Steroidal saponins from the rhizome of Polygonatum sibiricum

Article abstract of DOI:10.1080/10286020.2018.1478815

Four new steroidal saponins, 3-O-β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl -(25R)-spirost-5-en-3β,17α-diol (1), 3-O-β-D-glucopyranosyl(1→4)-β-D- fucopyranosyl-(25S)-spirost-5-en-3β,17α-diol (2), 3-O-β-D-glucopyranosyl(1→2) -β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl-(25R)-spirost-5-en-3β,17α-diol (3), 3-O-β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl-(25R/S)-spirost-5-en-3β,12β-diol (4), together with five known steroidal saponins were isolated from the ethanolic extract of the rhizome of Polygonatum sibiricum. Chemical structures of these compounds were elucidated by spectroscopic techniques. Anti-inflammatory activities of these new compounds were evaluated.

Full text of DOI:10.1080/10286020.2018.1478815

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Steroidal saponins from the rhizome of
Polygonatum sibiricum
Cheng Tang, Ya-Ming Yu, Qing-Ling Qi, Xiao-Dan Wu, Jia Wang & Sheng-An
Tang
To cite this article: Cheng Tang, Ya-Ming Yu, Qing-Ling Qi, Xiao-Dan Wu, Jia Wang & Sheng-
An Tang (2018): Steroidal saponins from the rhizome of Polygonatum sibiricum, Journal of Asian
Natural Products Research, DOI: 10.1080/10286020.2018.1478815
To link to this article: https://doi.org/10.1080/10286020.2018.1478815
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Journal of asian natural Products research, 2018
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1478815
ꢀ
Steroidal saponins from the rhizome of Polygonatum sibiricum
a
a
c
a
a
Cheng Tang , Ya-Ming Yu , Qing-Ling Qi , Xiao-Dan Wu , Jia Wang and
a,b
Sheng-An Tang
ꢇ
tꢅꢇꢈjꢅꢈ Kꢉy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢈ tꢉꢊꢀꢈꢄꢋꢄgꢅꢉꢂ eꢈꢇbꢋꢅꢈg dꢉvꢉꢋꢄpmꢉꢈꢁ cꢋꢅꢈꢅꢊꢇꢋ tꢀꢉꢆꢇpꢉꢌꢁꢅꢊꢂ ꢇꢈꢃ dꢅꢇgꢈꢄꢂꢁꢅꢊꢂ
tꢀꢉꢆꢇꢈꢄꢂꢁꢅꢊꢂ), sꢊꢀꢄꢄꢋ ꢄꢍ Pꢀꢇꢆmꢇꢊy, tꢅꢇꢈjꢅꢈ Mꢉꢃꢅꢊꢇꢋ uꢈꢅvꢉꢆꢂꢅꢁy, tꢅꢇꢈjꢅꢈ 300070, cꢀꢅꢈꢇ; sꢁꢇꢁꢉ Kꢉy lꢇbꢄꢆꢇꢁꢄꢆy
b
(
ꢄꢍ Bꢅꢄꢇꢊꢁꢅvꢉ sꢌbꢂꢁꢇꢈꢊꢉ ꢇꢈꢃ fꢌꢈꢊꢁꢅꢄꢈ ꢄꢍ nꢇꢁꢌꢆꢇꢋ Mꢉꢃꢅꢊꢅꢈꢉꢂ, iꢈꢂꢁꢅꢁꢌꢁꢉ ꢄꢍ Mꢇꢁꢉꢆꢅꢇ Mꢉꢃꢅꢊꢇ, cꢀꢅꢈꢉꢂꢉ aꢊꢇꢃꢉmy ꢄꢍ
ꢊ
Mꢉꢃꢅꢊꢇꢋ sꢊꢅꢉꢈꢊꢉꢂ ꢇꢈꢃ Pꢉkꢅꢈg uꢈꢅꢄꢈ Mꢉꢃꢅꢊꢇꢋ cꢄꢋꢋꢉgꢉ, Bꢉꢅjꢅꢈg 100050, cꢀꢅꢈꢇ; dꢉpꢇꢆꢁmꢉꢈꢁ ꢄꢍ aꢈꢉꢂꢁꢀꢉꢂꢅꢄꢋꢄgy,
tꢀꢉ fꢅꢆꢂꢁ affiꢋꢅꢇꢁꢉꢃ hꢄꢂpꢅꢁꢇꢋ ꢄꢍ tꢅꢇꢈjꢅꢈ uꢈꢅvꢉꢆꢂꢅꢁy ꢄꢍ tꢆꢇꢃꢅꢁꢅꢄꢈꢇꢋ cꢀꢅꢈꢉꢂꢉ Mꢉꢃꢅꢊꢅꢈꢉ, tꢅꢇꢈjꢅꢈ 300193, cꢀꢅꢈꢇ
ABSTRACT
ARTICLE HISTORY
Four new steroidal saponins, 3-O-β-D-glucopyranosyl(1→4)-β-
rꢉꢊꢉꢅvꢉꢃ 26 dꢉꢊꢉmbꢉꢆ 2017
aꢊꢊꢉpꢁꢉꢃ 16 Mꢇy 2018
D-fucopyranosyl -(25R)-spirost-5-en-3β,17α-diol (1), 3-O-β-D-
glucopyranosyl(1→4)-β-D-
fucopyranosyl-(25S)-spirost-5-en-3β,
KEYWORDS
Polygonatum sibiricum;
ꢂꢁꢉꢆꢄꢅꢃꢇꢋ ꢂꢇpꢄꢈꢅꢈꢂ;
ꢂꢁꢆꢌꢊꢁꢌꢆꢇꢋ ꢉꢋꢌꢊꢅꢃꢇꢁꢅꢄꢈ; ꢇꢈꢁꢅ-
ꢅꢈflꢇmmꢇꢁꢄꢆy ꢇꢊꢁꢅvꢅꢁy
1
7α-diol (2), 3-O-β-D-glucopyranosyl(1→2) -β-D-glucopyranosyl(1→4)-
β-D-fucopyranosyl-(25R)-spirost-5-en-3β,17α-diol (3), 3-O-β-D-
glucopyranosyl(1→4)-β-D-fucopyranosyl-(25R/S)-spirost-5-en-
3β,12β-diol (4), together with five known steroidal saponins were
isolated from the ethanolic extract of the rhizome of Polygonatum
sibiricum. Chemical structures of these compounds were elucidated by
spectroscopic techniques. Anti-inflammatory activities of these new
compounds were evaluated.
CONTACT sꢀꢉꢈg-aꢈ tꢇꢈg
ꢁꢇꢈgꢂꢀꢉꢈgꢇꢈ@ꢁmꢌ.ꢉꢃꢌ.ꢊꢈ
sꢌppꢋꢉmꢉꢈꢁꢇꢋ ꢃꢇꢁꢇ ꢍꢄꢆ ꢁꢀꢅꢂ ꢇꢆꢁꢅꢊꢋꢉ ꢊꢇꢈ bꢉ ꢇꢊꢊꢉꢂꢂꢉꢃ ꢇꢁ ꢀꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1478815.
©
2018 iꢈꢍꢄꢆmꢇ uK lꢅmꢅꢁꢉꢃ, ꢁꢆꢇꢃꢅꢈg ꢇꢂ tꢇyꢋꢄꢆ & fꢆꢇꢈꢊꢅꢂ Gꢆꢄꢌp

