Three new cytotoxic oleanane triterpenoid saponins from Cylindrokelupha dalatensis

Article abstract of DOI:10.1080/10286020.2017.1391227

Three new minor oleanane triterpenoid saponins, cylindrosides B (1), C (2), and D (3), were isolated from the seed of Cylindrokelupha dalatensis using chromatographic method. Their structures were established on the basis of the chemical and spectroscopic evidences. They displayed significant antitumor activity in vitro against HL60 cancer cell lines and IC₅₀ values were 7.15?±?0.63, 10.07?±?0.97, and 4.74?±?0.57?μM, respectively, by MTT method.

Full text of DOI:10.1080/10286020.2017.1391227

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Three new cytotoxic oleanane triterpenoid
saponins from Cylindrokelupha dalatensis
Meng-Yun Yan, Jie Liu, Shuang Xing, Guo-Ling Xiong, Ling-Peng Pei, Bai-Ping
Ma, Ping Liu & Wen-Bin Zhou
To cite this article: Meng-Yun Yan, Jie Liu, Shuang Xing, Guo-Ling Xiong, Ling-Peng Pei,
Bai-Ping Ma, Ping Liu & Wen-Bin Zhou (2017): Three new cytotoxic oleanane triterpenoid
saponins from Cylindrokelupha dalatensis, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2017.1391227
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1391227
Published online: 07 Nov 2017.
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Date: 08 November 2017, At: 01:49

Journal of asian natural Products research, 2017
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2017.1391227
ꢀ
Three new cytotoxic oleanane triterpenoid saponins from
Cylindrokelupha dalatensis
a
b
c
c
a
c
Meng-Yun Yan , Jie Liu , Shuang Xing , Guo-Ling Xiong , Ling-Peng Pei , Bai-Ping Ma ,
d
a
Ping Liu and Wen-Bin Zhou
ꢇ
b
iꢈꢂꢁꢅꢁꢉꢁꢊ ꢄꢋ Mꢅꢈꢄꢆꢅꢁy tꢆꢇꢃꢅꢁꢅꢄꢈꢇꢌ Mꢊꢃꢅꢍꢅꢈꢊ rꢊꢂꢊꢇꢆꢍꢀ, Mꢅꢈzꢉ uꢈꢅvꢊꢆꢂꢅꢁy ꢄꢋ cꢀꢅꢈꢇ, Bꢊꢅjꢅꢈg 100081, cꢀꢅꢈꢇ; Bꢊꢅjꢅꢈg
rꢊꢂꢊꢇꢆꢍꢀ iꢈꢂꢁꢅꢁꢉꢁꢊ ꢄꢋ cꢀꢅꢈꢊꢂꢊ Mꢊꢃꢅꢍꢅꢈꢊ, Bꢊꢅjꢅꢈg uꢈꢅvꢊꢆꢂꢅꢁy ꢄꢋ cꢀꢅꢈꢊꢂꢊ Mꢊꢃꢅꢍꢅꢈꢊ, Bꢊꢅjꢅꢈg 100029, cꢀꢅꢈꢇ;
ꢍ
dꢊpꢇꢆꢁmꢊꢈꢁ ꢄꢋ expꢊꢆꢅmꢊꢈꢁꢇꢌ tꢀꢊꢆꢇpꢊꢉꢁꢅꢍ ꢄꢋ rꢇꢃꢅꢇꢁꢅꢄꢈ sꢅꢍkꢈꢊꢂꢂ, Bꢊꢅjꢅꢈg iꢈꢂꢁꢅꢁꢉꢁꢊ ꢄꢋ rꢇꢃꢅꢇꢁꢅꢄꢈ Mꢊꢃꢅꢍꢅꢈꢊ,
ꢃ
Bꢊꢅjꢅꢈg 100850, cꢀꢅꢈꢇ; dꢊpꢇꢆꢁmꢊꢈꢁ ꢄꢋ Pꢀꢇꢆmꢇꢍy, hꢇꢅꢈꢇꢈ Bꢆꢇꢈꢍꢀ ꢄꢋ cꢀꢅꢈꢊꢂꢊ Pꢊꢄpꢌꢊ’ꢂ lꢅbꢊꢆꢇꢁꢅꢄꢈ aꢆmy Gꢊꢈꢊꢆꢇꢌ
hꢄꢂpꢅꢁꢇꢌ, hꢇꢅꢈꢇꢈ 572013, cꢀꢅꢈꢇ
ABSTRACT
ARTICLE HISTORY
Three new minor oleanane triterpenoid saponins, cylindrosides B
rꢊꢍꢊꢅvꢊꢃ 25 Mꢇꢆꢍꢀ 2017
aꢍꢍꢊpꢁꢊꢃ 9 oꢍꢁꢄbꢊꢆ 2017
(
1), C (2), and D (3), were isolated from the seed of Cylindrokelupha
dalatensis using chromatographic method. Their structures were
established on the basis of the chemical and spectroscopic evidences.
