Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity against HepG2 cells

Article abstract of DOI:10.1016/j.steroids.2020.108587

A phytochemical study on the rhizomes of Trillium tschonoskii led to the isolation of fourteen new steroidal saponins, trillitschosides S1-S14 (1–14), along with ten known analogues (15–24). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence. All isolated compounds were screened for the cytotoxicity against HepG2 cells, and the results demonstrated that only the known compounds 21–24 exhibited the remarkable cytotoxic activity against HepG2 cells which is much better than the positive control of 5-FU.

Full text of DOI:10.1016/j.steroids.2020.108587

Steroids156(2020)108587
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity
against HepG2 cells
Yin-Jun Yanga,b,1, Xu Panga,1, Bei Wanga, Jie Yanga, Xiao-Juan Chena, Xin-Guang Suna, Qi Lia,
Jie Zhanga, Bao-Lin Guob, Bai-ping Maa,⁎
a Beijing Institute of Radiation Medicine, Beijing 100850, China
b Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Trillium tschonoskii
Liliaceae
Steroidal saponin
Trillitschoside
HepG2 cell
A phytochemical study on the rhizomes of Trillium tschonoskii led to the isolation of fourteen new steroidal
saponins, trillitschosides S1-S14 (1–14), along with ten known analogues (15–24). Their structures were es-
tablished mainly by spectroscopic analyses as well as necessary chemical evidence. All isolated compounds were
screened for the cytotoxicity against HepG2 cells, and the results demonstrated that only the known compounds
21–24 exhibited the remarkable cytotoxic activity against HepG2 cells which is much better than the positive
control of 5-FU.
1. Introduction
structure elucidation of these compounds and their cytotoxicity against
HepG2 cells.
Trillium tschonoskii Maxim (Liliaceae) is a perennial herb mainly
distributed in Shan’xi, Sichuan, Yuannan, and Hubei provinces in
China. In many regions, the dried rhizomes of T. tschonoskii were used
as a kind of often-applied folk medicine (Yan-ling-cao in Chinese) for
the purposes of treating neurasthenia, headache, traumatic injury and
bleeding, cancer, and various inflammatory, etc. [1,2]. To Tujia na-
tionality, T. tschonoskii is honored as one of four magical herbs due to its
obvious curative effects for a series of diseases. T. tschonoskii and two
other plants of same genus, T. kamtschaticum and T. govanianum, are
only three species of genus Trillium distributed in China. Previous
phytochemical investigations on these plants indicated that, all the
time, there is lack of systematic chemical study on T. tschonoskii, which
is not conductive to elucidate the active constituents of this herb
medicine. Recently, a phytochemical study was carried out on T.
tschonoskii, which let us finally obtain total twenty-four spirostanol
saponins (1–24) from its rhizomes, of which 1–14 were new com-
pounds (Fig. 1). A preliminary screening displayed that the extract of T.
tschonoskii affect cytotoxic activity of HepG2 cells obviously after
treatment for 24 h and in a dose-dependent manner (Supplementary
Fig. S1). As our effort to search for anti-liver cancer bioactive com-
pounds, all isolated compounds were tested for their individual cyto-
toxic activities in vitro using the human hepatocellular carcinoma
(HepG2) cell line. Herein, we mainly described the isolation and
2. Experimental
2.1. General methods
HRESIMS was recorded on a Synapt MS (Waters Corporation, USA).
The NMR experiments were performed on Varian
UNITY
INOVA 600
spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR). The
optical rotations were measured with a JASCO J-810 polarimeter. IR
spectra were recorded on a Bruker VERTEX 70 FT infrared spectro-
meter. HPLC analyses were performed on Agilent 1100 system
equipped with an Alltech 2000 evaporative light scattering detector
(Temperature: 110 °C, Gas: 2.4 L/min). Preparative HPLC was per-
formed on an NP7000 module (Hanbon Co. Ltd., China) equipped with
a Shodex RID 102 detector (Showa Denko Group, Japan). Silgreen
HPLC C18 columns (4.6/10.0/20.0 × 250 mm, 5 μm, Greenherbs
Science and Technology, China) were used for HPLC and preparative
HPLC. TLC was performed on silica gel GF254 plates (Qingdao Marine
Chemical, China). Macroporous resin SP825 (Mitsubishi Chemicals,
Japan), silica gel H (Qingdao Marine Chemical, China), and MCI gel
(Mitsubishi Chemicals, Japan) were applied for column chromato-
graphy.
⁎ Corresponding author.
E-mail address: mabaiping@sina.com (B.-p. Ma).
1 These authors contribute equal to this work.
https://doi.org/10.1016/j.steroids.2020.108587
Received 14 June 2019; Received in revised form 2 January 2020; Accepted 20 January 2020
Availableonline23January2020
0039-128X/©2020ElsevierInc.Allrightsreserved.

Y.-J. Yang, et al.
Steroids156(2020)108587
Fig. 1. Structures of 1–24.
2.2. Plant material
6/3, Fr.C3/7–9/3, Fr.C3/12/3 and Fr.C3/13–14/3 were together se-
parated by pHPLC with (CH3)2CO-H2O (v/v, 22:78) to give 8 (49.0 mg)
and 9 (19.5 mg). Fr.C3/6/4–6, Fr.C3/10–11/4, Fr.C3/12/2, and Fr.C3/
13–14/2 were separated by pHPLC with (CH3)2CO-H2O (v/v, 22:78) to
give 18 (25.0 mg). Fr.C3/12/4 and Fr.C3/13–14/4 were separated by
pHPLC with (CH3)2CO-H2O (v/v, 24:76) to give 1 (26.0 mg) and 4
(62.0 mg). Fr.C4 (35 g) was subjected to a MCI gel column eluted with
(CH3)2CO-H2O (v/v, 10:90 → 15:85 → 20:80 → 30:70 → 50:50) to
afford nine fractions (Fr.C4/1 ~ Fr.C4/9). Fr.C4/1 was separated by
pHPLC with CH3CN-H2O (v/v, 20:80) to give six fractions (Fr.C4/1/
1 ~ Fr.C4/1/6). Fr.C4/2 was separated by pHPLC with CH3CN-H2O (v/
v, 20:80) to give six fractions (Fr.C4/2/1 ~ Fr.C4/2/6). Fr.C4/3 was
separated by pHPLC with CH3CN-H2O (v/v, 21:79) to give three frac-
tions (Fr.C4/3/1 ~ Fr.C4/3/3). Fr.C4/4 was separated by pHPLC with
CH3CN-H2O (v/v, 22:78) to give four fractions (Fr.C4/4/1 ~ Fr.C4/4/
4). Fr.C4/5 was separated by pHPLC with CH3CN-H2O (v/v, 22:78) to
give five fractions (Fr.C4/5/1 ~ Fr.C4/5/5). Fr. C4/6 was separated by
pHPLC with CH3CN-H2O (v/v, 23:77) to give five fractions (Fr. C4-
6–1 − Fr.C4-6–5). Fr.C4/1/1–3 together with Fr.C4/2/2–4 were se-
parated by pHPLC with (CH3)2CO-H2O (v/v, 23:77) to give 6 (12.3 mg)
and 19 (56.5 mg). Fr.C4/1/6, Fr.C4/2/6 and Fr.C4/3/2 were together
separated by pHPLC with (CH3)2CO-H2O (v/v, 25:75) to give 11
(16.0 mg). Fr.C4/5/5 and Fr.C4/6/5 were separated by pHPLC with
(CH3)2CO-H2O (v/v, 28:72) to give 5 (10.0 mg) and 17 (49.0 mg). Fr.C5
(35 g) was subjected to ODS column with (CH3)2CO-H2O (v/v, 24:76) to
afford six fractions (Fr.C5/1 ~ Fr.C5/6). Fr.C5-2 was separated by
pHPLC with (CH3)2CO-H2O (v/v, 22:78) to give four fractions (Fr.C/5/
2/1 ~ Fr.C5/2/4). Fr.C5/3 was separated by pHPLC with (CH3)2CO-
H2O (v/v, 22:78) to give five fractions (Fr.C5/3/1 ~ Fr.C5/3/5). Fr.C5/
4 was separated by pHPLC with (CH3)2CO-H2O (v/v, 22:78) to give five
fractions (Fr.C5/4/1 ~ Fr.C5/4/5). Fr.C5/5 was separated by pHPLC
with CH3CN-H2O (v/v, 20:80) to give six fractions (Fr.C5/5/1 ~ Fr.C5/
The rhizomes of T. tschonoskii were collected from the Shennongjia
of Hubei province and 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 author’s la-
boratory in the Beijing Institute of Radiation Medicine.
