Three new sesquiterpene glycosides from the rhizomes of trillium tschonoskii

Article abstract of DOI:10.3390/molecules22081283

Three new sesquiterpene glycosides, possessing a rare aglycone with a sulfonyl between C-1 and C-15 positions, named 3-(3E-7R,8-dihydroxy-4,8-dimethyl-3-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (1), 3-(3E-7R,8- dihydroxy-4,8-dimethyl-3-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (2), and 3-(3E-7R,8-dihydroxy-4,8-dimethyl-3-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7-O-β-D-glucopyranosyl-6-O-acetyl-(1→4)-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (3), respectively, were isolated from the rhizomes of Trillium tschonoskii. Their structures were established on the basis of spectroscopic data, including HR-ESI-MS, IR, 1D and 2D NMR. The cytotoxic properties of the three compounds were investigated using human hepatic L02 cells.

Full text of DOI:10.3390/molecules22081283

molecules
Article
Three New Sesquiterpene Glycosides from the
Rhizomes of Trillium tschonoskii
Jie Yang 1,2, Yin-Jun Yang 2,3, Xin-Guang Sun 2, Jie Zhang 2, Yang Zhao 2, Bei Wang 1,2
,
Qian-Zhi Ding 2, Bao-Lin Guo 3 and Bai-Ping Ma 1,2,
*
1
School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China;
yangjie0528@163.com (J.Y.); wbshell125@126.com (B.W.)
Beijing Institute of Radiation Medicine, No. 27, Taiping Road, Beijing 100850, China;
iyangyinjun@163.com (Y.-J.Y.); sxgzhwu07@163.com (X.-G.S.); zhangjie061003@163.com (J.Z.);
mmyzhao@163.com (Y.Z.); dqz1990@163.com (Q.-Z.D.)
2
3
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, China;
guobaolin010@163.com
*
Correspondence: mabaiping@sina.com; Tel.: +86-10-6821-0077 (ext. 930265)
Received: 24 June 2017; Accepted: 1 August 2017; Published: 2 August 2017
Abstract: Three new sesquiterpene glycosides, possessing a rare aglycone with a sulfonyl between
C-1 and C-15 positions, named 3-(30E-70R,80-dihydroxy-40,80-dimethyl-30-nonenyl)-2,5-
dihydro-1,1-dioxo-thiophen 70-O-
glucopyranoside (
β
-D-glucopyranosyl-(1
→
4)-O-
β
-D-glucopyranosyl-(1
→
4)-O-β-D-
1
), 3-(30E-70R,80- dihydroxy-40,80-dimethyl-30-nonenyl)-2,5-dihydro-1,1-dioxo-
thiophen 70-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (2), and 3-(30E-70R,80-dihydroxy-
40,80-dimethyl-30-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 70-O- -D-glucopyranosyl-60-O-acetyl-
β
(1
→
4)-O-
β
-D-glucopyranosyl-(1
→
4)-O-β-D-glucopyranoside (3), respectively, were isolated from the
rhizomes of Trillium tschonoskii. Their structures were established on the basis of spectroscopic data,
including HR-ESI-MS, IR, 1D and 2D NMR. The cytotoxic properties of the three compounds were
investigated using human hepatic L02 cells.
Keywords: Trillium tschonoskii; sesquiterpene glycosides; separation
1. Introduction
Trillium tschonoskii Maxim is perennial herb, mainly distributed in Hubei, Shanxi and Anhui
provinces of China at an altitude of 1600–3200 m [
Yan Ling Cao, have been used as a traditional Chinese medicine (TMC) for the treatment of
headache, traumatic injury, and neurasthenia [ ]. Recent pharmacological studies have shown that
Yan Ling Cao possesses anti-tumor, anti-inflammatory, analgesic and blood coagulation activities [ ].
