Two new cytotoxic glycosides isolated from the green walnut husks of Juglans mandshurica Maxim.

Article abstract of DOI:10.1080/14786419.2016.1233412

Two new glycosides including an alcohol glycoside and a phenolic glycoside: hexyl-1-O-α-d-arabinofuranosyl-(1?→?6)-β-d-glucopyranoside (1), 4-hydroxypropiophenone-4-O-β-d-glucopyranosyl(1?→?6)-β-d-glucopyranoside(2), along with six known naphthalenyl glucosides (3–8) were isolated from green walnut husks of Juglans mandshurica, and their structures were elucidated on the basis of spectroscopic studies. All compounds were evaluated for their inhibitory effects on tumour cells (BGC-823, HepG-2, MCF-7). The results showed that new compounds 1 and 2 had superior inhibitory activity in comparison with other naphthalenyl glucosides.

Full text of DOI:10.1080/14786419.2016.1233412

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Two new cytotoxic glycosides isolated from the
green walnut husks of Juglans mandshurica
Maxim.
Yuan-Yuan Zhou, Quan-Yu Liu, Bing-You Yang, Yan-Qiu Jiang, Yu-Xin Liu, Ying
Wang, Shuang Guo & Haixue Kuang
To cite this article: Yuan-Yuan Zhou, Quan-Yu Liu, Bing-You Yang, Yan-Qiu Jiang, Yu-Xin Liu,
Ying Wang, Shuang Guo & Haixue Kuang (2016): Two new cytotoxic glycosides isolated from
the green walnut husks of Juglans mandshurica Maxim., Natural Product Research, DOI:
10.1080/14786419.2016.1233412
To link to this article: http://dx.doi.org/10.1080/14786419.2016.1233412
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Published online: 26 Sep 2016.
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Date: 26 September 2016, At: 18:34

Natural Product research, 2016
ꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2016.1233412
ꢀ
Two new cytotoxic glycosides isolated from the green walnut
husks of Juglans mandshurica Maxim.
a
b
a
a
a
Yuan-Yuan Zhou , Quan-Yu Liu , Bing-You Yang , Yan-Qiu Jiang , Yu-Xin Liu ,
a
a
a
Ying Wang , Shuang Guo and Haixue Kuang
ꢅ
b
cꢃꢆꢆꢇgꢇ ꢃf Pꢀꢅꢄmꢅꢈy, hꢇiꢆꢃngjiꢅng univꢇꢄꢉiꢁy ꢃf cꢀinꢇꢉꢇ Mꢇꢂiꢈinꢇ, hꢅꢄbin, P.r. cꢀinꢅ; Qꢊꢅꢆiꢁy tꢇꢉꢁing Bꢄꢅnꢈꢀ,
Vꢇꢁꢇꢄinꢅꢄy dꢄꢊg ꢅnꢂ Fꢇꢇꢂ sꢊpꢇꢄviꢉiꢃn Inꢉꢁiꢁꢊꢁꢇ ꢃf hꢇiꢆꢃngjiꢅng Pꢄꢃvinꢈꢇ, hꢅꢄbin, P.r. cꢀinꢅ
ARTICLE HISTORY
rꢇꢈꢇivꢇꢂ 7 Mꢅy 2016
aꢈꢈꢇpꢁꢇꢂ 16 aꢊgꢊꢉꢁ 2016
ABSTRACT
Two new glycosides including an alcohol glycoside and a phenolic
glycoside: hexyl-1-O-α-
d
-arabinofuranosyl-(1 →6)-β-d-glucopyranoside
(
1), 4-hydroxypropiophenone-4-O-β-d-glucopyranosyl(1 → 6)-β-d-
glucopyranoside(2), along with six known naphthalenyl glucosides
3–8) were isolated from green walnut husks of Juglans mandshurica,
KEYWORDS
Juglans mandshurica;
ꢅꢆꢈꢃꢀꢃꢆ gꢆyꢈꢃꢉiꢂꢇ; pꢀꢇnꢃꢆiꢈ
gꢆyꢈꢃꢉiꢂꢇ; ꢈyꢁꢃꢁꢃxiꢈ ꢅꢈꢁiviꢁy
(
and their structures were elucidated on the basis of spectroscopic
studies. All compounds were evaluated for their inhibitory effects on
tumour cells (BGC-823, HepG-2, MCF-7). The results showed that new
compounds 1 and 2 had superior inhibitory activity in comparison with
other naphthalenyl glucosides.