They displayed significant antitumor activity in vitro against HL60
cancer cell lines and IC50 values were 7.15 ± 0.63, 10.07 ± 0.97, and
KEYWORDS
Cylindrokelupha dalatensis;
ꢁ
ꢆꢅꢁꢊꢆpꢊꢈꢄꢅꢃ ꢂꢇpꢄꢈꢅꢈ;
ꢍyꢌꢅꢈꢃꢆꢄꢂꢅꢃꢊ B; ꢍyꢌꢅꢈꢃꢆꢄꢂꢅꢃꢊ
c; ꢍyꢌꢅꢈꢃꢆꢄꢂꢅꢃꢊ d
4.74 ± 0.57 ꢀM, respectively, by MTT method.
CONTACT Wꢊꢈ-Bꢅꢈ Zꢀꢄꢉ
zꢀꢄꢉ_wꢊꢈbꢅꢈ@mꢉꢍ.ꢊꢃꢉ.ꢍꢈ; Pꢅꢈg lꢅꢉ
ꢌꢅꢉpꢅꢈg13601203271@163.ꢍꢄm
©
2017 iꢈꢋꢄꢆmꢇ uK lꢅmꢅꢁꢊꢃ, ꢁꢆꢇꢃꢅꢈg ꢇꢂ tꢇyꢌꢄꢆ & fꢆꢇꢈꢍꢅꢂ Gꢆꢄꢉp

2
M.ꢁY. YAN ET AL.
1
. Introduction
Cylindrokelupha dalatensis (Kosterm.) T. L.Wu. belongs to Cylindrokelupha Kosterm. of the
Leguminosae Albizzia, which is an endemic plant distributed in south China. Cylindrokelupha
Kosterm. have about thirteen species in the world and eleven species in China, and belong to
the endangered species [1]. e seeds of Cylindrokelupha dalatensis are used as folk medicine
for antibacterial and difficult miscellaneous diseases in the countryside. Cylindrokelupha
Kosterm. should comprise a number of chemical constituents, such as triterpenoid genins
and saponins, flavonoids, lignans, and tannins. However, there is hardly any research report
about chemical composition of the seed of Cylindrokelupha dalatensis (Kosterm.) T. L.Wu.
[
2–9]. e present study describes the purification, structural elucidation, and cytotoxic
activity of three new triterpenoid saponins from the 70% ethanol extracts, cylindrosides B
(
1), C (2), and D (3), which showed significant inhibitory activity in vitro against human
tumor cell lines (HL60)(Figure 1). eir structures were determined by comprehensive
spectroscopic analysis (HR-ESI-MS (high-resolution electrospray ionization mass spec-
1
13
1
1
trometry), H NMR (nuclear magnetic resonance), C NMR, H– H COSY (correlation
spectroscopy), HSQC (heteronuclear singular quantum correlation), HMBC (heteronuclear
multiple bond correlation)), and chemical methods.
2
. Results and discussion
Cylindroside B (1), white amorphous powder, gave positive Molish reaction and Liebermann–
−1
Burchard reaction. e IR spectrum of 1 showed absorptions bands of carbonyl (1723 cm )
−1
and α, β-unsaturated carbonyl groups (1643 cm ). e negative ESI-TOF(electrospray
−
ionisation time of flight) MS of 1 gave quasi-molecular ion peak at m/z 2301.1003 [M−H] .
1
Its H NMR spectrum showed seven methyl signals at δ 0.92 (3H, s), 0.97 (6H, s), 1.15 (3H,
s), 1.19 (3H, s), 1.29 (3H, s), and 1.37 (3H, s), one olefinic proton signal at δ 5.46 (1H, br
1
3
s), and sugar proton signals at δ 3.5–6.0. C NMR spectrum showed two olefinic carbon
signals at δ 123.2 and 143.1, suggesting that 1 was an oleanane type triterpenoid sapo-
1
13
nin. When the H- and C-NMR signals of 1 were compared with those of julibroside J
1
2
1
3
[
10], they were in good agreement for those of the aglycon moieties. From the C-NMR
spectrum, the aglycon moiety of 1 was identified to be machaerinic acid, which is a deoxy
derivative of acacic acis. Upon acid hydrolysis with CF COOH, 1 gave D-glucose, D-xylose,
3
1
3
D-fucose, D-quinovose, L-arabinose, and L-rhamnose [11]. e C-NMR spectrum gave
Figure 1. tꢀꢊ ꢂꢁꢆꢉꢍꢁꢉꢆꢊꢂ ꢄꢋ ꢍꢄmpꢄꢉꢈꢃꢂ 1–3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
ten anomeric carbon signals at δ 95.6, 99.4 (×2), 101.8, 103.5, 104.9, 105.8, 106.0, 107.2,
and 111.0, four methyl carbon signals at δ 17.3, 18.9, 18.5, and 18.9. e anomeric pro-
ton signals at δ 6.08 (d, J = 7.8 Hz), 4.88 (d, J = 6.6 Hz)/4.86 (d, J = 7.2 Hz), 6.01(br s),
4
.99 (d, J = 7.2 Hz), 4.87 (d, J = 6.6 Hz), 5.32 (d, J = 7.8 Hz), 5.39 (d, J = 7.8 Hz), 5.02 (d,
J = 6.6 Hz), and 6.22 (br s) were assigned by direct correlation from HSQC spectrum. e
configurations of the anomeric carbons of sugars were determined to be β-D for glucose,