2.3. Extraction and isolation
T. tschonoskii rhizomes (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 to get the supernatants and
sediments. The supernatants were subjected to a SP825 macroporous
resin column eluted with EtOH-H2O (v/v, 5:95 → 30:70 → 50:50 →
75:25 → 95:5) to yield five factions (A ~ E). Fr.C (120 g) was subjected
to silica-gel column chromatography (CC) eluted with a gradient mix-
ture of CHCl3-MeOH-H2O (v/v/v, 5:1:0.1 → 2:1:0.1) to give five frac-
tions (Fr.C1 ~ Fr.C5). Fr.C3 (35 g) was subjected to MCI CC eluted with
(CH3)2CO-H2O (v/v, 10:90 → 50:50). As a result, a total of 30 fractions
were collected (Fr.C3/1 ~ Fr.C3/30). Fr.C3/6 was separated by pre-
parative HPLC (pHPLC) with CH3CN-H2O (v/v, 20:80) to give seven
subfractions (Fr.C3/6–1 ~ Fr.C3/6–7). Fr.C3/7–9 was separated by
pHPLC with CH3CN-H2O (v/v, 20:80) to yield three fractions (Fr.C3/
7–9/1 ~ Fr.C3/7–9/3). Fr.C3/10–11 was separated by pHPLC with
CH3CN-H2O (v/v, 22:78) to yield five fractions (Fr.C3/10–11/
1 ~ Fr.C3/10–11/5). Fr.C3/12 was separated by pHPLC with CH3CN-
H2O (v/v, 22:78) to give five subfractions (Fr.C3/12/1 − Fr.C3/12/5).
Fr.C3/13–14 were separated by pHPLC with CH3CN-H2O (v/v, 22:78) to
yield five subfractions (Fr.C3/13–14/1 ~ Fr. C3/13–14/5). Then,
Fr.C3/6/2 together with Fr.C3/7–9/2 were separated by pHPLC with
(CH3)2CO-H2O (v/v, 20:80) to give 7 (31.0 mg) and 10 (6.3 mg). Fr.C3/
2

Y.-J. Yang, et al.
Steroids156(2020)108587
5/6). Fr.C5/6 was separated by pHPLC with CH3CN-H2O (v/v, 22:78) to
give seven fractions (Fr.C5/6/1 ~ Fr.C5/6/7). Fr.C5/2/2 together with
Fr.C5/3/2 and Fr.C5/4/1 were separated by pHPLC with CH3CN-H2O
(v/v, 17:83) to give 3 (24.7 mg). The sediments (133 g) together with
Fr.D (95 g) were separated on ODS CC with CH3CN-H2O (v/v, 45:55) to
afford 23 (6.8 g), 22 (10.3 g) and 21 (1.2 g), along with a mix fraction
(Fr.F). Fr.F was further subjected to ODS CC eluting with (CH3)2CO-
H2O (v/v, 40:60) to afford a five fractions (Fr.F1 ~ Fr.F5). Fr.F2 was
separated by pHPLC with CH3CN-H2O (v/v, 22:78) to give five fractions
(Fr.F2/1 ~ Fr.F2/5). Fr.F3 was separated by pHPLC with CH3CN-H2O
(v/v, 24:76) to give six fractions (Fr.F3/1 ~ Fr.F3/6). Fr.F4 was sepa-
rated by pHPLC with CH3CN-H2O (v/v, 26:74) to give six fractions
(Fr.F4/1 ~ Fr.F4/6). Fr.F5 was separated by pHPLC with CH3CN-H2O
(v/v, 30:70) to give six fractions (Fr.F5/1 ~ Fr.F5/5). Fr.F2/2, Fr.F2/3
and Fr.F3/1 were separated by pHPLC with (CH3)2CO-H2O (v/v, 25:75)
to give 13 (20.0 mg). Fr.F2/5, Fr.F3/2, Fr.F3/4 and Fr.F4/1 were to-
gether separated by pHPLC with (CH3)2CO-H2O (v/v, 29:71) to give 14
(37.0 mg) and 20 (25.0 mg). Fr.F3/5 together with Fr.F4/4, and Fr. F5/
2 were separated by pHPLC with (CH3)2CO-H2O (v/v, 33:67) to give 2
(127.0 mg), 12 (120.0 mg), 16 (167.0 mg), and 15 (71.0 mg). Fr.F4/5,
and Fr.F5/4 were separated by pHPLC with (CH3)2CO-H2O (v/v, 36:64)
to give 24 (21.8 mg).
for C45H71O18, 899.4640). 1H NMR (600 MHz, pyridine‑d5): δ 3.98 (1H,
m, H-3), 0.94 (3H, s, H-18), 1.07 (3H, s, H-19), 1.19 (3H, d, J = 7.2 Hz,
H-21), 4.11 (1H, m, H-27a), 3.91 (1H, m, H-27b), 4.93 (1H, overlap, H-
1′ of 3-O-Glc), 6.39 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.0 Hz,
H-6′' of 2′-O-Rha), 5.84 (1H, d, J = 6.6 Hz, H-1′″ of 4′-O-Rha), 1.62
(3H, d, J = 6.0 Hz, H-6′″ of 4′-O-Rha). 13C NMR (150 MHz, pyridine‑d5)
spectroscopic data see Tables 1 and 2.