1
. Introduction
Juglans mandshurica Maxim. (Juglandaceae) is one of the rare species of trees for pharmacy
resources and distributed throughout urban and rural areas in north-east of Asia (Wu &
Raven 1999). Its leaves, roots, fruit and barks have been used as medicinal parts in use or
development (Li et al. 2003; Liu et al. 2004; Lin et al. 2014; Yao et al. 2015a). Recent pharma-
cological studies of medicinal parts from J. mandshurica have reported they had many bio-
logical properties, including antitumour, of green husks, roots, barks and leaves (Lee et al.
2002; Li et al. 2008; Liu et al. 2010; Guo et al. 2015; Zhou et al. 2015a; Gao et al. 2016), anti-in-
flammatory, of barks (Ju et al. 2009), anticomplement activity, of stem-barks (Min et al. 2003),
antioxidant, of stem-barks (Ngoc et al. 2008), as well as protection of skin fibroblasts from
CONTACT hꢅixꢊꢇ Kꢊꢅng
ꢀxkkꢊꢅng0451@ꢃꢊꢁꢆꢃꢃk.ꢈꢃm, kꢊꢅngꢀꢅixꢊꢇ@ꢀꢆjꢊꢈm.nꢇꢁ
sꢊppꢆꢇmꢇnꢁꢅꢆ ꢂꢅꢁꢅ fꢃꢄ ꢁꢀiꢉ ꢅꢄꢁiꢈꢆꢇ ꢈꢅn bꢇ ꢅꢈꢈꢇꢉꢉꢇꢂ ꢅꢁ ꢀꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2016.1233412
©
2016 Infꢃꢄmꢅ uK limiꢁꢇꢂ, ꢁꢄꢅꢂing ꢅꢉ tꢅyꢆꢃꢄ & Fꢄꢅnꢈiꢉ Gꢄꢃꢊp

2
Y.ꢀY. ZHꢁꢂ ꢃT AL.
damage by regulating the oxidative defence system of leaves (Park et al. 2012), anti-human
immunodeficiency virus-type 1 of stem-barks (Min et al. 2002). In addition, the types of
components isolated from different parts of J. mandshurica were multiple and different.
Naphthoquinones and their derivatives widely existed in the roots, stem barks, leaves and
green husks (Lee et al. 2000; Min et al. 2002; Li et al. 2008; Lin et al. 2014; Wang et al. 2014;
Yao et al. 2015b; Zhou et al. 2015a). Diarylheptanoids were distributed among leaves, green
husks and roots (Li et al. 2003; Chen et al. 2015; Jin et al. 2015;Yao et al. 2015b), also including
flavonoids from stem-barks (Min et al. 2003), phenolic glycoside from barks and stem-barks
(
(
Machida et al. 2009; Yao et al. 2014), fatty acid from walnuts, barks and stem-barks
Bouabdallah et al. 2014;Yao et al. 2015a; Gao et al. 2016) and so on. According to the reports,
the types of components isolated from green walnut husks of this plant mainly focused on
naphthoquinones, α-tetralones and their glycosides, triterpenes and diarylheptanoids (Liu
et al. 2004, 2010; Chen et al. 2015; Zhou et al. 2015b). As part of our continuing search for
biologically active compounds from green walnut husks of J. mandshurica Maxim, we inves-
tigated the BuꢁH fraction of the ꢃtꢁH extract which was barely reported, since this polarity
fraction exhibited potential cytotoxic activity in our screening procedures. Indeed, we iso-
lated two new glycosides including an alcohol glycoside (1) and a phenolic glycoside (2),
together with six known naphthalenyl glucosides (3–8). The isolated compounds were tested
for their cytotoxic activities against three human cancer cell lines in vitro. The results showed
that new compounds 1, 2 showed superior inhibitory activity and the other naphthalenyl
glucosides showed almost no activity.
