Oxygenated pentacyclic triterpenoids from the stems and branches of Enkianthus chinensis

DOI: 10.1016/j.bioorg.2021.104866

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Bioorganic Chemistry (2021)

Update date:2022-08-17

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Authors:

Wang, Hai-Qiang

Ma, Shuang-Gang

Zhang, Dan

Li, Yu-Huan

Qu, Jing

Li, Yong

Liu, Yun-Bao

Yu, Shi-Shan

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Article abstract of DOI:10.1016/j.bioorg.2021.104866

Thirty new pentacyclic triterpenoids, including five oleanane-type (1–5), twenty-three ursane-type (9–23, 26–33) and two taraxerane-type (24 and 25), along with fourteen known triterpenoids, were isolated from the stems and branches of Enkianthus chinensis. Their structures were elucidated by extensive spectroscopic analyses, X-ray crystallographic data and electronic circular dichroism (ECD) techniques. Sixteen compounds (1–5, 9–13, 20, 22, 32, 34–36) bearing a gem-hydroxymethyl group at C-4 represent rare examples of pentacyclic triterpenoids. In the in vitro biological activity evaluation, compounds 8, 9, 12–14, 17, 24, and 44 exhibited potent hepatoprotective effects at 10 μM. Moreover, compound 25 showed latent activity against HSV-1 with an IC₅₀ value of 6.4 μM.

Full text of DOI:10.1016/j.bioorg.2021.104866

Bioorganic Chemistry 111 (2021) 104866

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Oxygenated pentacyclic triterpenoids from the stems and branches of

Enkianthus chinensis

Hai-Qiang Wang , Shuang-Gang Ma , Dan Zhang , Yu-Huan Li , Jing Qu , Yong Li ,

a,*

Yun-Bao Liu , Shi-Shan Yu

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union

Medical College, Beijing 100050, People’s Republic of China

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China

A R T I C L E I N F O

A B S T R A C T

Keywords:

Thirty new pentacyclic triterpenoids, including ﬁve oleanane-type (1–5), twenty-three ursane-type (9–23,

Enkianthus chinensis

Triterpenoids

6–33) and two taraxerane-type (24 and 25), along with fourteen known triterpenoids, were isolated from the

stems and branches of Enkianthus chinensis. Their structures were elucidated by extensive spectroscopic analyses,

X-ray crystallographic data and electronic circular dichroism (ECD) techniques. Sixteen compounds (1–5, 9–13,

Hepatoprotective activity

Antiviral activity

0, 22, 32, 34–36) bearing a gem-hydroxymethyl group at C-4 represent rare examples of pentacyclic tri-

terpenoids. In the in vitro biological activity evaluation, compounds 8, 9, 12–14, 17, 24, and 44 exhibited potent

hepatoprotective effects at 10

value of 6.4 M.

M. Moreover, compound 25 showed latent activity against HSV-1 with an IC50

Introduction

Triterpenoids, comprising of 30-carbon compounds originated from

hydroxymethyl group at C-4. Furthermore, the hepatoprotective effects

and antiviral activities of the isolated triterpenoids were evaluated in

vitro.

the C5 isoprene units, represent a large and structurally diverse class of

natural products. Hundreds of different triterpene backbones have been

found in plants, animals and microorganisms [1]. Due to a wide range of

pharmacological activities, including anticancer [2], anti-inﬂammatory

2. Material and methods

2.1. Plant material

[

3], antidiabetic [4], immunomodulatory [5], antiviral [6], and anti-

microbial properties [7], the continuing investigation on novel plant

triterpenoids is valuable and signiﬁcant for discovering drug candidates.

Enkianthus chinensis Franch., a toxic shrub of the family Ericaceae, is

distributed mainly in the southern and southwestern parts of the Peo-

ple’s Republic of China. Extract of this plant has been used as a folk

medicine for the treatment of phlebitis [8]. Phytochemical investigation

on this plant by our group has preliminarily resulted in the isolation and

identiﬁcation of several hydroxylated ethacrylic acid and tiglic acid

derivatives, of which some derivatives have exhibited signiﬁcant anti-

inﬂammatory activity [9]. As part of our continued study on the

chemical composition and biological activities, we report herein the

isolation and structural elucidation of triterpenoids from E. chinensis.

Thirty new oxygenated pentacyclic triterpenoids were obtained (Fig. 1),

including sixteen rare examples of pentacyclic triterpenoids with a gem-

The stems and branches of E. chinensis grown in the county of

Wuning, Jiangxi Province were collected and identiﬁed by Prof. Lin Ma

(the Chinese Academy of Medical Sciences and Peking Union Medical

College) in October 2017. A voucher specimen of E. chinensis (accession

number 2017004) was deposited in the herbarium of our institute.

.2. General experimental procedures

UV and ECD spectra were recorded on a JASCO V-650 spectropho-

tometer and a JASCO J-815 spectropolarimeter (JASCO, Japan),

respectively. IR were performed on a Nicolet 5700 FT-IR instrument

(

Thermo Electron Corporation, USA). Optical rotations were detected on

Rudolph Autopol automatic polarimeter (Rudolph Research

Analytical, USA). HRESIMS data were obtained on a Thermo Scientiﬁc

Corresponding author.

E-mail address: yushishan@imm.ac.cn (S.-S. Yu).

https://doi.org/10.1016/j.bioorg.2021.104866

Received 25 January 2021; Received in revised form 20 March 2021; Accepted 22 March 2021

Available online 26 March 2021

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Fig. 1. Structures of compounds 1–44.

Q-Exactive Focus LC/MS spectrometer (Thermo Fisher Scientiﬁc, USA).

Haiyang Chemical Co., Ltd., P.R. China), Sephadex LH-20 (GE Health-

H, C and 2D NMR spectra were obtained on Bruker Avance III 500 and

00 MHz spectrometers (Bruker, Germany). X-ray crystallographic data

care Bio-Sciences Corp., USA), ODS C18 (50

μm, Merck, Germany),

macroporous resin (D101 type, Chemical Plant of Nankai University, P.

R. China), and polyamide resin (30–60 mesh, Jiangsu Linjiang Chemical

Reagents Factory, P.R. China).

were obtained using a XtaLAB Synergy R, HyPix single-crystal X-ray

diffractometer (Rigaku, Japan). Semi-preparative HPLC was carried out

on a Shimadzu LC-6AD apparatus with an SPD-20A detector using a

SiGreen Pack C18 column (250 mm × 10 mm, 5

m; Greenherbs Science

.3. Extraction and isolation

and Technology Development Co., Ltd., P.R. China) or a YMC-Pack ODS-

A column (250 × 10 mm, 5 µm; YMC Co., Ltd., Japan). Column chro-

matography was performed using silica gel (200–300 mesh, Qingdao

Dried and powdered plant material (54.0 kg) were extracted three

times (each 3 h) with 95% EtOH under reﬂux conditions. The resulting

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Table 1

H NMR (600 MHz) Data of compounds 1–5, 9–15 (δ in ppm, J in Hz).

No.

1.65,

1.92, overlap

1.43, overlap

1.77, overlap

1.48, m

1.93,

1.85,

1.84, m

1.54, m

1.86,

1.91, overlap

1.39, overlap

1.42,

overlap

1.43,

overlap

1.40,

overlap

1.53, m

overlap

1.47, m

overlap

1.53, m

overlap

1.48, m

overlap

1.29,

.46,

overlap

1.96, m

overlap

2.60, m

2.30, m

overlap

2.08,

overlap

1.98, m

1.44,

2.62, m

2.73, m

2.72, m

2.71, m

2.58, m

2.08,

2.71, m

2.59, m

2.08,

2.68, m

.61,

2.32, ddd

(16.6,6.1,2.5)

2.52, ddd

(16.5,6.2,2.8)

overlap

1.85,

2.56, ddd

(16.7,5.2,2.5)

overlap

1.84,

overlap

1.85,

2.23, ddd

overlap

(16.4,11.6,3.2)

overlap

4.74,

overlap

4.71,

overlap

4.71,

4.05, dd

3.58,

brd

(

2.8,

.8)

brs

brd

(2.7)

(3.0)

(3.1)

1.75,

overlap

1.76,

1.95, overlap

2.44, dd

1.95,

2.26,

2.46, m

2.43, m

2.19,

2.44,

2.20,

1.91, overlap

4.34, brs

overlap

(12.4, 2.6)

overlap

d (12.0)

overlap

1.54,

1.61, overlap

1.56, overlap

1.90, m

1.59,

1.85,

1.91, m

1.88,

1.81,

1.86,

1.82,

4.35,

brs

overlap

1.77, overlap

overlap

1.59,

overlap

1.66,

1.76, overlap

overlap

1.69,

overlap

1.58, m

overlap

1.69,

overlap

1.58, m

.48,

overlap

1.52,

overlap

1.59,

overlap

1.73,

overlap

1.68,

overlap

1.68,

1.74, overlap

1.59, overlap

1.68, m

1.41, m

1.76, overlap

1.47, m

1.73,

1.85, m

1.84,

overlap

1.35, m

overlap

1.41,

overlap

1.45, m

overlap

1.43, m

overlap

1.45, m

overlap

1.43, m

1.57, overlap

overlap

1.50,

.45,

overlap

2.19,

overlap

1.91,

2.31, brs

1.77, overlap

2.00, overlap

2.05, overlap

1.77,

2.06, overlap

2.09, overlap

1.72,

1.85,

1.71,

1.83,

1.91, overlap

2.12, overlap

overlap

1.99,

overlap

2.14,

overlap

1.98, m

overlap

2.04,

overlap

1.98,

overlap

2.07,

overlap

2.05, m

5.85, dd

(

10.2,

.2)

overlap

2.07,

overlap

1.97,

overlap

2.09,

overlap

1.96,

overlap

5.60,

overlap

5.73, dd

(3.7,

overlap

5.75, dd

(3.7,

overlap

5.73,

6.25, dd

10.2,

.0)

5.28, dd (3.7,

3.7)

5.59, dd (3.1,

3.1)

5.29, brs

5.65, dd (2.9,

2.9)

5.74, dd

(3.7,

5.35, dd (3.7,

3.7)

5.34, dd

(3.7,

(

brs

overlap

3.7)

1.60,

1.79, overlap

1.11, overlap

2.14, overlap

1.25, overlap

1.79,

2.13,

2.30, overlap

1.32, m

2.26, m

1.26, m

2.25, m

1.25,

2.25,

2.23,

1.90, overlap

1.02, m

1.89,

overlap

1.11,

overlap

1.24,

overlap

1.25,

overlap

1.25, m

overlap

1.01, m

.32,

overlap

2.24, m

overlap

2.03,

overlap

2.82, m

2.13,

overlap

2.09,

2.00, overlap

1.78, overlap

2.82, m

3.23, m

2.15,

2.13,

2.07,

2.57, m

2.58,m

1.51,

.61,

2.14, overlap

overlap

1.61,

2.16, overlap

overlap

2.10,

overlap

2.08,

overlap

2.03,

1.52, overlap

overlap

2.88, dd

(13.6,

overlap

3.71, s

overlap

3.70, s

overlap

3.68, s

2.87, dd

3.64, brs

3.64,

brs

3.26, s

3.69, s

2.54, s

2.52, s

(

13.9, 4.0)

.8)

4.84,

1.70, overlap

1.14, overlap

1.81,

3.62,

brs

overlap

.10,

overlap

1.36, overlap

1.36,

overlap

1.74,

1.52,

1.36, overlap

1.21, overlap

2.14, overlap

1.17, overlap

1.49,

2.17,

3.17, m

2.59, m

2.59,

2.44,

2.44, m

2.22,

1.72, overlap

1.26, overlap

overlap

1.15,

overlap

1.16,

2.30, overlap

d (8.8)

overlap

2.33,

overlap

1.25,

.45,

overlap

1.76,

overlap

1.77,

overlap

2.21,

overlap

2.10,

overlap

1.73,

1.59, overlap

1.37, overlap

2.21, overlap

2.05, overlap

2.34, overlap

2.14, overlap

2.15,

2.14,

2.09,

1.73, overlap

1.64, overlap

overlap

1.60,

overlap

2.04,

overlap

2.06,

overlap

2.05,

overlap

2.01,

overlap

2.01,

overlap

1.64,

.70,

overlap

3.89,

overlap

4.08,

overlap

4.58,

overlap

4.69,

overlap

4.55,

overlap

4.70,

overlap

4.57,

overlap

3.69,

4.08, d (11.1)

