A new steroidal saponin from Polygonatum sibiricum

Article abstract of DOI:10.1080/10286020.2017.1351436

A new furostan type steroidal saponin, kingianoside Z (1), along with four known compounds (2–5), was isolated from the ethanolic extract of Polygonatum sibiricum Delar. ex Redoute. Their structures were determined by spectroscopical method and by comparison with previously reported spectroscopic data. Compounds 3–5 showed significant cytotoxicity against HepG2 cell lines with IC₅₀ values of 14.2, 12.1 and 8.5?μM, respectively.

Full text of DOI:10.1080/10286020.2017.1351436

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
A new steroidal saponin from Polygonatum
sibiricum
Hong-Yang Zhang, Wei-Cheng Hu, Guo-Xu Ma, Nai-Liang Zhu, Xiao-Bo Sun,
Hai-Feng Wu & Xu-Dong Xu
To cite this article: Hong-Yang Zhang, Wei-Cheng Hu, Guo-Xu Ma, Nai-Liang Zhu, Xiao-Bo Sun,
Hai-Feng Wu & Xu-Dong Xu (2017): A new steroidal saponin from Polygonatum sibiricum, Journal
of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1351436
To link to this article: https://doi.org/10.1080/10286020.2017.1351436
Published online: 23 Nov 2017.
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Date: 24 November 2017, At: 04:05

Journal of asian natural Products research, 2017
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2017.1351436
ꢀ
A new steroidal saponin from Polygonatum sibiricum
a,b‡
c,d‡
b
b
b
Hong-Yang Zhang , Wei-Cheng Hu , Guo-Xu Ma , Nai-Liang Zhu , Xiao-Bo Sun ,
b
b
Hai-Feng Wu and Xu-Dong Xu
ꢇ
cꢈꢉꢁꢈꢆ ꢄꢊ rꢈꢂꢈꢇꢆꢋꢀ ꢇꢉꢃ dꢈvꢈꢌꢄpmꢈꢉꢁ ꢄꢉ lꢅꢊꢈ sꢋꢅꢈꢉꢋꢈꢂ ꢇꢉꢃ eꢉvꢅꢆꢄꢉmꢈꢉꢁ sꢋꢅꢈꢉꢋꢈꢂ, hꢇꢆbꢅꢉ uꢉꢅvꢈꢆꢂꢅꢁy ꢄꢊ
b
cꢄmmꢈꢆꢋꢈ, hꢇꢆbꢅꢉ 150076, cꢀꢅꢉꢇ; Kꢈy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢊ Bꢅꢄꢇꢋꢁꢅvꢈ sꢍbꢂꢁꢇꢉꢋꢈ ꢇꢉꢃ rꢈꢂꢄꢍꢆꢋꢈ uꢁꢅꢌꢅzꢇꢁꢅꢄꢉ ꢄꢊ cꢀꢅꢉꢈꢂꢈ
hꢈꢆbꢇꢌ Mꢈꢃꢅꢋꢅꢉꢈ, Mꢅꢉꢅꢂꢁꢆy ꢄꢊ eꢃꢍꢋꢇꢁꢅꢄꢉ, iꢉꢂꢁꢅꢁꢍꢁꢈ ꢄꢊ Mꢈꢃꢅꢋꢅꢉꢇꢌ Pꢌꢇꢉꢁ dꢈvꢈꢌꢄpmꢈꢉꢁ, cꢀꢅꢉꢈꢂꢈ aꢋꢇꢃꢈmy ꢄꢊ Mꢈꢃꢅꢋꢇꢌ
ꢋ
sꢋꢅꢈꢉꢋꢈꢂ ꢇꢉꢃ Pꢈkꢅꢉg uꢉꢅꢄꢉ Mꢈꢃꢅꢋꢇꢌ cꢄꢌꢌꢈgꢈ, Bꢈꢅjꢅꢉg 100193, cꢀꢅꢉꢇ; Jꢅꢇꢉgꢂꢍ cꢄꢌꢌꢇbꢄꢆꢇꢁꢅvꢈ iꢉꢉꢄvꢇꢁꢅꢄꢉ cꢈꢉꢁꢈꢆ
ꢄꢊ rꢈgꢅꢄꢉꢇꢌ Mꢄꢃꢈꢆꢉ agꢆꢅꢋꢍꢌꢁꢍꢆꢈ & eꢉvꢅꢆꢄꢉmꢈꢉꢁꢇꢌ Pꢆꢄꢁꢈꢋꢁꢅꢄꢉ, hꢇꢅyꢅꢉ nꢄꢆmꢇꢌ uꢉꢅvꢈꢆꢂꢅꢁy, hꢍꢇꢅꢇꢉ 223300, cꢀꢅꢉꢇ;
ꢃ
Jꢅꢇꢉgꢂꢍ Kꢈy lꢇbꢄꢆꢇꢁꢄꢆy ꢊꢄꢆ eꢋꢄ-agꢆꢅꢋꢍꢌꢁꢍꢆꢇꢌ Bꢅꢄꢁꢈꢋꢀꢉꢄꢌꢄgy ꢇꢆꢄꢍꢉꢃ hꢄꢉgzꢈ lꢇkꢈ, hꢇꢅyꢅꢉ nꢄꢆmꢇꢌ uꢉꢅvꢈꢆꢂꢅꢁy,
hꢍꢇꢅꢇꢉ 223300, cꢀꢅꢉꢇ
ABSTRACT
ARTICLE HISTORY
A new furostan type steroidal saponin, kingianoside Z (1), along
with four known compounds (2–5), was isolated from the ethanolic
extract of Polygonatum sibiricum Delar. ex Redoute. Their structures
were determined by spectroscopical method and by comparison with
previously reported spectroscopic data. Compounds 3–5 showed
significant cytotoxicity against HepG2 cell lines with IC50 values of
rꢈꢋꢈꢅvꢈꢃ 15 fꢈbꢆꢍꢇꢆy 2017
aꢋꢋꢈpꢁꢈꢃ 2 Jꢍꢌy 2017
KEYWORDS
aꢂpꢇꢆꢇgꢇꢋꢈꢇꢈ; Polygonatum
sibiricum; ꢊꢍꢆꢄꢂꢁꢇꢉ ꢂꢁꢈꢆꢄꢅꢃꢇꢌ
ꢂꢇpꢄꢉꢅꢉ; ꢋyꢁꢄꢁꢄxꢅꢋ ꢇꢋꢁꢅvꢅꢁꢅꢈꢂ
14.2, 12.1 and 8.5 μM, respectively.
OH
O
HO
HO
O
OH
OH
O
O
OH
O
O
HO
O
OOH
HO
O
HO
HO
O
OH
OH
HO
O
O
OH
HO
OH
1
. Introduction