2.3.3. Compound 3
C
51H82O24; white amorphous powder; [α] = − 100.5 (c = 0.11,
MeOH). IR (KBr, cm−1): 3416, 2934, 1637, 1454, 1383, 1221, 1044,
−
917. HRESIMS (negative) m/z: 1077.6578 [M − H]
(Calcd for
C
51H81O24, 1077.6516). 1H NMR (600 MHz, pyridine‑d5): δ 3.98 (1H,
m, H-3), 0.89 (3H, s, H-18), 1.06 (3H, s, H-19), 1.17 (3H, d, J = 7.2 Hz,
H-21), 4.96 (2H, m, H-27), 4.94 (1H, d, J = 7.2 Hz, H-1′ of 3-O-Glc),
6.32 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.6 Hz, H-6′' of 2′-O-
Rha), 5.08 (1H, d, J = 6.6 Hz, H-1′″ of 6′-O-Glc), 4.94 (1H, d,
J = 7.8 Hz, H-1′″ of 27-O- Glc). 13C NMR (150 MHz, pyridine‑d5)
spectroscopic data see Tables 1 and 2.
2.3.4. Compound 4
C
45H72O19; white amorphous powder; [α] = − 100.8 (c = 0.09,
MeOH). IR (KBr, cm−1): 3416, 2934, 1638, 1454, 1382, 1245, 1133,
2.3.1. Compound 1
1046, 911. HRESIMS (positive) m/z: 917.4801 [M + H]+ (Calcd for
C
45H72O19; white amorphous powder; [α] = − 110.9 (c = 0.08,
MeOH). IR (KBr, cm−1): 3402, 2933, 1649, 1454, 1379, 1050, 898.
HRESIMS (positive) m/z: 917.4803 [M + H]+ (Calcd for C45H73O19
C
45H73O19, 917.4746). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H, m,
H-3), 0.92 (3H, s, H-18), 1.06 (3H, s, H-19), 1.18 (3H, d, J = 7.2 Hz, H-
21), 3.75 (1H, m, H-27a), 3.64 (1H, m, H-27b), 4.92 (1H, d, J = 6.6 Hz,
H-1′ of 3-O-Glc), 6.32 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.6 Hz,
H-6′' of 2′-O-Rha), 5.04 (1H, d, J = 6.6 Hz, H-1′″ of 6′-O-Glc). 13C NMR
(150 MHz, pyridine‑d5) spectroscopic data see Tables 1 and 2.
,
917.4746). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H, m, H-3), 0.92
(3H, s, H-18), 1.06 (3H, s, H-19), 1.18 (3H, d, J = 7.2 Hz, H-21), 4.11
(1H, m, H-27a), 3.91 (1H, m, H-27b), 4.92 (1H, d, J = 6.6 Hz, H-1′ of 3-
O-Glc), 6.32 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.6 Hz, H-6′' of
2′-O-Rha), 5.04 (1H, d, J = 6.6 Hz, H-1′″ of 6′-O-Glc). 13C NMR
(150 MHz, pyridine‑d5) spectroscopic data see Tables 1 and 2.
2.3.5. Compound 5
C
51H82O23; white amorphous powder; [α] = − 97.9 (c = 0.10,
MeOH). IR (KBr, cm−1): 3415, 2934, 1637, 1455, 1383, 1052, 906.
2.3.2. Compound 2
HRESIMS (positive) m/z: 1063.5375 [M + H]+ (Calcd for C51H83O23
,
C
45H72O18; white amorphous powder; [α] = −129.7 (c = 0.10,
1063.5325). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H, m, H-3), 0.93
(3H, s, H-18), 1.07 (3H, s, H-19), 1.18 (3H, d, J = 7.2 Hz, H-21), 3.76
(1H, m, H-27a), 3.70 (1H, m, H-27b), 4.94 (1H, d, J = 6.0 Hz, H-1′ of 3-
MeOH). IR (KBr, cm−1): 3404, 2931, 1639, 1452, 1379, 1220, 1128,
1069, 991, 917. HRESIMS (negative) m/z: 899.4671 [M − H]− (Calcd
Table 1
13C NMR data for aglycones of 1–14 (δ in pyridine‑d5).
POS.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
2
3
4
5
6
7
8
37.6
30.3
76.8
39.2
141.0
121.7
32.4
31.8
50.1
37.2
20.9
32.1
45.3
53.0
32.3
90.3
90.1
17.2
19.5
45.1
9.6
37.6
30.2
77.8
39.0
140.8
121.8
32.5
32.3
50.3
37.2
21.0
32.1
45.1
53.1
31.8
90.2
90.1
17.2
19.5
45.3
9.6
37.6
30.3
76.8
39.2
141.0
121.7
32.4
31.8
50.1
37.2
20.9
32.1
45.2
53.0
32.3
90.2
90.0
17.1
19.5
45.1
9.5
37.6
30.3
76.8
39.2
141.0
121.7
32.4
31.9
50.1
37.2
20.9
32.1
45.2
53.0
32.4
90.2
90.1
17.2
19.5
44.9
9.8
37.6
30.2
77.8
39.0
140.8
121.9
32.5
32.4
50.3
37.2
21.0
32.1
45.2
53.1
31.8
90.1
90.1
17.1
19.5
44.8
9.7
37.6
30.2
78.0
39.0
140.8
121.9
32.5
32.4
50.3
37.2
21.0
32.3
45.8
53.2
31.9
90.9
90.2
17.5
19.5
38.8
9.4
37.6
30.2
77.8
39.0
140.8
121.8
32.5
32.4
50.2
37.2
21.0
32.2
45.7
53.2
31.8
91.0
90.0
17.4
19.4
39.0
9.2
37.6
30.2
77.8
39.0
140.8
121.8
32.5
32.4
50.3
37.2
21.0
32.1
45.2
53.1
31.8
90.3
89.9
17.1
19.5
45.1
9.8
37.6
30.2
77.8
39.0
140.9
121.8
32.5
32.4
50.2
37.2
21.0
32.1
45.2
53.1
31.8
90.3
89.9
17.1
19.5
45.1
9.8
37.6
30.3
76.8
39.2
140.9
121.7
32.4
32.4
50.1
37.2
20.9
32.4
45.2
53.0
31.8
90.4
89.9
17.1
19.5
45.1
9.8
37.6
30.2
78.0
39.0
140.8
121.8
32.5
32.4
50.3
37.2
21.0
32.1
45.2
53.1
31.8
90.3
89.9
17.1
19.5
45.1
9.8
37.6
30.2
77.8
39.0
140.8
121.8
32.5
32.4
50.3
37.2
21.0
32.1
45.0
53.1
31.8
90.3
89.9
17.1
19.5
45.2
9.8
33.1
29.7
75.6
38.8
75.9
76.2
35.8
31.4
45.7
39.3
21.4
32.7
45.8
52.8
31.8
90.1
90.1
17.6
17.2
44.8
9.8
33.1
29.7
75.6
38.7
75.6
76.3
35.8
31.4
45.7
39.3
21.4
32.7
45.9
52.8
31.8
90.1
90.1
17.6
17.2
44.8
9.8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
110.5
27.5
21.2
36.1
61.4
60.6
110.5
27.5
21.2
36.1
61.4
60.6
110.4
27.3
20.9
33.3
60.8
69.4
110.3
31.9
23.6
39.1
64.0
64.4
110.1
31.6
23.5
36.6
63.5
71.7
112.7
68.1
33.1
40.4
63.2
64.0
113.1
64.3
31.1
39.4
59.7
62.3
112.1
42.1
66.5
47.7
62.3
61.5
112.1
42.1
66.5
47.7
62.3
61.5
112.1
42.1
66.5
47.7
62.3
61.5
112.1
42.1
66.5
47.7
62.3
61.5
112.4
42.2
70.3
39.9
65.3
13.6
109.8
32.1
28.8
30.5
66.7
17.3
109.8
32.1
28.8
30.5
66.7
17.3
3

Y.-J. Yang, et al.