1
13
fucose, xylose and quinovose, and α-L for rhamnose and arabinose from its H and C
1
13
NMR data. Except for the resonances of H and C NMR data belonging to aglycone and
13
sugar moieties, C NMR spectrum of 1 also showed eight olefinic carbon signals at δ 144.2,
1
44.0, 143.4, 142.9, 128.2, 128.0, 115.1, and 114.8, two α, β-unsaturated carbonyl carbons
1
at δ 167.8 and 167.6. H NMR spectrum showed four methyl proton signals at δ 1.90 (3H,
s), 1.85 (3H, s), 1.56 (3H, s), and 1.44 (3H, s), two olefinic proton signals at δ 7.03 (1H, t,
J = 6.6 Hz) and 6.91 (1H, t, J = 6.9 Hz), indicating 1 had two monoterpene moiety (Tables
3
1
–3). In a comparison of the C NMR data of 1 with those of julibroside J [12], all of the
3
6
signals due to sugar moieties of 1 were identical with those of julibroside J , indicating
3
6
that the sugars of 1 were the same as those of julibroside J . e linkages among aglycone,
sugars, and monoterpene moieties were determined on the basis of HMBC experiments
3
6
(
(
Figure 2). erefore, the structure of 1 was determined as 3-O-{β-ꢀ-glucopyranosyl-
1→2)-[β-ꢀ-xylopyranosyl-(1→2)-β-ꢀ-fucopyranosyl-(1→6)]-β-ꢀ-glucopyranosyl}-
2
1-O-{(6S)-2-trans-2,6-dimethyl-6-O-[4-O-(6R)-2-trans-2,6-dimethyl-6-O-β-ꢀ-
quinovopyranosyl-2,7-octadienoyl]-β-ꢀ-quinovopyranosyl-2,7-octadienoyl}-machaerinic
acid-28-O-β-ꢀ-glucopyranosyl(1→3)-[α-ꢁ-arabinofuranosyl-(1→4)]-α-ꢁ-rhamnopyranosyl-
(
1→2)]-β-ꢀ-glucopyranosyl ester. Compound 1 was a new saponin, named cylindroside B.
Cylindroside C (2) was obtained with retention time of 2.1 min longer than that of 1 by
HPLC (high performance liquid chromatography) (23% CH CN), also was white amorphous
3
powder, gave positive Molish reaction and Liebermann–Burchard reaction. e ESI-TOF
1
13
MS, H, and C NMR spectra of 2 were good agreement with those of 1, except that
1
proton and carbon-13 signals due to MT′ group. A comparison of the H NMR spectral
data of 2 with those of 1 showed that the signals for H-7 and H-10 of MT′ of 2 undergo
upfield shif of 0.14 ppm and downfield shif of 0.10 ppm, respectively (Table 1). And
1
3
the C NMR spectral data of 2 with those of 1 showed that the signals for C-5 and C-10
of MT′ of 2 undergo a downfield shif of 2.0 ppm and upfield shif of 1.10 ppm, respec-
tively (Table 2). According to the rules reported in the literature of Kun Zou [10], 1 and
2
were a pair of diastereoisomers, and the absolute configuration of 2 for C-6 of MT′ was
1
3
determined to be 6S by a comparison of markedly different C NMR signals due to C-5
and C-10 of MT′. us, the structure of 2 was determined as 3-O-{β-ꢀ-glucopyranosyl-
(
1→2)-[β-ꢀ-xylopyranosyl-(1→2)-β-ꢀ-fucopyranosyl-(1→6)]-β-ꢀ-glucopyranosyl}-
2
1-O-{(6S)-2-trans-2,6-dimethyl-6-O-[4-O-(6S)-2-trans-2,6-dimethyl-6-O-β-ꢀ-
quinovopyranosyl-2,7-octadienoyl]-β-ꢀ-quinovopyranosyl-2,7-octadienoyl}-machaerinic
acid-28-O-β-ꢀ-glucopyranosyl(1→3)-[α-ꢁ-arabinofuranosyl-(1→4)]-α-ꢁ-rhamnopyranosyl-
(
1→2)]-β-ꢀ-glucopyranosyl ester. Compound 2 was a new saponin, named cylindroside C.
Cylindroside D (3) was obtained with retention time of 3.2 min longer than that of 2
by HPLC (23% CH CN), also was white amorphous powder, gave positive Molish reaction
3
and Liebermann–Burchard reaction. e negative ESI-TOF MS of 3 gave quasi-molecular
−
ion peak at m/z 2155.0388 [M−H] , with m/z 146 less than that of 1 and 2. In a comparison
1
3
of the C NMR data of 3 with those of 1, 3 only have nine anomeric carbon signals, and

4
M.ꢁY. YAN ET AL.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
1
3
Table 2. c nMr ꢂpꢊꢍꢁꢆꢇꢌ ꢃꢇꢁꢇ ꢄꢋ mꢄꢈꢄꢁꢊꢆpꢊꢈꢄꢅꢃꢇꢌ mꢄꢅꢊꢁꢅꢊꢂ ꢄꢋ 1–3 (Pyꢆꢅꢃꢅꢈꢊ-d ).