2
. Results and discussion
Compound 1 was obtained as white amorphous powder. The positive-mode-HR-ꢃSI-MS data
+
showed a quasi-molecular ion peak [M + Na] at m/z 419.4137, suggesting a molecular mass
1
of 396.2011 and thus a molecular formula of 1 as C H ꢁ . The H NMR data of 1 (Table S1
1
7
32 10
and Figure S2) showed the methyl group at δ 0.90 (3H, t, J = 6.8 Hz), and four methylene
H
proton signals at δ 1.61 (2H, dt, J = 14.9, 6.8 Hz) and 1.28–1.35 (6H, m) indicating the pres-
H
1
ence of saturated straight-chain paraffin. Further, the H NMR data revealed two anomeric
proton signals [δ 4.24 (d, J = 7.8 Hz, Glc H-1), δ 4.95 (d, J = 1.1 Hz, Ara H-1)] implying the
H
H
occurrence of two sugar units: one β-glucopyranose and one α-arabinofuranose which were
determined by the coupling constant of the anomeric proton. The proton signals for the
disaccharide sugar unit of 1 were identical to the published compound Bumaldoside A
1
3
(
Hideaki et al. 2010) The C NMR data (Table S1 and Figure S3) and distortionless enhanced
polarisation transfer spectra (DꢃPT, Table S1 and Figure S4) of 1 showed 17 carbons including
1
3
seven methylene carbons, nine methine carbons and one methyl group. The C NMR data
also revealed two anomeric carbon signals at δ 104.4 (Glc C-1) and 110.0 (Ara C-1) in accord-
C
1
ance with the two anomeric signals in the H NMR spectrum. In the HMBC spectrum (Figure
S6), a suggestive correlation was observed between the anomeric proton signal of glucopyra-
nose and an oxymethylene carbon signal at δ 71.1 (C-1) of the aglycone, indicating that the
C
sugar moieties were linked at the C-1 position. Moreover, the connectivity of the arabino-
furanose with the glucopyranose was indicted by the cross-peak between the anomeric
proton H-1 of arabinofuranose and C-6 of glucopyranose. The sugar moieties of 1 linked at
1-O- were assigned as 1-O-α-l-arabinofuranosyl (1 → 6)-β-d-glucopyranoside, combined with
the result of acid hydrolysis of 1. The aqueous layer was separated by HPLC to give
d-glucose

NATꢂRAL PRꢁDꢂCT RꢃSꢃARCH
3
Figure 1. sꢁꢄꢊꢈꢁꢊꢄꢇꢉ ꢃf nꢇw ꢈꢃmpꢃꢊnꢂꢉ 1 ꢅnꢂ 2.
and l-arabinose. ꢁn the basis of the above evidence, the structure of 1 was established as
the new compound hexyl-1-O-α-l-arabinofuranosyl (1 → 6)-β-d-glucopyranoside (Figure 1).
