Lanostane triterpene glycosides from the flowers of Lyonia ovalifolia var. hebecarpa and their antiproliferative activities

Article abstract of DOI:10.1016/j.bioorg.2020.103598

Sixteen lanostane-type triterpene glycosides including eight new ones, named lyonicarposides A–H (1–8), were isolated from the flowers of Lyonia ovalifolia var. hebecarpa (Franch. ex F.B. Forbes & Hemsl.) Chun (Ericaceae). The chemical structures of the new compounds were elucidated by the comprehensive spectroscopic techniques and chemical methods. The Mo₂(OAc)₄–induced electronic circular dichroism method was used to determine the absolute configurations of C-24 in lyonicarposides A (1), C (3), and E (5). This is the first phytochemical study on the flowers of L. ovalifolia var. hebecarpa. All the isolates were evaluated for their antiproliferative activities against SMMC-7721, HL-60, SW480, MCF-7, and A-549 cell lines. Lyonicarposides A (1) and B (2) showed moderate antiproliferative activities against five cancer cell lines with IC₅₀ values ranging from 12.39 to 28.71 μM. Lyonicarposides C (3) and G (7) and lyonifoloside M (12) selectively inhibited the proliferation of HL-60 and MCF-7 cell lines with IC₅₀ values ranging from 13.03 to 17.71 μM. Interestingly, lyonifoloside L (13) selectively inhibited the proliferation of MCF-7 cell line with an IC₅₀ value of 16.27 μM. Their structure-activity-relationships were discussed.

Full text of DOI:10.1016/j.bioorg.2020.103598

BioorganicChemistry96(2020)103598
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Lanostane triterpene glycosides from the flowers of Lyonia ovalifolia var.
hebecarpa and their antiproliferative activities
Hanqi Zhanga, Xiang Penga,b, Xiaofeng Zhenga, Shunyi Lia, Yang Tengb, Junjun Liua,
⁎
Congming Zouc, Guangmin Yaoa,
a Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, PR China
b College of Pharmacy, Jiamusi University, Jiamusi 154007, PR China
c Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Sixteen lanostane-type triterpene glycosides including eight new ones, named lyonicarposides A–H (1–8), were
isolated from the flowers of Lyonia ovalifolia var. hebecarpa (Franch. ex F.B. Forbes & Hemsl.) Chun (Ericaceae).
The chemical structures of the new compounds were elucidated by the comprehensive spectroscopic techniques
and chemical methods. The Mo2(OAc)4–induced electronic circular dichroism method was used to determine the
absolute configurations of C-24 in lyonicarposides A (1), C (3), and E (5). This is the first phytochemical study on
the flowers of L. ovalifolia var. hebecarpa. All the isolates were evaluated for their antiproliferative activities
against SMMC-7721, HL-60, SW480, MCF-7, and A-549 cell lines. Lyonicarposides A (1) and B (2) showed
moderate antiproliferative activities against five cancer cell lines with IC50 values ranging from 12.39 to
28.71 μM. Lyonicarposides C (3) and G (7) and lyonifoloside M (12) selectively inhibited the proliferation of HL-
60 and MCF-7 cell lines with IC50 values ranging from 13.03 to 17.71 μM. Interestingly, lyonifoloside L (13)
selectively inhibited the proliferation of MCF-7 cell line with an IC50 value of 16.27 μM. Their structure-activity-
relationships were discussed.
Lyonia ovalifolia var. hebecarpa (Franch. ex F.B.
Forbes & Hemsl.) Chun (Ericaceae)
Lanostane triterpene glycosides
Mo2(OAc)4−induced electronic circular
dichroism
Chemical methods
Antiproliferative activity
1. Introduction
studied, leading to the isolation of 16 antiproliferative triterpene gly-
cosides classified into lanostane, oleanane, and 9,10-seco-cycloarane
Plants of the Ericaceae family, comprising of 103 genera and 3350
species, are widely distributed in temperate regions and sub-frigid zone.
Lyonia is a small genus of the Ericaceae family, and there are only 35
species all over the world. In China, there are only six species and five
varieties in the Lyonia genus. Previous phytochemical studies on the
Lyonia genus mainly focused on L. ovalifolia and L. ovalifolia var. ellip-
tica., leading to the isolation of flavonoids [1,2], phenylpropanoids
[2–6], monoterpenoids [7], sesquiterpenoids [7,8], diterpenoids
[9–13], and triterpenoids [2,14–16]. Some of them exhibited various
biological activities, such as nervous system inhibitory [17], cAMP in-
hibitory [12], antibacterial [13], antiviral [16], and antihyperglycemic
activities [18]. However, the phytochemical study on Lyonia ovalifolia
var. hebecarpa (Franch. ex F.B. Forbes & Hemsl.) Chun is relatively rare.
Lyonia ovalifolia var. hebecarpa is a tree or shrub, 8–16 m height, and
is mainly distributed in Southern China [19]. In a course of search for
biological terpenoids from the Ericaceae plants in China [20–31], the
leaves of Lyonia. ovalifolia var. hebecarpa have been phytochemically
types [32,33]. However, heretofore, there is no report of phytochemical
studies on the flowers of L. ovalifolia var. hebecarpa. Expecting the other
plant parts to be chemically distinct from the leaves, the flowers of L.
ovalifolia var. hebecarpa were collected and phytochemically in-
vestigated, leading to the isolation of a total of 16 lanostane triterpene
glycosides including eight new ones, named lyonicarposides A–H (1–8)
(Fig. 1). This is the first phytochemical study on the flowers of L. ova-
lifolia var. hebecarpa. Herein, the extraction, isolation, structure eluci-
dation, and the antiproliferative activities of 16 lanostane triterpene
glycosides from the flowers of L. ovalifolia var. hebecarpa were de-
scribed.
2. Materials and methods
2.1. General experimental procedures
Optical rotations were measured with a Rudolph Autopol IV
⁎ Corresponding author.
E-mail address: gyap@mail.hust.edu.cn (G. Yao).
https://doi.org/10.1016/j.bioorg.2020.103598
Received 8 December 2019; Received in revised form 14 January 2020; Accepted 19 January 2020
Availableonline21January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

H. Zhang, et al.
BioorganicChemistry96(2020)103598
Fig. 1. Chemical structures of compounds 1–16.
automatic polarimeter. The IR, UV, and ECD data were recorded by a
Bruker Vertex 70 instrument, a Varian Cary 50 spectrophotometer, and
a JASCO J–810 spectrometer, respectively. The NMR data were ob-
tained through a Bruker AM–400 spectrometer operating at 400 (1H)
and 100 (13C) MHz with referencing to the residuals 1H (δH 3.31 for
methanol‑d4) or 13C (δC 49.15 for methanol‑d4). HRESIMS were per-
formed on a Bruker micrOTOF II spectrometer. Semi-preparative HPLC
was accomplished on a Dionex 680 system with a UV detector or
Agilent 1260 with a DAD detector using an RP C18 column (5 μm,
10 × 250 mm, Welch Ultimate XB–C18). GC was analyzed using the
Agilent 7820A gas chromatography and the capillary column
(30 m × 0.25 mm × 0.5 μm, Welch WM-1). The cytotoxicity assay was
performed using Multiskan FC multifunctional microplate reader.
with MeCN–H2O (80:20, v/v, 1.5 mL/min) from Fr.4D1 (102 mg).