e genus Polygonatum (Asparagaceae) of more than 60 species is mainly distributed in
the temperate and subtropical regions in the world, among which 31 species are widely
growing in Northern China [1]. Polygonatum sibiricum Delar. ex Redoute is herbaceous
perennial plant traditionally used as anti-cancer, anti-inflammatory, analgesia, hypotensive,
anti-atherogenetic, anti-senility, anti-tumor agents [2,3]. Previous chemical research on the
genus Polygonatum disclosed the presence of triterpenes, steroidal saponins, flavonoids,
alkaloids, lignanoid, polysaccharides [4,5]. However, there are few chemical studies on P.
sibiricum. Steroidal saponins are characteristic constituents of the genus, exhibiting diverse
biological activities including immune-regulation, antitumor, and improvement of learning
CONTACT hꢇꢅ-fꢈꢉg Wꢍ
tꢀꢈ fiꢆꢂꢁ ꢁwꢄ ꢇꢍꢁꢀꢄꢆꢂ ꢋꢄꢉꢁꢆꢅbꢍꢁꢈꢃ ꢈqꢍꢇꢌꢌy ꢁꢄ ꢁꢀꢅꢂ wꢄꢆk.
ꢀꢊwꢍ@ꢅmpꢌꢇꢃ.ꢇꢋ.ꢋꢉ; Xꢍ-dꢄꢉg Xꢍ
xꢃxꢍ@ꢅmpꢌꢇꢃ.ꢇꢋ.ꢋꢉ
‡
©
2017 iꢉꢊꢄꢆmꢇ uK lꢅmꢅꢁꢈꢃ, ꢁꢆꢇꢃꢅꢉg ꢇꢂ tꢇyꢌꢄꢆ & fꢆꢇꢉꢋꢅꢂ Gꢆꢄꢍp