Compound 2 was obtained as white amorphous powder. The positive-mode HR-ꢃSI-MS
+
data showed a quasi-molecular ion peak [M + H] at m/z 475.1865, indicating a molecular
1
mass of 474.1768 and the molecular formula of 2 as C H ꢁ . In the H NMR spectrum (Table
2
1 30 12
S2 and Figure S7), it showed that AA′BB′-type aromatic proton signals at δ 7.98 (2H, d,
H
1
J = 8.9 Hz) and 7.19 (2H, d, J = 8.9 Hz) implying the presence of p-substituted phenyl. The H
NMR data of 2 appeared one terminal methyl protons at δ 1.17 (3H, t, J = 7.2 Hz) and one
H
1
connected methylene protons at δ 3.02 (2H, q, J = 7.2 Hz). The H NMR data also showed
H
two anomeric proton signals [δ 5.02 (d, J = 7.6 Hz, H-1′), δ 4.37 (d, J = 7.7 Hz, H-1″)] sug-
H
H
1
3
gesting the presence of two β-glucopyranose units. The C NMR data (Table S2 and Figure
S8) and DꢃPT (Table S2 and Figure S9) of 2 showed 21 different carbons signals, comprised
1
carbonyl carbon, 6 aromatic carbons, 3 methylene aliphatic carbons, 10 methane aliphatic
carbons and 1 methyl carbon. The above data also revealed that 2 contained two typical
glucopyranose units [δ 101.5, 75.2, 78.0, 71.4, 77.9, 69.8] and [δ 104.7, 74.8, 78.0, 71.6, 77.8,
C
C
62.7], which were confirmed by acid hydrolysis and HPLC as the same as 1. HMBC data(Table
S2 and Figure S11) of 2, revealed the presence of the propionyl group in the aglycone as
correlations between the signals: (i) δ 3.02 (2H, q, J = 7.2 Hz) to the carbonyl carbon at δC
H
2
02.2 (C-7) and one methyl group at δ 8.8 (C-9); (ii) the methyl proton at δ 1.17 (H-9) to δ
C
H
C
202.2 (C-7) and 32.4 (C-8). HMBC correlations also showed the sugar unit connectivities as
correlations between the signals: the anomeric proton δ 5.02 (1H, d, J = 7.6 Hz) of Glc I to
H
δ 162.8 (C-4) of the aglycone indicating Glc I H-1 to be linked to C-4 of the aglycone; δ 4.37
C
H
(
1H, d, J = 7.7 Hz, Glc II H-1) to δ 69.8 (Glc I C-6) showing Glc II H-1 to be connected to Glc I
C
C-6. The sugar moieties of 2 linked at 4-O- were assigned as 4-O-β-d-glucopyranosyl
1→6)-β-d-glucopyranoside. Thus, the structure of 2 was identified as 4-hydroxypropiophe-
(
none-4-O-β-d-glucopyranosyl (1→6)-β-d-glucopyranoside (Figure 1).
The identity of the known naphthalenyl glucosides 3–8 (Figure 2) was established by
comparison with previously reported spectroscopic data as follows: 1, 4, 8-trihydroxy-

4
Y.ꢀY. ZHꢁꢂ ꢃT AL.
Figure 2. sꢁꢄꢊꢈꢁꢊꢄꢇꢉ ꢃf ꢈꢃmpꢃꢊnꢂꢉ 3–8.
3-naphthalene-carboxylic acid-1-O-β-d-glucopyranoside ethyl ester (3) (Zhou et al. 2015a),
(4S)-4-hydroxy-α-tetralone-4-O-β-d-glucopyranoside (4) (Liu et al. 2004), (4S)-45-dihydroxy-
α-tetralone-4-O-β-d-glucopyranoside (5) (Liu et al. 2004) (4S)-4-hydroxy-α-tetralone-4-O-β-
d-(6′-O-4″-hydroxylbenzoyl)glucopyrano-side (6) (Zhou et al. 2015a), (4S)-45-dihydroxy-
α-tetralone-4-O-β-
d
-(6′-O-4″-hydro-xylbenzoyl) glucopyranoside (7) (Zhou et al. 2015a), (4S)-
458-thihydroxy-α-tetralone-5-O-β-
d-(6′-O-4″-hydroxylbenzoyl) glucopyranoside (8) (Zhou et
al. 2015a). Among them, 3 and 6–7 were first discovered by our research group.
The isolated compounds (1–8) were assessed for cytotoxic activity against three human
tumour cell lines, BGC-823 (gastric carcinoma), HepG-2 (liver carcinoma), MCF-7 (breast car-
cinoma) and cisplatin as positive control using the MTT method. The in vitro cytotoxic activity
of these compounds and cisplatin was evaluated at different concentrations against
above-mentioned tumour cells, and the IC50 values are shown (Table S3). Among all the
compounds tested, only the new compounds 1 and 2 showed superior cytotoxic activity
against tumour cell, but the remaining compounds of naphthalenyl glucosides 3–8 had less
or no activity. These results were in accordance with previous reports (Liu et al. 2010).