Previous phytochemical studies on the Trillium reveals that steroidal saponins constitute the main
chemical components [ 12]. Besides, it also contains a few phenylpropanoid glycosides [13] and
sesquiterpenoid glycosides [ 13 14]. In this study, a chemical investigation on T. tschonoskii led to the
1]. The rhizomes of T. tschonoskii named
2
3–7
6,8–
3
,
,
isolation of three new sesquiterpene glycosides that have a rare aglycone with a sulfonyl between C-1
and C-15 positions. Their structures were identified using the spectroscopic techniques of HR-ESIMS,
IR, and NMR. The cytotoxic activity of the three compounds were evaluated against L02 cells.
2. Results and Discussion
The rhizomes of T. tschonoskii were extracted using 50% aq. EtOH. The extract was subjected
to macroporous resin SP825 column chromatography to afford five fractions (Fr. A Fr. E). Fraction
−
C was subsequently separated on silica-gel, MCI, ODS, preparative and semi-preparative HPLC to
provide three new sesquiterpene glycosides (Figure 1), named 3-(30E-70R,80-dihydroxy-40,80-dimethyl-
Molecules 2017, 22, 1283; doi:10.3390/molecules22081283
www.mdpi.com/journal/molecules

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30-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 70-O-
β
-D-glucopyranosyl-(1
→
4)-O-
β
-D-glucopyranosyl-
(1
→
4)-O-
β
-D-glucopyranoside (
1
), 3-(30E-70R,80-dihydroxy-40,80-dimethyl-30-nonenyl)-2,5-dihydro-
0
0 0 0
2), 3-(3 E-7 R,8 -dihydroxy-
1,1-dioxo-thiophen 7 -O-
β-D-glucopyranosyl-(1
→
4)-O-
β
-D-glucopyranoside (
-D-glucopyranosyl-60-O-acetyl-
4)-O-β-D-glucopyranoside (3), which were identified by NMR, IR
40,80-dimethyl-30-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 70-O-
β
(1
→
4)-O-
β
-D-glucopyranosyl-(1
→
and HR-ESI-MS techniques.
Figure 1. The chemical structures of compounds 1–3.
Compound
1
was obtained as a white amorphous powder and the molecular formula C33H56O19S
was indicated by HR-ESIMS at m/z 787.3065 [M
1H-NMR (600 MHz) spectrum of
(Table 1) showed three tertiary methyl group signals (δH 1.37,
1.32, 1.61), two olefinic protons (δH 5.58, 5.34 (br t, J = 7.2 Hz)), as well as signals for three
anomeric protons at (δH 4.89 (d, J = 7.9 Hz), 5.12 (d, J = 7.8 Hz), and 5.15 (d, J = 7.8 Hz)). The 13
combined with HSQC NMR spectra of indicated a structure with a total of 33 C-atom signals.