2
H.ꢀY. ZHANG ET AL.
and memory abilities [6–9]. During our investigation of bioactive constituents from natural
products, a new steroidal saponin, kingianoside Z (1), and four known homoisoflavonoids,
were isolated from the 95% ethanolic extract of the rhizomes of P. sibiricum (Figure 1). is
paper describes the isolation, structural elucidation of the new compound, and biological
activities of several known compounds.
2
. Results and discussion
Compound 1 was isolated as a white amorphous powder and gave positive Liebermann–
Burchard and Ehrlish reactions, suggesting 1 to be a furostanol saponin. Its molecular formula
was C H O as determined by the HR-ESI-MS quasi-molecular ion peak at m/z 1261.5466
57
90 29
+
[
M + Na] , requiring 13 degrees of unsaturation. UV λmax (MeOH) nm (log ε): 208 (4.24).
−1

e IR spectrum showed an absorption band at 3431 cm for hydroxyl group, a character-
−1
−1
istic absorption band at 1706 cm for carbonyl, an absorption band at 1550 cm for double
−1
−1
bond, a characteristic absorption band at 1072 cm for glucosidic bond, methyl (2929 cm ),
−1
−1
methylene (2878 cm ), C-H vibration area of methyl and methylene (1305–1383 cm ). e
1
H NMR spectrum (Table 1) showed four methyl signals at δ 0.91 (H-18), 0.90 (H-19), 1.72
H
(
H-21), 1.01 (d, J = 6.6 Hz, H-27), five anomeric protons at δ 4.83 (1H, d, J = 7.8 Hz, H-1′′′′′),
H
4
.86 (1H, d, J = 7.8 Hz, H-1′), 5.13 (1H, d, J = 7.8 Hz, H-1′′), 5.29 (1H, br d, J = 7.8 Hz, H-1′′′′)
and 5.58 (1H, br d, J = 7.8 Hz, H-1′′′), and an olefinic proton at δ 5.26 (1H, br s, H-6). e
H
13
C APT NMR spectrum (Table 1) of 1 displayed 57 carbon resonances including a carbonyl
carbon signal at δ 213.2 (C-12), two double bonds at δ 141.2 (C-5), 121.8 (C-6), 103.4
C
C
(
C-20), and 153.5 (C-22), and three oxymethine carbons at δ 77.8 (C-3), 83.4 (C-16), and 75.6
C
(
C-26), five anomeric carbons at δ 103.1 (C-1′), 105.5 (C-1′′), 105.3 (C-1′′′), 104.9 (C-1′′′′)
C
and 105.6 (C-1′′′′′). e above evidences indicated that compound 1 was a furostanol sapo-
1
13
nin with five sugar units. e H and C NMR spectroscopic data of 1 was highly similar to
those of kingianoside E with the differences that an extra sugar unit was attached at C-3′′ and
another double bond was formed between C-20 and C-22 in 1 [10]. Combination of HSQC,
COSY, TOCSY, and HSQC-TOCSY experiments allowed the sequential assignments of all
proton resonances within each sugar residue, starting from the readily identifiable anomeric
OH
O 1'''''
O
HO
HO
27
OH
2
1
2
6
20
22
25
OH
O 18
23
24
1
1
O
O
OH
12
17
HO
19
13
14
16
OH
O
1
O
O
O 1
''
2
15
9
HO
HO
8
7
O
10
OH1''''
O
3
HO
5
OH
OH1'
4
6
O
HO
HO
OH 1'''
Figure 1. tꢀꢈ ꢋꢀꢈmꢅꢋꢇꢌ ꢂꢁꢆꢍꢋꢁꢍꢆꢈ ꢄꢊ ꢋꢄmpꢄꢍꢉꢃ 1.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
13
Table 1. h (600 Mhz) ꢇꢉꢃ c (150 Mhz) nMr ꢃꢇꢁꢇ ꢄꢊ ꢋꢄmpꢄꢍꢉꢃ 1 ꢅꢉ c d n (δ ꢅꢉ ppm).
5
5
No.
δH (J in Hz)
δC, type
No.
δH (J in Hz)
δC, type
1
2
3
4
5
6
7
8
9
0.84–0.87, m; 1.46–1.49, m
1.66–1.69, m; 2.06–2.08, m
3.83–3.85, m
37.4, ch2
30.2, ch2
77.8, ch
39.4, ch2
141.2, c
121.8, ch
32.2, ch2
31.0, ch
52.7, ch
37.9, c
Gꢇꢌ-1′
2′
3′
4′
5′
6′
Gꢌꢋ-1′′
2′′
3′′
4′′
5′′
6′′
Gꢌꢋ-1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
Gꢌꢋ-1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
Gꢌꢋ-1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′