3.44, d (11.1)

4.75, d (10.8)

3.98, d (10.8)

4.71, d (10.7)

4.00, d (10.7)

3.82, d (11.0)

3.43, d (11.0)

d (11.2)

d (11.1)

3.43,

d (11.0)

4.37,

d (10.7)

3.99,

d (11.5)

4.36,

d (10.6)

3.99,

d (11.0)

4.37,

d (11.0)

3.34,

.72,

d (11.2)

3.66,

d (11.1)

3.89,

d (11.0)

4.16,

d (10.7)

4.27,

d (11.5)

4.15,

d (10.6)

4.27,

d (11.0)

4.15,

d (11.0)

1.10, s

3.90, d (11.7)

3.57, d (11.7)

4.28, d (11.2)

4.07, d (11.2)

4.28, d (11.2)

4.09, d (11.2)

1.25, s

d (11.5)

d (11.7)

3.57,

d (11.0)

4.04,

d (11.2)

4.09,

d (10.9)

4.02,

d (11.3)

4.08,

d (11.0)

4.02,

.59,

d (11.5)

1.00, s

0.81, s

1.09, s

0.97, s

d (11.7)

1.14, s

0.89, s

1.21, s

3.19, s

0.94, s

d (11.0)

1.04, s

1.11, s

1.61, s

1.20, s

1.13, s

d (11.2)

1.16, s

1.10, s

1.12, s

1.86, s

4.58,

d (10.9)

1.03, s

1.07, s

1.08, s

1.83, s

4.57,

d (11.3)

1.15, s

1.09, s

1.08, s

1.80, s

4.02,

d (11.0)

1.03, s

1.06, s

1.05, s

1.78, s

4.02,

1.14, s

0.88, s

1.19, s

0.91, s

0.95, s

1.18, s

1.13, s

1.61, s

1.21, s

1.13, s

1.17, s

1.18, s

1.73, s

1.67, s

5.03, brs

1.47, s

1.35, s

1.09, s

1.35, s

1.21, s

0.94,

1.18, s

1.35, s

1.20, s

0.93, d (6.7)

.83, brs

d (12.0)

d (11.5)

4.35,

d (11.0)

3.98,

d (11.2)

3.96,

d (6.6)

.37,

d (12.0)

d (11.5)

d (11.0)

3.31, s

d (11.2)

3.31, s

OMe

Recorded in CD OD.

Recorded in Pyridine‑d .

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Table 2

H NMR (600 MHz) Data of compounds16 ꢀ 25 (δ in ppm, J in Hz).

a a b b a

No.

1.40,

1.44,

1.95,

1.87,

2.17,

1.53,

1.90,

1.75,

1.79, overlap

1.70, overlap

overlap

1.28,

overlap

1.42,

overlap

1.42,

overlap

1.47,

overlap

1.45,

overlap

1.54,

overlap

1.19,

.31,

overlap

2.01,

overlap

2.01,

overlap

2.20,

overlap

2.88, m

2.40, m

overlap

2.68, m

2.38, m

overlap

2.08,

overlap

2.75, m

2.60, m

overlap

1.89,

2.76, ddd

2.80, ddd

overlap

1.52,

overlap

1.83,

overlap

1.59,

overlap

1.46,

(15.9,11.7,5.6)

2.23, ddd

(16.0,11.6,5.7)

2.40, ddd

.51,

overlap

3.68, dd

(2.9,2.9)

2.07,

overlap

4.17, brs

overlap

3.67, m

overlap

3.94, dd

(11.8, 5.6)

1.61,

(14.4,9.8,4.6)

(15.4,9.9,4.6)

3.61, dd

(

2.9,2.9)

1.52,

2.75, brs

5.10, brs

2.57, brs

4.42, brs

2.01, m

2.07,

2.43,

1.71, overlap

1.88, d (11.0)

overlap

4.20, brs

overlap

4.35 brs

overlap

4.42, brs

overlap

1.91,

overlap

1.55,

1.65,

3.92, ddd (11.1,

11.1, 4.2)

4.24, ddd (10.9,

10.9, 4.1)

overlap

1.80,

overlap

1.46,

.65,

overlap

1.50,

overlap

1.62,

overlap

1.61,

1.78,

1.92,

2.30,

2.04,

1.70,

2.33, dd (12.4, 4.2)

1.44, dd (12.4,

11.9)

2.62, dd (12.2, 4.1)

1.77, dd (12.4,

11.9)

overlap

1.47,

overlap

1.95,

overlap

1.87,

overlap

1.40,

overlap

1.53,

overlap

1.57,

overlap

1.35,

.52,

overlap

1.91, m

overlap

1.99,

overlap

2.34,

overlap

2.15,

overlap

2.17,

overlap

2.31,

overlap

1.84,

overlap

1.86,

1.61, overlap

1.62, m

overlap

2.05,

overlap

2.34,

overlap

2.31,

overlap

5.68, dd

(10.2, 2.0)

overlap

5.71, dd

(10.2, 2.3)

overlap

2.15,

overlap

2.12, m

2.00, m

2.05,

1.79, overlap

1.66, overlap

1.70, overlap

1.55, overlap

overlap

2.17,

overlap

2.05,

.27,

overlap

5.70, dd

(4.5, 4.5)

2.65, m

1.40,

overlap

5.71, dd

(3.6, 3.6)

2.48, m

1.32,

overlap

5.53,

5.32, dd

5.33, dd

(3.7, 3.7)

1.89,

6.02, dd

(10.2, 3.1)

1.82, m

5.98, dd

(10.2, 2.7)

1.88,

5.31, dd

(3.7, 3.7)

1.83,

1.83, overlap

1.70, overlap

(

3.7, 3.7)

overlap

2.15,

1.66, overlap

1.54, overlap

1.89,

5.71, dd (8.2, 3.1)

5.72, dd (8.1, 3.2)

overlap

1.01, m

1.18, m

overlap

1.17,

overlap

0.85,

overlap

1.04, m

.00, m

overlap

2.17,

overlap

2.40,

2.57, m

.52,

2.58, m

1.52,

2.98, m

2.74,

2.04,

2.17,

2.59,

2.27, overlap

1.80, overlap

2.19, m

overlap

1.39,

overlap

1.48,

overlap

1.47,

overlap

1.54,

1.70, overlap

overlap

3.12, s

overlap

5.42, s

overlap

5.40, s

overlap

2.52, s

3.11, s

1.36, dd (13.6, 4.2)

2.46, t (13.6)

1.28, dd (13.8, 4.0)

1.96, d (13.7)

.40, dd (13.6, 4.2)

1.47, dd (13.7, 4.0)

1.36,

1.72,

1.53,

2.55, m

2.55,

1.36,

overlap

1.73,

overlap

1.73,

overlap

2.46, m

1.90,

overlap

2.13,

overlap

1.69,

overlap

1.74,

1.69,

7.01,

overlap

1.25,

overlap

1.42,

overlap

1.32,

overlap

1.32,

d (7.4)

overlap

1.25,

.25,

overlap

1.73,

overlap

1.75,

overlap

2.15,

overlap

1.68,

overlap

1.69,

overlap

1.74,

4.44, dd

6.91,

2.70, d (12.9)

1.94, d (12.9)

2.77, d (13.0)

1.99, d (13.0)

overlap

1.63, m

(11.6, 4.4)

overlap

1.41,

overlap

1.40,

d (7.4)

overlap

1.63,

.63,

overlap

4.09,

overlap

4.11,

overlap

5.05,

4.70,

1.32, s

1.34, s

1.70, s

1.78, s

d (10.2)

d (11.1)

3.48,

d (10.8)

3.98,

d (2.0)

4.64,

.00,

d (10.2)

1.30, s

d (11.1)

3.88,

d (10.8)

4.28,

d (2.0)

1.54, s

2.00, s

2.13, s

1.51, s

d (11.6)

d (11.3)

4.09,

.59,

d (11.6)

1.11, s

0.79, s

0.99, s

d (11.3)

1.18, s

1.03, s

0.83, s

1.36, s

1.08, s

1.34, s

1.38, s

1.11, s

1.39, s

1.73, s

1.78, s

1.68, s

1.73, s

1.74, s

1.75, s

1.34, s

1.06, s

1.00, s

0.77, s

0.86, s

1.38, s

0.88, s

0.94, s

1.18, s

1.10, s

0.85, s

1.19, s

1.16, s

0.83, s

1.21, s

1.44, s

1.49, s

2.14, s

2.27, s

2.22, s

1.21, s

3.65, dd (10.4)

.28, dd (10.4)

0.93, d (6.6)

2.06, s

0.93, d (6.7)

3.66 s

1.18,

1.14,

1.08, d (7.0)

1.08,

0.94,

0.95, s

1.20, s

d (6.6)

3.61, s

d (6.6)

3.71, s

d (7.3)

3.67, s

d (6.7)

OMe/

OAc

Recorded in CD

OD.

Recorded in Pyridine‑d

extract (2.6 kg) was mixed with kieselguhr and loaded on a Soxhlet

extractor, and then extracted successively with petroleum ether (PE, 10

a silica gel column eluting with CH

-MeOH (100:1 to 1:1 v/v).

2 2

Fraction S2 (21.4 g) was submitted to a silica gel column using CH Cl -

L), CH

(10 L), EtOAc (10 L), and MeOH (10 L). Six fractions (S1 ꢀ

Cl fraction (150 g) by

acetone (20:1 to 1:1 v/v) as eluent to generate fractions S2-1 ꢀ S2-7.

S6) were obtained from the puriﬁcation of the CH

Subsequently, further puriﬁcation of fraction S2-2 (0.7 g) yielded 13

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Table 3

H NMR Data of compounds 26–33 in CD

OD (δ in ppm, J in Hz).

No.

1.63, overlap

1.93, overlap

1.27, overlap

2.21, overlap

1.76, overlap

1.84, overlap

1.37, overlap

2.69, m

1.42, overlap

1.30, overlap

1.98, overlap

1.46, overlap

3.58, dd (5.3,

1.43, overlap

1.28, overlap

2.01, overlap

1.53, overlap

3.68, m

1.97, overlap

1.41, overlap

2.80, m

1.94, overlap

1.44, overlap

2.62, overlap

2.28, m

1.68, overlap

1.08, m

.95, overlap

1.64, overlap

.64, overlap

1.72, overlap

1.61, overlap

3.90, dd

2.22, m

2.23, m

3.81, m

.0)

(11.9,4.2)

2.07, overlap

1.24, overlap

1.66, overlap

2.83, m

1.09, overlap

4.12, brs

1.90, overlap

4.33, brs

1.73, overlap

4.34, brs

2.05, overlap

4.34, brs

2.17, overlap

3.88, brs

1.96, overlap

1.57, overlap

1.48, overlap

3.83, brs

.26, overlap

1.65, overlap

.31, overlap

1.71, overlap

1.60, overlap

1.79, overlap

2.14, overlap

1.91, overlap

1.57, overlap

2.14, overlap

2.12, overlap

1.83, overlap

1.50, overlap

1.89, overlap

2.01, overlap

1.92, overlap

1.47, overlap

1.97, overlap

2.05, overlap

1.73, overlap

1.51, overlap

1.89, overlap

2.17, overlap

1.65, overlap

1.36, overlap

2.06, overlap

2.04, overlap

1.64, overlap

1.45, m

1.67, overlap

1.97, overlap

1.77, overlap

2.05, overlap

.12, overlap

5.30, dd (3.7, 3.7)

1.83, overlap

5.33, dd (3.7,

5.37, dd (3.7,

3.7)

5.35, dd (3.7,

3.7)

5.35, dd (3.7,

3.7)

5.37, dd (3.7,

3.7)

5.32, dd (3.7,

3.7)

5.34, dd (3.8,

3.8)

.7)

1.93, overlap

1.00, m

1.92, overlap

1.03, m

1.89, overlap

1.01, m

1.89, overlap

1.02, m

1.89, overlap

1.01, m

1.85, overlap

1.03, m

1.86, overlap

0.98, m

.00, m

2.60, m

.64, overlap

2.59, m

2.61, m

2.60, m

2.61, overlap

1.64, overlap

2.53, s

2.60, m

1.63, overlap

2.52, s

1.65, overlap

2.55, s

1.64, overlap

2.54, s

1.64, overlap

2.53, s

1.64, overlap

2.55, s

1.63, overlap

2.53, s

2.51, s

1.34, overlap

1.72, overlap

1.33, overlap

1.70, overlap

1.21, overlap

1.71, overlap

1.60, overlap

1.00, d (6.6)