Fraction Fr.4D2 (48.6 mg) was separated by a semipreparative HPLC
eluting with MeCN–H2O (84:16, v/v, 1.5 mL/min) to give compound 8
(12.2 mg, tR = 28.6 min). Compounds 1 (3.0 mg, tR = 49.6 min), 2
(7.0 mg, tR = 45.5 min), and 5 (5.0 mg, tR = 40.5 min) were obtained
by semipreparative HPLC eluting with MeCN–H2O (70:30, v/v, 1.5 mL/
min) from the fraction Fr.4D3 (142 mg). Fr.5 (129.5 g) was chroma-
tographed over a C18 MPLC eluting with MeOH–H2O (from 50:50 to
100:0, v/v) to obtain the major fraction Fr.5C (2.7 g). Fr.5C was applied
to
a
Sephadex LH-20 column to afford five subfractions
(Fr.5C1–Fr.5C5). Compound 13 (15.1 mg, tR = 33.3 min) was obtained
from Fr.5C1 (148.7 mg) by a semipreparative HPLC eluting with
MeCN–H2O (80:20, v/v, 1.5 mL/min). Compounds 14 (12.6 mg,
tR = 24.3 min) and 16 (7.0 mg, tR = 38.2 min) were isolated from
Fr.5C2 (102.5 mg) by a semipreparative HPLC with MeCN–H2O (82:18,
v/v, 1.5 mL/min). Purification of Fr.5C3 (14.2 mg) by semipreparative
HPLC with MeCN–H2O (77:23, v/v, 1.5 mL/min) to yield compound 6
(2.6 mg, tR = 45.3 min). Fraction Fr.5C4 (102.3 mg) was separated by a
semipreparative HPLC eluting with MeCN–H2O (80:20, v/v, 1.5 mL/
min) to give compound 3 (3.6 mg, tR = 33.3 min). Further purification
of the fraction Fr.5C5 (248.6 mg) by a semipreparative HPLC eluting
with MeCN–H2O (78:22, v/v, 2.0 mL/min) afforded compounds 15
(7.0 mg, tR = 20.6 min) and 6 (5.2 mg, tR = 25.4 min). The n-BuOH
fraction (49.3 g) was subjected to a silica gel (100–200 mesh) column
and eluted with CH2Cl2–MeOH (30:1 to 0:1, v/v) to afford eight frac-
tions Fr.1–Fr.8. Fr.6 (4.9 g) was isolated by an RP C18 MPLC eluting
with MeOH–H2O (from 50:50 to 100:0, v/v) to yield seven subfractions
Fr.6A–Fr.6G. Fr.6E (0.8 g) was subjected to a silica gel (200–300 mesh)
column and eluted with CH2Cl2–MeOH (40:1 to 0:1, v/v) to afford five
subfractions Fr.6E1–Fr.6E5. Purification of Fr.6E3 (59.6 mg) by a
semipreparative HPLC eluting with MeCN–H2O (67:33, v/v, 1.5 mL/
min) to yield compounds 4 (1.6 mg, tR = 57.4 min), 9 (3.8 mg,
tR = 49.3 min), and 11 (3.6 mg, tR = 53.3 min). Fraction Fr.6E4
(32.3 mg) was separated by semipreparative HPLC with MeCN–H2O
(65:35, v/v, 1.5 mL/min) to give compound 10 (5.6 mg,
tR = 46.8 min).
2.2. Plant materials
The flowers of Lyonia ovalifolia var. hebecarpa (Franch. ex F.B.
Forbes & Hemsl.) Chun (Ericaceae) were collected in Jinyun County
(latitude 28°26′29″ North and longitude 120°8′59″ East), Zhejiang
province, China, in June 2015, and authenticated by Professor Bing-
Yang Ding at Wenzhou University. A voucher specimen (No. 2015-
0617) has been deposited at School of Pharmacy, Tongji Medical
College, Huazhong University of Science and Technology.
2.3. Extraction and isolation
The air-dried flowers of L. ovalifolia var. hebecarpa (1.5 Kg) were
extracted (5 × 10 L) with MeOH at 40 °C. After the removal of the
solvent under the reduced pressure, the crude extract (386.3 g) was
suspended in H2O (1 L) and then successively partitioned with
chloroform (5 × 2 L) and n-BuOH (5 × 2 L). The chloroform fraction
(186.7 g) was subjected to a silica gel (100–200 mesh) column and
eluted with CH2Cl2–MeOH (50:1 to 0:1, v/v) to afford nine fractions
Fr.1–Fr.9. Fr.4 (3.5 g) was isolated by RP C18 MPLC eluting with
MeOH–H2O (from 60:40 to 100:0, v/v) to yield the major fraction Fr.4D
(1.2 g). Fr.4D was subjected to a silica gel (200–300 mesh) column and
eluted with CH2Cl2–MeOH (50:1 to 0:1, v/v) to obtain three subfrac-
tions Fr.4D1–Fr.4D3. Compounds 7 (5.2 mg, tR = 33.5 min) and 12
(3.6 mg, tR = 36.1 min) were purified by semipreparative HPLC eluted
2

H. Zhang, et al.
BioorganicChemistry96(2020)103598
Table 1
Table 2
1H NMR spectroscopic data for compounds 1–4 in methanol‑d4 (δ in ppm, J in
13C NMR spectroscopic data for compounds 1–8 in methanol‑d4 (δ in ppm and
Hz, and 400 MHz).
100 MHz).
No.
1
2
3
4
No.