2
C. TANG ET AL.
1
. Introduction
Plants of the genus Polygonatum, comprising about 60 species, distribute widely in the most
area of northern temperature zone [1]. e rhizomes of Polygonatum sibiricum, one of the
original plants known as Huang-jing in traditional Chinese medicine, were used as a tonic
and a remedy for lung troubles and ringworm for thousands of years [2].
In recent years, phytochemical investigations on P. sibiricum have revealed the existence
of polysaccharides [3], alkaloids [4], steroidal saponins [5], and triterpenoid saponins [6].
Among the isolated compounds from P. sibiricum, steroidal saponins were the most potential
bioactive compounds for medical use. Steroidal saponins possess various biological activities
such as anti-inflammatory, immunoregulation, anti-diabetic [7–9]. However, steroidal sap-
onins separated from this plant were still very limited. erefore, a phytochemical analysis
has been carried out on the rhizome of P. sibiricum with particular attention to the steroidal
saponins. is study resulted in the isolation of four new steroidal saponins (1–4) and five
known compounds (Figure 1). And the anti-inflammatory activities of these compounds
were also investigated in this study.
2
. Results and discussion
Compound 1 was obtained as white powder. e molecular formula was established to be
+
1
13
C H O from the [M + Na] ion at m/z 761.4060. e H NMR and C NMR spectra of
3
9
62 13
compound 1 showed four methyl signals [δ 0.67 (3H, d, J = 5.6 Hz, 27-CH ), 0.95 (3H, s,
H
3
1
9-CH ), 0.92 (3H, s, 18-CH ), 1.22 (3H, d, J = 7.2 Hz, 21-CH )], one olefinic proton signal
3
3
3
at δ 5.30 (1H, br s, H-6), together with two typical olefinic carbon signals resonated at δ
H
C
1
22.2 and 141.5, and one quaternary carbon signal resonated at δ 110.4 was the charac-
C
teristic signal of spiro ketal. One hydroxyl group attached to the quaternary carbon at δC
9
0.7 was assigned at C-17 (Tables 1–4). All of this NMR data revealed that the aglycone of 1
1
3
was pennogenin [10]. e C NMR and DEPT spectra showed the presence of five methyl,
eleven methylene, eighteen methine, and five quaternary carbons. e NOESY spectrum
Figure 1. sꢁꢆꢌꢊꢁꢌꢆꢉꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃꢂ 1–4.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
Table 1. h nMr (400 Mhz) ꢂpꢉꢊꢁꢆꢇꢋ ꢃꢇꢁꢇ ꢍꢄꢆ ꢇgꢋyꢊꢄꢈꢉ mꢄꢅꢉꢁꢅꢉꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃꢂ 1–4 (pyꢆꢅꢃꢅꢈꢉ- d , δ ꢅꢈ
5
ppm, J ꢅꢈ hz).ꢇ
4
No
1
2
3
R
S
1
1.71–1.73 (m)
1.70–1.72 (m)
0.96–0.98 (m)
2.06–2.08 (m)
1.71–1.73 (m)
3.85 (bꢆ ꢂ)
2.63–2.65 (m)
2.38–2.40 (m)
–
1.68–1.70 (m)
1.00–1.02 (m)
2.07–2.09 (m)
1.68–1.70 (m)
3.85 (bꢆ ꢂ)
2.64–2.66 (m)
2.39–2.41 (m)
–
1.70–1.72 (m)
0.97–0.99 (m)
2.10–2.12(m)
1.64–1.66 (m)
3.70 (bꢆ ꢂ)
2.64–2.66 (m)
2.34–2.36 (m)
–
0
.96–0.98 (m)