3
. Experimental
3
.1. Plant material
The green husks of J. mandshurica were collected in Changbai Mountains (Jilin, China), in
late July 2014 and identified by one of us (Z.Y.Y.). The dried samples were grounded into fine
powder (60 mesh), dried thoroughly in an oven at 40 °C for 3 days. A voucher specimen (QLY
20140112) has been stored at the College of Pharmacy, Heilongjiang ꢂniversity of Chinese
Medicine.
3
.2. Chemicals and general experiments procedures
High-resolution electrospray ionisation (HR-ꢃSI) mass spectra were obtained on a micromass
LCT spectrometer (Waters, Milford, ꢂSA). The IR spectra were obtained on a Shimadzu

NATꢂRAL PRꢁDꢂCT RꢃSꢃARCH
5
FTIR-8400S spectrometer (Shimadzu, Tokyo, Japan). Melting points are uncorrected and were
1
13
obtained on Hoover capillary melting point (Thomas-Hoover, New Jersey, ꢂSA). H, C NMR,
DꢃPT, HSQC, HMBC were obtained on Bruker DPX 400 NMR instrument (Bruker, Rheinstetten,
Germany). Chemical shifts (δ) are expressed in parts per million (ppm) using TMS as an
internal standard. HPLC chromatograms were obtained with an Agilent Technologies 1260
infinity HPLC system (Agilent Technologies, Germany) and semi-preparative HPLC (Waters,
515–2414, ꢂSA) was performed using a Hypersil-ꢁDS II column (300 × 20 mm i.d., 10 μm,
Ylite, Dalian, China). Deionised water was prepared a Milli-Q system (Milford, MA, ꢂSA). An
automatic polarimeter (WZZ-2B, Chengdu, China) was used for the determination of specific
rotation. A multiscan microplate reader (Thermo Labsystems, Helsinki, Finland) was used
for the MTT assays. BGC-823, HepG-2 and MCF-7 cell lines obtained from Institute of bio-
chemistry and cell biology (Shanghai, China) were grown in Dulbecco’s modified ꢃagle’s
medium (DMꢃM) and foetal bovine serum (FBS) from Sigma (St. Louis, Mꢁ, ꢂSA). Column
chromatography (CC) was performed using Si gel (80–300 mesh) from HaiYang Co. Ltd
(Qingdao, China) and ꢁDS from DAISꢁGꢃL (Beijing, China). All solvents used were of analyt-
ical grade.
3
.3. Extraction and isolation
The air-dried parts of materials (10.0 kg) were powdered and soaked in 80 L of ꢃtꢁH for
days. The extraction was repeated three times and then concentrated under reduced pres-
7
sure to afford the ꢃtꢁH extract (637 g). The extracts were concentrated and then suspended
in H ꢁ followed by successive partitioning with n-hexane, CH Cl , ꢃtꢁAc and n-BuꢁH, respec-
2
2
2
tively. The BuꢁH extract (122 g) was subjected to silica gel (200–300 mesh) column chroma-
tography (CC), eluted with CH Cl –MeꢁH (30:1 → 1:1, v/v), to afford six fractions (Fr1-Fr6).
2
2
Fraction 3 (5.15 g) was fractionated and purified by ꢁDS CC with MeꢁH/H ꢁ (2:8 → 1:0) to
2
yield 1 (10.8 mg) and 3 (5.5 mg). Fraction 4 (4.88 g) was separated twice by ꢁDS CC with
MeꢁH/H ꢁ (2:8 → 1:0) to afford a number of subfractions 4a, 4b, 4c and 4d. Subfraction 4b
2
(
0.56 g) was purified by semi-preparative HPLC chromatography (MeꢁH/H ꢁ 45:55, v/v, flow
2
rate 3 mL/min) to yield 2 (6.4 mg), 6 (10.2 mg), 7 (8.1 mg) and 8 (12.4 mg). Subfraction 4c
0.56 g) was purified by semi-preparative HPLC chromatography (MeꢁH/H ꢁ 55:45, v/v, flow
(
2
rate 3 mL/min) to yield 4 (4.9 mg), 5 (9.3 mg).