1
2
3
4
5
6
7
8
1α
1.48, m
1.81, m
1.83, m
1.75, m
3.39, br s
1.55, m
1.55, m
1.62, m
1.88, m
1.53, m
2.12, m
2.30, m
1.93, m
2.10, m
2.07, m
1.41, m
2.10, m
1.54, m
1.05, m
0.81, s
1.40, m
1.81, m
1.84, m
1.75, m
3.39, br s
1.55, m
1.56, m
1.62, m
1.89, m
1.55, m
2.12, m
2.24, m
1.93, m
2.11, m
2.05, m
1.45, m
1.92, m
1.82, m
1.06, m
0.81, s
1.42, m
1.79, m
1.78, m
1.74, m
3.49, br s
1.65, m
1.55, m
1.64, m
1.71, m
1.56, m
2.14, m
2.20, m
1.71, m
2.22, m
2.00, m
1.94, m
2.10, m
1.54, m
1.54, m
0.82, s
1.29, m
2.10, m
1.78, m
1.60, m
3.39, br s
1.65, m
1.55, m
1.65, m
1.95, m
2.05, m
2.03, m
2.10, m
1.70, m
2.26, m
1.56, m
1.48, m
1.40, m
1.70, m
1.57, m
0.82, s
1
31.6
22.7
82.5
38.3
46.1
19.3
27.4
123.5
146.2
39.1
23.1
32.0
47.3
68.5
28.8
29.1
54.5
17.5
20.1
37.7
19.4
34.6
28.8
80.6
74.0
25.9
24.9
29.1
23.1
200.8
101.9
75.1
78.3
72.1
75.2
64.9
31.6
22.3
82.2
38.4
46.0
19.3
27.4
123.5
146.2
39.2
23.1
32.0
47.3
68.5
28.8
29.1
54.5
17.5
20.1
37.7
19.4
34.6
28.8
80.6
74.0
25.9
24.9
29.2
23.1
200.8
101.9
72.6
74.7
69.9
67.0
31.7
22.7
82.6
38.3
46.1
19.3
27.4
128.9
141.7
39.1
23.2
32.0
47.3
64.1
28.8
30.0
52.2
18.3
20.3
37.7
19.4
34.6
29.2
80.6
74.0
25.9
24.9
29.1
23.1
180.3
101.9
74.1
75.8
73.2
75.3
62.8
31.8
22.5
82.3
38.3
47.5
19.4
28.5
128.9
141.7
38.7
23.4
32.7
48.1
64.0
29.6
31.1
52.2
18.4
20.3
37.0
19.2
34.3
32.7
89.4
73.7
26.7
25.0
29.3
23.1
180.2
102.2
72.9
73.4
74.4
71.9
19.6
105.5
72.5
75.4
70.0
67.5
31.1
22.3
82.3
38.3
45.7
19.4
28.9
128.9
141.8
38.7
23.4
32.7
48.3
64.0
28.5
30.5
52.4
18.3
20.3
37.8
19.4
34.9
29.2
79.9
74.0
25.7
25.0
29.1
23.0
181.0
101.7
75.3
75.9
73.3
76.0
62.9
31.1
22.6
82.6
38.2
45.8
19.4
28.5
128.8
142.0
38.7
23.4
32.7
48.3
64.0
29.2
30.5
52.3
18.3
20.3
37.8
19.2
34.9
31.1
79.9
74.0
25.8
25.1
29.1
23.1
180.3
101.9
75.1
78.1
72.2
75.2
65.0
31.1
22.4
82.6
38.3
45.7
19.1
29.1
129.0
141.7
38.7
23.4
32.7
48.2
64.2
28.5
30.4
52.2
18.4
20.3
37.0
19.1
30.1
34.0
218.0
78.1
26.9
26.9
29.1
23.0
181.7
101.7
73.6
79.4
70.2
77.8
62.8
31.6
22.4
82.0
38.4
46.0
19.4
28.5
123.5
146.3
39.2
23.4
32.7
47.3
68.5
29.2
30.5
54.3
17.4
20.1
36.8
19.0
34.9
31.1
217.8
78.5
26.9
26.9
29.1
23.0
200.8
101.9
75.1
78.1
72.2
78.2
62.5
1β
2
2α
3
2β
4
3β
5
5α
6
6α
7
6β
8
7α
9
7β
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
11α
11β
12α
12β
15α
15β
16α
16β
17α
18β
19β
20β
21α
22a
22b
23a
23b
24
1.10, s
1.11, s
1.07, s
1.06, s
1.47, m
0.96, d (6.5)
1.40, m
1.40, m
1.71, m
1.71, m
3.14, dd (10.2,
1.6)
1.48, m
0.96, d (6.6)
1.78, m
1.75, m
1.67, m
1.62, m
3.14, dd (10.3,
1.5)
1.48, m
0.96, d (6.2)
1.80, m
1.73, m
1.79, m
1.70, m
3.14, dd (10.3,
1.8)
1.45, m
0.95, d (5.0)
1.48, m
1.48, m
2.26, m
2.26, m
3.40, overlap
26
27
28α
29β
30
1′
1.15, s
1.15, s
1.16, s
1.16, s
1.13, s
0.97, s
0.90, s
1.11, s
1.11, s
1.07, s
0.92, s
0.97, s
0.99, s
2′
0.90, s
0.91, s
0.91, s
3′
9.46, s
9.46, s
4′
4.25, d (7.8)
3.17, dd (9.3,
7.8)
4.21 d (6.4)
3.53, overlap
4.30, d (7.8)
3.27, dd (9.3,
7.8)
4.18, br s
5′
2′
3.57, overlap
6′
1″
2″
3″
4″
5″
OAc
3′
3.55, t (9.3)
3.27, t (9.3)
3.39, ddd (9.3,
6.0, 2.4)
3.52, overlap
3.80, m
3.55, t (9.3)
4.73, t (9.3)
3.39, ddd (9.3,
6.0, 2.8)
3.59, overlap
3.54, overlap
3.56, overlap
4′
5′a
3.85 dd (12.4,
2.9)
5′b
6′a
3.50, overlap
20.9
20.9
21.1
20.9
21.3
4.37, dd (11.7,
2.4)
3.86, dd (11.7,
2.8)
1.25, d (6.3)
172.8
172.4
172.4
172.9
172.8
6′b
4.23 dd (11.7,
6.0)
3.66, dd (11.7,
6.0)
see Tables 1 and 2; HRESIMS m/z 717.4248 [M + Na]+ (calcd for
1″
4.26, d (6.9)
3.57, overlap
3.46, overlap
3.81, overlap
3.89, dd (12.5,
2.2)
C38H62O11Na, 717.4190).
2″
25
Lyonicarposide D (4): white amorphous powder; [α]D −33 (c 0.1,
3″
4″
MeOH); UV (MeOH) λmax (log ε) 231.0 (2.74), 278.7 (1.94) nm; IR
(KBr) νmax 3418, 2936, 1633, 1539, 1455, 1384, 1067 cm−; 1 1H NMR
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 791.4540
[M + Na]+ (calcd for C41H68O13Na, 791.4558).
5″a
5″b
3.56, overlap
OAc 2.09, s
2.09, s
25
Lyonicarposide E (5): white amorphous powder; [α]D −46 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 231.2 (2.83) nm; IR (KBr) νmax 3414,
2942, 1728, 1630, 1596, 1376, 1250, 1030 cm−; 1 1H NMR and 13C NMR
data, see Tables 2 and 3; HRESIMS m/z 717.4128 [M + Na]+ (calcd for
2.4. Spectroscopic data
25
Lyonicarposide A (1): white amorphous powder; [α]D −14 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 238.2 (2.92), 301.0 (2.28) nm; IR
C38H62O11Na, 717.4190).