5
1
2
3
MT
c-1
c-2
c-3
c-4
c-5
c-6
c-7
c-8
c-9
c-10
MT′
c-1
c-2
c-3
c-4
c-5
c-6
c-7
c-8
c-9
c-10
167.6
128.2
142.9
23.8
40.5
79.8
144.0
115.1
12.6
167.6
128.2
142.9
23.7
40.5
79.8
144.0
115.1
12.6
167.6
128.2
142.9
23.7
40.5
79.8
144.0
115.1
12.6
23.7
23.7
23.7
167.8
128.0
143.4
23.8
38.5
79.5
144.2
114.8
12.7
167.6
128.2
143.1
23.6
40.5
79.8
143.8
115.1
12.7
167.8
127.9
143.7
24.1
40.5
72.1
146.6
111.7
12.6
24.8
23.7
28.6
tꢀꢊ ꢂꢅgꢈꢅfiꢍꢇꢈꢍꢊ ꢄꢋ bꢄꢌꢃ vꢇꢌꢉꢊꢂ ꢂꢀꢄwꢊꢃ 1 ꢇꢈꢃ 2 wꢊꢆꢊ ꢇ pꢇꢅꢆ ꢄꢋ ꢃꢅꢇꢂꢁꢊꢆꢊꢄꢅꢂꢄmꢊꢆꢂ, ꢇꢈꢃ ꢁꢀꢊ ꢇbꢂꢄꢌꢉꢁꢊ ꢍꢄꢈfigꢉꢆꢇꢁꢅꢄꢈ ꢄꢋ 1 ꢇꢈꢃ 2
ꢋꢄꢆ c-6 ꢄꢋ Mt′ wꢊꢆꢊ 6r ꢇꢈꢃ 6s, ꢆꢊꢂpꢊꢍꢁꢅvꢊꢌy.
less oneanomeric carbon signal of quinovosyl. All other signals due to the aglycone, one
monoterpenoid moiety (MT), and sugar moieties attached in 3 were good agreement with
those in 1, except for signals of another monoterpenoid moiety (MT′) (Tables 1–3). Another
monoterpenoid moiety (MT′) signals, the HMBC correlations between δ 5.35(Quip 4-H)
and δ 167.8 (MT′ 1-C), between δ 1.91 (MT′ 9-H) and δ 167.8 (MT′ 1-C), between δ 1.91
(
MT′ 9-H) and δ 143.7 (MT′ 3-C), between δ 5.17/5.56 (MT′ 8a/8b-H) and δ 72.1 (MT′ 6-C),
and between δ 1.43 (MT′ 10-H) and δ 146.4 (MT′ 7-C) indicated that the MT′ linked to 4-C
of the quinovopyranosyl moiety (Figure 1). e HMBC spetrum showed no correlation sig-
nals between all anomeric proton signal and carbon-13 signals of the MT′, which indicated
1
3
that no sugar moiety linked with this MT′. e C NMR spectrum of MT′ showed that C-6
shif ed from δ 79.8 to δ 72.1, C-7 shif ed from δ 143.8 to δ 146.6, C-8 shif ed from δ 115.1 to δ
1
11.7, and C-10 shif ed from δ 23.7 to δ 28.6, indicating that the hydroxy group of C-6 wasn’t
1
substituted by any group. And the H NMR signals of MT′ were in good agreement with those
of julibroside J and julibroside J [10]. ese results indicated that MT′ is (6R)-2-trans-2,
5
12
6
-dimethyl-6-hydroxyl-2, 7-octadienoyl moiety. us, the structure of 3 was determined as
-O-{β-ꢀ-glucopyranosyl-(1→2)-[β-ꢀ-xylopyranosyl-(1→2)-β-ꢀ-fucopyranosyl-(1→6)]-β-
3
ꢀ
-glucopyranosyl}-21-O-{(6S)-2-trans-2,6-dimethyl-6-O-[4-O-(6R)-2-trans-2,6-dimethyl-
-hydroxyl-2,7-octadienoyl]-β-ꢀ-quinovopyranosyl-2,7-octadienoyl}-machaerinic
acid-28-O-β-ꢀ-glucopyranosyl(1→3)-[α-ꢁ-arabinofuranosyl-(1→4)]-α-ꢁ-rhamnopyranosyl-
1→2)]-β-ꢀ-glucopyranosyl ester. Compound 3 was a new saponin, named cylindroside D.
ree new triterpenoid saponins were evaluated for their cytotoxic activity against HL60
6
(

human cancer cells in vitro by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide) method [13]. In particular, cylindroside D (3) was highly effective at
inhibiting the cell lines tested, its IC value reaching 4.74 ± 0.57 μM. cylindroside B (1)
5
0
was also greatly effective at inhibiting the cell lines tested, just slightly lower than 3, its IC
5
0

6
M.ꢁY. YAN ET AL.