1.37, overlap

1.65, overlap

1.24, overlap

1.77, overlap

1.64, overlap

3.82, d (11.4)

1.36, overlap

1.73, overlap

1.25, overlap

1.79, overlap

1.63, overlap

3.69, d (11.5)

3.43, d (11.5)

1.10, s

1.36, overlap

1.74, overlap

1.26, overlap

1.80, overlap

1.64, overlap

1.36, overlap

1.73, overlap

1.25, overlap

1.80, overlap

1.64, overlap

1.35, overlap

1.73, overlap

1.24, overlap

1.80, overlap

1.62, overlap

4.11, d (11.1)

3.43, d (11.1)

3.92, d (11.6)

3.55, d (11.6)

1.15, s

1.36, overlap

1.71, overlap

1.23, m

.25, overlap

1.79, overlap

.62, overlap

1.78, overlap

1.63, overlap

.42, d (11.4)

3.92,dd (10.9,9.3)

.56,dd (10.9,2.2)

1.24, s

1.53, s

1.69, s

1.48, s

0.79, s

1.41, s

1.48, s

1.34, s

1.38, s

1.54, s

1.33, s

0.77, s

1.10, s

1.14, s

1.05, s

1.08, s

1.12, s

0.84, s

1.04, s

1.34, s

1.29, s

1.34, s

1.33, s

1.37, s

1.33, s

1.36, s

1.31, s

1.20, s

1.18, s

1.22, s

1.21, s

1.20, s

1.21, s

0.92, d (6.6)

0.90, d (6.6)

0.93, d (6.6)

0.94, d (6.7)

3.66, s

0.93, d (6.7)

3.69, s

0.93, d (6.7)

3.68, s

OMe

5.32, d (8.2)

5.30, d (8.2)

3.31, overlap

3.37, overlap

3.34, overlap

3.31, overlap

3.77, dd (12.0,

2.3)

5.32, d (8.2)

3.34, overlap

3.40, overlap

3.37, overlap

3.34, overlap

3.81, dd (12.2,

2.3)

5.31, d (8.2)

3.33, overlap

3.40, overlap

3.37, overlap

3.33, overlap

3.81, dd (12.0,

2.3)

5.32, d (8.1)

3.33, overlap

3.40, overlap

3.36, overlap

3.33, overlap

3.79, dd (12.1,

2.2)

5.32, d (8.1)

3.33, overlap

3.40, overlap

3.37, overlap

3.34, overlap

3.80, dd (12.0,

2.2)

5.32, d (8.1)

3.31, overlap

3.37, overlap

3.31, overlap

3.80, dd (12.0,

2.2)

5.30, d (8.1)

3.32, overlap

3.40, overlap

3.36, overlap

3.33, overlap

3.79, dd (12.0,

2.3)

3.32, overlap

3.39, overlap

3.35, overlap

3.32, overlap

3.79, dd (12.0, 2.3) 3.68, dd

(

12.0, 4.8)

.66, dd (12.0,

.6)

3.69, dd (12.2,

4.6)

3.68, dd (12.0,

4.4)

3.69, m

3.68, m

3.68, dd (12.0,

4.6)

3.69, m

Recorded at 600 MHz.

Recorded at 500 MHz.

fractions (S2-2–1 ꢀ S2-2–13) by ODS-MPLC eluting with MeOH-H

2 2

Sephadex LH-20 column with PE-CH Cl -MeOH (5:5:1 v/v/v) as eluent

gradient system (60%, 70%, 80%, 90%, and 100%). The main fraction

S2-2–6 (50 mg) was separated on a semi-preparative RP-HPLC column

with 80% MeOH as mobile phase (2 mL/min) to give compounds 23

to produce seven fractions (S3-1-S3-7) and ﬁve fractions (S4-1-S4-5),

respectively. Fractions S3-6-1-S3-6-6 were obtained from the puriﬁca-

tion of the fraction S3-6 (1.3 g) using an OSD-MPLC with a gradient

(

3.1 mg, t

36 min) and 41 (16.0 mg, t

–9 (50 mg) via semi-preparative RP-HPLC with 90% MeOH at 2 mL/

32 min) and 40 (2.0 mg, t

44 min). Compounds 7 (12.0 mg,

elution of MeOH-H

Fraction S3-6–5 (45 mg) was then separated by semi-preparative RP-

HPLC with 65% MeOH-H O to give compounds 9 (3.2 mg, t 34 min)

and 14 (1.2 mg, t 55 min). Eight fractions (S4-4-1-S4-4-8) was obtained

from fraction S4-4 (3 g) using a silica gel column with a gradient elution

of CH Cl

-MeOH (100:1 to 1:1 v/v). Then, fraction S4-4–5 yielded

compounds 20 (2.3 mg, t 45 min) and 3 (2.8 mg, t 48 min) as well as

10 (1.6 mg, t 51 min) using semi-preparative HPLC with MeOH-H O-

TFA (67:33:0.01, 2 mL/min).

The EtOAc-soluble extract (85.0 g) obtained above was applied to a

polyamide resin column using a successive elution of H O, 30% EtOH,

O system (30%, 40%, 50%, 60%, 70%, and 100%).

38 min) were isolated from fraction S2-

min. Fraction S2-3 (1 g) was loaded onto a Sephadex LH-20 column

eluting with PE-CH Cl -MeOH (5:5:1 v/v/v) to generate six fractions

S2-3–1 ꢀ S2-3–6). Fraction S2-3–4 (170 mg) was subjected to an ODS-

MPLC eluting with MeOH-H O gradient system (30%, 50%, 70%, 90%,

and 100%). Then, compounds 19 (3.1 mg, t 30 min) and 24 (17.3 mg, t

6 min) were puriﬁed from 70% MeOH fraction by semi-preparative RP-

(

HPLC with 70% MeOH as mobile phase. Fraction S2-6 (5.7 g) was

separated on an ODS-MPLC (eluted successively with 50%, 60%, 70%,

0%, 90%, 95%, and 100% MeOH-H

O) to give 14 fractions (S2-6-1-S2-

60% EtOH, and 95% EtOH. The 30% EtOH fraction extract (13 g) was

loaded on a Sephadex LH-20 column to produce fractions (E1ꢀ E9) using

90% MeOH as eluent. The 60% EtOH fraction (18 g) was chromato-

-14). Fraction S2-6–6 was further puriﬁed by a Sephadex LH-20 column

and separated chromatographically via semi-preparative RP-HPLC with

MeOH-H O-TFA (73:27:0.01, 2 mL/min) to afford compounds 16 (6.5

mg, t 55 min), 37 (3.3 mg, t 69 min), and 42 (1.5 mg, t 67 min).

Fractions S3 (5.6 g) and S4 (10 g) obtained above were subjected to a

2 2

graphed over a silica gel column eluting with CH Cl -MeOH (100:1 to

1:1 v/v) to afford 11 fractions (F1-F11). Fractions E5 (2.9 g) and E7 (0.5

g) were subjected to silica gel columns to obtain six fractions (E5-1-E5-6)

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Table 4

C NMR (150 MHz) Data of compounds 1–5, 9–18 (δ in ppm).

No.

34.3,

26.5,

39.5,

37.2,

38.7,

37.1,

39.5,

37.2,

33.9,

26.7,

38.9,

37.2,

39.1,

37.3,

34.5,

26.9,

39.2,

37.3,

34.6,

26.9,

41.2,

36.3,

36.4,

26.8,

36.3,

26.0,

35.6,

26.1,

35.3,

26.5,

71.1,

217.2,

215.7,

217.2,

70.2,

216.0,

216.1,

70.1,

216.1,

70.1,

219.2,

78.6,

73.0,

75.7,

75.0,

46.6, C

45.2,

59.7, C

49.9,

59.5, C

49.8,

59.7, C

49.9,

46.4, C

45.6,

59.4, C

49.8,

59.3, C

49.6,

46.5, C

45.6,

59.3, C

49.6,

46.5, C

45.6,

54.9, C

49.7,

41.9, C

44.9,

41.9, C

48.1,

53.4, C

46.1,

53.5, C

46.0,

19.6,

20.5,

20.4,

20.5,

19.6,

20.4,

20.2,

19.4,

20.2,

19.4,

69.3,

69.4,

69.3,

71.6,

70.7,

34.5,

33.8,

33.4,

33.8,

34.0,

33.9,

34.4,

34.9,

34.4,

34.9,

41.6,

41.8,

41.5,

42.4, C

54.5,

40.7, C

48.2,

40.3, C

47.8,

40.7, C

48.2,

40.5, C

48.9,

40.5, C

47.0,

40.0, C

47.4,

40.1, C

48.7,

40.0, C

47.4,

40.1, C

48.7,

40.5, C

48.0,

40.4, C

48.8,

40.2, C

48.7,

40.9, C

48.9,

40.9, C

48.7,

37.9, C

131.1,

37.6, C

25.0,

36.1, C

24.8,

37.6, C

25.0,

37.8, C

24.7,

37.0, C

24.8,

36.8, C

24.5,

37.5, C

24.3,

36.8, C

24.5,

37.5, C

24.4,

37.1, C

25.0,

37.7, C

24.7,

37.8, C

24.6,

37.6, C

24.7,

37.5, C

24.4,

124.3,

123.6,

123.7,

124.2,

128.5,

127.8,

127.9,

128.1,

129.8,

129.9,

129.8,

129.9,

128.7,

136.7,

145.5,

145.3,

145.4,

145.2,

140.1,

138.7,

138.5,

138.4,

139.7,

139.4,

139.6,

139.7,

41.9, C

26.8,

43.2, C

29.0,

42.6, C

29.5,

43.2, C

29.0,

42.5, C

29.6,

42.7, C

29.6,

44.3, C

29.0,

44.2, C

29.0,

44.3, C

29.0,

44.2, C

28.9,

43.6, C

29.7,

43.3, C

29.7,

43.2, C

29.7,

43.5, C

29.7,

43.7, C

29.2,

25.5,

24.2,

28.7,

24.2,

28.7,

27.2,

24.3,

24.2,

24.4,

24.2,

26.8,

19.7,

45.5, C

133.8,

47.8, C

42.9,

46.4, C

45.2,

48.1, C

42.2,

46.4, C

45.2,

48.7, C

55.7,

47.3, C

51.5,

47.2, C

51.4,

47.2, C

51.4,

47.2, C

51.4,

49.2, C

55.4,

49.1, C

55.3,

49.1, C

55.2,

49.2, C

55.4,

54.9, C

55.9,

86.7,

47.4,

81.5, C

41.5,

81.6, C

73.3, C

132.0,

132.1,

134.8,

135.0,

73.7, C

73.8, C

72.9, C

36.9, C

31.8, C

36.1, C

37.0, C

36.1, C

156.9,

130.1,

130.0,

126.6,

43.2,

43.3,

41.0,

33.9,

35.0,

29.5,

29.4,

29.5,

29.3,

25.0,

25.3,

27.4,

27.5,

36.3,

35.7,

68.8,

64.6,

18.7,

17.5,

19.7,

33.9,

64.3,

63.6,

15.9,

17.9,

26.5,

34.0,

64.7,

63.5,

15.6,

17.8,

25.1,

33.2,

64.2,

63.6,

15.9,

17.9,

26.5,

34.0,

69.5,

64.9,

15.9,

17.8,

25.1,

39.8,

65.0,

63.6,

15.9,

17.7,

24.3,

33.9,

65.1,

63.6,

16.4,

18.4,

22.5,

34.0,

69.5,

65.0,

16.5,

18.3,

22.6,

33.7,

65.1,

63.6,

16.4,

18.4,

22.4,

33.7,

69.5,

65.0,

16.5,

18.3,

22.5,

39.1,

66.9,

20.5,

17.3,

19.1,

25.0,

39.2,

72.1,

20.1,

17.6,

18.6,

25.1,

39.2,

73.0,

19.9,

17.5,

18.5,

25.0,

39.2,

75.4,

179.1,

178.0,

20.1,

20.4,

17.4,

18.9,

25.2,

17.5,

18.8,

25.4,

180.4,

181.9,

181.3,

181.8,

181.3,

180.5,

180.2,

180.1,

182.4,

182.5,

182.4,

180.2,

28.2,

33.7,

29.2,

74.6,

29.2,

27.9,

17.3,

17.5,

27.2,

23.6,

24.1,

25.2,

19.7,

25.2,

105.7,

63.5,

73.8,

57.9,

73.8,

57.9,

16.7,

16.8,

16.7,

52.2,

17.0,

51.7,

OMe/-

OAc

173.7,

1.1,

Recorded in CD

OD.