2.07–2.09 (m)
.72–1.74 (m)
2
1
3
4
3.85 (bꢆ ꢂ)
2.63–2.65 (m)
2
.39–2.41 (m)
–
5
6
7
5.30 (bꢆ ꢂ)
1.90–1.92 (m)
5.31 (bꢆ ꢂ)
5.28 (bꢆ ꢂ)
5.55 (bꢆ ꢂ)
1.89–1.91 (m)
1.55–1.57 (m)
1.64–1.66 (m)
0.95–0.97(m)
–
1.56–1.58 (m)
1.42–1.44 (m)
2.17–2.19 (m)
1.46–1.48 (m)
–
2.02–2.04 (m)
2.15–2.17 (m)
1.47–1.49 (m)
4.40 (m)
1.89–1.91 (m)
1.54–1.56 (m)
2.14–2.16 (m)
1.00–1.02 (m)
–
1.54–1.56 (m)
1.44–1.46 (m)
1.89–1.91 (m)
1.49–1.51 (m)
–
2.00–2.02 (m)
1.89–1.91 (m)
1.49–1.51 (m)
4.44 (m)
1.84–1.86 (m)
1.47–1.49 (m)
1.54–1.56 (m)
1.02–1.04 (m)
–
2.03–2.05 (m)
1.85–1.87 (m)
3.49–3.51 (m)
1
.56–1.58 (m)
8
9
1.64–1.66 (m)
0.94–0.96 (m)
–
10
1
1
1.56–1.58 (m)
1
.43–1.45 (m)
12
2.17–2.19 (m)
1
.47–1.49 (m)
–
13
14
15
–
2.00–2.02 (m)
2.15–2.17 (m)
1.07–1.09 (m)
2.03–2.05 (m)
1.85–1.87 (m)
4.50 (m)
2.14–2.16 (m)
1.20(ꢂ)
1
.47–1.49 (m)
16
17
18
19
20
21
22
4.44 (m)
–
–
–
0.92 (ꢂ)
0.93(ꢂ)
0.93(ꢂ)
0.95 (ꢂ)
0.91(ꢂ)
0.89(ꢂ)
0.89(ꢂ)
2.26–2.28 (m)
1.22 (ꢃ, 7.2)
–
2.25–2.27 (m)
1.22 (ꢃ, 7.2)
–
2.19–2.21 (m)
1.20 (ꢃ, 7.2)
–
1.88–1.90 (m)
1.43 (ꢃ, 7.2)
–
–
2
3
1.91–1.93 (m)
1.89–1.91 (m)
1.43–1.45 (m)
2.09–2.11 (m)
1.43–1.45 (m)
1.54–1.56 (m)
4.00 (m)
1.89–1.91 (m)
1.47–1.49 (m)
2.14–2.16 (m)
1.46–1.48 (m)
1.54–1.56 (m)
3.49 (m)
1.89–1.91 (m)
1.55–1.47 (m)
2.16–2.18 (m)
1.54–1.56 (m)
1.54–1.56 (m)
3.55 (m)
1.89–1.91 (m)
1.41–1.43 (m)
2.09–2.11 (m)
1.31–1.33 (m)
1.54–1.56 (m)
4.00 (m)
1
.46–1.48 (m)
2.15–2.17 (m)
.43–1.45 (m)
24
1
2
2
5
6
1.51–1.53 (m)
3.50 (m)
3
.45 (m)
3.20 (m)
1.03 (ꢃ, 7.2)
3.47 (m)
0.64 (ꢃ, 5.6)
3.47 (m)
0.67 (ꢃ, 5.2)
3.35 (m)
1.04 (ꢃ, 7.2)
27
0.67 (ꢃ, 5.6)
ꢇovꢉꢆꢋꢇppꢉꢃ pꢆꢄꢁꢄꢈ nMr ꢂꢅgꢈꢇꢋꢂ ꢇꢆꢉ ꢆꢉpꢄꢆꢁꢉꢃ wꢅꢁꢀꢄꢌꢁ ꢃꢉꢂꢅgꢈꢇꢁꢉꢃ mꢌꢋꢁꢅpꢋꢅꢊꢅꢁy.
provided certain information for the stereostructure assignments. e clear NOESY corre-
lations, H-3/H-4, H-5, H-24/H-25, H-26, H-18/H-20, and H-21/H-23 were found (Figure
2
). e upfield shif for H-27 was δ 0.67, indicated the (S) configuration of C-25 [11]. e
H
aglycone of the other new saponins from P. sibiricum was similar with 1. e difference
between 1 and them only existed in the configuration of C-25 and the substituent group
of C-17, which resulted in significantly different chemical shif s for the carbons of the C/D
rings in these compounds. Acid hydrolysis of compound 1 gave the aglycone, along with
1
D-fucose and D-glucose. e H NMR spectrum also showed the proton at δ 1.52 (3H,
H
d, J = 6.4 Hz). It was similar with the tertiary methyl signal of fucose [12]. e 2D-NMR
spectra showed two anomeric carbons resonated at δ 103.2, 107.5 and their correspond-
C
ing protons were deduced from HSQC spectrum, and were attached to anomeric protons
resonated at δ 4.79 (1H, d, J = 7.6 Hz), 5.16 (1H, d, J = 7.6 Hz), respectively. e complete
H
1
1
assignments of each glycosidic proton system were achieved by analysis of H- H COSY and