Table 3. nMr ꢂpꢊꢍꢁꢆꢇꢌ ꢃꢇꢁꢇ ꢄꢋ ꢂꢉgꢇꢆꢂ mꢄꢅꢊꢁꢅꢊꢂ ꢄꢋ 1–3 (Pyꢆꢅꢃꢅꢈꢊ-d5).
1
2
3
Position
δC
δH (J in Hz)
δC
δC
sꢉgꢇꢆ(c-3)
Gꢌꢍ c-1
c-2
c-3
c-4
c-5
c-6
104.9
83.4
76.8
71.9
77.6
69.8
4.87 (ꢃ, 6.6)
4.25 (ꢄ)
4.11 (ꢄ)
4.26 (ꢄ)
4.04 (ꢄ)
104.9
83.4
76.9
71.9
77.6
69.8
104.9
83.4
76.7
71.8
77.6
69.8
4.30, 4.73 (ꢄ)
fꢉꢍ(1→6)Gꢌꢍ
c-1
103.5
82.5
75.2
72.2
71.5
17.3
4.99 (ꢃ, 7.2)
4.40 (ꢄ)
4.13 (ꢄ)
4.02 (ꢄ)
4.10 (ꢄ)
103.5
82.5
75.2
72.2
71.5
17.3
103.5
82.5
75.2
72.2
71.5
17.3
c-2
c-3
c-4
c-5
c-6
1.49 (ꢃ, 6.0)
Xyꢌ(1→2)fꢉꢍ
c-1
107.2
76.0
75.5
70.9
67.2
5.02 (ꢃ, 6.6)
4.06 (ꢄ)
3.99 (ꢄ)
4.12 (ꢄ)
3.56, 4.42 (ꢄ)
107.2
76.0
75.4
70.8
67.2
107.2
76.0
75.4
70.9
67.2
c-2
c-3
c-4
c-5
Gꢌꢍ′(1→2)Gꢌꢍ
c-1
106.0
75.3
78.0
71.3
78.1
62.5
5.39 (ꢃ, 7.8)
4.13 (ꢄ)
4.23 (ꢄ)
4.19 (ꢄ)
3.91 (ꢄ)
106.0
75.2
78.0
71.3
78.1
62.5
106.0
75.2
78.0
71.3
78.1
62.5
c-2
c-3
c-4
c-5
c-6
4.17, 4.30 (ꢄ)
sꢉgꢇꢆ(c-28)
Gꢌꢍ″c-1
c-2
c-3
c-4
c-5
c-6
95.6
76.9
78.4
71.3
79.1
62.1
6.08 (ꢃ, 7.8)
4.11 (ꢄ)
4.16 (ꢄ)
4.19 (ꢄ)
3.95 (ꢄ)
95.6
76.9
78.4
71.9
79.1
62.1
95.6
76.8
78.4
71.3
79.1
62.1
4.21, 4.35 (ꢄ)
rꢀꢇ(1→2)Gꢌꢍ″
c-1
101.8
70.8
82.1
79.2
69.0
18.9
6.01 (bꢆ ꢂ)
5.17 (ꢄ)
4.90 (ꢄ)
4.48 (ꢄ)
4.55 (ꢄ)
101.8
70.9
82.1
79.2
68.9
18.9
101.8
70.8
82.1
79.2
69.0
18.9
c-2
c-3
c-4
c-5
c-6
1.79 (ꢃ, 6.0)
Gꢌꢍ″′(1→3)rꢀꢇ
c-1
105.8
75.7
78.2
71.5
78.1
62.9
5.32 (ꢃ, 7.8)
4.02 (ꢄ)
4.18 (ꢄ)
4.10 (ꢄ)
3.98 (ꢄ)
105.8
75.6
78.4
71.5
78.2
62.9
105.8
75.7
78.2
71.5
78.1
62.9
c-2
c-3
c-4
c-5
c-6
4.21, 4.41 (ꢄ)
aꢆꢇ(1→4)rꢀꢇ
c-1
111.0
84.7
78.5
84.9
62.5
6.22 (bꢆ ꢂ)
4.72 (ꢄ)
4.77 (ꢄ)
4.94 (ꢄ)
4.16, 4.28 (ꢄ)
110.9
84.7
78.4
84.9
62.5
111.0
84.7
78.4
84.9
62.5
c-2
c-3
c-4
c-5
sꢉgꢇꢆ(c-28)
Qꢉꢅ
c-1
c-2
c-3
c-4
c-5
99.4
75.6
75.7
77.2
70.2
4.88 (ꢃ, 6.6)
4.01 (ꢄ)
4.20 (ꢄ)
5.35 (ꢄ)
3.69 (ꢄ)
99.4
73.5
79.4
75.0
72.6
99.4
75.5
75.7
77.2
70.2
(
Continued)

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Table 3. (Continued).