Recorded in Pyridine‑d

and 10 fractions (E7-1-E7-10), respectively. Fraction E7-9 was further

puriﬁed by semi-preparative RP-HPLC eluting with MeOH-H O-TFA

63:37:0.02) to generate compound 15 (2.7 mg, t 43 min). Separation

of fractions E5-6 (0.5 g) and E5-2 (0.2 g) via an ODS column (eluted with

0%, 50%, 60%, 70%, 80%, and 100% MeOH-H O) produced eight

fractions (E5-6-1-E5-6-8) and 10 fractions (E5-2-1-E5-2-10), respec-

tively. Compounds 12 (65% MeOH-0.0.5% TFA, 1.5 mg, t 45 min) and

5 (75% MeCN-0.0.5% TFA, 2.8 mg, t 28 min) were isolated, respec-

mg, t

42 min) and 28 (40% MeOH-0.0.2% TFA, 3.2 mg, t

69 min) were

obtained from fractions E5-6-4 (98 mg) and E5-6–5 (56 mg) using a

semi-preparative RP-HPLC, respectively. Fraction E6 (2 g) was chro-

matographed on an ODS column to obtain eight major fractions (E6-1-

(

E6-8). Subsequently, compound 18 (2.0 mg, t

from fraction E6-4 (20 mg) by semi-preparative RP-HPLC (MeOH-H

TFA, 65:35:0.02). Puriﬁcation of fraction E6-6 (70 mg) using semi-

preparative RP-HPLC with MeOH-H O-TFA (62:38:0.05) as mobile

phase led to the isolation of compounds 34 (5.3 mg, t 43 min) and 11

(20.0 mg, t 50 min). Fraction E6-7 (200 mg) was applied to a silica gel

28 min) was isolated

O-

tively, from fractions E5-2-5 (43 mg) and E5-2-9 (18 mg) via semi-

preparative RP-HPLC. Compounds 32 (58% MeOH-0.0.2% TFA, 13

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

column with a gradient elution of CH

-MeOH (50:1 to 1:1 v/v) to

2.3.4. 23,24,29-trihydroxy-3-oxo-olean-12-en-28-oic acid (4)

afford one terpenoid-containing fraction, which was then separated by

semi-preparative RP-HPLC to obtain compounds 5 (2.7 mg, t 26 min),

5 (6.1 mg, t 28 min), and 13 (4.0 mg, t 28 min). Subsequent sepa-

ration of fraction F2 (50 mg) afforded compounds 44 (3 mg, t 38 min),

3 (2 mg, t 43 min), and 8 (4.5 mg, t 45 min) by semi-preparative RP-

HPLC (MeOH-H O-TFA, 99:1:0.02). Fractions F3 (0.25 g), F5 (0.4 g) and

F9 (0.5 g) were subjected to silica gel columns to obtain 10 fractions (F3-

ꢀ F3-10), 11 fractions (F5-1-F5-11), and seven fractions (F9-1-F9-7),

respectively. Fraction F3-8 (30 mg) was chromatographed by semi-

preparative RP-HPLC (MeOH-H O-TFA, 90:10:0.05) to furnish com-

pound 22 (6 mg, t 35 min). Puriﬁcation of fractions F5-4 (30 mg) and

F5-6 (30 mg) resulted in the isolation of compounds 2 (85% MeOH-

.02% TFA, 18 mg, t 22 min) and 38 (65% MeOH-0.02% TFA, 3.4 mg,

40 min) by semi-preparative RP-HPLC. Compounds 39 (3.4 mg, t 42

min) and 6 (1.3 mg, t 44 min) were acquired from fraction F9-7 using

semi-preparative RP-HPLC (MeOH-H O-TFA, 70:30:0.02).

The MeOH-soluble extract (2.7 kg) was directly subjected to sepa-

ration over a polyamide resin column eluting successively with H O,

0% EtOH, and 60% EtOH. The 30% EtOH fraction (112 g) was frac-

tionated on a D101 macroporous resin column eluting successively with

0% EtOH, 50% EtOH, 70% EtOH, and 95% EtOH. The 70% EtOH

fraction (10 g) obtained was then resolved on an MCI gel column (eluted

with 30%, 50%, 60%, 70%, and 100% MeOH-H O) to furnish 10 frac-

tions (M1-M10). Fractions M4 and M5 were further puriﬁed using semi-

white powder; [

]

+ 55.3 (c 0.21, MeOH); UV (MeOH) λmax (log

) 205 (3.81) nm; IR (KBr) max 3459, 2920, 1695, 1459, 1383, 1272,

ꢀ

1232, 1203, 1181, 1046, 1009 cm ; H and C NMR data, see Tables 1

ꢀ

and 4; HRESIMS m/z 501.3225 [M-H] (calcd for C30

, 501.3211).

2.3.5. 3

white powder; [

) 206 (3.84) nm; IR (KBr)

,19 ,23,24-tetrahydroxyolean-12-en-28-oic acid (5)

]

+ 12.7 (c 0.43, MeOH); UV (MeOH) λmax (log

max 3459, 3382, 2930, 1704, 1451, 1382,

1 1

ꢀ

1197, 1078, 1046, 990 cm ; H and C NMR data, see Tables 1 and 4;

ꢀ

HRESIMS m/z 503.3372 [M-H] (calcd for C30

, 503.3367).

2.3.6. 19

white powder; mp 201–202 C; [

(MeOH) λmax (log ) 204 (3.75) nm; ECD (MeOH) λmax (Δ

264 (1.1) nm; IR (KBr) max 3504, 2940, 1701, 1455, 1378, 1205, 1038,

,23,24-trihydroxyurs-12,20(30)-dien-3-one-28-oic acid (9)

◦

]

+ 54.4 (c 0.38, MeOH); UV

) 231 (-0.5),

ꢀ

907 cm

;

H and C NMR data, see Tables 1 and 4; HRESIMS m/z

ꢀ

499.3050 [M-H] (calcd for C30

, 499.3054).

2.3.7. 23,24,30-trihydroxyurs-12,19-dien-3-one-28-oic acid (10)

white powder; [

]

+ 16.4 (c 0.17, MeOH); UV (MeOH) λmax (log

) 205 (3.51) nm; ECD (MeOH) λmax (Δ

3417, 2939, 1700, 1452, 1378, 1200, 1036, 804 cm ; H and C NMR

) 212 (–22.6) nm; IR (KBr)

max

ꢀ 1

ꢀ

data, see Tables 1 and 4; HRESIMS m/z 499.3064 [M-H] (calcd for

30 43 6

C H O , 499.3054).

preparative RP-HPLC (MeOH-H

mg, t 45 min) and 33 (6.1 mg, t

O, 60:40) to obtain compounds 4 (2.7

39 min), respectively. Fraction M6 was

2.3.8. 3

white powder; [

206 (3.79) nm; IR (KBr)

,23,24,30-tetrahydroxyurs-12,19-dien-28-oic acid (11)

submitted to an MCI gel column and further puriﬁed with MeOH-H

TFA (60:40:0.01) as mobile phase to afford compounds 29 (6.4 mg, t

3 min) and 30 (2.3 mg, t 63 min). Similar to fraction M6, fraction M7

was fractionated by an MCI gel column and puriﬁed to yield compounds

1 (65% MeOH-0.01% TFA, 3.6 mg, t 37 min), 27 (65% MeOH-0.01%

TFA, 6.8 mg, t 32 min), and 26 (62% MeOH, 2.4 mg, t 32 min). The

5% EtOH fraction (5 g) obtained above was subjected to a silica gel

column with CH Cl -MeOH (50:1 to 1:1 v/v) as eluent to produce nine

O-

]

ꢀ 1.4 (c 0.15, MeOH); UV (MeOH) λmax (log

)

max 3381, 2939, 1701, 1448, 1381, 1207, 1033,

ꢀ 1

991 cm

;

H and C NMR data, see Tables 1 and 4; HRESIMS m/z

ꢀ

501.3224 [M-H] (calcd for C30

, 501.3211).

2.3.9. 23,24-dihydroxyurs-12,19-dien-3-one-28-oic acid 30-methyl ether

(12)

white powder; [

204 (3.72) nm; ECD (MeOH) λmax (Δ

3448, 2926, 1716, 1691, 1450, 1378, 1217, 1196, 1083, 1041, 803

]²⁰

+ 0.9 (c 0.22, MeOH); UV (MeOH) λmax (log

max

)

fractions (N1 ꢀ N9). Fraction N4 (0.6 g) was submitted to an ODS col-

) 211 (ꢀ 26) nm; IR (KBr)

umn with MeOH-H

O gradient system (60%, 70%, 80%, 90%, and

ꢀ 1

00%) to give four fractions (N4-1-N4-4). Compounds 17 (15.6 mg, t

cm ; H and C NMR data, see Tables 1 and 4; HRESIMS m/z 513.3224

ꢀ

4 min) and 21 (2.0 mg, t

41 min) were obtained from fraction N4-1 by

[M-H] (calcd for C31

, 513.3211).

semi-preparative HPLC (MeOH-H

1.3 mg, t 36 min) was puriﬁed from fraction N4-4 with MeOH-H

TFA (68:32:0.01) as mobile phase.

O-TFA, 69: 31:0.01). Compound 1

(

O-

2.3.10. 3

,23,24-trihydroxyurs-12,19-dien-28-oic acid 30-methyl ether

(13)

white powder; [

]²⁰

ꢀ 3.4 (c 0.18, MeOH); UV (MeOH) λmax (log

)

.3.1. 3

white powder; [

) 203 (3.40), 252 (3.74) nm; ECD (MeOH) λmax (Δ

nm; IR (KBr) 3587, 3304, 2938, 1784, 1683, 1452, 1389, 1373, 1310,

,23,24-Trihydroxyolean-11,13(18)-dien-28,19β-olide (1)

206 (3.47) nm; ECD (MeOH) λmax (Δ

2939, 1702, 1451, 1378, 1261, 1211, 1070, 1025, 898 cm ; H and

) 212 (-8) nm; IR (KBr)

max 3454,

ꢀ 1

]

+ 34.8 (c 0.07, MeOH); UV (MeOH) λmax (log

ꢀ

) 229 (-8), 255 (-5)

NMR data, see Tables 1 and 4; HRESIMS m/z 515.3372 [M-H] (calcd

47 6

for C31H O , 515.3367).

ꢀ 1

208, 1138, 1084, 1027, 935 cm ; H and C NMR data, see Tables 1

and 4; HRESIMS m/z 507.3082 [M + Na] (calcd for C30

Na,

2.3.11. 6β,19

white powder; [

06 (3.05) nm; ECD (MeOH) λmax (Δ

,23-trihydroxyurs-12-en-3-one-28-oic acid (14)

07.3081).

]

+ 2.9 (c 0.07, MeOH); UV (MeOH) λmax (log

) 219 (-1.6), 297 (-0.2) nm; IR

max 3575, 3509, 2921, 1733, 1639, 1464, 1379, 1262, 1102,

)

.3.2. 23,24-Dihydroxy-3-oxo-olean-12-en-28-oic acid (2)

(KBr)

ꢀ 1

white powder; [

04 (3.74) nm; ECD (MeOH) λmax (Δ

max 3462, 2941, 2565, 1694, 1461, 1382, 1210, 1179, 1034, 978

]

+ 47.4 (c 0.1, MeOH); UV (MeOH) λmax (log

ε)

1041, 803 cm ; H and C NMR data, see Tables 1 and 4; HRESIMS m/

ꢀ

) 223 (ꢀ 2.3), 308 (ꢀ 0.2) nm; IR

z 501.3219 [M-H] (calcd for C30

, 501.3211).

(

KBr)

ꢀ

1 1

cm ; H and C NMR data, see Tables 1 and 4; HRESIMS m/z 485.3277

2.3.12. 3

white powder; [

06 (3.66) nm; IR (KBr)

α,6β,19α,23-tetrahydroxyurs-12-en-28-oic acid (15)

ꢀ

[

M-H] (calcd for C30

, 485.3262).