4.86, ꢃ (7.8)
4.41–4.43, m
4.08–4.10, m
4.57–4.59, m
3.97–3.99, m
103.1, ch
73.2, ch
75.6, ch
80.5, ch
75.3, ch
60.9, ch2
105.5, ch
81.8, ch
88.8, ch
70.6, ch
77.4, ch
63.2, ch2
105.3, ch
76.2, ch
77.9, ch
70.9, ch
78.6, ch
62.7, ch2
104.9, ch
75.4, ch
76.7, ch
72.1, ch
77.3, ch
63.4, ch2
105.6, ch
75.3, ch
78.2, ch
71.7, ch
78.7, ch
62.2, ch2
2.40, ꢁ (11.4); 2.66, ꢃ (12.6)
5.26, (bꢆ ꢂ)
1.41–1.43, m; 1.84–1.86, m
1.83–1.85, m
4.66–4.68, m; 4.20–4.22, m
5.13, ꢃ (7.8)
4.35–4.36, m
4.15, ꢃ (9.0)
3.72–3.75, m
3.85–3.88, m
1.28–1.30, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.28, ꢃꢃ (5.4, 14.4); 2.50–2.53, m
38.1, ch2
213.2, c
57.5, c
3.98–3.99, m; 4.40–4.42, m
5.58, bꢆ ꢃ (7.8)
4.05–4.07, m
1.41–1.44, m
1.58–1.60, m; 2.04–2.07, m
4.83–4.85, m
3.42, ꢃ (10.2)
0.91, ꢂ
54.6, ch
34.3, ch2
83.4, ch
56.4, ch
14.3, ch3
19.2, ch3
103.4, c
12.0, ch3
153.5, c
24.1, ch2
31.7, ch2
34.1, ch
75.6, ch2
17.5, ch3
4.14–4.16, m
4.23–4.26, m
3.86–3.88, m
4.36–4.38, m; 4.51–4.52, m
5.29, bꢆ ꢃ (7.8)
3.96–3.99, m
0.90, ꢂ
1.72, ꢂ
4.10–4.12, m
4.33–4.36, m
3.80–3.82, m
1.94–1.97, m; 2.03–2.06, m
1.66–1.68, m; 2.03–2.04, m
1.92–1.95, m
3.47, ꢃꢃ (7.8, 7.8); 4.04–4.06, m
1.01, ꢃ (6.6)
4.22–4.25, m; 4.05–4.07, m
4.83, ꢃ (7.8)
4.01–4.03, m
4.23–4.25, m
4.23–4.25, m
3.95–3.97, m
4.54–4.57, m; 4.39–4.41, m
protons (Figure 2). us, on the basis of chemical shif s, the multiplicity of the signals and the
coupling constants, the extra sugar residue was identified as glucose [11]. Furthermore, the
sugar was also identified as glucose and galactose by acid hydrolysis followed by comparison
with an authentic sample via TLC. e common D-configurations for β-galactopyranosyl
and β-glucopyranose were assumed, according to those most of en encountered among the
plant glycosides in each case. e HMBC experiment was performed to establish the location
of the functional groups and the full structure of 1. Key correlation peaks observed in the
HMBC spectrum between H-1′′′′′ of the glucosyl at δ 4.83 (d, J = 7.8 Hz) and C-26 (δ 75.6)
H
C
of the aglycon, between H-1′ of the galactose at δ 4.86 (1H, d, J = 7.8 Hz) and C-3 (δ 77.8)
H
C
of the aglycon, between H-1′′ of the glucosyl at δ 5.13 (d, J = 7.8 Hz) and C-4′ (δ 80.5) of
H
C
the galactose residue, between H-1′′′ of the glucosyl at δ 5.58 (d, J = 7.8 Hz) and C-2′′ (δ
H
C
8
1.8) of the glucosyl residue, between H-1′′′′ of the glucosyl at δ 5.29 (d, J = 7.8 Hz) and C-3′′
H
(δC 88.8) of the glucosyl residue allowed the tetrasccharide chain at C-3 to be determined as
β-ꢀ-glucopyranosyl-(1→3)-O-[O-β-ꢀ-glucopyranosyl-(1→2)]-O-β-ꢀ-glucopyranosyl-(1→4)-
β-ꢀ-galacto-pyranoside. e HMBC experiment also showed the presence of key correlations
between H-18 and C-13, C-14, C-17, and C-12; between H-19 and C-1, C-5, C-9. In HSQC and
1
1
H– H COSY spectra, the proton signals of C-26 were observed at δ 4.05 (1H, m, 26-H ) and
H
a
3
.47 (1H, dd, J = 7.8, 7.8 Hz, 26-H ) in the spirostanol, which indicated that the absolute con-
b
figuration of C-25 was S (Δa-b = 4.05–3.47 > 0.57) [12]. erefore, the structure of compound
1
was established as 3β,25(S)-[3-O-β-ꢀ-glucopyranosyl-(1→3)-β-ꢀ-glucopyranosyl-(1→2)-
β-ꢀ-glucopyranosyl-(1→4)-β-ꢀ-galactopyranosy)oxy]-26-[(β-ꢀ-glucopyranosyl)oxy]-20(22)-
furost-5-en-12-one, and named for kingianoside Z.