Steroids156(2020)108587
Table 2
13C NMR data for sugar units of 1–14 (δ in pyridine‑d5).
POS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
3-O-β-D-Glcp-1′
100.7
79.6
77.5
71.7
78.4
69.9
102.0
72.6
72.8
74.2
69.5
18.7
105.5
75.2
78.5
71.7
78.5
62.8
100.3
78.1
78.7
78.0
76.9
61.3
102.0
72.6
72.8
73.9
69.5
18.7
102.9
72.6
72.8
74.2
70.4
18.5
100.7
79.5
77.5
71.7
78.4
69.9
102.0
72.6
72.8
74.2
69.5
18.7
105.5
75.2
78.5
71.8
78.6
62.8
105.0
75.3
78.4
71.7
78.6
63.0
100.7
79.6
77.5
71.7
78.3
69.9
102.0
72.6
72.8
74.2
69.5
18.7
105.5
75.2
78.5
71.7
78.5
62.8
100.3
78.1
78.6
78.0
76.9
61.3
102.0
72.6
72.8
74.0
69.5
18.7
102.9
72.6
72.9
74.2
70.4
18.5
105.1
75.2
78.6
71.9
78.6
62.9
100.3
78.2
78.7
78.0
77.0
61.4
102.1
72.6
72.8
74.0
69.6
18.7
103.0
72.6
72.9
74.2
70.5
18.6
100.3
78.1
78.7
78.0
76.9
61.3
102.0
72.5
72.8
73.9
69.5
18.6
102.9
72.6
72.8
73.9
70.4
18.5
100.3
78.1
78.7
78.0
76.9
61.3
102.0
72.6
72.8
74.0
69.5
18.7
102.9
72.6
72.8
74.2
70.4
18.5
100.4
79.7
78.0
71.9
78.3
62.7
102.1
72.6
72.9
74.2
69.5
18.7
100.7
79.6
77.5
71.7
78.4
69.9
102.0
72.6
72.8
74.2
69.5
18.7
105.5
75.2
78.5
71.7
78.5
62.8
100.4
77.8
77.0
78.1
77.8
61.2
102.3
72.7
72.9
74.2
69.6
18.7
103.3
73.3
72.9
80.4
68.4
18.9
102.2
72.5
72.9
74.1
70.4
18.5
100.3
78.1
78.7
78.0
76.9
61.3
102.0
72.6
72.8
73.9
69.5
18.7
102.9
72.6
72.8
74.2
70.4
18.5
100.6
78.1
78.5
77.9
76.9
61.2
101.8
72.6
72.8
73.9
69.3
18.6
102.9
72.4
72.7
74.4
70.4
18.5
100.5
79.8
77.9
71.7
78.2
62.6
101.9
72.7
72.5
74.5
69.3
18.7
2′
3′
4′
5′
6′
2′-O-α-L-Rhap-1′'
2′'
3′'
4′'
5′'
6′'
6′-O-β-D-Glcp/4′-O-α-L-Rhap-1′″
2′″
3′″
4′″
5′″
6′″
27-O-β-D-Glcp/4′″-O-α-L-Rhap-1′″′
2′″′
3′″′
4′″′
5′″′
6′″′
O-Glc), 6.41 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.0 Hz, H-6′' of
2′-O-Rha), 5.84 (1H, d, J = 6.6 Hz, H-1′″ of 4′-O-Rha), 1.62 (3H, d,
J = 6.0 Hz, H-6′″ of 4′-O-Rha), 4.73 (1H, d, J = 6.6 Hz, H-1′″ of 27-O-
Glc). 13C NMR (150 MHz, pyridine‑d5) spectroscopic data see Tables 1
and 2.
spectroscopic data see Tables 1 and 2.
2.3.9. Compound 9
C
39H62O15; white amorphous powder; [α] = −115.7 (c = 0.09,
MeOH). IR (KBr, cm−1): 3416, 2933, 1636, 1454, 1383, 1056, 976.
HRESIMS (positive) m/z: 771.4196 [M + H]+ (Calcd for C39H63O15
,
771.4167). 1H NMR (600 MHz, pyridine‑d5): δ 3.88 (1H, m, H-3), 0.93
(3H, s, H-18), 1.07 (3H, s, H-19), 1.27 (3H, d, J = 7.2 Hz, H-21), 4.38
(1H, m, H-27a), 4.03 (1H, m, H-27b), 4.93 (1H, d, J = 6.6 Hz, H-1′ of 3-
O-Glc), 6.32 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.6 Hz, H-6′' of
2′-O-Rha). 13C NMR (150 MHz, pyridine‑d5) spectroscopic data see
Tables 1 and 2.
2.3.6. Compound 6
C
45H72O19; white amorphous powder; [α] = − 98.7 (c = 0.101,
MeOH). IR (KBr, cm−1): 3406, 2935, 1640, 1456, 1384, 1130, 1043,
911. HRESIMS m/z: 917.4785 [M + H]+ (Calcd for C45H73O19
,
917.4746). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H, m, H-3), 1.01
(3H, s, H-18), 1.16 (3H, s, H-19), 1.30 (3H, d, J = 7.2 Hz, H-21), 3.76
(1H, m, H-27a), 3.70 (1H, m, H-27b), 4.92 (1H, d, J = 6.0 Hz, H-1′ of 3-
O-Glc), 6.26 (1H, s, H-1′' of 2′-O-Rha), 1.74 (3H, d, J = 6.0 Hz, H-6′' of
2′-O-Rha), 5.83 (1H, d, J = 6.6 Hz, H-1′″ of 4′-O-Rha), 1.60 (3H, d,
J = 6.0 Hz, H-6′″ of 4′-O-Rha). 13C NMR (150 MHz, pyridine‑d5)
spectroscopic data see Tables 1 and 2.