]

+ 8.7 (c 0.07, MeOH); UV (MeOH) λmax (log

)

max 3435, 2930, 1693, 1448, 1377, 1206, 1148,

ꢀ 1

.3.3. 19

white powder; [

) 205 (3.51) nm; ECD (MeOH) λmax (Δ

KBr) max 3488, 2941, 1687, 1455, 1384, 1234, 1206, 1045 cm ; H

,23,24-trihydroxy-3-oxo-olean-12-en-28-oic acid (3)

1044, 929 cm ; H and C NMR data, see Tables 1 and 4; HRESIMS m/

ꢀ

]

+ 10.1 (c 0.56, MeOH); UV (MeOH) λmax (log

z 503.3384 [M-H] (calcd for C30

, 503.3378).

(

) 219 (-5.83), 303 (-0.43) nm; IR

ꢀ 1 1

2.3.13. 23-acetoxy-3

,6β,19

-trihydroxyurs-12-en-28-oic acid (16)

ꢀ 7.9 (c 0.58, MeOH); UV (MeOH) λmax (log

max 3503, 2931, 1717, 1456, 1376, 1256, 1156,

ꢀ

and C NMR data, see Tables 1 and 4; HRESIMS m/z 501.3216 [M-H]

calcd for C30 , 501.3211).

white powder; [

α]

)

(

204 (3.72) nm; IR (KBr)

ꢀ 1

034, 932 cm ; H and C NMR data, see Tables 2 and 4; HRESIMS m/

ꢀ

z 545.3486 [M-H] (calcd for C32

, 545.3473).

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Table 5

C NMR Data of compounds 19–33 (δ in ppm).

b,c

a,c

b,c

a,c

b,c

a,c

a,d

a,c

a,d

a,c

No.

41.2,

35.2,

39.2,

37.0,

34.9,

25.9,

38.9,

36.9,

40.3,

33.3,

38.8,

34.1,

38.1,

33.6,

39.9,

27.6,

43.3,

38.4,

41.3,

36.3,

36.5,

26.7,

35.6,

26.1,

42.0,

35.5,

39.4,

36.9,

41.9,

27.7,

211.3,

216.7,

75.7,

215.9,

74.0,

222.8,

218.8,

75.3,

217.4,

219.1,

78.6,

75.7,

213.5,

217.2,

77.4,

64.3, C

59.9, C

53.3, C

59.5, C

154.6,

40.1, C

39.1, C

50.2,

43.8,

54.9, C

41.9, C

53.4, C

64.8, C

59.8, C

56.4, C

53.3,

49.3,

46.2,

49.5,

51.7,

60.0,

59.3,

49.6,

56.7,

48.9,

44.9,

46.1,

53.9,

49.8,

54.1,

70.9,

20.5,

71.0,

19.8,

22.6,

68.3,

67.1,

25.5,

68.8,

69.4,

71.6,

71.8,

20.6,

72.0,

33.0,

34.0,

32.9,

33.9,

34.0,

41.7,

40.4,

51.7,

51.8,

39.6,

41.5,

41.7,

42.0,

40.6, C

48.2,

41.7, C

55.0,

41.3, C

56.0,

40.3, C

47.1,

41.3, C

46.3,

41.1, C

48.7,

40.4, C

47.9,

41.9, C

47.7,

40.4, C

46.3,

40.7, C

48.1,

40.6, C

48.8,

41.1, C

49.0,

41.1, C

48.7,

41.2, C

47.9,

41.0, C

49.4,

36.5, C

24.5,

37.4, C

128.0,

37.6, C

128.4,

36.6, C

24.1,

39.9, C

25.7,

40.1, C

18.3,

38.0, C

17.7,

37.2, C

25.1,

37.2, C

25.3,

37.1, C

25.0,

37.7, C

24.8,

37.6, C

24.7,

36.9, C

24.9,

37.5, C

25.2,

37.3, C

24.8,

34.8,

34.0,

128.3,

131.4,

131.0,

125.6,

129.8,

129.7,

129.9,

130.1,

130.2,

130.1,

129.8,

129.6,

129.9,

139.9,

144.0,

144.2,

139.2,

140.3,

39.2, C

38.1, C

139.8,

139.4,

139.3,

139.0,

139.2,

139.9,

139.1,

43.2, C

42.5, C

43.1, C

44.5, C

43.0, C

159.8,

158.8,

42.9, C

43.5, C

43.6, C

43.3, C

43.6, C

43.5, C

42.9, C

43.3, C

29.6,

27.3,

27.2,

32.6,

29.7,

117.9,

117.0,

29.8,

26.7,

28.0,

31.4,

26.8,

26.7,

26.8,

26.7,

38.8,

38.1,

48.7, C

48.2, C

138.8,

49.1, C

38.4, C

38.0, C

49.6, C

49.7, C

49.6, C

49.7, C

49.5, C

49.6, C

55.1,

128.9,

128.6,

139.5,

55.4,

50.1,

49.8,

55.2,

55.3,

55.1,

55.2,

55.1,

73.0, C

215.3,

133.9,

73.7, C

32.5,

37.9,

73.8, C

73.7, C

73.8, C

73.7, C

73.8, C

42.7,

48.4,

135.4,

43.2,

50.3, C

43.6, C

43.1,

43.7,

43.1,

27.3,

28.8,

28.9,

128.0,

27.4,

221.4,

218.1,

27.4,

27.3,

27.4,

38.8,

39.1,

103.0,

38.4,

11.6,

39.5,

39.4,

123.5,

55.6,

52.1,

38.4,

38.3,

38.4,

32.4,

20.5,

16.8,

26.4,

20.9,

33.6,

71.4,

19.4,

32.3,

20.9,

16.7,

26.3,

20.9,

33.2,

28.6,

23.7,

175.1,

64.0,

178.9,

64.5,

66.9,

72.1,

179.1,

176.1,

64.4,

180.4,

63.2,

17.9,

17.0,

20.4,

63.3,

16.2,

17.0,

27.5,

20.5,

17.4,

19.3,

24.9,

20.1,

17.6,

18.9,

24.9,

63.6,

16.0,

17.8,

24.8,

19.8,

19.7,

61.2,

15.7,

17.7,

24.8,

20.1,

19.7,

12.8,

16.5,

18.9,

25.1,

19.8,

17.9,

20.5,

17.5,

19.2,

25.1,

16.7,

19.1,

24.9,

17.6,

18.8,

24.8,

14.1,

17.6,

25.0,

15.8,

19.2,

24.9,

181.0,

179.2,

179.3,

182.4,

178.7,

178.6,

178.7,

178.6,

178.7,

178.6,

27.4,

28.5,

17.1,

21.0,

27.2,

17.1,

52.6,

16.7,

52.2,

16.7,

16.8,

16.7,

52.2,

16.7,

52.9,

16.8,

16.7,

52.5,

OMe

95.9,

96.0,

95.9,

96.0,

74.0,

74.1,

74.0,

74.1,

74.0,

78.5,

78.4,

78.5,

78.4,

71.3,

71.4,

71.3,

71.4,

71.3,

71.2,

71.3,

78.7,

78.8,

78.7,

62.6,

62.5,

62.6,

62.5,

62.6,

Recorded in CD OD.

Recorded in Pyridine‑d .

Recorded 150 MH.

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Recorded at 125 MHz.

.3.14. 3

methyl ester (17)

white powder; [

06 (3.91) nm; IR (KBr)

,6β,19

-trihydroxyurs-12-en-28-oic acid-23-carboxylic acid

) 203 (3.73) nm; ECD (MeOH)λmax (Δ

) 223 (-0.36), 242 (0.51), 273

(0.29), 316 (-0.12) nm; IR (KBr) max 3457, 2960, 1702, 1468, 1423,

]²⁰

ꢀ 17.7 (c 0.81, MeOH); UV (MeOH) λmax (log

ε)

1382, 1261, 1210, 1170, 1147, 1110, 1044, 1006, 980 cm ; H and

ꢀ 1

;

max 3462, 2933, 1709, 1451, 1377, 1236, 1154,

NMR data, see Tables 2 and 5; HRESIMS m/z 455.3512 [M+H] (calcd

ꢀ

044, 1033, 930 cm

H and C NMR data, see Tables 2 and 4;

47 3

for C30H O , 455.3520).

ꢀ

HRESIMS m/z 531.3328 [M-H] (calcd for C31

, 531.3316).

.3.23. 3β,19

ester (26)

white powder; [

) 204 (3.76) nm; IR (KBr)

,24-trihydroxy-23-norurs-12-en-28-O-β-D-glucopyranosyl

.3.15. 3

acid methyl ester (18)

white powder; [

]²⁰

03 (3.34) nm; IR (KBr)

,6β,19

,22 -tetrahydroxyurs-12-en-28-oic acid-23-carboxylic

]²⁰

+ 29.9 (c 0.11, MeOH); UV (MeOH) λmax (log

max 3379, 2921, 2855, 1735, 1676, 1577,

ꢀ 5.9 (c 0.29, MeOH); UV (MeOH) λmax (log

)

ꢀ 1

max 3448, 2940, 1685, 1453, 1378, 1206, 1144,

1456, 1379, 1320, 1261, 1173, 1075, 1031, 803 cm ; H and C NMR

ꢀ

055, 1028, 932 cm

;

H and C NMR data, see Tables 2 and 4;

data, see Tables 3 and 5; HRESIMS m/z 659.37659 [M + Na] (calcd for

ꢀ

HRESIMS m/z 547.3281 [M-H] (calcd for C31

, 547.3265).

35 56

C H O10Na, 659.37657).

.3.16. 6β,19

-dihydroxyurs-12-en-3-one-28-oic acid-23-carboxylic acid

2.3.24. 6β,19

glucopyranosyl ester (27)

α-dihydroxy-24-norurs-12-en-3-one-28-O-β-D-

methyl ester (19)

]²⁰

ꢀ 6.4 (c 0.11, MeOH); UV (MeOH) λmax (log

)

white powder; [

]

+ 12.0 (c 0.13, MeOH); UV (MeOH) λmax (log

max 3392, 2919, 1731, 1693, 1596, 1456,

white powder; [

06 (3.13) nm; IR (KBr)

max 3573, 2929, 1720, 1688, 1451, 1378, 1261,

) 203 (3.72) nm; IR (KBr)

ꢀ 1

210, 1144, 1104, 1075, 802 cm ; H and C NMR data, see Tables 2

1427, 1378, 1262, 1204, 1181, 1074, 930, 804 cm ; H and C NMR

ꢀ

and 5; HRESIMS m/z 529.3176 [M-H] (calcd for C31

, 529.3160).

data, see Tables 3 and 5; HRESIMS m/z 657.3611 [M+Na] (calcd for

35 54

C H O10Na, 657.3609).

.3.17. 23,24-dihydroxy-3,19-dioxo-18,19-seco-11,13(18)-urs-diene-28-

oic acid (20)

white powder; [

03 (4.14), 241 (4.18), 281 (3.36) nm; ECD (MeOH) λmax (Δ

-7.6), 300 (-0.4) nm; IR (KBr) max 3358, 2921, 1702, 1660, 1633, 1468,

423, 1413, 1201, 1136, 1051, 721 cm

2.3.25. 6β,19

ester (28)

α,23-trihydroxyurs-12-en-3-one-28-O-β-D-glucopyranosyl

]²⁰

ꢀ 15.5 (c 0.1, MeOH); UV (MeOH) λmax (log

)

) 243.5

white powder; [

]²⁰

ꢀ 1.1 (c 0.28, MeOH); UV (MeOH) λmax (log

)

(

207 (3.66) nm; IR (KBr)

max 3413, 2934, 1733, 1691, 1457, 1379, 1265,

ꢀ 1

;

H and C NMR data, see

1227, 1204, 1175, 1140, 1070, 934 cm

;

H and C NMR data, see

—

Tables 2 and 5; HRESIMS m/z 523.3029 [M + Na] (calcd for

Tables 3 and 5; HRESIMS m/z 709.3814 [M + HCOO] (calcd for

Na, 523.3030).

37 57

C H O13, 709.3749).