25
Lyonicarposide F (6): White amorphous powder; [α]D −55 (c 0.1,
(KBr) νmax 3401, 2940, 1744, 1703, 1630, 1453, 1384 cm−1
;
1H NMR
MeOH); UV (MeOH) λmax (log ε) 231.2 (2.89) nm; IR (KBr) νmax 3418,
2938, 1723, 1632, 1456, 1384, 1224, 1045 cm−; 1 1H NMR and 13C NMR
data, see Tables 2 and 3; HRESIMS m/z 717.4182 [M + Na]+ (calcd for
C38H62O11Na, 717.4190).
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 701.4253
[M + Na]+ (calcd for C38H62O10Na, 701.4241).
25
Lyonicarposide B (2): white amorphous powder; [α]D −69 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 239.0 (2.87), 302.0 (2.22) nm; IR
Lyonicarposide G (7): White amorphous powder; [α]D25 −30 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 234.7 (3.45), 285.0 (2.47) nm; IR
(KBr) νmax 3410, 2964, 1693, 1632, 1603, 1461, 1385 cm−1
;
1H NMR
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 629.4025
(KBr) νmax 3409, 2939, 1708, 1595, 1461, 1377, 1255, 1036 cm;−1 1
H
[M + Na]+ (calcd for C35H58O8Na, 629.4029).
NMR and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 715.3979
[M + Na]+ (calcd for C38H60O11Na, 715.4033).
25
Lyonicarposide C (3): white amorphous powder; [α]D −47 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 230.7 (2.91) nm; IR (KBr) νmax 3419,
Lyonicarposide H (8): White amorphous powder; [α]D25 −22 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 239.0 (3.11), 301.0 (2.50) nm; IR
2960, 1727, 1631, 1459, 1384, 1248 cm−1; 1H NMR and 13C NMR data,
3

H. Zhang, et al.
BioorganicChemistry96(2020)103598
Table 3
at 2 °C/min. In the same way, the trimethylsilylthiazolidine derivatives
of the standards D- and L-glucose were prepared and analyzed by GC.
Retention times of trimethylsilylthiazolidine derivatives of the hydrate
of 1, D- and L-glucose were 15.946, 16.042, and 16.349 min, respec-
tively.
1H NMR spectroscopic data for compounds 5–8 in methanol‑d4 (δ in ppm, J in
Hz, and 400 MHz).
No.
5
6
7
8
1α
1.42, m
1.79, m
1.78, m
1.74, m
3.49, br s
1.65, m
1.55, m
1.64, m
1.71, m
1.56, m
2.14, m
2.20, m
1.71, m
2.22, m
2.00, m
1.94, m
2.10, m
1.54, m
1.54, m
0.82, s
1.42, m
1.78, m
1.78, m
1.72, m
3.38, br s
1.62, m
1.56, m
1.62, m
1.96, m
2.08, m
2.13, m
2.24, m
1.71, m
2.20, m
2.11, m
1.71, m
1.56, m
2.11, m
1.53, m
0.82, s
1.42, m
1.79, m
1.79, m
1.74, m
3.50, br s
1.63, m
1.56, m
1.64, m
1.56, m
1.62, m
2.13, m
2.23, m
1.68, m
2.26, m
2.08, m
1.98, m
2.10, m
1.54, m
1.55, m
0.81, s
1.49, m
1.93, m
1.93, m
1.81, m
3.50, br s
1.57, m
1.57, m
1.65, m
1.96, m
2.08, m
2.13, m
2.24, m
1.71, m
2.20, m
2.11, m
1.71, m
1.56, m
2.11, m
1.53, m
0.81, s
In the same way, lyonicarposides B–H (2–8) were hydrolyzed by
2 mM HCl, and the trimethylsilylthiazolidine derivatives of the hydrates
of compounds 2–8 and the standards D- and L-glucoses, D- and L-ara-
binoses, and L-rhamnose were prepared. The trimethylsilylthiazolidine
derivatives of the hydrates of compounds 3 and 5–8 gave GC retention
times at 16.040, 15.978, 16.100, 15.999, and 15.913 min, respectively,
suggesting the presence of D-glucose. Detection conditions of the deri-
1β
2α
2β
3β
5α
6α
6β
7α
vatives of standards
L-arabinose, L-rhamnose, and hydrates of
compounds 2 and 4 Dw- earneddifferent at column temperature: initially
220 °C for 5 min, raised to 270 °C at 5 °C/min. The retention times of
the derivatives of standards D- and L-arabinoses and L-rhamnose were
11.255, 10.702, and 12.198 min, respectively. The trimethylsilylthia-
zolidine derivative of the hydrate of compound 2 was 10.628 min, in-
dicating the presence of
7β
11α
11β
12α
12β
15α
15β
16α
16β
17α
18β
19β
20β
21α
22a
22b
23a
23b
24
derivative of the standardL-La-rrhaabminnooseseinwa2s. 1T2h.1e9r8etmeninti.oTnwtoimseugoafrsthine
compound 4 were determined to be L-arabinose and L-rhamnose by the
GC retention times of their trimethylsilylthiazolidine derivatives at
10.626 and 12.298 min, respectively.
1.07, s
1.06, s
1.06, s
1.10, s
1.48, m
0.96, d (6.2)
1.80, m
1.73, m
1.79, m
1.70, m
3.22, dd (10.3,
1.8)
1.47, m
0.96, d (6.2)
1.75, m
1.48, m
1.42, m
2.26, m
3.21, dd (10.4,
1.9)
1.46, m
0.93, d (5.9)
1.42, m
1.68, m
2.02, m
2.05, m
1.47, m
0.93, d (6.6)
1.75, m
1.48, m
1.42, m
2.26, m
2.6. Preparation of the aglycone of lyonicarposides A (1), C (3), and E (5)
Lyonicarposide A (1) (3.0 mg) was dissolved in MeOH (0.5 mL) and
then 0.6 mM TsOH·H2O (40 μL) was added, and the mixture was stirred
at 65 °C for 72 h. After the removal of MeOH by evaporation, the re-
action mixture was diluted with H2O (3.0 mL) and extracted with ethyl
acetate (3 × 1.0 mL). The ethyl acetate fraction was subjected to a
silica gel (200–300 mesh) CC eluting with a CH2Cl2–CH3OH (from 50:1
to 20:1, v/v) gradient system to yield the aglycone 1a (1.2 mg). In the
same way, lyonicarposides C (3) (3.6 mg) and H (5) (5.0 mg) were
hydrolyzed to give the aglycones 3a and 5a.