2.3.10. Compound 10
C
45H72O20, white amorphous powder, [α] = −100.8 (c = 0.11,
MeOH). IR (KBr, cm−1): 3404, 2933, 1642, 1454, 1382, 1253, 1136,
1046, 905. HRESIMS (positive) m/z: 933.4767 [M+H]+ (Calcd for
C
45H73O20, 933.4695). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H, m,
H-3), 0.93 (3H, s, H-18), 1.07 (3H, s, H-19), 1.28 (3H, d, J = 7.2 Hz, H-
21), 4.38 (1H, m, H-27a), 4.03 (1H, m, H-27b), 4.93 (1H, d, J = 6.6 Hz,
H-1′ of 3-O-Glc), 6.32 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.6 Hz,
H-6′' of 2′-O-Rha), 5.07 (1H, d, J = 6.6 Hz, H-1′″ of 6′-O-Glc); 13C NMR
(150 MHz, pyridine‑d5) spectroscopic data see Tables 1 and 2.
2.3.7. Compound 7
C45H72O19; white amorphous powder; [α] = −119.5 (c = 0.08,
MeOH). IR (KBr, cm−1): 3416, 2935, 1735, 1642, 14556, 1381, 1248,
1136, 1046, 915. HRESIMS (positive) m/z: 917.4785 [M+H]+ (Calcd
for C45H73O19, 917.4746). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H,
m, H-3), 1.01 (3H, s, H-18), 1.13 (3H, s, H-19), 1.26 (3H, d, J = 7.2 Hz,
H-21), 4.18 (1H, m, H-27a), 4.01 (1H, m, H-27b), 4.94 (1H, d,
J = 6.0 Hz, H-1′ of 3-O-Glc), 6.38 (1H, s, H-1′' of 2′-O-Rha), 1.75 (3H, d,
J = 6.0 Hz, H-6′' of 2′-O-Rha), 5.84 (1H, d, J = 6.6 Hz, H-1′″ of 4′-O-
Rha), 1.59 (3H, d, J = 6.0 Hz, H-6′″ of 4′-O-Rha). 13C NMR (150 MHz,
pyridine‑d5) spectroscopic data see Tables 1 and 2.
2.3.11. Compound 11
C51H82O23; white amorphous powder; [α] = −114.3 (c = 0.08,
MeOH). IR (KBr, cm−1): 3415, 2934, 1637, 1455, 1383, 1129, 1052,
906. HRESIMS (positive) m/z: 1063.5375 [M+H]+ (Calcd for
C
51H83O23, 1063.5325). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H,
m, H-3), 0.93 (3H, s, H-18), 1.07 (3H, s, H-19), 1.27 (3H, d, J = 7.2 Hz,
H-21), 4.38 (1H, m, H-27a), 4.03 (1H, m, H-27b), 4.94 (1H, d,
J = 6.0 Hz, H-1′ of 3-O-Glc), 6.38 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d,
J = 6.6 Hz, H-6′' of 2′-O-Rha), 5.81 (1H, s, H-1′″ of 4′-O-Rha), 1.58 (3H,
d, J = 6.0 Hz, H-6′″ of 4′-O-Rha), 6.27 (1H, s, H-1′″' of 4′'-O-Rha), 1.60
(3H, d, J = 6.0 Hz, H-6′″' of 4′'-O-Rha). 13C NMR (150 MHz, pyr-
idine‑d5) spectroscopic data see Tables 1 and 2.
2.3.8. Compound 8
C45H72O19; white amorphous powder; [α] = −86.2 (c = 0.09,
MeOH). IR (KBr, cm−1): 3416, 2933, 1636, 1454, 1383, 1245, 1050,
898. HRESIMS (positive) m/z: 917.4769 [M
45H73O19, 917.4746). 1H NMR (600 MHz, pyridine‑d5): δ 3.97 (1H, m,
+
H]+ (Calcd for
C
H-3), 0.93 (3H, s, H-18), 1.07 (3H, s, H-19), 1.28 (3H, d, J = 7.2 Hz, H-
21), 4.38 (1H, m, H-27a), 4.03 (1H, m, H-27b), 4.94 (1H, d, J = 6.0 Hz,
H-1′ of 3-O-Glc), 6.41 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.0 Hz,
H-6′' of 2′-O-Rha), 5.84 (1H, d, J = 6.6 Hz, H-1′″ of 4′-O-Rha), 1.59
(3H, d, J = 6.0 Hz, H-6′″ of 4′-O-Rha). 13C NMR (150 MHz, pyridine‑d5)
2.3.12. Compound 12
C45H72O18; white amorphous powder; [α] = −112.2 (c = 0.08,
MeOH). IR (KBr, cm−1): 3417, 2933, 1637, 1455, 1380, 1044, 981.
4

Y.-J. Yang, et al.
Steroids156(2020)108587
HRESIMS (negative) m/z: 899.4603 [M−H]− (Calcd for C45H71O18
,
were placed at 37 °C, and cultured in a 5% CO2 incubator for 48 h.
10 µL CCK-8 solutions was added into each well, and further cultured
for 4 h at 37 °C. The absorbance of each group at 450 nm was detected
(n = 5) using an absorbance microplate reader (Sunrise, Tecan,
Germany). The optical density values of each well represented the
survival/proliferation of cells. The inhibition rates of all test com-
pounds on HepG2 were calculated.
899.4640). 1H NMR (600 MHz, pyridine‑d5): δ 3.98 (1H, m, H-3), 0.95
(3H, s, H-18), 1.07 (3H, s, H-19), 1.26 (3H, d, J = 7.2 Hz, H-21), 1.09
(1H, d, J = 6.0 Hz, H-27), 4.93 (1H, overlap, H-1′ of 3-O-Glc), 6.39
(1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.0 Hz, H-6′' of 2′-O-Rha),
5.84 (1H, s, H-1′″ of 4′-O-Rha), 1.62 (3H, d, J = 6.0 Hz, H-6′″ of 4′-O-
Rha); 13C NMR (150 MHz, pyridine‑d5) spectroscopic data see Tables 1
and 2.
3. Results and discussion
2.3.13. Compound 13
C
45H74O19; white amorphous powder; [α] = −84.2 (c = 0.10,
Through series of purification steps over macroporous resin, MCI
gel, silica gel, ODS and preparative HPLC, twenty-four steroidal sapo-
nins were isolated from 50% ethanol extract of T. tschonoskii rhizomes.