−
H]− (calcd. for C33H55O19S 787.3058). The
1
C
1
Fifteen of them were attributed to the aglycone carbons including four olefinic carbons (δC 117.8,
138.7, 123.6, 136.8), one oxygenated methine carbon (δC 90.0), one oxygenated quaternary carbon
(δC 71.9), two sulfonated methylene carbons (δC 57.4, 58.0), four sp3 methylene carbons (δC 25.6,
33.1, 36.2, 30.8), and three tertiary methyl carbons (δC 16.1, 25.3, 26.8), while the remaining carbon
signals 0were character0ist0ic to three glucosyl moieties. By comparing the NMR data of
1 with
0
0
3-(3 E-7 ,8,-dihydroxy-4 ,8 -dimethyl-3 -nonenyl)-2,5-dihydro-1,1-dioxo-thiophen [15], the structure of
1
was similar to 3-(30E-70,8,-dihydroxy-40,80-dimethyl-30-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen
except for one sugar moiety at C-10 of 1, which was further supported by 1H 1H COSY correlation
−
(Figure 2) of H-1/H-2, H-5/H-4 and H-6, H-9/H-8 and H-10, and HMBC correlation of H-2/C-3,
C-4 and C-15, H-6/C-8 and C-14, H-10/C-11 and C-12, H-12/C-10, C-11 and C-13. Comparison
the NMR data of
1
with (2,3-S-trans,10R,6E)-7,11-dimethyl-3-methylene-1,6-dodecadien-10,11-diol
4)-O- -D-glucopyranosyl-(1 4)-O- -D-glucopyranoside [14] suggested
10-O- -D-glucopyranosyl-(1
β
→
β
→
β
that they shared the same sugar chain. The sugar moiety was further assigned by HSQC,
1
HMBC and H–1H COSY experiments. Furthermore, the HMBC correlations between H-1-Glc0
(
(
δ
4.89) and C-10 (
δ 80.9) (Figure 2) verified that the linkage of the sugar unit and its location at C-10 of
was confirmed as R by the values of glycosylation shift
δ
90.0), H-1-Glc” (
δ
5.12) and C-4-Glc0” (δ 80.9), H-1-Glc0 (
δ
5.15) and C-4-Glc”
1. The
absolute configuration at C-10 of
1

Molecules 2017, 22, 1283
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of
α
-,
β
-(pro-S side), and
β
-(pro-R side) carbons of secondary alcohols to which glucosyl
moieties were attached [16]. Furthermore, the 13C chemical shifts at C-8 and C-12 of
1
were
quite similar to those of (2,3-S-trans,10R,6E)-7,11-dimethyl-3-methylene-1,6-dodecadien-10,11-diol
10-O- -D- glucopyranosyl-(1 4)-O- -D-glucopyranosyl-(1 4)-O- -D-glucopyranoside (10R) [14],
7,11-dimethyl-3-methylene-1,6-dodecadien-10,11-diol 10-O- -D-glucopyranosyl-(1 4)-O- -D-
glucopyranoside (10R) [13], and icariside C4 (10R) [17], while different from those of icariside
C1 [17]. The assignments at C-8 and C-9 of icariside C4 and icariside C1 in the literature [17
β
→
β
→
β
β
→
β
]
should be interchanged. Based on the above evidence,
1
was defined as 3-(30E-70R,80-dihydroxy-
-D-glucopyranosyl-(1 4)-O- -D-
40,80-dimethyl-30-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 70-O-
glucopyranosyl-(1→4)-O-β-D-glucopyranoside.
β
→
β
1
Figure 2. Key HMBC (arrows) and 1H− H COSY (thick lines) correlations of compounds 1–3.
Compound
2
was obtained as a white amorphous powder and its molecular formula C27H46O14S
−
was established by HR-ESIMS at m/z 625.2515 [M
the NMR and MS data of with
resonances of the sugar unit were identified by HSQC and further confirmed by HMBC experiments.
The sugar moieties of were the same as those of 7,11-dimethyl-3-methylene-1,6-dodecadien-10,11-diol
10-O- –D-glucopyranosyl-(1 4)-O- -D-glucopyranoside [13], as revealed by comparing the NMR
data. The HMBC spectrum showed long-range correlations between H-1-Glc0 (
4.91) and C-10
90.0), H-1-Glc” ( 81.3) (Figure 2), which assigned the linkage of the
5.18) and C-4-Glc0 (
sugar moiety. Therefore,
−
H] (calcd. for C27H46O14S 625.2530). Comparing
2
1, it was determined that 2 had the same aglycone as 1
. The 13C-NMR
2
β
→
β
δ
(δ
δ
δ
2
was defined as 3-(30E-70R,80-dihydroxy-40,80-dimethyl-30-nonenyl)-
2,5-dihydro-1,1-dioxo-thiophen 70-O-β-D-glucopyranosyl-(1→4)-O- β-D-glucopyranoside.