1
2
3
Position
δC
δH (J in Hz)
δC
δC
c-6
Qꢉꢅ′
c-1
c-2
c-3
c-4
c-5
c-6
18.5
1.35 (ꢃ, 6.6)
18.7
18.5
99.4
75.4
78.4
76.9
72.6
18.9
4.86 (ꢃ, 7.2)
3.98 (ꢄ)
4.13 (ꢄ)
3.70 (ꢄ)
3.71 (ꢄ)
99.3
75.4
78.5
76.6
72.6
18.9
1.59 (ꢃ, 5.4)
nꢄꢁꢊ: (ꢄ) ovꢊꢆꢌꢇppꢊꢃ wꢅꢁꢀ ꢄꢁꢀꢊꢆ ꢂꢅgꢈꢇꢌꢂ.
Figure 2. tꢀꢊ mꢇꢅꢈ hMBc ꢍꢄꢆꢆꢊꢌꢇꢁꢅꢄꢈꢂ ꢄꢋ ꢍyꢌꢅꢈꢃꢆꢄꢂꢅꢃꢊꢂ B ꢇꢈꢃ c (h→c).
value reaching 7.15 ± 0.63 μM. cylindroside C (2) also presented moderate action on HL60
cell lines, its IC value reaching 10.07 ± 0.97 μM.
5
0
3
. Experimental
3
.1. General experimental procedures
Optical rotations were measured with a Perkin-Elmer 343 polarimeter (Waltham,
Massachusetts, U.S.A.). e IR spectra were obtained using a FT/IR-4100 Fourier transform
infrared spectrometer (JASCO Corporation, Tokyo, Japan) with a KBr disk. e NMR spec-
UNITY
1
tra were recorded with Varian
INOVA 600 (599.8 MHz for H NMR and 150.8 MHz
1
3
for C NMR) (Palo Alto, CA, U.S.A.) and the chemical shif s were given in δ (ppm) scaled
with tetramethylsilane (TMS) as an internal standard. HR-ESI-MS were recorded on a
Synapt MS System (Waters Corporation, Milford, MA, U.S.A.). HPLC was performed on
an Agilent 1100 system (quaternary pump; Santa Clara, CA, U.S.A.) with a YMC-Pack
ODS-A C (YMC, 4.6 mm i.d. × 250 mm, and 10 mm i.d. × 250 mm, ODS, 5 μm; Kyoto,
1
8
Japan); detectors were RID (refractive index detector) and ELSD (evaporative light scattering
detector) (Alltech 3300 Evaporative Light Scattering Detector, temperature: 70 °C, gas flow:
1
.6 liters/min). Macroporous resin SP825 (Mitsubishi chemical, Tokyo, Japan), silica gel
(
SiO ; Qingdao Haiyang Chemical Co. Ltd, Qingdao, China), and ODS(operational data
2
store) silica gel (120 Å. 50 μm, YMC) were used for column chromatography.

8
M.ꢁY. YAN ET AL.
HPLC grade acetonitrile (CH CN) and methanol (MeOH) were purchased from Fisher
3
Scientific (Fair Lawn, NJ). Deionized water (H O) was purified by Milli-Q system (Bedford,
2
MA, U.S.A.). Other reagents for purification, such as methanol (MeOH), chloroform
(
CHCl ), tetrahydrofuran (THF), and n-butanol (n-BuOH), were of analytical grade bought
3
from Beijing Fine Chemicals (Beijing, China).
3
.2. Plant material
Fresh seeds of Cylindrokelupha dalatensis (Kosterm.) T. L.Wu. were plucked from
Xishuangbanna Yunan Province in August 2010, and identified by Professor Ping Liu. A
sample was deposited in the Herbarium of Pharmacy of Traditional Chinese Medicine,
Chinese People’s Liberation Army (PLA) General Hospital.
3
.3. Extraction and isolation
e seeds of C. dalatensis (7 kg) were extracted thrice with 70% ethanol under reflux for 1 h

and the solvent was evaporated under reduced pressure to give a brown residue (860 g). Af er
the residual extract was isolated on macroporous resin SP 825 and eluted with a gradient
mixture of EtOH:H O (5:95; 15:85; 45:55 and 85:15) to give four fractions (Fr. A, B, C and
2
D). Fr. D (40 g) was subjected to silica gel column chromatography eluting with gradient
mixture of CHCl :MeOH:H O (70:26:6; 65:35:10 and 60:40:12, the underlayer) to yield 9
3
2
fractions (Fr. I-IX). Fraction VI amount was separated by repeated chromatography over
RP C18 eluting with MeOH:H O (40:60→70:30) and preparative HPLC with YMC-Pack
2
ODS-A C (YMC, 10 mm i.d. × 250 mm, ODS, 5 μm), eluting with CH CN∶H O (23∶77;
1
8
3
2
7
ml/min) to give compound 1 (23 mg; Rt 33.2 min), compound 2 (28 mg; Rt 35.3 min),
and compound 3 (19 mg; Rt 38.5 min).