26
27
28α
29β
30
1′
1.16, s
1.16, s
1.29, s
1.29, s
0.98, s
0.91, s
1.29, s
1.07, s
1.13, s
1.29, s
0.99, s
0.93, s
0.97, s
0.91, s
0.89, s
0.92, s
9.47, s
4.30, d (7.8)
3.27, dd (9.3,
7.8)
4.24, d (7.6)
3.25, dd (9.1,
7.8)
4.35, d (7.8)
3.29, dd (9.4,
7.8)
4.30, d (7.8)
3.17, dd (9.0,
7.8)
2′
3′
4′
5′
3.55, t (9.3)
4.73, t (9.3)
3.39, ddd (9.3,
6.0, 2.8)
3.55, t (9.1)
3.27, t (9.1)
3.38, ddd (9.1,
6.3, 2.6)
4.39, t (9.4)
3.34, t (9.4)
3.30, ddd (9.4,
5.5, 2.1)
3.34, t (9.0)
3.26, t (9.0)
3.22, ddd (9.0,
5.6, 2.0)
3.86, dd (11.7,
2.0)
2.6.1. Lyonicarpol A (1a)
25
White amorphous powder; [α]D −43 (c 0.1, MeOH); 1H NMR
6′a
6′b
3.86, dd (11.7,
2.8)
4.37, dd (11.8,
2.6)
3.86, dd (11.8,
2.1)
(CD3OD, 400 MHz) δ: 3.35 (1H, br s, H-3β); 0.81 (3H. s, H-18β), 1.10
(3H. s, H-19β), 0.96 (3H. d, J = 6.5 Hz, H-21α), 3.14 (1H. dd, J = 10.2,
1.6 Hz, H-24), 1.15 (3H. s, H-26), 1.11 (3H. s, H-27), 0.93 (3H. s, H-
28α), 0.87 (3H. s, H-29β). HRESIMS m/z 497.3594 [M + Na]+ (calcd
for C30H50O4Na, 497.3607).
3.66, dd (11.7,
2.8)
4.23 dd (11.8,
6.3)
3.68, dd (11.8,
5.5)
3.56, dd (11.7,
5.6)
OAc 2.09, s
2.05, s
2.10, s
(KBr) νmax 3441, 2942, 1706, 1467, 1384, 1017 cm−; 1 1H NMR and 13
C
2.6.2. Hebecarpolic acid A (3a)
NMR data, see Tables 2 and 3; HRESIMS m/z 657.3981 [M + Na]+
The spectroscopic data of the aglycone 3a were identical to those of
hebecarpolic acid A [33].
(calcd for C36H58O9Na, 657.3979).
2.6.3. Hebecarpolic acid E (5a)
2.5. Acid hydrolysis and determination of the absolute configurations of
The spectroscopic data of the aglycone 5a were identical to those of
hebecarpolic acid E [33].
sugar moieties in lyonicarposides A–H (1–8)
Lyonicarposide A (1) (0.5 mg) was hydrolyzed by 2 mM HCl
(1.5 mL) for 6 h at 80 °C. After extraction with ethyl acetate
(3 × 2.0 mL), the aqueous layer was evaporated to dryness and dis-
solved in anhydrous pyridine (0.2 mL). Then L-cysteine methyl ester
hydrochloride (0.2 mg) was added to the solution. The mixture was
kept at 60 °C for 1.5 h and subsequently was trimethylsilylated with N-
trimethylsilylimidazole (0.1 mL) without any isolation at 60 °C for
1.5 h. The reaction mixture was suspended in H2O (1.0 mL) and ex-
tracted with n-hexane (3 × 1.0 mL). The layer of n-hexane was directly
analyzed by GC with WM-1 capacity column. Nitrogen was used as a
carrier gas at a flow rate of 1.0 mL/min. Flame ionization detector (FID)
and injection were set at 250 °C. The split ratio was 10:1 and the in-
jection volume was 1 μL. The oven temperature was programmed as
follows: the initial temperature at 220 °C for 5 min, increased to 260 °C
2.7. Preparation of the methyl esters of the aglycones 3a and 5a
A mixture of 3a and K2CO3 (1.0 mg) in anhydrous DMF (0.5 mL)
was added to MeI (4.6 μL) and then the mixture was stirred at room
temperature for 2 h. The reaction mixture was neutralized with Na2SO4
(0.5 M) and diluted in H2O (1.0 mL), and extracted with ethyl acetate
(3 × 1.0 mL). After concentration, the ethyl acetate fraction was sub-
jected to silica gel CC (200–300 mesh) eluting with a CH2Cl2–CH3OH
(from 50:1 to 30:1, v/v) gradient system to yield the ester 3b. In the
same way, the ester 5b was prepared from 5a.
The spectroscopic data of the esters 3b and 5b were identical to
those of hebecarpolic acid A methyl ester and hebecarpolic acid E
methyl ester, respectively [33].
4

H. Zhang, et al.
BioorganicChemistry96(2020)103598
Fig. 2. 1H−1H COSY and the key HMBC correlations of lyonicarposides A–H (1–8).
2.8. Determination of the absolute configuration of the 24,25-diol moiety in
The molecular formula of C38H62O10 was assigned to 1 based on the 13
C
compounds 1a, 3b, and 5b
NMR data and HRESIMS ion at m/z 701.4253 [M + Na]+ (calcd for
C38H62O10Na, 701.4241), indicating eight indices of hydrogen defi-
According to the method previously described [33], Mo2(OAc)4 was
dissolved in dry DMSO to prepare a stock solution (1.0 mg/mL), then
freshly prepared solution of Mo2(OAc)4 were added to compound 1a
(0.6 mg) so that the molar ratio of stock complex to ligand was about
1.2:1. The Mo2(OAc)4-induced ECD spectrum was immediately re-
corded every ten minutes until the IECD spectrum was constant. After
subtracting the ECD spectrum of 1a, the correlation between the ab-
solute configuration of C-24 in 1a and the Cotton effects in IECD
spectrum were inferred using Snatzke’s regulations [34]. Compounds
3b and 5b were treated in the same way.
ciency. Absorption bands at 1384 cm−1 and 1744 cm−1 in the IR
spectrum suggested the presence of a double bond and a carbonyl
group, respectively. 1H NMR data of 1 (Table 1) showed resonances for
eight methyl groups at δH 0.81 (3H, s, CH3-18), 1.10 (3H, s, CH3-19),
0.96 (3H, d, J = 6.5 Hz, CH3-21), 1.15 (3H, s, CH3-26), 1.11 (3H, s,
CH3-27), 0.92 (3H, s, CH3-28), 0.90 (3H, s, CH3-29), and an acetyl at δH
2.09 (3H, s, 6′-OAc), two oxymethines at δH 3.39 (1H, br s, H-3) and
3.14 (1H, dd, J = 10.2, 1.6 Hz, H-24), an aldehyde group at δH 9.46
(1H, s, H-30), and an glucopyranosyl unit at δH 4.25 (1H, d, J = 7.8 Hz,
H-1′), 3.17 (1H, dd, J = 9.3, 7.8 Hz, H-2′), 3.55 (1H, t, J = 9.3 Hz, H-
3′), 3.27 (1H, t, J = 9.3 Hz, H-4′), 3.39 (1H, ddd, J = 9.3, 6.0, 2.4 Hz,
H-5′), 4.37 (1H, dd, J = 11.7, 2.4 Hz, H-6′a), and 4.23 (1H, dd,
J = 11.7, 6.0 Hz, H-6′b) [16]. Combined with the DEPT and HSQC
data, a total of 38 carbon resonances in the 13C NMR data (Table 2)
were assigned to be a glucopyranosyl unit at δC 101.9 (C-1′), 75.1 (C-
2′), 78.3 (C-3′), 72.1 (C-4′), 75.2 (C-5′), and 64.9 (C-6′) [16], eight
methyls, ten methylenes, five methines including two oxymethines at δC
82.5 (C-3) and 80.6 (C-24), four quaternary carbons, one oxygenated
tertiary carbon at δC 74.0 (C-25), a tetrasubstituted double bond at δC
123.5 (C-8) and 146.2 (C-9), an ester carbonyl group at δC 172.8 (C-
OAc), and an aldehyde group at δC 200.8 (C-30). A tetrasubstituted
double bond, an aldehyde group, an acetyl group, and a glucopyranosyl
unit account for four indices of hydrogen deficiency, and the remaining
four indices of hydrogen deficiency suggested the presence of a tetra-
cyclic system in 1. Thus, lyonicarposide A (1) should be a tetracyclic
triterpene glycoside.