4
C. TANG ET AL.
1
Table 2. h nMr (400 Mhz) ꢂpꢉꢊꢁꢆꢇꢋ ꢃꢇꢁꢇ ꢍꢄꢆ ꢂꢌgꢇꢆ mꢄꢅꢉꢁꢅꢉꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃꢂ 1–4 (pyꢆꢅꢃꢅꢈꢉ-d , δ ꢅꢈ ppm,
5
J ꢅꢈ hz)ꢇ
No
1
2
3
4
1
2
3
4
5
6
1
2
3
4
5
6
′
′
′
′
′
′
″
″
″
″
″
″
4.79 (ꢃ, 7.6)
4.78 (ꢃ,7.6)
4.79 (ꢃ, 7.6)
4.81 (ꢃ, 7.6)
4.34–4.36 (m)
4.03–4.05 (m)
4.05–4.07 (m)
3.75–3.77 (m)
1.52 (ꢃ, 6.4)
4.34–4.36 (m)
4.02–4.04 (m)
4.08–4.10 (m)
3.75–3.77 (m)
1.58 (ꢃ, 6.4)
4.49–4.51 (m)
3.99–4.01 (m)
3.95–3.97 (m)
3.73–3.75 (m)
1.62 (ꢃ, 5.9)
4.34–4.36 (m)
4.02–4.04 (m)
4.11–4.13 (m)
3.73–3.75 (m)
1.56 (ꢃ, 6.4)
5.16 (ꢃ, 7.6)
5.19 (ꢃ, 7.6)
5.13 (ꢃ, 8.0)
5.17 (ꢃ, 7.6)
4.14–4.16 (m)
4.19–4.21 (m)
4.19–4.21 (m)
3.83–3.85 (m)
4.45–4.47 (m)
4.14–4.16 (m)
4.19–4.21 (m)
4.19–4.21 (m)
3.83–3.85 (m)
4.45–4.47 (m)
4.28–4.30 (m)
4.11–4.13 (m)
4.20–4.22 (m)
4.22–4.24 (m)
3.83–3.85 (m)
4.29–4.31 (m)
4.44–4.46 (m)
5.24 (ꢃ, 7.2)
4.05–4.07 (m)
4.24–4.26 (m)
4.19–4.21 (m)
3.85–3.87 (m)
4.45–4.47 (m)
4.11–4.13 (m)
4.19–4.21 (m)
4.19–4.21 (m)
3.82–3.84 (m)
4.46–4.48 (m)
4.33–4.35 (m)
4
.30–4.32 (m)
1
2
3
4
5
6
‴
‴
‴
‴
‴
‴
4.32–4.34 (m)
ꢇovꢉꢆꢋꢇppꢉꢃ pꢆꢄꢁꢄꢈ nMr ꢂꢅgꢈꢇꢋꢂ ꢇꢆꢉ ꢆꢉpꢄꢆꢁꢉꢃ wꢅꢁꢀꢄꢌꢁ ꢃꢉꢂꢅgꢈꢇꢁꢉꢃ mꢌꢋꢁꢅpꢋꢅꢊꢅꢁy.
Table 3. 13c nMr (100 Mhz) ꢂpꢉꢊꢁꢆꢇꢋ ꢃꢇꢁꢇ ꢍꢄꢆ ꢇgꢋyꢊꢄꢈꢉ mꢄꢅꢉꢁꢅꢉꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃꢂ 1–4 (pyꢆꢅꢃꢅꢈꢉ-d , δ ꢅꢈ
5
ppm).
4
No
1
2
3
R
S
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
38.1
30.9
79.0
39.8
141.5
122.2
32.8
32.3
50.8
37.6
21.5
32.9
45.3
53.5
32.6
90.5
90.7
17.7
19.9
45.6
10.3
110.4
27.9
27.1
30.7
67.2
18.2
38.0
30.7
78.3
39.7
141.5
122.1
32.7
30.2
50.7
37.5
21.4
32.9
45.7
53.5
32.7
90.4
90.6
17.2
19.9
45.5
14.6
110.8
26.8
26.2
27.8
65.4
16.7
38.0
30.7
79.3
39.7
141.5
122.1
32.5
32.2
50.7
37.5
21.4
32.8
45.5
53.5
32.8
90.4
90.6
17.8
19.9
45.2
10.3
110.3
30.9
29.3
30.4
67.1
17.8
36.3
32.3
78.3
40.2
139.8
125.5
32.9
33.4
51.7
38.7
32.9
81.6
40.7
57.3
32.7
81.6
63.6
14.2
17.1
42.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
18.1
109.7
29.7
29.8
31.1
67.3
17.8
14.6
110.2
26.9
26.7
28.0
65.5
16.8
HMBC experiments and the sugar part of 1 was identified as D-fucose and D-glucose. e
HMBC correlations (Figure 3) showed a set of correlations which confirmed the attachment
of sugars; the correlation of the proton resonated at δ 4.79 (H-1′) with the carbon signal
H