.3.18. 3

,6β-dihydroxy-19-oxo-18,19-seco-11,13(18)-urs-dien-28-oic

2.3.26. 3

ester (29)

white powder; [

207 (4.15) nm; IR (KBr)

α,6β,19α,23-tetrahydroxyurs-12-en-28-O-β-D-glucopyranosyl

acid-23-carboxylic acid methyl ester (21)

]²⁰

white powder; [

]

ꢀ 75.4 (c 0.11, MeOH); UV (MeOH) λmax (log

ε)

ꢀ 2.9 (c 0.31, MeOH); UV (MeOH) λmax (log

)

03 (4.02), 241 (4.25) nm; ECD (MeOH) λmax (Δ

) 244 (-1.9) nm; IR

max 3402, 2933, 1731, 1672, 1452, 1376, 1320,

ꢀ

(

KBr)

max 3487, 2936, 1702, 1453, 1378, 1265, 1204, 1186, 1154,

1264, 1227, 1205, 1074, 1033, 930 cm

;

H and C NMR data, see

ꢀ 1

097, 1072, 1045, 1017, 981, 858 cm

;

H and C NMR data, see

Tables 3 and 5; HRESIMS m/z 689.3873 [M + Na] (calcd for

Tables 2 and 5; HRESIMS m/z 553.3136 [M + Na] (calcd for

36 58

C H O11Na, 689.3871).

Na, 553.3136).

.3.27. 3

α,6β,19α-trihydroxyurs-12-en-23,28-dioic acid 23-methyl ester

.3.19. 23,24-dihydroxy-28-norursane-3-one-12,17,19,21-tetraene (22)

28-O-β-D-glucopyranosyl ester (30)

white powder; [

]

+ 13.2 (c 0.17, MeOH); UV (MeOH) λmax (log

) 219 (-3.5), 245

white powder; [

]

–23.0 (c 0.19, MeOH); UV (MeOH) λmax (log

)

) 213 (4.16), 244 (3.76) nm; ECD (MeOH) λmax (Δ

14.5) nm; IR (KBr)

203 (3.91) nm; IR (KBr)

max 3448, 2932, 1723, 1453, 1377, 1262, 1238,

ꢀ 1

(

max 3392, 2950, 1687, 1453, 1374, 1203, 1177,

1205, 1178, 1072, 1031, 929 cm ; H and C NMR data, see Tables 3

ꢀ 1

050 cm

;

H and C NMR data, see Tables 2 and 5; HRESIMS m/z

and 5; HRESIMS m/z 717.3815 [M + Na] (calcd for C37

H O12Na,

37.3044 [M + H] (calcd for C29

, 437.3050).

717.3820).

.3.20. 3β,19

white powder; [

) 203 (3.66) nm; ECD (MeOH) λmax (Δ

KBr) max 3401, 2938, 1691, 1451, 1383, 1262, 1206, 1142, 1051,

-dihydroxy-24-norurs-4(23),12-dien-28-oic acid (23)

2.3.28. 6β,19

ester 28-O-β-D-glucopyranosyl ester (31)

-dihydroxyurs-12-en-3-one-23,28-dioic acid 23-methyl

]

+ 50.3 (c 0.33, MeOH); UV (MeOH) λmax (log

) 224 (-0.2), 276 (-0.5) nm; IR

(

white powder; [

α]

ꢀ 7.1 (c 0.38, MeOH); UV (MeOH) λmax (log

)

204 (3.65) nm; IR (KBr)

max 3436, 2932, 1729, 1702, 1455, 1375, 1260,

ꢀ 1

019, 802 cm ; H and C NMR data, see Tables 2 and 5; HRESIMS m/

1177, 1075, 1031, 929, 803 cm

;

H and C NMR data, see Tables 3

ꢀ

z 455.3152 [M ꢀ H] (calcd for C29

, 455.3156).

and 5; HRESIMS m/z 715.3666 [M + Na] (calcd for C37

H O12Na,

15.3664).

.3.21. 6

white powder; mp 239–240 C; [

MeOH) λmax (log ) 203 (3.80) nm; ECD (MeOH)λmax (Δ

94 (17.3) nm; IR (KBr) max 3473, 3402, 2946, 1698, 1683, 1462, 1417,

,29-dihydroxy-3,21-dioxo-taraxer-14-ene (24)

◦

α]

+ 155.7 (c 0.42, MeOH); UV

) 218 (ꢀ 0.9),

2.3.29. 19

,23,24-trihydroxyurs-12-en-3-one-28-O-β-D-glucopyranosyl

]²⁰

(

ester (32)

white powder; [

+ 17.1 (c 0.28, MeOH); UV (MeOH) λmax (log

max 3415, 2931, 1732, 1686, 1454, 1380,

ꢀ 1

376, 1257, 1202, 1173, 1145, 1045, 1005, 980 cm ; H and C NMR

) 204 (3.87) nm; IR (KBr)

ꢀ 1

data, see Tables 2 and 5; HRESIMS m/z 471.3469 [M+H] (calcd for

1261, 1203, 1177, 1138, 1074 cm ; H and C NMR data, see Tables 3

, 471.3469).

and 5; HRESIMS m/z 665.3887 [M + H] (calcd for C36

57 11

H O ,

65.3895).

.3.22. 6

-hydroxy-3,21-dioxo-taraxer-14-ene (25)

white powder; [

]

+ 152.7 (c 0.20, MeOH); UV (MeOH) λmax (log

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

H- H COSY

HMBC

ROESY

OH OH

Fig. 2. Key H– H COSY, HMBC, and ROESY correlations of 1.

.3.30. 3β,6β,19

8-O-β-D-glucopyranosyl ester (33)

-trihydroxyurs-12-en-23,28-dioic acid 23-methyl ester

2.5. Acid hydrolysis and determination of the absolute conﬁgurations of

the sugar units of compounds 26–33

white powder; [

α]

+ 14.9 (c 0.3, MeOH); UV (MeOH) λmax (log

)

05 (3.87) nm; IR (KBr)

;

max 3386, 2935, 1714, 1452, 1372, 1261, 1187,

Compounds 26–33 (each 1.0 mg) were dissolved in MeOH and

ꢀ

¹H and C NMR data, see Tables 3 and 5;

◦

treated with 2 M HCl (1.0 mL). After heating at 90 C for 10 h, each

142, 1071, 929 cm

HRESIMS m/z 717.3819 [M + Na] (calcd for C37

12Na, 717.3820).

solution was diluted with H O (2.0 mL) and extracted with EtOAc three

times. The H O-soluble layer was then concentrated and dried in vacuo

.4. X-ray crystallographic analysis

to give the sugar residue. The residue was dissolved in anhydrous pyr-

idine (1.0 mL), and L-cysteine methyl ester hydrochloride (1.0 mg) was

◦

Single crystals of 9 and 24 were cultured from isopropanol–MeOH

5:1) and MeOH solutions, respectively. Their X-ray crystallographic

added. The mixture was kept at 60 C for 2 h. Then, N-trimethylsilyli-

◦

(

midazole (0.2 mL) was added and stirred at 60 C for 2 h. The reaction

structures were acquired by anomalous scattering of Cu Ka radiation.

Crystallographic data for 9 and 24 were deposited at the Cambridge

Crystallographic Data Centre (CCDC 2050806 and 2050807).

was quenched by the addition of 1.0 mL of water. After extraction with

n-hexane three times, the n-hexane layer was detected by gas chroma-

tography under the following conditions: SE-54 column (30 m × 0.25

◦

mm), FID detector (280 C), and N

as the carrier gas. The initial oven

◦

.4.1. Crystallographic data of 9

, M = 560.74, a = 7.97913(13) Å, b = 11.6325(2) Å, c =

temperature of 200 C was raised to 280 C at a rate of 10 C/min, and

held for 35 min. Finally, D-glucose was deﬁned by comparing the

retention time of its derivative with that of the standard D-glucose

derivative.

33 52 7

C H O

◦

1.5927(5) Å,

α = 90 , β = 90 , γ = 90 , V = 2932.34(8) Å , T = 100 K,

orthorhombic, space group P2

2 2

1 1

(no. 19), Z = 4,

(Cu K

α) = 0.700

ꢀ

◦

mm , 20445 reﬂections measured (8.1 ≤ 2θ ≤ 144.214 ), 5672 unique

(

int = 0.0399, Rsigma = 0.0346) which were used in all calculations. The

ﬁnal R was 349 (I > 2 (I)) and wR was 0.0893 (all data). The goodness

of ﬁt on F was 1.030. Flack parameter = ꢀ 0.08(7).

2.6. Hepatoprotective assay

As previously described, the MTT assay method was used to test the

hepatoprotective effects of the isolated compounds (3, 5, 7–9, 12–14,

16–19, 22, 24, 25, 32, 34–41, and 44) [10]. The detailed process is

described in the Supporting Information.

.4.2. Crystallographic data of 24

, M = 470.67, a = 8.88371(14) Å, b = 11.82467(19) Å, c =

30 46 4

C H O

◦

5.3311(4) Å,

= 90 , β = 90 , γ = 90 , V = 2660.95(7) Å , T = 108.55

(

10) K, orthorhombic, space group P2

(no. 19), Z = 4,

(Cu K

) =

.7. In vitro anti-HSV-1 and anti-CVB3 activity assays

ꢀ 1

◦

.593 mm , 9202 reﬂections measured (8.252 ≤ 2θ ≤ 142.154 ),

027 unique (Rint = 0.0256, Rsigma = 0.0369) which were used in all

Anti-HSV-1 and anti-CVB3 activities of the isolated compounds were

calculations. The ﬁnal R

was 0.0396 (I > 2 (I)) and wR was 0.1022 (all

tested by the Reed-Muench method [11], as described in the Supporting

Information.

data). The goodness of ﬁt on F was 1.030. Flack parameter = 0.02 (10).

. Results and discussion

Compound 1 was obtained as a white powder. Its molecular formula

Fig. 3. Experimental ECD spectrum of 1 (left) and the Beecham’s rules for the bridged-ring lactone of 1 (right).

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Fig. 4. ORTEP diagram of 9.

was determined as C30

based on HRESIMS analysis (507.3082 [M

from δ

49.9) and C-24 (δ

4.08 and 3.44 (H

2 C C C

-23) to C-3 (δ 217.2), C-4 (δ 59.7), C-5 (δ

Na] , calcd for C30

Na, 507.3081) and C NMR data, indicating

63.6) suggested that the hydroxy group is located at

nine degrees of unsaturation. The IR spectrum showed absorption bands

C-23. The relative conﬁguration of 2 was also similar to that of 24-hy-

droxy-3-oxo-olean-12-en-28-oic acid according to their similar ROESY

correlations. In the ECD spectrum of 2 (Fig. S24), a weak (–)-CE at 307.5

ꢀ 1

for hydroxy (3587, 3304 cm ) and carbonyl (1784 cm ) groups. The

H NMR data (Table 1) clearly displayed ﬁve methyl groups (δ

.09, 1.00, 0.97, and 0.81), two oleﬁnic protons [δ

1.09,

6.25 (1H, dd, J =

0.2, 3.0 Hz), 5.85 (1H, dd, J = 10.2, 2.2 Hz)], two oxygenated meth-

3.89 (1H, d, J = 11.2 Hz), 3.72 (1H, d, J = 11.2 Hz), δ 3.66

1H, d, J = 11.5 Hz), and 3.59 (1H, d, J = 11.5 Hz)], and two oxygenated

nm for the n–

* transition of the C-3 carbonyl group suggested a

(5R,8R,9R,10R,14S,17S,18S) conﬁguration based on the cyclohexanone

octant rule [18]. Thus, the structure of 2 was considered as 23,24-dihy-

droxy-3-oxo-olean-12-en-28-oic acid.

ylenes [δ

(

methines [δ

4.84 (overlap), 4.05 (1H, dd, J = 2.8, 2.8 Hz)]. The

Compounds 3 and 4 showed the same molecular formula of

NMR data (Table 4) revealed 30 carbon signals attributed to ﬁve

methyls, 10 methylenes (including two oxygenated), six methines

30 46 6 45 6

C H O by HRESIMS analysis (calcd for C30H O , 501.3211). Their

1D NMR data (Tables 1 and 4) were similar to those of 2, except that

compounds 3 and 4 each possessed an additional hydroxy group. In the

(

including two oleﬁnic and two oxygenated), and nine quaternary car-

bons (including one carbonyl and two oleﬁnic). These characteristics of

were consistent with those of oleanane-type triterpenoids. The HMBC

correlation (Fig. 2) of H-19 (δ 4.84)/C-28 (δ 180.4) indicated that 1

possessed a bridged-ring lactone unit between C-28 and C-19. The above

data of 1 were similar to those of 2 ,3β,23-trihydroxyolean-11,13(18)-

dien-28,19β-olide [12], except for the disappearance of the 2 -OH and

the presence of an additional hydroxy group at C-24, which was

conﬁrmed by the HMBC cross-peaks from H -23 (δ 3.89, 3.72) to C-24

64.6), and from H -24 (δ 3.66, 3.59) to C-3 (δ 71.1), along with the

coupling system [–CH (1)–CH (2)–CH(3)-] in the H– H COSY spec-

trum. The 3β-H conﬁguration was established by the small coupling

constant (J = 2.8, 2.8 Hz) between H-3 and H -2. This assignment was

further supported by a ROESY correlation of H-3 (δ 4.05)/H-24 (δ

.66) (Fig. 2). Additional ROESY correlations of H -24/H -25 (δ 1.00),

-25/H -26 (δ 0.81), H -23/H-5 (δ 1.76), H-5/H-9 (δ 2.31), and H-

/H -27 (δ 1.09) indicated that H -24, H -25 and H -26 are β-oriented,

while H-5, H-9, H -23, and H -27 are -oriented.