2.9. Cytotoxicity assays
The cytotoxicity assays were performed according to the method
reported previously [32,33] and the IC50 values of the compounds were
calculated by Reed and Muench method.
3. Results and discussion
3.1. Structure elucidation
The air-dried flowers of L. ovalifolia var. hebecarpa (1.5 Kg) were
powdered and extracted with MeOH. The crude extract was suspended
in H2O and then partitioned with chloroform and n-BuOH. The
chloroform fraction was repeatedly subjected to silica gel, reversed
phase (RP) C18 silica gel, and Sephadex LH–20 column chromato-
graphy, as well as HPLC to afford seven new lanostane-type triterpene
glycosides (1–3 and 5–8), named lyonicarposides A–C and E–H, along
with five known analogues (12–16). The n-BuOH fraction was treated
in a similar procedure to give one new lanostane-type triterpene gly-
coside lyonicarposide D (4) and three known analogues (9–11). The
known triterpene glycosides 9–16 were identified to be lyonifolosides P
(9), J (10), R (11), M (12), and L (13) [16], hebecarposides A (14), E
(15), and H (16) [33], respectively, by their NMR data analysis and
comparison with literature data.
A comparison of the 1H and 13C NMR data of lyonicarposide A (1)
with those of lyonifoloside M (12) [16] revealed their structural simi-
larity. The major difference was the presence of an aldehyde group (δH
9.46, s, H-30; δC 200.8, C-30) in 1, replacing the carboxyl group (δC
180.5, C-30) in 12. This was further confirmed by HMBC correlations
from H-30 (δH 9.46, s) to C-14 (δC 68.5) and C-15 (δC 28.8) and from
H2-15 (δH 2.07, m; 1.41, m) to C-30. The glycosidation at C-3 was
proved by the HMBC cross-peaks from H-1′ to C-3 and H-3 to C-1′. The
planar structure of 1 was confirmed by the detailed 2D NMR analysis
(Fig. 2).
Lyonicarposide A (1) was obtained as a white amorphous powder.
5

H. Zhang, et al.
BioorganicChemistry96(2020)103598
Fig. 3. Key NOESY correlations of lyonicarposide A (1).
Similar to 12, H3-19 in 1 was assigned as β-orientation. The broad
The molecular formula of lyonicarposide B (2) was determined to be
C35H58O8 based on the 13C NMR data and HRESIMS ion at m/z
629.4025 [M + Na]+ (calcd for C35H58O8Na, 629.4029). Analysis of
the 1H and 13C NMR data (Tables 1 and 2) revealed that the NMR data
of 2 were similar to those of hebecarposide A (14) [33], and the major
difference was that an aldehyde group (δH 9.46, s, H-30; δC 200.8, C-30)
in 2 replaced the carboxyl group (δC 180.3, C-30) in 14, which was
supported by the HMBC correlations from H-30 (δH 9.46, s) to C-14 (δC
68.5) and C-15 (δC 28.8) and from H2-15 (δH 2.05, m; 1.45, m) to C-30.
Further 2D NMR data analysis (Fig. 2 and Supporting Information, Fig.
S1) established the structure of lyonicarposide B (2). To determine the
absolute configuration of the arabinose moiety, lyonicarposide B (2)
was hydrolyzed, and the trimethylsilylthiazolidine derivatives of the
hydrolysate of 2 and the standards, D- and L-arabinoses, were prepared.
The GC retention time of the trimethylsilylthiazolidine derivative of the
hydrolysate of 2 (tR = 10.628 min) was close to that of
single peak of H-3 (δH 3.39, br s) suggested the equatorial position of H-
3 in chair conformation of the cylcohexane in ring A (Fig. 3), and a β-
orientation of H-3 was assigned. The NOESY correlation between H3-
19β and H3-29 indicated the β-orientation of H3-29 and the α-orienta-
tion of H3-28. The strong NOESY correlation between H3-28α and H-5
and the lack of NOESY correlation between H3-19β and H-5 suggested
the α-orientation of H-5. The β-orientation of H3-18 was determined by
the NOESY correlations between H3-19β and H-11β (δH 2.12, m) and
between H-11β and H3-18. The NOESY correlations between H-11α (δH
2.30, m) and CHO-30 and between CHO-30 and H-17 suggested the α-
orientations of CHO-30 and H-17. The α-orientation of H-7 and the
configuration of C-20 in 1 were established to be identical to that of 12
by their biogenetic relationships and the biosynthetic pathway of la-
nostane triterpenoids [35,36].
The absolute configuration of C-24 in 1 was determined by a
Mo2(OAc)4–induced electronic circular dichroism (IECD) method [34].
According to the method previously described [32,33], 1 was hydro-
lyzed by TsOH to yield the aglycone 1a. The Mo2(OAc)4-IECD spectrum
of the 1a (Fig. 4) showed the positive Cotton effect at 316 nm, sug-
gesting the 24S absolute configuration of C-24 [34], which was iden-
tical to that of lyonifoloside M (12).
(tR = 10.702 min), instead of D-arabinose (tR = 11.255 Lm-airna)b,insousge-
gesting the L-configuration of the arabinose unit in 2. The large cou-
pling constant of H-1′ (J = 6.4 Hz) revealed the α-L-arabinopyranosyl
linkage in 2. Therefore, the structure of 2 was defined as 3α-[α-L-ara-
binopyranosyl)-oxy]-24(S),25-dihydroxy-30-oxo-lanost-8-ene.