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 4. 13c nMr (100 Mhz) ꢂpꢉꢊꢁꢆꢇꢋ ꢃꢇꢁꢇ ꢍꢄꢆ ꢂꢌgꢇꢆ mꢄꢅꢉꢁꢅꢉꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃꢂ 1–4 (pyꢆꢅꢃꢅꢈꢉ-d , δ ꢅꢈ ppm).
5
No
1
2
3
4
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
′
′
′
′
′
′
″
″
″
″
″
″
″′
″′
″′
″′
″′
″′
103.2
73.4
76.7
83.8
71.0
17.9
107.5
76.0
79.1
72.1
78.4
63.3
103.1
73.4
76.7
83.8
71.0
18.1
107.8
76.0
79.1
72.1
79.0
63.3
102.8
73.1
77.1
84.5
70.9
18.6
105.2
86.7
78.7
71.3
78.4
63.1
107.2
76.4
78.3
70.7
78.9
62.0
103.4
75.4
76.7
83.8
71.0
17.8
107.5
76.0
79.1
72.0
79.0
63.3
Figure 2. impꢄꢆꢁꢇꢈꢁ noesY ꢊꢄꢆꢆꢉꢋꢇꢁꢅꢄꢈꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃ 1.
resonated at δ 79.0 (C-3) confirmed the attachment of fucose-1 to C-3 of pennogenin, while
C
the correlation of the proton resonated at δ 5.16 (H-1″) with the carbon signal resonated at
H
δC 83.8 (C-4′) confirmed the attachment of glucose-1 to C-4′ of fucose. e up-field shif ed
carbon signals at δ 72.1 and δ 63.3 for C-4″ and C-6″ of glucose, confirmed that the glucose
C
C
was terminal and not attached to another sugars. Additionally, the chemical shif of H-27
(δH 0.67) in 1 suggested that the H-27 in 1 was on equatorial bond. is indicated that 1
had the same 25R configurations as pennogenin [12]. us, the structure of compound 1
was proved to be a new compound (3-O-β-D-glucopyranosyl (1→4)-β-D-fucopyranosyl-
(
25R)-spirost-5-en-3β,17α-diol) (Figure 1).
Compound 2 was obtained as white powder, whose molecular formula was established
+
to be C H O from the [M + Na] ion at m/z 761.4060. Acid hydrolysis of 2 yielded agly-
3
9
62 13
cone and two sugars, which was identified directed by the GC analysis as D-fucose and
D-glucose. e analysis of the NMR data for compound 2 confirmed that it was similar
with 1. e major difference was the H-27, the chemical shif of H-27 (δ 1.03) in 2 was
H
different with δ 0.67 in 1, suggesting that the H-27 in 2 was on axial bond while H-27
H
in 1 was on equatorial bond [13]. us, the structure of compound 2 was proved to be a