H– H COSY spectrum of 3, the fragment [ꢀ CH(18) ꢀ CH(19) ꢀ ] was

evidence of the hydroxy group attached at C-19. The 19

-OH conﬁgu-

3.64. This

assignment was also supported via a ROESY correlation of H-19/H -30

(δ 1.13). The ECD spectrum of 3 (Fig. S35) exhibited a weak negative

sign at 303.5 nm due to the n– * transition of the C-3 carbonyl group,

which indicated the absolute conﬁguration of to be

ration of 3 was determined by the H-19 broad singlet at δ

5R,8R,9R,10R,14S,17R,18S,19S using the cyclohexanone octant rule

and the observed ROESY correlations. In the HMBC spectrum of 4, the

(

long-range correlations of the hydroxymethyl group (δ

(δ 41.5), C-20 (δ 37.0) and C-21 (δ 29.4), together with the ROESY

correlation of H-18 (δ 2.88)/H -30 (δ 0.94), indicated that the hy-

droxy group is attached to C-29 in 4. According to the above evidence,

the structures of 3 and 4 were considered as 19 ,23,24-trihydroxy-3-

3.19) with C-19

oxo-olean-12-en-28-oic acid and 23,24,29-trihydroxy-3-oxo-olean-12-

en-28-oic acid, respectively.

Compound 5 exhibited an [M – H] ion at m/z 503.3372 (calcd for

C H O , 503.3367), and its molecular formula, C30H O , was estab-

30 47 6 48 6

To determine its absolute conﬁguration, the ECD spectrum (Fig. 3) of

was measured in MeOH solution, and strong Cotton effects (CEs) were

lished by HRESIMS analysis. The H and C NMR data (Tables 1 and 4)

of 5 displayed a resemblance to those of 1 for the AB rings, while the

CDE rings were the same as 3. This deduction was conﬁrmed by the 2D

NMR data (Figs. S51-S54). The ROESY spectrum of 5 showed the cor-

observed at 229 and 255 nm for the n ꢀ

lactone moiety and for the * transition of a conjugated diene

moiety, respectively. According to Beecham’s rules [13–15], the sign of

the n ꢀ * CE of the bridged-ring lactone mainly depends upon the

location of C-β relative to the lactone ring plane. Based on this rule, the

–)-CE at 229 nm implied that the C-β of the lactone ring was below the

C-CO C lactone plane, suggesting a 19S conﬁguration (Fig. 3).

Additionally, the sign of the * transition (255 nm) was deduced by

* transition of a bridged-ring

ꢀ π

relations of H-3 (δ

suggesting that the relative conﬁguration of H-3 is β-oriented. The 19

OH conﬁguration was undoubtedly conﬁrmed by the broad singlet of H-

19 in the ¹H NMR spectrum. Thus, the structure of 5 was identiﬁed as

H H 3 H

4.74)/H-24 (δ 4.16) and H-24/H -25 (δ 1.04),

(

–

ꢀ

3α

,19

,23,24-tetrahydroxyolean-12-en-28-oic acid.

the diene helicity rule [16], which allowed for elucidation of the con-

ﬁgurations 8R,9R,14S,17R. In combination with the observed ROESY

correlations, the absolute conﬁgurations of the remaining chiral carbons

were determined to be 3R,5R,10S. Therefore, the structure of 1 was

The molecular formula of 9 was determined to be C30H O by

44 6

HRESIMS analysis (m/z 499.3050 [M – H] , calcd for C30

499.3054). In the 1D NMR spectroscopic data (Tables 1 and 4) of 9,

there were clearly observed signals from four methyls (δ 1.73, 1.67,

1.18, 1.17; δ 27.9, 24.3, 17.7, 15.9), one exo-oleﬁn (δ 5.03, 4.83; δ

156.9, 105.7), one trisubstituted double bond (δ 5.65; δ 140.1, 128.5),

two hydroxymethyl groups (δ 4.71, 4.00, 4.28, 4.09; δ 65.0, 63.6), and

one carbonyl carbon (δ 216.0). The above data were very similar to

those of the known compound 19 ,23,24-trihydroxyurs-3-oxo-12-en-

43 6

H O ,

identiﬁed as 3

,23,24-trihydroxyolean-11,13(18)-dien-28,19β-olide.

ꢀ

Compound 2 displayed an [M ꢀ H] ion at m/z 485.3277 (calcd for

, 485.3262), which corresponded to a molecular formula of

by HRESIMS analysis. The H and C NMR data (Tables 1 and

) of 2 were structurally similar to those of 24-hydroxy-3-oxo-olean-12-

en-28-oic acid [17], except for the presence of an additional hydrox-

ymethyl group (δ 4.08, 3.44; δ 64.3) in 2. The key HMBC correlations

28-oic acid (34), except for the presence of an exo-oleﬁn on the E ring in

9 instead of a methyl group in 34. According to the key HMBC

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

correlations of H

-30 (δ

5.03, 4.83) with C-19 (δ

73.3) and C-21 (δ

methine signal (δ

3.58, δ

78.6) in 15 instead of the carbonyl group at

0(30)

9.3), this exo-oleﬁn was determined to be located at △

. In the

C-3 in 14. In the HMBC spectrum of 15, the long-range correlations from

-23 (δ 3.69, 3.34) and H -24 (δ 1.10) to C-3 (δ 78.6) indicated that

the oxygenated methine is attached to C-3. In the NOESY experiment,

the key correlations of H-3 (δ 3.58)/H -24, H -24/H -25 (δ 1.35), and

H-6 (δ 4.35) /H-23 (δ 3.34) suggested that H-3 was assigned to be

β-oriented, whereas H-6 was assigned to be -oriented. Therefore, the

,23-tetrahydroxyurs-12-en-

ECD data of 9 (Fig. S66), a (–)-CE at 309 nm attributed to n ꢀ

* tran-

sition of C-3 carbonyl group was observed. From the cyclohexanone

octant rule and observed ROESY data, the absolute conﬁguration of 9

was assigned as 5R,8R,9R,10R,14S,17S,18S,19S, which was corrobo-

rated by the X-ray crystallographic data [Flack parameter of ꢀ 0.08(7)]

(

Fig. 4). Based on this evidence, the structure of 9 was unambiguously

structure of 15 was determined as 3α,6β,19α

established as 19

,23,24-trihydroxyurs-12,20(30)-dien-3-one-28-oic

28-oic acid.

acid.

Compound 16 had a molecular formula of C32

based on

HRESIMS analysis (m/z 545.3486 [M - H] , calcd for C32

545.3473). The 1D NMR data (Tables 2 and 4) of 16 were structurally

similar to those of 15, except for an additional acetoxy group (δ 2.06, δ

173.7, 21.1) at C-23 in 16. This assignment was unambiguously

conﬁrmed by the HMBC correlations from H -23 (δ 4.09, 4.00) to C-1ʹ

(δ 173.7). Furthermore, the 3 -OH and 6β-OH conﬁgurations were

deduced from the NOE signals of H-3 (δ 3.61)/H -24 (δ 1.30), H -24/

-25 (δ 1.36), and H-6 (δ 4.20)/H-23 (δ 4.09). Hence, the structure

,6β,19 -trihydroxyurs-12-en-28-

Compound 10 had the same molecular formula (C30

based on HRESIMS analysis (m/z 499.3064[M ꢀ H] , calcd for

, 499.3054). The 1D NMR spectroscopic data (Tables 1 and 4)

) as that of

49 7

H O ,

of 10 displayed high similarity to those of 9, except for the E ring signals.

The E ring of 10 possessed a hydroxy group at C-30 and a double bond

between C-19 and C-20, which was determined by the HMBC correla-

tions from H

130.1) and from H

5.0). Consequently, the structure of 10 was deﬁned as 23,24,30-trihy-

-29 (δ

1.86) to C-18 (δ

51.5), C-19 (δ

132.0), and C-20

(

-30 (δ 4.58, 4.37) to C-19, C-20, and C-21 (δ

of 16 was determined as 23-acetoxy-3

droxyurs-12,19-dien-3-one-28-oic acid.

oic acid.

The molecular formula of 11 was determined to be C30

Compound 17 possessed a molecular formula of C31

by HRE-

HRESIMS analysis. The 1D NMR spectroscopic data (Tables 1 and 4)

SIMS analysis (m/z 531.3328 [M - H] , calcd for C31H O , 531.3316)

47 7

and ¹³C NMR data. The H and C NMR data (Tables 2 and 4) of 17 were

highly similar to those of the reported compound elatumic acid [20],

except for the broad singlet of H-6 in 17, which was assigned to be the

were highly similar to those of 10, except that the carbonyl signal for C-3

was replaced by an oxygen-bearing methine (δ

speculation was conﬁrmed by the [ꢀ CH (1) ꢀ CH

fragment in the ¹H– H COSY data in combination with the HMBC cor-

relations from H -23 (δ 4.55, 4.36) and H -24 (δ 4.15, 4.02) to C-3 (δ

0.1). Finally, the NOE correlation of H-3 (δ 4.71)/H-24 (δ 4.15) and

4.71, δ 70.1). This

(2) ꢀ CH(3) ꢀ ]

-H conﬁguration. Furthermore, the NOE correction of H-3 (δ

with H -24 (δ 1.54) revealed that H-3 is β-oriented. Finally, the

structure of 17 was elucidated as 3 ,6β,19 -trihydroxyurs-12-en-28-oic

acid-23-carboxylic acid methyl ester.

The HRESIMS spectrum of 18 showed an [M - H]^-ion at m/z

547.3281 (calcd for C31 , 547.3265), which gave a molecular for-

mula of C31 . The 1D NMR spectroscopic data (Tables 2 and 4) of

18 displayed similarity to those of 17 except for an additional oxy-

methine at C-22 (δ 4.44, δ 75.4). This assumption was established

based on the HMBC correlation of H-22 (δ 4.44)/C-28 (δ 180.2) along

with the fragment [ꢀ CH(20) ꢀ CH (21) ꢀ CH(22) ꢀ ] in the H– H

COSY spectrum. Moreover, the 22 -OH conﬁguration was determined

by the splitting pattern of H-22 (J = 11.6, 4.4 Hz), which was also

supported by the ROESY correlations of H-22 with H-18 (δ 3.11) and

-29 (δ 1.44). Therefore, the structure of 18 was assigned as

,6β,19 ,22 -tetrahydroxyurs-12-en-28-oic acid-23-carboxylic acid

methyl ester.

The molecular formula of 19 was determined to be C31

3.68)

the splitting pattern of H-3 (4.71, brd, J = 2.7 Hz) revealed that H-3 is

β-oriented. Thus, the structure of 11 was identiﬁed as 3 ,23,24,30-tet-

rahydroxyurs-12,19-dien-28-oic acid.

47 8

H O

The HRESIMS ion of 12 at m/z 513.3224 [M ꢀ H] revealed a mo-

48 8

H O

lecular formula of C31

scopic data (Tables 1 and 4) indicated that 12 were extremely similar to

0. The only difference was the presence of an additional oxygen-

bearing methoxyl group (δ 3.31, δ 57.9) in 12. In the HMBC spec-

trum, a key correlation from the oxygen-bearing methoxyl group to C-30

73.8) suggested that this group was attached to the C-30 position.

46 6

H O . Detailed analysis of the 1D NMR spectro-

(δ

Thus, the structure of 12 was identiﬁed as 23,24-dihydroxyurs-12,19-

dien-3-one-28-oic acid 30-methyl ether.