Compound 3 was obtained as a white amorphous powder and the molecular formula C35H58O20S
was deduced by HR-ESIMS at m/z 829.3126 [M
−
H]− (calcd. for C35H58O20S 829.3164). The NMR
data of were very similar to 1, except for the presence of the CH3CO group. Furthermore, the
3
CH3CO group located at the OH group of C-6 positio0n in the inner Glc0, which turned into ester, was
supported by the HMBC correlations between H-6-Glc (δ 5.13, 4.77) and C-CH3CO (δ 170.8). The sugar
moieties of
3
were further assigned by the HSQC, HMBC, and 1H 1H COSY experiments. Therefore,
−

Molecules 2017, 22, 1283
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3
was defined as 3-(30E-70R,80-dihydroxy-40,80-dimethyl-30-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen
70-O-β-D-glucopyranosyl-60-O-acetyl-(1→4)-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside.
1
Table 1. H-NMR, 13C-NMR data for compounds 1–3.
No.
1
2
3
δC
δ
δC
δ
δC
δ
H
H
H
2
3
4
5
58.0
138.7
117.8
57.4
33.1
25.6
123.6
136.8
36.2
3.85 (2H, m)
58.0
138.7
117.8
57.4
33.1
25.6
123.6
136.8
36.1
3.85 (2H, m)
58.0
138.7
117.8
57.4
33.1
25.6
123.6
136.8
36.1
3.85 (2H, m)
5.58 (1H, m)
3.89 (2H, m)
2.07 (2H, m)
5.58 (1H, m)
3.89 (2H, m)
2.07 (2H, m)
5.58 (1H, m)
3.89 (2H, m)
2.07 (2H, m)
0
1
2
3
4
0
2.09 (2H, m)
2.09 (2H, m)
2.09 (2H, m)
0
5.34 (1H, br t, J = 7.2Hz)
5.34 (1H, br t, J = 7.2Hz)
5.34 (1H, br t, J = 7.2Hz)
0
0
5
2.74 (1H, m)
2.50 (1H, m)
1.84 (1H, m)
2.74 (1H, m)
2.50 (1H, m)
1.84 (1H, m)
2.74 (1H, m)
2.50 (1H, m)
1.84 (1H, m)
0
6
30.8
30.8
30.8
1.76 (1H, m)
3.75 (1H, dd, J = 1.5, 9.5Hz)
1.76 (1H, m)
3.75 (1H, dd, J = 1.5, 9.5Hz)
1.76 (1H, m)
3.75 (1H, dd, J = 1.5, 9.5Hz)
0
7
8
9
90.0
71.9
25.3
26.8
16.1
90.0
71.9
25.3
26.8
16.1
90.4
71.9
25.5
26.7
16.1
0
0
1.37 (3H, s)
1.32 (3H, s)
1.61 (3H, s)
1.37 (3H, s)
1.32 (3H, s)
1.61 (3H, s)
1.37 (3H, s)
1.32 (3H, s)
1.61 (3H, s)
0
0
8 -CH3
4 -CH3
0
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
105.7
74.3
76.5
80.9
76.4
61.9
4.89 (1H, d, J = 7.8 Hz)
4.05 (1H, o)
105.7
74.8
78.5
81.3
76.8
62.0
4.91 (1H, d, J = 7.8Hz)
4.05 (1H, o)
105.6
71.9
76.5
81.3
76.8
62.1
4.86 (1H, d, J = 7.8 Hz)
4.23 (1H, o)
0
0
4.22 (1H, o)
4.28 (1H, o)
3.95 (1H, o)
4.47 (1H, o)
4.00 (1H, o)
4.31 (1H, o)
4.28 (1H, o)
4.50 (2H, o)
4.22 (1H, o)
4.23 (1H, o)
4.00 (1H, o)
4.58 (1H, o)
0
0
0
4.47 (1H, o)
5.12 (1H, d, J = 7.8 Hz)
4.44 (1H, o)
5.00 (1H, d, J = 7.8 Hz)
00
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
105.0
74.7
76.7
80.9
78.5
61.8
105.0
75.0
78.3
71.6
76.6
62.5
5.18 (1H, d, J = 7.8Hz)
4.05 (1H, o)
105.0
74.2
78.5
81.5
73.2