3
.3.1. Hexyl-1-O-α-l-arabinofuranosyl (1 → 6)-β-d-glucopyranoside (1)
25
−1
White amorphous powder,[ꢀ] − 22.4(c = 0.50, MeꢁH); IRνmax (film) cm : 3395, 2961, 2928,
D
1
2855, 1453, 1078, 1004. H NMR (MeꢁH, 400 MHz) δ: 4.95 (1H, d, J = 1.1 Hz, H-1′′), 4.24 (1H,
d, J = 7.8 Hz, H-1′), 4.04 (1H, dd, J = 11.0, 2.3 Hz, H-6′a), 3.98 (1H, m, H-4′′), 3.96 (1H, m, H-2′′),
3
5
.86(1H, dt, J = 9.6, 6.8 Hz, H-1a), 3.81 (1H, dd, J = 6.2, 3.6 Hz, H-3′′), 3.73 (1H, dd, J = 11.6,
.3 Hz, H-5′′a), 3.63 (1H, dd, J = 11.6, 3.4 Hz, H-5′′b), 3.53 (1H, dt, J = 9.6, 6.8 Hz, H-1b), 3.50
(
1H, dd, J = 11.0, 5.0 Hz, H-6′b), 3.42 (1H, ddd, J = 9.0, 5.9, 2.3 Hz, H-5′), 3.34 (1H, t, J = 8.8 Hz,
H-3′), 3.25 (1H, t, J = 9.0 Hz, H-4′), 3.15 (1H, dd, J = 8.8, 7.8 Hz, H-2′), 1.61 (2H, dt, J = 14.9,
1
3
6
1
7
.8 Hz, H-2), 1.28–1.35 (6H, m, H-3, 4, 5), 0.90 (3H, t, J = 6.8 Hz, H-6). C NMR (125 MHz, MeꢁH):
10.0 (C-1′′), 104.4 (C-1′), 86.0 (C-2′′), 83.3 (C-4′′), 78.1 (C-3′), 76.7 (C-5′), 75.1 (C-2′), 72.0 (C-4′),
1.1 (C-1), 68.1 (C-6′), 63.1 (C-5′′), 32.9 (C-4), 30.8 (C-2), 26.8 (C-3), 23.7 (C-5), 14.4 (C-6).
+
HR-ꢃSI-MS (positive): m/z 419.4137 [M + Na] (Calcd for C H ꢁ , 396.2011).
1
7
32 10

6
Y.ꢀY. ZHꢁꢂ ꢃT AL.