6
C. TANG ET AL.
Figure 3. impꢄꢆꢁꢇꢈꢁ hMBc ꢊꢄꢆꢆꢉꢋꢇꢁꢅꢄꢈꢂ ꢄꢍ ꢊꢄmpꢄꢌꢈꢃꢂ 1–4.
new compound (3-O-β-D-glucopyranosyl (1→4)-β-D-fucopyranosyl-(25S)-spirost-5-en-
3
β,17α-diol) (Figure 1).
Compound 3 was obtained as white powder. e molecular formula of 3 was established
+
to be C H O from the [M + Na] ion at m/z 923.4590. e analysis of the NMR data for
4
5
72 18

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
compound 3 confirmed that it was very close to the structure of 1. Acid hydrolysis of com-
1
pound 3 gave the aglycone pennogenin, along with D-fucose and D-glucose. e H NMR
spectrum also showed δ 1.62 (3H, d, J = 5.9 Hz), which was similar with the tertiary methyl
H
signal of fucose [13,14]. e 2D-NMR spectra showed three anomeric carbons resonated at
δC 102.8, 105.2, 107.2 and their corresponding protons were deduced from HSQC spectrum,
and were attached to anomeric protons resonated at δ 4.79 (1H, d, J = 7.6 Hz), 5.13 (1H,
H
d, J = 8.0 Hz), and 5.24 (1H, d, J = 7.2 Hz), respectively. e units with anomeric protons
corresponded to three hexoses and were identified as D-fucose and D-glucoses. e HMBC
correlations confirmed that the sugars were connected to C-3 of the aglycone (Figure 3).

e correlation of the proton resonated at δ 4.79 (H-1′) with the carbon signal resonated
H
at δ 79.3 (C-3) confirmed the attachment of fucose-1 to C-3 of pennogenin, while the
C
correlations of the proton resonated at δ 5.13 (H-1″) and δ 5.24 (H-1‴) with the carbon
H
H
signals resonated at δ 84.5 (C-4′) and δ 86.7 (C-2″), respectively. All of these confirmed the
C
C
attachment of glucose-1 to C-4′ of fucose and glucose-2 to C-2″ of glucose-1. e up-fielded
carbon signals at δ 70.7 and δ 62.0 for C-4‴ and C-6‴ of glucose-2, confirmed that the
C
C
glucose was terminal and not attached to another sugars. Additionally, the chemical shif
of H-27 (δ 0.64) in 3 suggested that H-27 in 3 was on equatorial bond. is indicated that
H
3
had the same 25R configurations as pennogenin [10]. us, the structure of compound 3
proved to be a new compound (3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl(1→4)-
β-D- fucopyranosyl-(25R)-spirost- 5-en-3β,17α-diol) (Figure 1).
Compound 4 was obtained as white powder, whose molecular formula was established to
+
1
13
be C H O from the [M + Na] ion at m/z 761.4051. e H NMR and C NMR spectra
3
9
62 13
of compound 4 showed five tertiary methyl signals [δ 0.89（3H, s, 19-CH ), 1.20 (3H, s,
H
3
1
8-CH ), 1.04 (3H, d, J = 7.2 Hz, 27-CH ), 0.67 (3H, d, J = 5.2 Hz, 27-CH ), 1.43 (3H, d,
3
3
3
J = 7.2 Hz, 21-CH )], together with one olefinic proton signal resonated at δ 5.55 (1H, br
3
H
s, H-6); two typical olefinic carbon signals resonated at δ 125.5 and δ 139.8, and one qua-
C
C
ternary carbon signal resonated at δ 110.2 was the characteristic signal of spiro ketal. And
C
the carbon signals resonated at δ 81.6 indicated a hydroxyl group attached with C-12 [15].
C
All of these data revealed that its aglycone was similar with those of the previous report [16].
Acid hydrolysis of compound 4 gave the aglycone of polygodoside F, along with D-fucose
and D-glucose. e HMBC correlations (Figure 3) showed a set of correlations which con-
firmed the attachment of sugars. e correlation of the proton resonated at δ 4.81 (H-1′)
H
with the carbon signal resonated at δ 78.3 (C-3) confirmed the attachment of fucose-1 to
C
C-3 of that aglycone, while the correlation of the proton resonated at δ 5.17 (H-1″) with
H
the carbon signal resonated at δ 83.8 (C-4′) confirmed the attachment of glucose-1 to C-4′
C
of fucose. e up-fielded carbon signals at δ 72.0 and δ 63.3 for C-4″ and C-6″ of glucose,
C
C
1
3
confirmed that the glucose was terminal and not attached to another sugars [12]. e C
NMR signals ascribable to ring F appeared as pairs of signals, indicating that 4 was obtained
as a C-25 epimeric mixture. is showed close similarities to those of the previous report
[
15]. us, the structure of compound 4 was proved to be the new compound (3-O-β-D-
glucopyranosyl(1→4)- β-D-fucopyranosyl-(25R/S)-spirost -5- en-3β,12β-diol) (Figure 1).
Five known compounds were identified as 3-O-β-D-glucopyranosyl(1→4)-β-D-
fucopyranosyl-(25R/S)-spirost-5-en-3β,12β-diol (5) [16], 3-O-β-D-glucopyranosyl
(
1→4)-β-D-galactopyranosyl-(25S)-spirost-5-en-3β-ol (6) [12], 3-O-β-D- glucopyrano-
syl(1→2)-β-D-glucopyranosyl(1→4)-β-D-galactopyranosyl-(25R)-spirost-5-en-3β,17α-diol
7) [10], 3-O-β-D-glucopyranosyl(1→4)-β-D- fucopyranosyl- (25S)-spirost-5-en-3β-ol (8)
(