4
H.ꢀY. ZHANG ET AL.
OH
O
HO
HO
O
OH
OH
O
OH
O
O
O
HO
OH
O
O
O
HO
HO
O
OH
HO
O
OH
HMBC
OH
O
HO
HO
1
1
H- H COSY
OH
NOESY
OH
O
HO
O
HO
OH
H
OH
H
H
H
H
OH
O
O
H
H
O
HO
H
H
O
O
OH
H
H
O
H
H
HO
HO
H
O
H
O
OH
HO
O
H
H
OH
H
OH
H
H
O
HO
HO
OH
1
1
Figure 2. Kꢈy hMBc, h- h cosY ꢇꢉꢃ noesY ꢋꢄꢆꢆꢈꢌꢇꢁꢅꢄꢉꢂ ꢄꢊ ꢋꢄmpꢄꢍꢉꢃ 1.
Table 2. cyꢁꢄꢁꢄxꢅꢋ ꢇꢋꢁꢅvꢅꢁy ꢄꢊ ꢋꢄmpꢄꢍꢉꢃꢂ 3–5.
IC50 (μM)
HepG2
Compounds
3
4
5
14.2 ± 2.1
12.1 ± 1.3
8.5 ± 1.7
5
-FUꢇ
49.8 ± 3.6
nꢄꢁꢈ: Vꢇꢌꢍꢈꢂ pꢆꢈꢂꢈꢉꢁ mꢈꢇꢉ ± sd ꢄꢊ ꢁꢆꢅpꢌꢅꢋꢇꢁꢈ ꢈxpꢈꢆꢅmꢈꢉꢁꢂ.
ꢇ
Pꢄꢂꢅꢁꢅvꢈ ꢋꢄꢉꢁꢆꢄꢌ ꢂꢍbꢂꢁꢇꢉꢋꢈ.