Detailed comparison of NMR data with the reported values in the lit-
eratures allowed the ten known analogues to be identified as trikam-
steroside A (15) [3], (25S)-27-hydroxypenogenin-3-O-α-L-rhamnopyr-
MeOH). IR (KBr, cm−1): 3415, 2932, 2879, 1654, 1455, 1383, 1051,
979. HRESIMS (negative) m/z: 917.4775 [M − H]− (Calcd for
C
45H73O19, 917.4746). 1H NMR (600 MHz, pyridine‑d5): δ 4.10 (1H, m,
H-3), 1.02 (3H, s, H-18), 1.66 (3H, s, H-19), 1.22 (3H, d, J = 7.2 Hz, H-
21), 0.65 (1H, d, J = 6.0 Hz, H-27), 4.83 (1H, d, J = 7.2 Hz, H-1′ of 3-
O-Glc), 6.41 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.0 Hz, H-6′' of
2′-O-Rha), 5.84 (1H, s, H-1′″ of 4′-O-Rha), 1.62 (3H, d, J = 6.0 Hz, H-
6′″ of 4′-O-Rha). 13C NMR (150 MHz, pyridine‑d5) spectroscopic data
see Tables 1 and 2.
anosyl-(1
→
2)-O-β-D-glucopyranoside (16) [3], (25S)-27-[(β-D-
glucopyranosyl)oxy]-17α-hydroxyspirost-5-en-3β-O-α-L-rhamnopyr-
anosyl-(1 → 2)-O-β-D-glucopyranoside (17) [4], chonglouside SL-17
(18) [5], chonglouside SL-18 (19) [5], trikamsteroside B (20) [3], sa-
ponin Tg (21) [6], pennogenin 3-O-β-chacotrioside (22) [7], penno-
2.3.14. Compound 14
genin 3-O-α-L-rhamnopyranosyl-(1
→
2)-β-D-glucopyranoside (23)
C
39H64O15; white amorphous powder; [α] = −164.8 (c = 0.12,
[3,7], and pennogenin 3-O-β-D-glucopyranoside (24) [3].
MeOH). IR (KBr, cm−1): 3415, 2933, 2874, 1658, 1456, 1382, 1050,
Compound 1 had a molecular formula C45H72O19 as determined by
HRESIMS. In the 1H NMR spectrum, three typical methyl proton signals
at δ 0.92 (3H, s), 1.07 (3H, s), 1.18 (3H, d, J = 7.2 Hz), one olefinic
proton signal at δ 5.27 (1H, d, J = 6.0 Hz), and three anomeric proton
signals at δ 6.32 (1H, br s), 5.04 (1H, d, J = 6.6 Hz) and 4.92 (1H, d,
J = 6.6 Hz) were observed. The 13C NMR spectrum exhibited total 54
carbon resonances including 27 ones due to the aglycone part and 18
ones attributed to the sugar moiety consisting of three hexoses (Tables 1
and 2). Comparison of the NMR data of 1 with those of trikamsteroside
A [3] indicated that they shared the identical spirostanol skeleton of
(25R)-5-en-spirost-3β,17α,27-triol, which was further confirmed by
detailed analyses of 1H–1H COSY, HSQC and HMBC spectra of 1.
Starting from three anomeric proton signals, all proton signals of each
sugar unit were delineated using 1H–1H COSY analysis, and then the
carbon signals were assigned depending on the HSQC correlations.
Thus, the structures of all sugar units could be established, and the NMR
data suggested them to be two glucoses and a rhamnose. Acid hydro-
lysis experiment further proved that the sugar moiety of 1 comprised of
D-glucopyranose (Glcp) and L-rhamnopyranose (Rhap). The large cou-
pling constants (J1,2 greater than 7 Hz) indicated the β-configurations
of two D- Glcp, and the α-configurations of the L-Rhap was determined
by carbon signals of δ 72.8 (C-3′′) and δ 69.5 (C-5′′), respectively [8].
The connectivity and the sequence of the sugar moiety was determined
due to the HMBC correlations between δ 4.92 (H-1′ of 3-O-Glcp) and
76.8 (C-3), between δ 6.32 (H-1′′ of 2′-O-Rhap) and 79.6 (C-2′), and
between δ 5.04 (H-1′′′ of 6′-O-Glcp) and 69.6 (C-6′). Consequently, the
structure of 1 was elucidated as (25R)-5-en-spirost-3β,17α,27-triol 3-O-
α-L-rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 6)]-β-D-gluco-
pyranoside, named trillitschoside S1.
979, 919. HRESIMS (negative) m/z: 771.4222 [M−H]− (Calcd for
C
39H63O15, 771.4167). 1H NMR (600 MHz, pyridine‑d5): δ 4.10 (1H, m,
H-3), 1.02 (3H, s, H-18), 1.66 (3H, s, H-19), 1.22 (3H, d, J = 7.2 Hz, H-
21), 0.65 (1H, d, J = 6.0 Hz, H-27), 4.83 (1H, d, J = 7.2 Hz, H-1′ of 3-
O-Glc), 6.39 (1H, s, H-1′' of 2′-O-Rha), 1.76 (3H, d, J = 6.6 Hz, H-6′' of
2′-O-Rha); 13C NMR (150 MHz, pyridine‑d5) spectroscopic data see
Tables 1 and 2.
2.4. Acid hydrolysis and absolute configuration determination
Standard monosaccharides, L-rhamnose (5 mg), D-glucose (5 mg)
and L-cysteine methyl ester hydrochloride (5 mg) was dissolved in
pyridine (5 mL) and heated to 60 °C for 1 h. And then o-tolyl iso-
thiocyanate (10 μL) was added to the mixture and refluxed on a water
bath further for 1 h. The reaction mixture (1 μL) was analyzed by UPLC-
Q/TOFMS on a Acquity UPLC HSS T3 C18 column (2.1 × 100 mm,
1.7 μm) at 40 °C with gradient elution of CH3CN-H2O (containing 0.1%
FA) (v/v, 20:80 → 30:70) for 8 min at a flow rate of 0.6 mL/min.
Compounds 1–14 (each 1.0 mg) were individually hydrolyzed by
heating in 6 M TFA (1 mL) at 90 °C for 2 h. After cooling, the reaction
mixture was extracted with CHCl3. Then, each aqueous layer was
evaporated to dryness, and the residue was dissolved in pyridine (1 mL)
containing L-cysteine methyl ester hydrochloride (1 mg) and heated at
60 °C for 1 h. Following, o-tolyl isothiocyanate (5 μL) was added to each
mixture, and heated at 60 °C for another 1 h. The reaction mixture
(1 μL) was analyzed following the above procedure. As a result, the
sugars in the test compounds were identified as D-glucose and L-rham-
nose, respectively, by comparing their molecular weight and retention
time with the standards (tR 3.18 min for D-glucose; tR 5.38 min for L-
rhamnose).
Compound 2 had a molecular formula C45H72O18 as determined by
HRESIMS. The NMR data suggested that 2 had the same aglycone as 1.
In the 1H NMR spectrum, the observed three anomeric carbon re-
sonances at δ 4.83 (1H, d, J = 7.2 Hz), 6.41 (1H, s) and 5.84 (1H, s)
suggested the presence of three sugar units. Comparison of the NMR
data of sugar moiety between 2 and pennogenin 3-O-β-chacotrioside
[7] suggested they had the same sugar chain. By detailed analyses of
1H–1H COSY, HSQC and HMBC spectra, the structure of 2 was con-
firmed to be (25R)-5-en-spirost-3β,17α,27-triol 3-O-α-L-rhamnopyr-
anosyl-(1 → 4)-α-L-rhamnopyranosy1-(1 → 2)-β-D-glucopyranoside,
named trillitschoside S2.
2.5. HepG2 cell proliferation assay
Cell proliferation was measured by Cell Counting Kit-8 (CCK-8,
Dojindo, Japan) method. HepG2 cells in logarithmic phase were tryp-
sinized and seeded into 96 well plates at a density of 1 × 105 cells/mL.