8
C. TANG ET AL.
[
17], 3-O-β-D-glucopyranosyl(1→4)-β-D- galactopyranosyl-(25R/S)-spirost-5-en-3β-ol-12-
one (9) [18] respectively, by comparing their spectroscopic data with those literature data.
e secreted level of inflammatory cytokines such as TNF-α and NO of RAW264.7 cells

had been examined by treating with the four new steroidal saponins. e inhibitory activities
for TNF-α of compounds 1–4 (10 μM) were 30.0, 26.5, 33.7, and 23.1% respectively. Besides,
these compounds also could inhibit the NO release of RAW264.1 significantly (p < 0.05).
e inhibitory activities of compounds 1–4 at 10 μM were 17.1, 9.3, 15.2, 12.5%, respectively.
3
. Experimental
3
.1. General experimental procedures
Optical rotations were measured with a MC 241 digital polarimeter (Perkin-Elmer Co.
Ltd., MA, USA). IR spectra were acquired using a Nicolet 380 FT-IR spectrophotometer
(
ermo Electron Corporation, Sugar Land, TX, USA). NMR spectra were run on a Bruker
AVANCE III 400 instrument (Bruker Biospin, Fällanden, Switzerland). e high-resolution
mass spectra were recorded on a Waters Xevo G2-xs mass spectrometer (Waters Co. Ltd.,
MA, USA). Preparative HPLC was performed on a YMC-Pack ODS-A column (20*250 mm,
5
μm; YMC Co. Ltd., Kyoto, Japan), equipped with a SSI-201 pump (Science system Inc.
Co. Ltd., PA, USA), and with a differential refractive index detector (Science system Inc.
Co. Ltd., PA, USA). Sugar analysis was performed by 7890A -5975C series GC/MS (Agilent
Inc. Co. Ltd., CA, USA).
3
.2. Plant material

e plant was collected from Guiyang Guizhou province of China. And it was authenticated
as Polygonatum sibiricum by Dr. Cheng Tang. e sample was deposited in the school of
Pharmacy, Tianjin Medical University, Tianjin, China.
3
.3. Extraction and isolation
1
8 kg air-dried rhizomes of P. sibiricum were ground into fine powder and were exhaustively
extracted by hot reflux with 70% ethanol for 3 times (every time 2 h). en, the extract
was evaporated under reduced pressure. And the residue was re-suspended in water. e
suspension was extracted by petroleum ether, ethyl acetate and n-butyl alcohol, respec-
tively. e sub-fraction of n-butyl alcohol was concentrated. And the residue (95.8 g) was
re-suspended in water. en it was subjected to AB-8 series macroporous adsorption resin
column chromatography. e sample was eluted by a series of concentrations of ethanol
(
20, 40, 60, 95%) to yield eight fractions according to the result of TLC analysis. Fraction
4
(8.8 g) was re-treated by a silica gel chromatography eluting with CH Cl :MeOH sys-
2
2
tem gradually to yield ten sub-fractions according to the result of TLC analysis. Fractions
such as F4-3 (780 mg), F4-5 (520 mg), and F4-6 (470 mg) were repeatedly purified by
HPLC (reversed phase silica gel, MeOH : H O = 80:20, flow rate was 3 ml/min, detective
2
wavelength was 210 nm) to yield 1 (76.6 mg, t = 85.0 min), 2 (15.8 mg, t = 87.2 min), 3
R
R
(
6.8 mg, t = 90.2 min), 5 (31.9 mg, t = 95.0 min)，6 (15.3 mg, t = 92.8 min), 7 (8.3 mg,
R R R
t = 97.5 min), and 9 (9.8 mg, t = 100.2 min). Fraction 6 (1.2 g) was treated by Sephadex
R
R