64.3
00
4.05 (1H, o)
4.22 (1H, o)
4.28 (1H, o)
3.96 (1H, o)
4.03 (1H, o)
4.01 (1H, o)
4.00 (1H, o)
4.13 (1H, o)
00
4.17 (1H, o)
4.17 (1H, o)
3.95 (1H, o)
00
00
00
4.49 (1H, o)
4.52 (1H, dd, J = 2.4, 11.4 Hz)
5.13 (1H, dd, J = 2.4, 11.4 Hz)
4.49 (1H, o)
5.15 (1H, d, J = 7.8 Hz)
4.29 (1H, o)
4.77 (1H, o)
5.13 (1H, d, J = 7.8 Hz)
4.05 (1H, o)
000
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
104.5
75.0
76.6
71.5
78.2
62.5
104.7
74.8
76.6
71.6
78.2
62.5
000
4.05 (1H, o)
4.17 (1H, o)
4.17 (1H, o)
3.95 (1H, o)
000
3.95 (1H, o)
4.15 (1H, o)
4.17 (1H, o)
000
000
000
4.52 (1H, dd, J = 2.4, 11.4 Hz)
4.52 (1H, dd, J = 2.4, 11.4 Hz)
4.29 (1H, o)
4.28 (1H, o)
CH3CO
CH3CO
20.8
170.8
2.08 (3H, s)
δ
in C5D5N, in ppm from tetramethylsilane (TMS), 1H-NMR at 600 MHz, 13C-NMR at 150 MHz; o: overlapped with
other signals; m: multiplet signals.
Compound 1–3 were evaluated for cytotoxicity against L02 cells. All three compounds showed
no cytotoxic activity at 100 µM.
3. Experimental
3.1. General Experimental Procedures
IR spectra, HR-ESIMS and NMR were recorded on a VERTEX 70 FT Infrared Spectrometer, Synapt
MS (Waters Corporation, Milford, MA, USA) and Varian UNITYINOVA 600 spectrometer (600 MHz
for 1H-NMR and 150 MHz for 13C-NMR, PaloAlto, CA, USA) in pyridine-d5 (Sigma-Aldrich, St. Louis,
MO, USA), respectively. HPLC analyses were performed on Agilent 1100 system (Agilent Technologies,
Santa Clara, CA, USA) equipped with a Silgreen C18 column (4.6 mm
×
250 mm, ODS, 5 µm, Silgreen
Co. Ltd., Beijing, China) and an Alltech 2000 evaporative light scattering detector (Temperature:
110 ◦C, Gas: 2.4 L/min, Alltech Corporation, Deerfield, IL, USA). Preparative HPLC was performed
on an NP7000 module (Hanbon Co. Ltd., Huaian, China) equipped with a Shodex RID 102 detector
(Showa Denko Group, Tokyo, Japan), and a Silgreen C18 column (20.0 mm
×
250 mm, ODS, 5 µm,

Molecules 2017, 22, 1283
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Silgreen Co. Ltd., Beijing, China). Semi-preparative HPLC was performed on a Waters 515 pump
(Waters Corporation, Milford, MA, USA) equipped with a Shodex RID 101 detector (Showa Denko
Group, Tokyo, Japan), using a Silgreen C18 column (10.0 mm
Beijing, China). TLC was performed on silica gel GF254 plates (Qingdao Marine Chemical, Qingdao,
China). Macroporous resin SP825 (Mitsubishi Chemicals, Tokyo, Japan), silica gel H (Qingdao Marine
×
250 mm, ODS, 5 µm, Silgreen Co. Ltd.,
Chemical, Qingdao, China), and MCI gel (50 µm, Mitsubishi Chemicals, Tokyo, Japan) were applied to
the performance of column chromatography.