100 μL of cell suspension was added to each well (1 × 104 cells per
well) and the plates were incubated at 37 °C for 24 h in a 5% CO2
incubator. After pre-incubation, 100 μL of the corresponding drug-
containing medium was added to each well. At the same time, a ne-
gative control group, a vehicle control group, and a positive control
group were set up, and 5 duplicate wells in each group; 96-well plates
Compound 3 had a molecular formula C51H82O24 as determined by
HRESIMS. The NMR data suggested it had the same aglycone and C-3
sugar chain with 1, but one glucose more than 1. In the 13C NMR
5

Y.-J. Yang, et al.
Steroids156(2020)108587
spectrum, the chemical shift of C-27 (δ 69.4) shifting to a lower field
suggested the linkage of the additional glucose to the C-27, which was
supported by the HMBC correlation of δ 4.94 (H-1′′′′ of 27-O-Glc) and δ
69.4 (C-27). The structure of 3 were finally confirmed by detailed
analyses of 1H–1H COSY, HSQC and HMBC spectra, and elucidated as
A [3]. By detailed analyses of 1H–1H COSY, HSQC and HMBC spectra,
the structure of 9 was confirmed to be (24R, 25S)-spirost-5-ene-
3β,17α,24α,27-tetraol 3-O-α-L-rhamnopyranosy1-(1
→
2)-β-D-gluco-
pyranoside, named trillitschonide S9.
Compound 10 had a molecular formula C45H72O20 as determined by
HRESIMS. The NMR data of 10 suggested it had the same spirotanol
skeleton with 8, but had the identical sugar chain with 1. Therefore,
structure of 10 was confirmed to be (24R, 25S)-spirost-5-ene-
(25R)-27-O-β-D-glucopyranosyl-5-en-spirost-3β,17α,27-triol
3-O-α-L-
rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 6)]-β-D-glucopyr-
anoside, named trillitschoside S3.
Compound 4 had a molecular formula C45H72O19 as determined by
HRESIMS, which was the same with 1. The NMR data of 4 showed the
pattern analogous to 1 except for the obvious differences in F-ring (C-22
to C-27), suggesting 4 was a pair of C-25 epimer of 1. Further com-
parison of the NMR data of 4 with those of (25S)-27-hydroxypenogenin-
3β,17α,24α,27-tetraol 3-O-α-L-rhamnopyranosy1-(1
→
2)-β-D-gluco-
pyranoside though combined analyses of 1H–1H COSY, HSQC and
HMBC spectra, named trillitschonide S10.
Compound 11 had a molecular formula C51H82O23 as determined by
HRESIMS. The NMR data suggested that 11 had an identical spirotanol
skeleton with 8, and had a same sugar chain with saponin Tg [6]. Thus,
by combined analyses of 1H–1H COSY, HSQC and HMBC spectra, the
structure of 11 was confirmed to be (24R, 25S)-spirost-5-ene-
3β,17α,24α,27-tetraol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamno-
pyranosyl-(1 → 4)-[α-L-rhamno-pyranosyl-(1 → 2)]-β-D-glucopyrano-
side, named trillitschonide S11.
3-O-α-L-rhamnopyranosyl-(1
→
2)-O-β-D-glucopyranoside [3] con-
firmed that the aglycone of 4 was (25S)-5-en-spirost-3β,17α,27-triol.
Finally, confirmed by detailed analyses of 1H–1H COSY, HSQC and
HMBC spectra, the structure of 4 were elucidated as (25S)-5-en-spirost-
3β,17α,27-triol 3-O-α-L-rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-
(1 → 6)]-β-D-glucopyranoside, named trillitschoside S4.
Compound 5 had a molecular formula C51H82O23 as determined by
HRESIMS. The NMR data of 5 suggested it had same C-3 sugar chain
with 2. And the aglycone together with the C-27-O-glucosyl was same
as (25S)-27-[(β-D-glucopyranosyl)oxy]-17α-hydroxyspirost-5-en-3β-O-
α-L-rhamnopyranosyl-(1 → 2)-O-β-D-glucopyranoside [4] by detailed
comparison of their NMR data. Though combined use of 1H–1H COSY,
HSQC and HMBC experiments, the structure of 5 was further confirmed,
Compound 12 possessed the molecular formula C45H72O18 de-
termined by HRESIMS. The 1H NMR spectrum of 12 showed four me-
thyl proton signals at δ 0.95 (3H, s), δ 1.07 (3H, s), 1.09 (3H, d,
J = 6.0 Hz), 1.26 (3H, d, J = 7.2 Hz), and three anomeric proton
signals at δ 4.93 (1H, overlap), 5.84 (1H, br s) and 6.39 (1H, br s). By
comparing of the NMR data, it was verified that 12 had the similar
structure with 8 except for the F-ring. The HMBC correlations of H-27 (δ
1.09) and C-24 (δ 70.3), C-25 (δ 39.9), and C-26 (δ 65.2) suggesting the
presence of 24-OH group in 12. When compared the NMR data of 12
with those of (24S,25S)-3β,17α,24-trihydroxy-5α-spirostan-6-one 3-O-
[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside [11], their F-ring
structure was verified to be identical. Therefore, the structure of 12 was
elucidated as (24S, 25R)-5-en-spirost-3β,17α,24β-triol 3-O-α-L-rhamno-
pyranosyl-(1 → 4)-α-L-rhamnopyranosy1-(1 → 2)-β-D-glucopyranoside,
named trillitschoside S12.
Compound 13 possessed the molecular formula C45H74O19 de-
termined by HRESIMS. The 1H NMR spectrum of 13 showed four ty-
pical methyl signals of steroidal saponin at δ 0.65 (3H, d, J = 6.0 Hz),
1.02 (3H, s) 1.22 (3H, d, J = 7.2 Hz), and 1.66 (3H, s). Moreover, three
anomeric proton signals at δ 4.82 (1H, d, J = 7.2 Hz), 5.84 (1H, br s),
and 6.41 (1H, br s) due to three sugar unites in the molecule were
obviously observed. The NMR data indicated that 13 had the identical
structure with pennogenin 3-O-β-chacotrioside [7] except for the ob-
viously differences in C-5 and C-6 positons. The HMBC correlations of
H-19 (δ 1.66)/C-5 (δ 75.4) and in the 1H–1H COSY spectrum a spin
system correlations of H-6 (δ 4.81) → H-7 (δ 1.75, 2.15) → H-8 (δ
2.15) → H-9 (δ 1.97) indicated that 13 presented two hydroxyl groups
at C-5 and C-6, respectively. The α- and β-orientations for 5-OH and 6-
OH, respectively, were determined by comparing the chemical shifts of
C-5 (δ 75.9) and C-6 (δ 75.6) with those of (3β,5α,6β,25R)-spirostane-
3,5,6-triol-3-O-a-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside [12].