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
LH-20 gel chromatography eluting with MeOH : H O (85: 15) to yield three fractions
2
according to the result of TLC analysis. Fraction F6-3 (170 mg) was repeatedly purified by
HPLC (reversed phase silica gel, MeOH : H O = 80 : 20, flow rate was 3 ml/min, detective
2
wavelength was 210 nm) to yield 4 (6.8 mg, t = 118.1 min) and 8 (6.3 mg, t = 107.7 min).
R
R
All of those compounds were identified by spectral management.
3
.3.1. Compound 1
20
White powder, [a] − 6.2 (MeOH, c 0.21). IR (KBr): ν 3400 (OH), 2940 (CH), 1650
D
max
−1
1
13
(
C=C), 1000–1100 (a glycosidic linkage) cm . H NMR (pyridine-d , 400 MHz) and C
5
NMR (pyridine- d , 100 MHz) spectral data, see Tables 1–4. HRESIMS: m/z 761.4060
5
+
[
M + Na] (calcd for C H O , 761.4083).
3
9
62 13
3
.3.2. Compound 2
20
White powder,[a] − 6.1(MeOH, c 0.20), IR (KBr) ν 3400 (OH), 2940 (CH), 1650 (ole-
D
max
−1 1
finic bond), 1000–1100 (glycosidic linkage) cm . H NMR (pyridine-d , 400 MHz) and
5
1
3
C NMR (pyridine- d , 100 MHz) spectral data, see Tables 1–4. HRESIMS: m/z 761.4060
5
+
[
M + Na] (calcd for C H O , 761.4083).
3
9
62 13
3
.3.3. Compound 3
20
White powder, [a] − 7.0 (MeOH, c 0.20). IR (KBr): ν 3400 (OH), 2910 (CH), 1620
D
max
−1 1
(
olefinic bond), 1000–1100 (glycosidic linkage) cm . H NMR (pyridine-d , 400 MHz) and
5
1
3
C NMR (pyridine-d , 100 MHz) spectral data, see Tables 1–4. HRESIMS: m/z 923.4590
5
+
[
M + Na] (calcd for C H O , 923.4611).
3
9
62 13
3
.3.4. Compound 4
20
White powder, [a] − 5.6 (MeOH, c 0.21), IR (KBr) ν 3421 (OH), 2900 (CH), 1650
D
max
−1 1
(
olefinic bond), 1000–1100 (glycosidic linkage) cm . H NMR (pyridine-d , 400 MHz) and
5
1
3
C NMR (pyridine- d , 100 MHz) spectral data, see Tables 1–4. HRESIMS: m/z 761.4051
5
+
[
M + Na] (calcd for C H O , 761.4083).
3
9
62 13
3
.4. Acid hydrolysis of the new compounds
Acid hydrolysis of these compounds was according to the previous report [15]. Briefly, it
was hydrolyzed with 2 M HCl (5 ml), heated for 2 h at 95 °C and was extracted with CHCl3
(
3 × 5 ml). e aqueous layer was concentrated to dryness under a stream of nitrogen. e
aqueous residue was redissolved in anhydrous pyridine (2 ml) and L-cysteine methyl ester
hydrochloride (2.5 mg) was added to the solution of pyridine. e mixture was heated at
6

0 °C for 2 h and acetic anhydride (2 ml) was added, followed by heating at 90 °C for 0.5 h.
e solution was concentrated to dryness under a stream of nitrogen. e reaction product
was subjected to analysis. An Agilent HP-5MS capillary column was used. e column
temperature started at 100 °C for injection, then programmed at 10 °C/min to 150 °C,
and programmed 15 °C/min to 280 °C. Helium was used as carrier gas. And the flow-rate
is 1.0 ml/min. e inlet ionization source temperature were 260 and 230 °C, respectively.

e absolute configurations of the monosaccharides of 1 were confirmed to be D-fucose
(
t = 11.076 min) and D-glucose (t = 13.089 min) by comparison of the retention times of
R
R

10
C. TANG ET AL.
monosaccharide derivatives with those of standard sugars (the standard sugars were sub-
jected to the same reaction): D-Fucose (t = 11.045 min) and D-glucose (t = 13.042 min).
R
R
3
.5. Procedure for biological evaluation

e activities of the new isolates were evaluated. MTT tests were carried out according to
the method described by Kwon with RAW264.1 cell lines [19]. e cell lines were provided
by ATCC, and the MTT of these new compounds were evaluated. en the TNF-α inhibit
ratio of them on RAW264.7 cells were evaluated in less-cytoxic dose which were obtained
6
by MTT tests. Briefly, the cells were seeded into 24-well plates at a density of 1 × 10 cells/
well and cultured overnight. Af er pretreatment with various concentrations of the isolates
or positive control for 1 h, the cells were stimulated with LPS (1 μg/ml) for the indicated
periods of time. e released levels of TNF-α in the culture media were detected by ELISA
kits (eBioscince Ltd. Co.) according to the manufacturers’ instructions. NOs level was deter-
mined in culture supernatant by using Griess assay.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was supported by National Natural Science Foundation of China [grant number 81303191];
Postdoctoral Foundation of China [grant number 2012M510759] for financial support of this project.
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