3
.3.1. Cylindroside B (1)
2
0
White amorphous powder.[ꢀ] −70.8 (c 0.033, pyridine). IR (KBr): v 3413, 2925, 1723,
D
−
max
1 1
1
1
5
643, 1555, 1380, 1073, 639 cm ; H NMR (600 MHz, Pyridine-d ) δ: 0.92, 0.97, 0.97, 1.15,
5
.19, 1.29 and 1.37 (each s, 3H, 7 × CH ), 3.59–3.61 (m, 1H, H-3), 5.46 (brs, 1H, H-12),
3
.17 (o, 1H, H-21), 4.87 (d, 1H, J = 6.6 Hz, glc H-1), 5.39 (d, 1H, J = 7.8 Hz, glc′ H-1), 4.99
(
d, 1H, J = 7.2 Hz, fuc H-1), 5.02 (d, 1H, J = 6.6 Hz, xyl H-1), 6.08 (d, 1H, J = 7.8 Hz, glc″
H-1), 6.01 (brs, 1H, rha H-1), 5.32 (d, 1H, J = 7.8 Hz, glc‴ H-1), 6.22 (brs, 1H, araf H-1),
.88 (d, 1H, J = 6.6 Hz, qui H-1), 4.86 (d, 1H, J = 7.2 Hz, qui′ H-1), 1.49 (d, 3H, J = 6.0 Hz,
4
fuc CH ), 1.79 (d, 3H, J = 6.0 Hz, rha CH ), 1.59 (d, 3H, J = 5.4 Hz, qui CH ), 1.35 (d, 3H,
3
3
3
J = 6.6 Hz, qui′ CH ), 6.91 (t, 1H, J = 6.9 Hz, MT H-3), 6.21 (dd, 1H, J = 11.4, 17.4 Hz, MT
3
H-7), 5.20 (d, 1H, J = 11.4 Hz, MT H-8a), 5.40 (d, 1H, J = 17.4 Hz, MT H-8b), 1.85(s, 3H,
MT H-9), 1.56 (s, 3H, MT H-10), 7.03 (t, 1H, J = 6.6 Hz, MT′ H-3), 6.31 (dd, 1H, J = 10.8,
1
7.4 Hz, MT′ H-7), 5.19 (d, 1H, J = 10.8 Hz, MT′ H-8a), 5.33 (d, 1H, J = 17.4 Hz, MT′ H-8b),
1
3
1
.90 (s, 3H, MT′ H-9), 1.44 (s, 3H, MT′ H-10); For C NMR spectral data, see Tables 1–3;
−
HR-ESI-MS (neg.): m/z 2301.1003 [M−H] (calcd for C H O , 2301.1071).
1
08 171 52
3
.3.2. Cylindroside C (2)
2
0
1
White amorphous powder.[ꢀ]D −18.3 (c 0.032, pyridine), H NMR (600 MHz, Pyridine-d )
δ: 0.92, 0.97, 0.97, 1.15, 1.19, 1.30 and 1.37 (each s, 3H, 7 × CH ), 3.57–3.61 (m, 1H, H-3),
5
3

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
5
.46 (brs, 1H, H-12), 5.17 (o, 1H, H-21), 4.88 (d, 1H, J = 6.0 Hz, glc H-1), 5.40 (d, 1H,
J = 7.2 Hz, glc′ H-1), 4.99 (d, 1H, J = 7.2 Hz, fuc H-1), 5.02 (d, 1H, J = 7.2 Hz, xyl H-1),
.07 (d, 1H, J = 8.4 Hz, glc″ H-1), 6.01 (brs, 1H, rha H-1), 5.32 (d, 1H, J = 6.0 Hz, glc‴
6
H-1), 6.20 (brs, 1H, araf H-1), 4.91 (d, 1H, J = 6.6 Hz, qui H-1), 4.83 (d, 1H, J = 7.2 Hz,
qui′ H-1), 1.49 (d, 3H, J = 6.6 Hz, fuc CH ), 1.79 (d, 3H, J = 6.0 Hz, rha CH ), 1.61 (d, 3H,
3
3
J = 6.6 Hz, qui CH ), 1.57 (d, 3H, J = 5.4 Hz, qui′ CH ), 6.91 (t, 1H, J = 6.6 Hz, MT H-3),
3
3
6
.17 (dd, 1H, J = 11.4, 17.4 Hz, MT H-7), 5.21 (d, 1H, J = 11.4 Hz, MT H-8a), 5.41 (d, 1H,
J = 17.4 Hz, MT H-8b), 1.84(s, 3H, MT H-9), 1.54 (s, 3H, MT H-10), 6.97 (t, 1H, J = 6.6 Hz,
MT′ H-3), 6.17 (dd, 1H, J = 11.4, 17.4 Hz, MT′ H-7), 5.20 (d, 1H, J = 11.4 Hz, MT′ H-8a),
5
.39 (d, 1H, J = 17.4 Hz, MT′ H-8b), 1.85 (s, 3H, MT′ H-9), 1.54 (s, 3H, MT′ H-10); For
1
3
−
C NMR spectral data, see Tables 1–3; HR-ESI-MS (neg.): m/z 2301.0933 [M−H] (calcd
for C H O , 2301.1071).