Lyonicarposide C (3) was obtained as white amorphous powder, and
a molecular formula of C38H62O11 was deduced from the HRESIMS ion
at m/z 717.4248 [M + Na]+ (calcd for C38H62O11Na, 717.4190) and
13C NMR data. Comparison of the NMR data of lyonicarposide C (3)
(Tables 1 and 2) with those of lyonifoloside L (13) [16] revealed the
presence of an additional acetyl group (δH 2.09, s; δC 172.8, 20.9) in 3,
suggesting that 3 was the acetyl derivative of 13. The significant de-
shielding of H-4′ (δH 4.73, t, J = 9.3 Hz) and C-4′ (δC 73.2) in 3
compared to those (δH 3.28, t, J = 8.6 Hz, H-4′; δC 72.2) in 13 indicated
that 3 should be a 4′-acetyl derivative of 13, which was further sup-
ported by the HMBC correlation from H-4′ to the acetyl carbonyl carbon
To determine the absolute configuration of the glucose moiety,
lyonicarposide A (1) was hydrolyzed, and the trimethylsilylthiazolidine
derivatives of the hydrolysate of 1 and the standards, D- and L-glucoses,
were prepared. The GC retention time of the trimethylsilylthiazolidine
derivative of the hydrolysate of 1 (tR = 15.946 min) was almost same
as that of D-glucose (tR = 16.042 min), while different from that of L-
glucose (tR = 16.349 min), indicating the D-glucose in 1. The large
coupling constant of H-1′ (J = 7.8 Hz) revealed the β-glucopyranosyl
linkage in 1. Thus, the structure of 1 was defined as 3α-[(6′-O-acetyl-β-
D-glucopyranosyl)-oxy]-24(S),25-dihydroxy-30-oxo-lanost-8-ene.
Fig. 4. Mo2(OAc)4 induced ECD spectra of compounds 1a, 3b, and 5b in DMSO.
6

H. Zhang, et al.
BioorganicChemistry96(2020)103598
at δC 172.8 in 3. To determine the absolute configuration of C-24,
lyonicarposide C (3) was hydrolyzed by TsOH to get the aglycone 3a,
and then 3a was methylated with CH3I to afford the ester 3b [33]. The
positive Cotton effect at 314 nm in the Mo2(OAc)4–IECD spectrum of 3b
verified the S configuration of C-24 (Fig. 3). Similar to lyonicarposide A
(1), a comparison of the GC retention time of the trimethylsilylthiazo-
lidine derivative of the hydrolysate of 3 with those of the standards
established the D-absolute configuration of the glucose moiety in 3. The
β- glucopyranosyl linkage in 3 was assigned by the large coupling
constant (J = 7.8 Hz) of H-1′. Further analysis of the 2D NMR data
(Fig. 2 and Supporting Information, Fig. S1) suggested that the aglycone
of 3 was the same to that of 13. Thus, the structure of 3 was identified
as 3α-[(4′-O-acetyl-β-D-glucopyranosyl)-oxy]-24(S),25-dihydroxylanost-
8-en-30-oic acid.
the same to that of 5. Accordingly, the structure of 6 was defined as 3α-
[6′-O-acetyl-β-D-glucopyranosyl-oxy]-24(R),25-dihydroxylanost-8-en-
30-oic acid by NMR data and chemical method.
The molecular formula of lyonicarposide G (7) was established as
C
38H60O11 by the HRESIMS ion at m/z 715.3979 [M + Na]+ (calcd for
C38H60O11Na, 715.4033) and 13C NMR data. The NMR data (Tables 2
and 3) of lyonicarposide G (7) were similar to those of hebecarposide H
(16) [33], and the major difference was the presence of an additional
acetyl group (δH 2.10, s; δC 21.3, 172.8) in 7. In addition, C-3′ (δC 79.4)
in 7 shifted to the downfield while C-2′ (δC 73.6) and C-4′ (δC 70.2) in 7
shifted to the upfield compared to C-3′ (δC 78.3), C-2′ (δC 75.2), and C-
4′ (δC 72.2) in 16, respectively, suggesting the location of the acetyl
group at C-3′ in 7. This deduction was supported by the HMBC corre-
lation from H-3′ (δH 4.39, t, J = 9.4 Hz) to the acetyl carbonyl (δC
172.8). Detailed analysis of 2D NMR data demonstrated that the agly-
cone of 7 was the same to that of hebecarposide H (16) (Fig. 2 and
Supporting Information, Fig. S1). Hence, the structure of 7 was defined
as 3α-[3′-O-acetyl-β-D-glucopyranosyl)-oxy]-25-hydroxy-24-oxo-lanost-
8-en-30-oic acid with chemical method.
Lyonicarposide D (4) possessed a molecular formula of C41H68O13 as
established by the HRESIMS ion at m/z 791.4540 [M + Na]+ (calcd for
C
41H68O13Na, 791.4558) and 13C NMR data. Comparison of the 1H and
13C NMR data of 4 (Tables 1 and 2) with those of lyonifolosides O (10)
[16] suggested their similarities and the major difference was the pre-
sence of an additional rhamnopyranosyl unit (δH 4.18, br s, H-1′; 3.57,
overlap, H-2′; 3.59, overlap, H-3′; 3.54, overlap, H-4′; 3.56, overlap, H-
5′; 1.25, d, J = 6.3 Hz, H-6′;δC 102.2, C-1′; 72.9, C-2′; 73.4, C-3′; 74.4,
C-4′; 71.9, C-5′; 19.6, C-6′) in 4, replacing the glucopyranosyl unit in
10. The HMBC correlation from the anomeric proton H-1′ (δH 4.18, br s)
of the rhamnopyranosyl unit to C-3 (δC 82.3) suggested the location of
the rhamnopyranosyl unit at C-3. Same to lyonicarposide B (2), The L-
absolute configurations of the arabinose and rhamnose units in 4 were
determined by comparison of the GC retention times of the tri-
methylsilylthiazolidine derivatives of the hydrolysate of 4 with those of
the standards. Therefore, the structure of 4 was defined as 3α-[(α-L-
rhamnopyranosyl)-oxy]-24(S)-[(α-L-arabinopyranosyl)-oxy]-25-hydro-
xylanost-8-en-30-oic acid by 2D NMR analysis (Fig. 2 and Supporting
Information, Fig. S1) and chemical methods.
Lyonicarposide H (8) was determined to have a molecular formula
of C36H58O9 by the HRESIMS ion at m/z 657.3981 [M + Na]+ (calcd
for C36H58O9Na, 657.3979) and 13C NMR data. The NMR data (Tables 2
and 3) of lyonicarposide H (8) were similar to those of hebecarposide H
(16) [33], except for an additional aldehyde (δH 9.47, s, H-30; δC 200.8,
C-30) in 8, replacing the carboxyl group (δC 181.2, C-30) in 16, which
was supported by the HMBC correlations from H-30 (δH 9.47, s) to C-14
(δC 68.5) and C-15 (δC 29.2) and from H2-15 (δH 2.11, m; 1.71, m) to C-
30. Finally, the structure of 8 was determined as 3α-[(β-D-glucopyr-
anosyl)-oxy]-25-hydroxy-24,30-dioxo-lanost-8-ene by 2D NMR data
analysis (Fig. 2 and Supporting Information, Fig. S1) and chemical
methods.
