco-chinaoside C (3)
White amorphous powder (MeOH); [ꢀ]2D5 − 44.94 (c 0.0178, MeOH); UV (MeOH) λmax
(log ε) 210 (0.66) nm; IR (KBr) vmax cm−1: 3426, 2924, 1742, 1041; 1H NMR and 13C NMR
spactral data see Tables 1 and 2; HR-ESI-MS: m/z 1073.5599 [M − H]− (calcd for C53H85O22,
1073.5532).
3.3.4. Glauco-chinaoside D (4)
White amorphous powder (MeOH);[ꢀ]2D5 − 35.9(c 0.0195, MeOH); UV (MeOH) λmax (log ε)
265 (0.67) nm; IR (KBr) vmax cm−1: 3431, 2925, 1737, 1164; 1H NMR and 13C NMR spactral
data see Tables 1 and 2; HR-ESI-MS: m/z 1097.5460 [M + Na]+ (calcd for C53H86O22Na,
1097.5508).
3.3.5. Glauco-chinaoside E (5)
White amorphous powder (MeOH);[ꢀ]2D5 − 28.57 (c 0.021, MeOH); UV (MeOH) λmax (log ε)
260 (0.31) nm; IR (KBr) vmax cm−1: 3432, 2924, 1736, 1163; 1H NMR and 13C NMR spactral
data see Tables 1 and 2; HR-ESI-MS: m/z 927.5032 [M − H]− (calcd for C47H75O18, 927.4953).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
3.3.6. Glauco-chinaoside F (6)
White amorphous powder (MeOH);[ꢀ]2D5− 29.85 (c 0.0067, MeOH); UV (MeOH) λmax (log ε)
210 (0.67) nm; IR (KBr) vmax cm−1: 3425, 2922, 1731, 1040; 1H NMR and 13C NMR spactral
data see Tables 1 and 2; HR-ESI-MS: m/z 1083.5369 [M + Na]+ (calcd for C52H84O22Na,
1083.5352).
3.4. Cytotoxic activity
e cytotoxicity against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, and
Lovo), with Vero as a positive control, was evaluated using the MTT method. Cells were
seeded in 96-well microplates at a density of 150 per well and were cultured in cell culture
medium (RPMI1640 medium supplemented with 10% FBS (fetal bovine serum), 100 U/
ml penicillin, and 100 g/ml streptomycin) for 12 h, then treated with the stock solution
of test compounds dissolved in DMSO. e final DMSO concentration never exceeded
0.2% (v/v). Afer 48 h of cultivation, cells were incubated with MTT (0.5 mg/ml, 4 h) and
subsequently resolved in DMSO. e absorbance in the control and drug-treated wells
was measured by an automated microplate reader at 570/630 nm. All experiments were
carried out in triplicate and repeated twice. e cytotoxicity was expressed as IC50 values
(50% inhibitory concentration).
3.5. Acid hydrolysis of 1–6
Compounds 1–6 (1.5 mg, each) were hydrolyzed with 2 M CF3COOH (5 ml), and the
hydrolyzed products were treated and detected through HPLC following the methods of
Lin et al., 2012 and Guo et al., 2004 [7,8].
Acknowledgments
e authors gratefully acknowledge grants from the National Natural Science Foundation of China
(NSFC) (Nos. 31370376 and 81360628) and the Science and Technology Projects of Jiangxi Province
(No. 20151BBG70141).
Disclosure statement
No potential conflict of interest was reported by the authors
Funding
is work was financially supported by the National Natural Science Foundation of China (NSFC)
[number 31370376], [number 81360628], [number 81560640]; and the Science and Technology
Projects of Jiangxi Province [number 20151BBG70141].
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