3
.3.2. 4-hydroxypropiophenone-4-O-β-d-glucopyranosyl (1 → 6)-β-d-
glucopyranoside (2)
2
5
−1
White amorphous powder,[ꢀ]D − 13.8(c = 0.75, MeꢁH); IRνmax (film) cm : 3287, 2923, 2916,
1
2915, 1725, 1688, 1095. H NMR (MeꢁH, 400 MHz) δ: 7.98 (2H, d, J = 8.9 Hz, H-2, 6), 7.19 (2H,
d, J = 8.9 Hz, H-3, 5), 5.02 (1H, d, J = 7.6 Hz, H-1′), 4.37 (1H, d, J = 7.7 Hz, H-1′′), 4.17 (1H, dd,
J = 11.6, 1.5 Hz, H-6′a), 3.81(1H, dd, J = 11.6, 5.8 Hz, H-6′b), 3.74 (1H, dd, J = 11.1, 5.3 Hz, H-6′′a),
3
.64 (1H, m, H-6′′b), 3.49 (2H, m, H-2′′, 5′′), 3.39 (1H, m, H-4′), 3.29 (3H, m, H-3′, 3′′, 4′′), 3.22
1
3
(
1H, m, H-2′), 3.17 (1H, m, H-5′), 3.02 (2H, q, J = 7.2 Hz, H-8), 1.17 (3H, t, J = 7.2 Hz, H-9). C
NMR (125 MHz, MeꢁH) δ: 202.2 (C-7), 162.8 (C-4),132.4 (C-1), 131.4 (C-2, 6), 117.4 (C-3, 5),
04.7 (C-1′′), 101.4 (C-1′), 78.0 (C-3′, 3′′), 77.9 (C-5′), 77.8 (C-5′′), 75.2 (C-2′), 74.8 (C-2′′), 71.6
1
(
4
C-4′′), 71.4 (C-4′), 69.8 (C-6′), 62.7 (C-6′′), 32.4 (C-8), 8.8 (C-9). HR-ꢃSI-MS (positive): m/z
+
75.1865 [M + H] (Calcd for C H ꢁ , 474.1768).
2
1
30 12
3
.4. Acid hydrolysis
Compounds 1 and 2 (each about 1.5 mg) were refluxed with 1.0 mol/L HCl (5 mL, dioxane-
H ꢁ, v/v) for 7 h under 80 °C water bath. After cooling down to room temperature, the reac-
2
tion mixture was extracted three times with CH Cl :H ꢁ (each 1:1, v/v, 1 mL). The aqueous
2
2
2
layer was neutralised with 5% NaꢁH and desalted with Sephadex LH-20 to obtain the sugar
residue (0.8 mg). The monosaccharides in 1 were first determined as l-arabinose and d-glu-
cose by co-TLC with authentic sugar, eluting with chloroform/n-BuꢁH/MeꢁH/acetic acid/
water 17:10:6:2:3 [Rf(l-Ara) = 0.41, Rf(d-Glc) = 0.38). In the same way, the monosaccharide in
2
was identified as d-glucose. Then, the PMP derivative of the monosaccharide mixture was
prepared as the past report (Honda et al. 1989). The resulted PMP-monosaccharide was
dissolved in ꢃtꢁH/n-hexane solution (1:4, v/v) and analysed by HPLC using CHRIALPAK AD-H
column, with an elution solvent system of n-hexane/2-propanol (87:13), at 25 °C, and
detected by DAD detector. The standard sugars were operated with the same methods. The
monosaccharides in 1 were determined to be l-arabinose (t = 11.3 min) and d-glucose
R
(
t = 9.6 min). The monosaccharide in 2 was determined to be d-glucose (t = 9.6 min).
R
R
3
.5. Cell cultures
HepG-2, BGC-823 and MCF-7 cell lines were maintained in DMꢃM supplemented with 10%
foetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco-BRL).
The cells were incubated in 5% Cꢁ humidified at 37 °C for growth.
2
3
.6. Cytotoxicity assay
Tumour cells in logarithmic growth phase were seeded in a 96-well microtiter plates and
kept overnight for attachment. ꢃight compounds and positive control (cisplatin), dissolved
in dimethyl sulfoxide and PB (phosphate buffer), were added at various concentrations from
200 to 0.5 μM for 24 h. The optical density was measured at 570 nm using a multiscan
microplate reader. All experiments were performed in triplicate. Data were expressed as the
concentration required for inhibiting growth of tumour cells by 50% (IC ).
5
0

NATꢂRAL PRꢁDꢂCT RꢃSꢃARCH
7
Supplementary material
Supplementary material relating to this paper is available online, alongside Table S1–S3 and Figure
S1–S11.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Science Foundation [grant number 81202890]; China
Postdoctoral Science Foundation funded project [grant number 2013M530164/2014T70374];
Heilongjiang Postdoctoral Fund [grant number LBH-Z13195]; Harbin city youth science and technol-
ogy innovation talents [grant number 2013RFQXJ052].
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