In a previous phytochemical study, 11 lanostane triterpene glyco-
sides were isolated from the leaves of L. ovalifolia var. hebecarpa [33]. In
the current study, 16 lanostane triterpene glycosides including eight
new ones were isolated from the flowers of L. ovalifolia var. hebecarpa.
In Ericaceae family, lanostane-type triterpenoids were only found in the
plants of Lyonia genera [16,33]. Therefore, lanostane triterpene gly-
cosides could be considered as a chemotaxonomic marker for the Lyonia
plants in the chemical classification of Ericaceae. Interestingly, seven
lanostane triterpene glycosides from the flowers of L. ovalifolia var.
hebecarpa were acetylated in the glucose units, which are different from
the leaves parts and could be the characteristic constituents of the
flowers of L. ovalifolia var. hebecarpa.
Lyonicarposide E (5) was found to possess the same molecular for-
mula as lyonicarposide C (3) by the HRESIMS peak at m/z 717.4128
[M + Na]+ (calcd for C38H62O11Na, 717.4190) and 13C NMR data. The
NMR data (Tables 2 and 3) of 5 also resemble to those of 3, and the
major differences were the deshielding of H-24 (δH 3.22, dd, J = 10.3,
1.8 Hz) and the shielding of C-24 (δC 79.9) in 5 compared to those (δH
3.14, dd, J = 10.3, 1.8 Hz, H-24; δC 80.6, C-24) in 3. Thus, lyoni-
carposide E (5) should be the 24-epimer of lyonicarposide C (3) [33].
To determine the absolute configuration of C-24, lyonicarposide E (5)
was hydrolyzed, and the resulted aglycone 5a was methylated to afford
the ester 5b using the same method as 3. The Mo2(OAc)4-IECD spec-
trum of 5b displayed a negative Cotton effect at 310 nm (Fig. 3), instead
of a positive Cotton effect in 3b, proving the R configuration of C-24.
The absolute configuration of the glucose unit in 5 was determined to
be D by comparison of the GC retention times of the trimethylsi-
lylthiazolidine derivative of the hydrolysate of 5 with those of the
standards, D and L-glucoses, and the β-glucopyranosyl linkage in 5 was
deduced from the large coupling constant (J = 7.8 Hz) of H-1′. Thus,
the structure of 5 was established as 3α-[4′-O-acetyl-β-D-glucopyr-
anosyl)-oxy]-24(R),25-dihydroxylanost-8-en-30-oic acid by 2D NMR
analysis (Fig. 2 and Supporting Information, Fig. S1) and chemical
methods.
3.2. Antiproliferative activities in vitro
All the isolated 16 lanostane-type triterpene glycosides (1–16) were
evaluated for their antiproliferative activities against five cancer cell
lines (SMMC-7721, HL-60, SW480, MCF-7, and A-549) by the MTT
method as described [32,33]. As shown in Table 4, lyonicarposides A
(1) and B (2) with an aldehyde group at C-30 exhibited moderate an-
tiproliferative activities against all the five cancer cell lines with IC50
values ranging from 12.3 to 28.7 μM. In contrast, lyonifoloside P (9)
and hebecarposide A (14) without the 30-aldehyde group did not ex-
hibit significant activity (IC50 > 40 μM), suggesting that the 30-alde-
hyde group is an important group to enhance the antiproliferative ac-
tivity. Lyonicarposide C (3) and lyonifoloside M (12) with glycosidation
at C-3 and a 24α-OH group selectively inhibited the proliferation of HL-
60 and MCF-47 cell lines with IC50 values ranging from 13.0 to 17.7 μM.
However, lyonicarposides E (5) and F (6) with glycosidation at C-3 but
without 24α-OH did not exhibit significant antiproliferative activity,
suggesting that the 24α-OH group may enhance the antiproliferative
activity. This may explain why lyonifolosides P (9) and J (10) without
24α-OH did not show significant antiproliferative activity. Interest-
ingly, lyonicarposide D (4) and lyonifolosides P (9), J (10), and R (11),
did not exhibited significant antiproliferative activity, suggesting that
Lyonicarposide F (6) possessed the same molecular formula as
lyonicarposide E (5), as determined by the HRESIMS ion at m/z
717.4182 [M + Na]+ (calcd for C38H62O11Na, 717.4190) and 13C NMR
data. The NMR data of 6 (Tables 2 and 3) were similar to 5, and the
major differences were the deshielding of H-6′a (δH 4.37, dd, J = 11.8,
2.6 Hz), H-6′b (δH 4.23, dd, J = 11.8, 6.3 Hz), and C-6′ (δC 65.0) in 6
compared to those (δH 3.86, dd, J = 11.8, 2.6 Hz, H-6′a; 3.66, overlap,
H-6′b; δC 62.9) in 5, indicating that the acetyl group was located at C-6′
in 6. This deduction was supported by the HMBC correlation from H2-6′
to acetyl carbonyl (δC 172.9). Further analysis of 2D NMR data (Fig. 2
and Supporting Information, Fig. S1) indicated the other parts of 6 to be
7

H. Zhang, et al.
BioorganicChemistry96(2020)103598
Table 4
Anti-proliferative activities of compounds 1–16 against five cancer cell lines (IC50 in μM).a,b
Compounds
HL-60
A-549
SMMC-7721
MCF-7
SW480
1
15.53
15.19
13.03
14.88
17.71
> 40
2.47
0.38
0.57
0.27
0.84
0.44
12.39
20.35
> 40
> 40
> 40
> 40
13.84
0.65
0.46
12.92
20.43
> 40
> 40
> 40
> 40
7.82
0.30
0.56
13.80
17.55
17.53
13.84
13.71
16.27
13.46
0.96
0.22
1.10
0.62
0.48
1.45
0.49
17.71
28.71
> 40
> 40
> 40
> 40
10.06
0.22
1.34
2
3
7
12
13
cis-platinc
0.12
0.47
0.62
0.30
a
b
c
Other compounds: IC50 > 40 μM.
Results were expressed as means
Positive control.
SD (n = 3).
diglycosidation of lanost-8-ene type triterpenoids may decrease the
antiproliferative activity. Furthermore, Lyonicarposide G (7) with an
acetyl group selectively inhibited the proliferation of HL-60 and A-549
cell lines with IC50 values of 14.9 and 13.8 μM, respectively, and
showed more potent antiproliferative activity than hebecarposide H
(16), suggesting the acetyl group in the glucose unit may enhance the
antiproliferative activities of the lanostane triterpene glycosides. This
deduction was supported by that lyonicarposide C (3) and lyonifoloside
M (12) with an acetyl group exhibited more potent antiproliferative
activities than lyonifoloside L (13) without an acetyl group. This pre-
liminary structure-activity-relationships analysis provided a basis to
optimize the lanost-8-ene type triterpene glycosides as antiproliferative
agents.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103598.
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