1
08 171 52
3
.3.3. Cylindroside D (3)
2
0
1
White amorphous powder.[ꢀ]D −38.3 (c 0.035, pyridine); H NMR (600 MHz, Pyridine-d )
δ: 0.92, 0.97, 0.97, 1.15, 1.19, 1.29 and 1.37 (each s, 3H, 7 × CH ), 3.59–3.61 (m, 1H, H-3),
5
3
5
.46 (brs, 1H, H-12), 5.17 (o, 1H, H-21), 4.88 (d, 1H, J = 6.0 Hz, glc H-1), 5.40 (d, 1H,
J = 7.2 Hz, glc′ H-1), 4.99 (d, 1H, J = 7.8 Hz, fuc H-1), 5.02 (d, 1H, J = 7.2 Hz, xyl H-1), 6.08
(
(
(
d, 1H, J = 7.8 Hz, glc″ H-1), 6.01 (brs, 1H, rha H-1), 5.32 (d, 1H, J = 6.0 Hz, glc‴ H-1), 6.22
brs, 1H, araf H-1), 4.87 (d, 1H, J = 7.8 Hz, qui H-1), 1.49 (d, 3H, J = 6.0 Hz, fuc CH ), 1.79
3
d, 3H, J = 6.0 Hz, rha CH ), 1.35 (d, 3H, J = 6.6 Hz, qui CH ), 6.91 (t, 1H, J = 6.6 Hz, MT
H-3), 6.25 (dd, 1H, J = 10.8, 17.4 Hz, MT H-7), 5.29 (d, 1H, J = 10.8 Hz, MT H-8a), 5.48
3
3
(d, 1H, J = 17.4 Hz, MT H-8b), 1.85(s, 3H, MT H-9), 1.56 (s, 3H, MT H-10), 7.12 (t, 1H,
J = 6.6 Hz, MT′ H-3), 6.12 (dd, 1H, J = 10.8, 17.4 Hz, MT′ H-7), 5.17 (d, 1H, J = 10.8 Hz, MT′
H-8a), 5.56 (d, 1H, J = 17.4 Hz, MT′ H-8b), 1.91 (s, 3H, MT′ H-9), 1.43 (s, 3H, MT′ H-10);
1
3
−
For C NMR spectral data, see Tables 1–3; HR-ESI-MS (neg.): m/z 2156.0300[M−H] (calcd
for C H O , 2156.0314)
1
02 163 48
3
.4. Acid hydrolysis of saponins and identification of the sugars of saponins
A solution of each saponin 10.0 mg in 2 N aq. CF COOH (5 ml) was heated for 3 h at
3
9
5 °C. Af er extraction with CH Cl (5 ml) for three times, the aq. layer was repeatedly
2 2
evaporated to dryness with EtOH until neutral. Af er that, the glucose, quinovose, fucose,
xylose, arabinose, and rhamnose in the monosaccharide mixture were detected by TLC over
silica gel (CHCl -MeOH-H O, 8:5:1) by comparison with authentic samples. Furthermore,
3
2
the residue of the sugars was dissolved in anhydrous pyridine (2 ml) and L-cysteine methyl
ester hydrochloride (12 mg) was added, then the mixture was stirred at 60 °C for 1 h. Af er
that, HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane 2:1; 6 ml) was added and
kept at again 60 °C for 0.5 h [11]. Finally, the supernatant was analyzed by GC under the
following conditions: Agilent Technologies 6890 gas chromatograph; 5973 mass spectro-
graph detector; HP-5 capillary column; column temperature: 180 °C–250 °C, programmed
increase, 15 °C/min; carrier gas: N (1 ml/min); injection and detector temperature: 250 °C;
2
injection volume: 1.0 μl, split ratio:1/50. Subsequently, the D-configurations of glucose,
quinovose, fucose, and xylose and the L-configuration of rhamnose and arabinose were
confirmed by comparing the retention times with those of standard samples, respectively
(all from Sigma, Shanghai, China).

10
M.ꢁY. YAN ET AL.
3
.5. Cytotoxicity bioassays against cancer cells
Exponentially growing cells were centrifuged at 2000 rpm for 5 min, washed with PBS
buffer and resuspended in culture media. For each assay, cells were plated at a cell density
5
of 1.0 × 10 cells/well in 96-well plates and then exposed to increasing concentration of
different compounds (0–100 μM). Following 48 h incubation, dye reduction was initiated
by adding 10 μl of 5 mg/ml MTT to each well. Af er 2 h, 200 μl of lysing buffer was added
and formazan crystals dissolved by trace vibrator before measuring A570 in an enzyme
micro-plate reader. e values from the treated cells were compared with the values gen-
erated from the untreated cells and reported as percent viability. Each experiment was
performed in triplicate.
Acknowledgments
We are grateful to Ms. Mei-feng Xu of the National Center of Biomedical Analysis (NCBA) for the
measurements of the NMR spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was financially supported by the Special Financial Grant from the China Postdoctoral
Science Foundation [grant number 201104783] and Collaborative Innovation Center for Ethnic
Minority Development.
ORCID
Wen-Bin Zhou
http://orcid.org/0000-0001-9961-0316
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