3.2. Plant Material
The rhizomes of T. tschonoskii were collected from the Shennongjia of Hubei province, were
identified by Professor Bao-Lin Guo (Institute of Medicinal Plant Development, Chinese Academy of
Medical Sciences). A voucher specimen (No. 151010) was deposited in the author0s laboratory in the
Beijing Institute of Radiation Medicine.
3.3. Extraction and Isolation
The rhizomes of T. tschonoskii (5 kg) were crushed and extracted with 50% aq. EtOH at reflux
three times (40 L, 30 L, and 30 L, each for 2 h). The filtered solution was concentrated in vacuo. The
supernatants were applied to a macroporous resin SP825 column, eluted with EtOH/H2O (5:95, 30:70,
50:50, 75:25 and 95:10, v/v) to yield five factions (Fr. A
CC with a gradient mixture of CHCl3:MeOH:H2O (5:1:0.01, 65:25:4, and 2:1:0.01) as the eluent, and five
subfractions were obtained (Fr. C-1 Fr. C-5). Fr. C-3 (35 g) was further subjected to MCI gel CC with a
gradient mixture of acetone/H2O (10:90, 15:85, 20:80, 30:70 and 50:50, v/v) as the eluent. As a result, a
total of 30 fractions were collected (Fr. C-3-1 Fr. C-3-30). Fr. C-3-6 was purified by preparative HPLC
with ACN/H2O (20:80, v/v) to obtain seven fractions (Fr. C-3-6-1 Fr. C-3-6-7). Fr. C-3-6-4 underwent
semi-preparative HPLC with acetone/H2O (25:75, v/v) to yield (55 mg) and (12 mg). Fr. C-3-12
was purified by preparative HPLC with ACN/H2O (22:78, v/v) to yield five fractions (Fr. C-3-12-1~Fr.
−
Fr. E). Fr. C (120 g) was subjected to silica-gel
−
−
−
1
2
C-3-12-5). Fr. C-1-12-5 was purified by semi-preparative HPLC with ACN/H2O (20:80, v/v) to yield
3
(15 mg).
0
0
0
0
0
0
0
3-(3 E-7 R,8 -Dihydroxy-4 ,8 -dimethyl-3 -nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7 -O-
(1 4)-O- -D-glucopyranosyl-(1 4)-O- -D-glucopyranoside (
(c 0.085, MeOH); IR (KBr)
3408, 2970, 2927, 1642, 1382, 1306, 1236, 1159, 1072, 1026; 1H-NMR and
β
-D-glucopyranosyl-
→
β
→
β
1
): White amorphous power; [α]D20
−
10.5◦
ν
max
13C-NMR spectroscopic data, see Table 1; HR-ESI-MS (positive) m/z 789.3241 [M + H]+ (calcd. for
C33H55O19S 789.3215), 811.3036 [M + Na]+, 627.2696 [M + H
−
Glc]+, 465.2148 [M + H
−
Glc
−
Glc]+,
303.1644 [M + H − Glc − Glc − Glc]+.
0
0
0
0
0
0
0
3-(3 E-7 R,8 -Dihydroxy-4 ,8 -dimethyl-3 -nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7 -O-
β
-D-glucopyranosyl-
): White amorphous power; [α]D20
−
10.3 (c 0.082, MeOH); IR (KBr)
ν
◦
(1
→
4)-O-
β
-D-glucopyranoside (
2
max
3433, 2928, 1642, 1383, 1306, 1237, 1074, 1041; 1H-NMR and 13C-NMR spectroscopic data, see Table 1;
HR-ESIMS (positive) m/z 627.2679 [M + H]+ (calcd. for C27H47O14S 627.2687), 649.2488 [M + Na]+
465.2147 [M + H − Glc]+, 303.1637 [M + H − Glc − Glc]+.