e known compounds, 4′-demethyl-3,9-dihydropunctatin (2) [13], (±)-5,7-dihydroxy-
6
,8-dimethyl-3-(2′-hydroxyl-4′-methoxybenzyl)-chroman-4-one (3) [14], (3R)-5,7-
dihydroxy-8-methyl-3-(2′,4′-dihydroxybenzyl)-chroman-4-one (4) [15], 3-(4′-hydroxyben-
zyl)-5,7-dihydroxy-6,8-dimethyl-chroman-4-one (5) [16] were identified by comparison
of their NMR spectral data with those in the literature.
Compounds 3–5 were tested in vitro for their cytotoxic activity against HepG2 human
cancer cell lines. e results showed that compounds 3–5 displayed significant cytotoxicity
against HepG2 cells with IC values of 14.2, 12.1, and 8.5 μM, respectively (Table 2).
5
0

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3
. Experimental
3
.1. General experimental procedures
Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (PerkinElmer,
Waltham, MA, USA). UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-
8
400S spectrometers (Bruker Optics, Billerica, Germany), respectively. NMR spectra were
obtained with a Bruker AV III 600 NMR spectrometer (chemical shif values are presented
as δ values with TMS as the internal standard) (Palo, Alto, CA, USA). HRESIMS were
performed on a LTQ-Obitrap XL spectrometer (Waters Corporation, Milford, MA, USA).
Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two
pumps of K-501, a UV detector of K-2600 (Agilent Technologies, Santa Clara, CA, USA),
and an YMC Pack C column (250 mm × 10 mm, i.d., 5 μM, YMC Co. Ltd., Japan) eluted
1
8
with CH OH-H O at a flow rate of 2 ml/min. C reversed-phase silica gel (40–63 μM,
3
2
18
Merk, Darmstadt, Germany), Toyopearl HW-40C (50–100 μM, Tosoh Corp., Tokyo, Japan),
Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75–150 μM, Mitsubishi
Chemical Corporation, Tokyo, Japan) and silica gel (100–200 and 300–400 mesh, Qingdao
Marine Chemical plant, Qingdao, China) were used for column chromatography. And pre-
coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai,
China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical
Works, Beijing, China).
3
.2. Plant material
e whole plants of Polygonatum sibiricum were collected in Nanning, Guangxi Province,
China, in July 2015, and were authenticated by Prof. Jun-Shan Yang. A voucher specimen
HJ20150710) has been deposited at the Institute of Medicinal Plant Development, Chinese
Academy of Medical Sciences.

(
3
.3. Extraction and isolation
e air-dried and powdered rhizomes of P. sibiricum (4.5 kg) were extracted three times

with 95% ethanol (20 L, three times) at room temperature. Af er filtration, the organic
solvent was removed from the combined extracts under vacuum to afford a crude extract
(
460 g). e extract was dissolved in water and partitioned with petroleum ether, EtOAc
and n-BuOH, successively. e EtOAc fraction (130 g) was subjected to silica gel column
chromatography (CC) (100–200 mesh), and eluted with a petroleum ether/EtOAc gradient
(15:1, 10:1, 5:1, 3:1, 1:1, 0:1, V/V) to yield five fractions (Fr. A–E). Fr. B (7.5 g) was repeat-
edly chromatographed on silica gel CC (200–300 mesh) using a petroleum ether–EtOAc
gradient (from 10:1 to 1:1). e obtained fractions were combined on the basis of silica gel
TLC and five fractions (Fr. B-a–Fr. B-e) were obtained. Fr. B-b (52 mg) was further purified
by semi-preparative HPLC (YMC C , 250 × 10 mm, 5 μm) isocratic elution using 60%
1
8
aqueous CH OH (2 ml/min) to obtain compound 5 (3.7 mg, t = 25 min). Fr. C (8.2 g) was
3
R
subjected to medium pressure liquid chromatography (MPLC, 36 mm × 310 mm) over
ODS using a MeOH/H O gradient system (30:70–70:30) to yield three fractions (Fr. C-a–Fr.
2
C-c). Fr. C-a (86 mg) was purified by HPLC (YMC C , 250 × 10 mm, 5 μm) using MeOH-
1
8
H O (45:55, v/v) gave compounds 2 (5.7 mg, t = 18 min) and 4 (3.1 mg, t = 23 min). e
2
R
R