By detailed analyses of the 1H–1H COSY, HSQC and HMBC spectra of
13, its structure were further confirmed. Consequently, the structure of
13 was established as (25R)-spirost-3β,5α,6β,17α-tetraol 3-O-α-L-
rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosy1-(1 → 2)]-β-D-gluco-
pyranoside, named trillitschoside S13.
and
elucidated
as
27-O-β-D-glucopyranosyl-(25S)-5-en-spirost-
3β,17α,27-triol 3-O-α-L-rhamnopyranosyl-(1
anosy1-(1 → 2)-β-D-glucopyranoside, named trillitschoside S5.
→
4)-α-L-rhamnopyr-
Compound 6 had a molecular formula C45H72O19 as determined by
HRESIMS. The NMR data suggested that it showed the pattern analo-
gous to 2 except for the obvious differences in F-ring. 6 had 16 Da mass
units more than of 2, deducing the presence of an additional hydroxyl
group at its F-ring. Further comparison of the NMR data of 6 and
borassoside B [9] confirmed that they had the same F-ring structure,
attributing to verify the presence of 23α-OH group and the C-25S
configuration for 6. Consequently, the structure of 6 was elucidated as
(23S, 25S)-5-en-spirost-3β,17α,23α,27-tetraol
3-O-α-L-rhamnopyr-
anosyl-(1 → 4)-α-L-rhamnopyranosy1-(1 → 2)-β-D-glucopyranoside,
named trillitschonide S6.
Compound 7, same with 6, also had a molecular formula C45H72O19
as determined by HRESIMS. The NMR data suggested that it showed the
pattern analogous to 6 except for the differences in F-ring, suggesting 7
and 6 was a pair of C-25 epimers. By comparing of the NMR data of 7
and borassoside C [9], the identical chemical shifts of F-ring verified the
C-25R configuration of 7. Subsequently, the structure of 7 was eluci-
dated as (25R)-5-en-spirost-3β,17α,23α,27-tetraol 3-O-α-L-rhamnopyr-
anosyl-(1 → 4)-α-L-rhamnopyranosy1-(1 → 2)-β-D-glucopyranoside,
named trillitschoside S7.
Compound 8 had a molecular formula C45H72O19 as determined by
HRESIMS, same with that of 6. When compared the NMR data of 8 with
those of 6, it was suggested that they had the almost identical structure
except for the slightly different in F-ring. The 1H–1H COSY correlations
of H-24 (δ 4.55)/H-25 (δ 2.25) and the HMBC correlation of H-27 (δ
4.18) and C-24 (δ 66.5) deduced the presence of a 24-OH group.
Comparison of the NMR data of 8 with those of (24R, 25S)-spirost-5-
ene-3β,17α,24,27-tetraol-3-O-β-D-glucopyranoside [10] deduced that
they had the same F-ring structure, attributing to further verify the
location of 24-OH group and the 24R, 25S configurations of 8. There-
fore, the structure of 8 was elucidated as (24R, 25S)-spirost-5-ene-
3β,17α,24α,27-tetraol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamno-
pyranosy1-(1 → 2)-β-D-glucopyranoside, named trillitschonide S8.
Compound 9 had a molecular formula C39H62O15 as determined by
HRESIMS. When comparing the NMR data, it was verified that of 9 to
have same spirotanol skeleton with 8. On the other hand, the NMR data
further suggested that 9 had the same sugar chain with trikamsteroside
Compound 14 possessed the molecular formula C39H64O15 de-
termined by HRESIMS. By comparing of the NMR data, it was verified
that 14 had the similar structure with 13 except for the sugar chain. In
the 1H NMR spectrum, the observed only two anomeric proton signals
at δ 4.83 (1H, d, J = 7.2 Hz) and 6.39 (1H, br s) suggested that 14 had a
rhamnose less than 13. Comparison of NMR data of 14 and 9 suggested
they had the same sugar chain. Therefore, the structure of 14 was
elucidated as (25R)-spirost-3β,5α,6β,17α-tetraol 3-O-α-L-rhamnopyr-
anosyl-(1 → 2) -β-D-glucopyranoside, named trillitschoside S14.
Compounds 1–24 were tested for their cytotoxic activities in vitro
using the HepG2 cell line. A preliminary screening displayed that, after
6

Y.-J. Yang, et al.
Steroids156(2020)108587
neuroprotection, etc. should be tested in the next, so as to illuminate the
Table 3
HepG2 cell survival rate after treatment of 1–24 and 5-Fu (each 100 μM).
material foundation of T. tschonoskii for its multiple activity.
Compounds
Cell survival rate (%)
Compounds
Cell survival rate (%)
Acknowledgement
1
2
3
4
5
6
7
8
98.36
97.42
97.85
90.99
91.65
101.36
94.70
97.71
107.33
90.88
98.16
99.33
95.42
6.10
6.13
3.91
6.99
8.36
6.09
0.48
3.75
14
15
16
17
18
19
20
21
22
23
24
5-FU
92.41
96.61
94.37
92.09
102.96
101.54
103.30
1.45
0.44
4.08
2.54
2.27
4.62
0.86
5.80
The authors will thank Prof. Xian-Zhong Yan of the National Center
of Biomedical Analysis (NCBA) for his great help in NMR spectra
measurements.
Statement clarifying
0.08
9
1.71
0.98
0.69
4.74
0.16
0.06
1.95
There are no conflicts of interests of all authors.
Appendix A. Supplementary data
10
11
12
13
7.95
5.32
1.02
3.92
21.09
0.49
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.steroids.2020.108587.
treatment with 21–24 (100 μM), the average survival rate of HepG2
cells was 0.69% to 4.74%, which were better than the positive control
5-FU (5-Fluorouracil), suggesting their remarkable cytotoxic activities
against HepG2 cells, while the other compounds displayed little in-
hibitory effects (Table 3). 21–24 have much better cytotoxic activity
against HepG2 cells than other compounds, which presumed that the
aglycone of pennogenin is critical for the cytotoxic activity, and the
structural changes on pennogenin due to substituents or the config-
uration difference could result in the activity disappeared. Not sur-
prisingly, HepG2 cells treated with 21 and 24 for 24 h could sig-
nificantly induce apoptosis (Supplementary Fig.S2). While it should be
noticed that 20 is also a pennogenin glycoside but displaying no activity
and it is speculated that this is caused by its different sugar chain, which
further indicated that the sugar moiety of steroidal saponin is also
important for their activity.
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4. Conclusion
This work presented a phytochemical study on T. tschonoskii rhi-
zomes, resulting in isolation of total twenty-four steroidal saponins with
multiple structural characteristics, which was helpful to understand the
structural composition of steroidal saponins as main constituents in this
medicine. The following cytotoxic activity assay showed that penno-
genin glycosides had the remarkable cytotoxic activities against HepG2
cells overall, suggesting the pennogenin glycosides, especially the high-
content ones, should be the basis of active material of this herb medi-
cine for playing an anti-tumor role. T. tschonoskii have various phar-
macological activities, therefore more biological activities of these
isolated compounds such as hemostasis, anti-inflammation and
7