,
0
0
0
0
0
0
0
3-(3 E-7 R,8 -Dihydroxy-4 ,8 -dimethyl-3 -nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7 -O-
60-O-acetyl-(1
4)-O- -D-glucopyranosyl-(1 4)-O- -D-glucopyranoside (
12.5◦ (c 0.088, MeOH); IR (KBr)
max 3428, 2970, 2926, 1736, 1644, 1383, 1307, 1239, 1161, 1072,
β
-D-glucopyranosyl-
→
β
→
β
3): White amorphous power;
[α]2D0
−
ν
1029; H-NMR and 13C-NMR spectroscopic data, see Table 1; HR-ESIMS (positive) m/z 831.3329
1
[M + H]+ (calcd. for C35H59O20S 831.3320), 853.3165 [M + Na]+, 669.2820 [M + H
−
Glc]+, 507.2261
[M + H − Glc − Glc]+, 303.1612 [M + H − Glc − Glc − (Ac-Glc)]+.

Molecules 2017, 22, 1283
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3.4. Acid Hydrolysis and GC-MS Analysis
Compound
1
(1.4 mg),
2
(1.6 mg), and
3
(1 mg) were hydrolyzed with 2 N aq. CF3COOH
◦
(5 mL) for 5 h at 95 C, respectively. After extraction with CH2Cl2 (3
×
5 mL), the aq. layer was
repeatedly evaporated to dryness with EtOH until neutral, and then analyzed by TLC over silica
gel (CHCl3:MeOH:H2O, 8:5:1) by comparison with authentic samples. Furthermore, the residue of
sugars was dissolved in anhydrous pyridine (2 mL), and L-cysteine methyl ester hydrochloride
(3 mg) was added. The mixture was stirred at 60 ◦C for 1 h, then 3 mL of HMDS-TMCS
(hexamethyldisilazane:trimethylchlorosilane, 2:1) was added, and the mixture was stirred at 60 ◦C
for 30 min. The precipitate was centrifuged off, and the supernatant was analyzed by GC-MS
(Agilent Technologies 5977A MSD). The absolute configurations were determined by comparing the
retention times with derivatives prepared in a similar way from standard D-glucose (Sigma-Aldrich).
Identification of D-glucose was carried out for compounds 1–3, giving two peaks at 3.75 min and
4.15 min which were two silylated derivatives (Supplementary Material, Figure S25).
3.5. Cytotoxicity Assay
The cytotoxic activity was measured by MTT assay [18]. L02 Cells were seeded in 96-well plates
and treated 24 h later with various (100
µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM) concentrations
of compounds . After 24 h of incubation, MTT was added to all wells. Plates were incubated for
1–3
another 24 h, and cell viability was measured by observing absorbance at 492 nm.
4. Conclusions
In conclusion, three new sesquiterpene glycosides were isolated from the rhizomes of T. tschonoskii.
Their structures were elucidated by extensive analysis of spectroscopic methods including 1D and 2D
NMR experiments (HSQC, HMBC, 1H–1H COSY), IR, and HR-ESI-MS. The aglycone of the compounds
found in this study was a rare aglycone which contains a sulfonyl between C-1 and C-15 positions.
Compounds 1–3 were investigated for their cytotoxic activity against L02 cells, and no obvious
cytotoxic activity was found.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Jie Yang, Yin-Jun Yang, and Xin-Guang Sun performed the isolation and structure
elucidation of the constituents. Jie Zhang, Bei Wang, and Qian-Zhi Ding contributed in the interpretation
of the spectra and also part of the preparation of the manuscript. Yang Zhao partially contributed the
structure elucidation, analyzed the data and together with Jie Yang and Bao-Lin Guo prepared the manuscript.
Bai-ping Ma planned, designed and organized the whole research study. All authors approved the final version of
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–3 are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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