6
H.ꢀY. ZHANG ET AL.
n-BuOH soluble fraction was separated by medium pressure liquid chromatography (MPLC,
6 mm × 310 mm) over ODS eluting with MeOH/H O gradient (30:70, 50:50, 70:30, 100:0,
3
2
V/V) to yield four fractions (Fr. 1–4). Fr. 1 (11.2 g) was loaded onto a Sephadex LH-20 col-
umn eluted with MeOH to give four fractions (Fr. 1-a–Fr. 1-d). Fr. 1-a (100 mg) was further
purified by semi-preparative HPLC (YMC C , 250 × 10 mm, 5 μm) isocratic elution using
1
8
MeOH-H O (25:75, v/v) to obtain compound 3 (4.3 mg). Fr. 3 (10.6 g) was subjected to
2
a reversed-phase C column chromatography eluted with MeOH-H O (30:70/70:30) and
1
8
2
then purified by HPLC (YMC C , 250 × 10 mm, 5 μm) isocratic elution using 20% aqueous
1
8
CH OH (2 ml/min) to obtain compound 1 (5.6 mg, t = 29 min).
3
R
3
.3.1. Kingianoside Z (1)
2
5
White amorphous powder;[ꢀ] −36.7 (c 0.1, CHCl ); IR (KBr) ν 3431, 1706, 1550, 1072,
D
−
3
max
1
1
13
2
929, 2878, 1383 and 1305 cm ; H and C NMR spectral data, see Table 1; HRESIMS:
+
m/z 1261.5466 [M + Na] (calcd for C H O Na, 1261.5465).
5
7
90 29
3
.4. Acid hydrolysis of compound 1
Compound 1 (2 mg) was heated in 2 ml of 2 M trifluoroacetic acid at 95 °C for 2 h. e
reaction mixture was extracted three times with 2 ml of CHCl . e remaining aqueous
3
layer was concentrated to dryness with EtOH to give a residue, and the residue was dis-
solved in anhydrous pyridine (2 ml). e sugar was derivatized with ꢁ-cysteine methyl ester
hydrochloride (3 mg, 60 °C, 1 h) and subsequently silylated with hexamethyldisilazane
and chlorotrimethylsilane (Fluka) (2:1, 1.5 mL; 60 °C, 30 min). Finally, the supernatant
(
1.0 ml) was analyzed by GC-MS (ermo Scientific Trace-1300 ISQ GC/MS, Waltham,
MA, USA) under the following conditions: capillary column ermo Scientific TR-5 MS
30 m × 0.32 mm × 0.25 μm); temperature gradient: 150 °C for 2 min, then 5 °C/min to
50 °C; carrier, nitrogen gas (20 ml/min); and injection volume: 1.0 μl. e injection and
detector temperature were set at 290 °C, and the splitting ratio was 1/5. e presence of
-glucose and ꢀ-galactose in the acid hydrolysate of 1 was confirmed by comparison of
their respective retention times with those of standard samples.
(
2
ꢀ
3
.5. Cytotoxicity assay

e cytotoxicity of compounds 3–5 was assessed by the MTT method. e cells were grown
4
in DMEM supplemented with 10% fetal bovine serum and cultured at a number of 1.2 × 10
cells in each well. Each compound at three different concentrations of 10, 1, and 0.1 μg/
ml dissolved in dimethyl sulfoxide (DMSO) was subsequently added to the wells. Each
concentration was tested in triplicate. Af er incubation under 5% CO at 37 °C for 48 h,
2
1
0 μl of MTT (4 mg/ml) was added to each well, and the cells were incubated for another
four hours. en, the liquid in each well was removed, and DMSO (200 μl) was added. e
absorbance was recorded using a microplate reader at a wavelength of 570 nm.
Disclosure statement
No potential conflict of interest was reported by the authors.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Funding

is work was financially supported from Huaian Key Laboratory of Food Components and
Functional Foods (HLFF1602), and the technological large platform for comprehensive research
and development of new drugs in the Twelf h Five-Year “Significant New Drugs Created” Science
and Technology Major Projects [grant number 2012ZX09301-002-001-026].
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