Compound 13 possessed an HRESIMS ion at m/z 515.3372 [M ꢀ H] ,

3α

48 6

which revealed a molecular formula of C31H O . The H and C NMR

data (Tables 1 and 4) closely resembled those of 12, except for the

presence of an oxygenated methine signal (δ 4.71, δ 70.1) in 13

instead of the carbonyl group at C-3 in 12. This signal assignable to C-3

46 7

H O based

on the HRESIMS ion at m/z 529.3176 [M - H] (calcd for C31

529.3160). The 1D NMR spectroscopic data (Tables 2 and 5) of 19 were

almost similar to those of 17 except for an additional carbonyl group (δ

211.3) at C-3 in 19 instead of the oxymethine in 17, which was

conﬁrmed by the HMBC correlations from H -24 (δ 2.13) and H-1 (δ

1.87) to C-3. Thus, the structure of 19 was elucidated as 6β,19 -dihy-

droxyurs-12-en-3-one-28-oic acid-23-carboxylic acid methyl ester.

45 7

H O ,

was deduced by the fragment [ꢀ CH

(1) ꢀ CH

-OH conﬁguration was assigned by a NOE

4.15), which was further supported by

(2) ꢀ CH(3) ꢀ ] in the

H– H COSY spectrum. The 3

signal of H-3 (δ

4.71)/H-24 (δ

the splitting pattern of H-3 (4.71, brd, J = 3.0 Hz). Therefore, the

structure of 13 was elucidated as 3

,23,24-trihydroxyurs-12,19-dien-

8-oic acid 30-methyl ether.

Compound 20 possessed a molecular formula of C30

as deter-

44 6

H O Na,

The HRESIMS spectrum of 14 displayed an [M ꢀ H] ion at m/z

mined by an [M + Na] ion at m/z 523.3029 (calcd for C30

01.3219 (calcd for C30

, 501.3211), which corresponded to a

. The 1D NMR data (Tables 1 and 4) of 14

was almost identical to those of the known compound silphanolic acid B

19]. The only difference was the presence of a hydroxy group at C-19,

as conﬁrmed by the HMBC correlations from H -29 (δ 1.20) and H -30

C-19 (73.7). Furthermore, the ROESY correlation of H-18

-29 indicated 19-OH to be -oriented. NOE correlations of

3.43)/H-6 (δ 4.34), and H -24 (δ 1.25)/H -25 (δ 1.47)

suggested that the 6-OH is β-oriented. Thus, the structure of 14 was

identiﬁed as 6β,19 ,23-trihydroxyurs-12-en-3-one-28-oic acid.

Compound 15 exhibited a molecular formula of C30

523.3030) in the HRESIMS spectrum. The 1D NMR data (Tables 2 and 5)

of 20 resembled to those of the known compound 3β-hydroxy-19-oxo-

18,19-seco-11,13(18)-ursa- diene-28-oic acid [21]. Signiﬁcant differ-

molecular formula of C30

[

ences included the presence of a keto carbonyl group (δ 216.7, C-3)

instead of an oxygenated methine and the presence of two additional

(

0.93) to δ

2.54)/H

hydroxy groups at C-23 and C-24, as conﬁrmed by the HMBC correla-

(

tions from H

-23 (δ

4.11, 3.48) to C-3 (δ

216.7) and C-5 (δ 49.3), and

H-23 (δ

from H -24 (δ

3.88, 3.59) to C-3 and C-5. The relative conﬁguration of

20 was determined to be the same as that of 9 by ROESY spectrum

analysis. In the experimental ECD data of 20, a strong (–)-CE at 243.5 nm

by HRE-

(Fig. S181) for the

ꢀ

* transition of the cyclic conjugated diene unit,

SIMS analysis (m/z 503.3384 [M - H] , calcd for C30

, 503.3378).

which revealed the absolute conﬁguration of 20 to be

5R,8R,9R,10S,14S,17S [22]. Thus, the structure of 20 was determined as

23,24-dihydroxy-3,19-dioxo-18,19-seco-11,13(18)-urs-diene-28-oic

The H and C NMR data (Tables 1 and 4) of 15 were found to be closely

comparable to those of 14, except for the appearance of an oxygenated

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

ROESY

H- H COSY

HMBC

Fig. 5. Key H– H COSY, HMBC, and ROESY correlations of 24.

acid.

¹³C NMR data (Table 5) revealed 29 carbon signals, including ﬁve

methyl carbons, ten methylenes (one oleﬁnic), six methines (one

oxygen-bearing and one oleﬁnic), and eight quaternary carbons (one

oxygenated, two oleﬁnic and one carboxyl). The above data closely

Compound 21 exhibited an [M + Na] ion at m/z 553.3136 (calcd

for C31 Na, 553.3136) in the HRESIMS spectrum, which revealed a

molecular formula of C31 . The 1D NMR data (Tables 2 and 5) of 21

46 7

H O

46 7

H O

were highly similar to those of 17 for the AB rings, while the remaining

resonances were the identical to those of 20. This deduction was further

corroborated by the 2D NMR data. The ECD spectrum of 21 exhibited a

resembled that of 2

,19 -trihydroxy-24-norurs-4(23),12-dien-28-oic

acid, a 24-nortriterpenoid isolated from Rumex japonicas [24]. These

compounds differed by the absence of the hydroxy group at C-2 in 23,

close resemblance to that of 20 (Fig. S192), suggesting

which was elucidated by the fragment [–CH

(1)–CH

H– H COSY spectrum. The NOE correlation of H-3 (δ

1.61) revealed a 3 -H conﬁguration. Consequently, the structure of 23

was deﬁned as 3β,19 -dihydroxy-24-norurs-4(23),12-dien-28-oic acid.

(2)–CH(3)-] in the

(

3R,4S,5R,6R,8R,9R,10R,14S,17S) conﬁguration [22]. Therefore, the

3.94)/H-5 (δ

structure of 21 was determined as 3

,6β-dihydroxy-19-oxo-18,19-seco-

1,13(18)-urs-dien-28-oic acid-23-carboxylic acid methyl ester.

The HRESIMS spectrum of 22 exhibited an [M + H] ion at m/z

37.3044 (calcd for C29 , 437.3050), which corresponded to a

. The 1D NMR data (Tables 2 and 5) of 22

closely resembled those of (3R)-3,23,24-trihydroxy-28-norursane-

2,17,19,21-tetraene, a 28-nortriterpenoid previously isolated from

Rhododendron latoucheae by our group [23], except for the presence of a

keto carbonyl group (δ 215.9) at C-3 in 22 instead of an oxygenated

methine. This assignment was further conﬁrmed by the HMBC correla-

tions from H -23 (δ 4.70, 3.98), H -24 (δ 4.28, 4.09), and H-1(δ 1.90)

to C-3 (δ 215.9). The experimental ECD spectrum of 22 showed an

* electronic

Compound 24 was obtained as a white powder with a molecular

formula of C30

for C30

(Table 2) clearly exhibited seven singlet methyl groups (δ

1.19, 1.16, 0.95, 0.88, and 0.83), one hydroxymethyl group [δ

(1H, d, J = 10.4 Hz), 3.28 (1H, d, J = 10.4 Hz)], one oxygenated methine

[δ

based on the [M + H] ion at m/z 471.3469 (calcd

molecular formula of C29

, 471.3469) in its HRESIMS spectrum. Its H NMR data

1.34, 1.32,

3.65

3.92, (1H, ddd, J = 11.1, 11.1, 4.2 Hz)] and one oleﬁnic proton [δ

5.71, (1H, dd, J = 8.2, 3.1 Hz)]. A total of 30 carbon signals, including

seven methyl carbons, nine methylenes (one oxygen-bearing), ﬁve

methines (one oxygen-bearing and one oleﬁnic), and nine quaternary

intense (+) CE at 244 nm (Fig. S203) due to the

ꢀ

carbons (one oleﬁnic and two carbonyl), were recognized in its C NMR

transition of the conjugated chromophores. This result revealed that the

absolute conﬁguration of 22 was 5R,8R,9R,10R,14S [23]. Therefore, the

structure of 22 was elucidated as 23,24-dihydroxy-28-norurs-3-one-

data (Table 5). The above data suggested that 24 belongs to the Δ

taraxerene-type triterpene skeleton. The key HMBC correlations (Fig. 4)

from H -2 (δ 2.76, 2.23), H -23 (δ 1.32) and H -24 (δ 1.34) to C-3 (δ

222.8) and from H -29 (δ 3.65, 3.28), H -30 (δ 0.95) and H -22 (δ

2.70, 1.94) to C-21 (δ 221.4) indicated the presence of two carbonyl

groups at C-3 and C-21 and the presence of a hydroxy group at C-29. In

2,17,19,21-tetraene.

Compound 23 gave a molecular formula of C29

based on the

, 455.3156).

1.38,

5.31 (1H, dd, J =

.7, 3.7 Hz), 5.05 (1H, d, J = 2.0 Hz), and 4.64 (1H, d, J = 2.0 Hz)], and

3.94 (1H, dd, J = 11.8, 5.6 Hz)]. The

HRESIMS ion at m/z 455.3152 [M ꢀ H] (calcd for C29

The H NMR data (Table 2) of 23 showed ﬁve methyl groups (δ

addition, the [ꢀ CH(5) ꢀ CH(6) ꢀ CH

COSY spectrum suggested that the other hydroxy group was attached to

C-6. The observed ROESY cross-peaks (Fig. 5) of H-6 (δ 3.92)/H -25 (δ

0.88) indicated that the 6-OH was assigned as -oriented. In the ECD

(7) ꢀ ] fragment in the H– H

.21, 0.94, 0.86, and 0.77), three oleﬁnic protons [δ

one oxygen-bearing methine [δ

Fig. 6. ORTEP diagram of 24.

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Fig. 7. Effects of the isolated compounds on the survival rates of APAP-treated HepG2 cells. Data are presented as the mean ± SD (n = 3). BIC was used as the

positive control.

data of 24 (Fig. S225), a strong (+)-CE at 294 nm mainly due to n ꢀ

the structure of 27 was identiﬁed as 6β,19

3-one-28-O-β-D-glucopyranosyl ester.

-dihydroxy-24-norurs-12-en-

transition of C-21 carbonyl group was observed, indicating a 20R

conﬁguration according to the cyclohexanone octant rule. Finally, The

absolute conﬁguration of 24 was unambiguously determined to be

Compounds 28–32 possessed the molecular formulas of C36

56 11

H O ,

11, C37

12, C37H

12 and C36 11, respectively, on the

H O

R,6S,8R,9R,13S,17S,18R,20R on the basis of the X-ray crystallographic

data [Flack parameter: 0.02(10)] (Fig. 6). Hence, the structure of 24 was

determined as 6 ,29-dihydroxy-3,21-dioxo-taraxer-14-ene.

basis of their respective HRESIMS and C NMR data. Detailed analyses

of their 1D and 2D NMR data (Tables 3 and 5) indicated that compounds

28–32 were glycosides of 14, 15, 17, 19, and 34, respectively. In the

HMBC spectra, the long-range correlations observed from the anomeric

proton to carbonyl carbon C-28 indicated that the sugar units are linked

to C-28 in each molecule. The large coupling constants (J = 8.1 ~ 8.2

Hz) of anomeric protons revealed that the relative conﬁgurations of the

glucopyranoses were all determined as β-oriented. Additionally, the acid

hydrolysis assays of 28–32 indicated each sugar unit to be D-conﬁgu-

ration. Thus, the structures of compounds 28–32 were identiﬁed as

Compound 25 gave a molecular formula of C30

(m/z 455.3512

[M + H] , calcd for C30

47 3

H O , 455.3520) using HRESIMS analysis and

C NMR data. Comparing the 1D NMR data (Tables 2 and 5) and the

molecular formula of 25 with those of 24 implied the presence of an

additional methyl group (δ 1.19; δ 28.6) at C-29 in 25 instead of the

hydroxymethyl group in 24. This deduction was further supported by

the key HMBC correlations of H -29 (δ 1.19) with C-19 (δ 37.9), C-20

43.6) and C-21 (δ 218.1) and of H -30 (δ 1.20) with C-19, C-20,

and C-21. Finally, the structure of 25 was determined as 6 -hydroxy-

(

6β,19

,23-trihydroxyurs-12-en-3-one-28-O-β-D-glucopyranosyl

ester,

,6β,19

,23-tetrahydroxyurs-12-en-28-O-β-D-glucopyranosyl

,21-dioxo-taraxer-14-ene.

The HRESIMS analysis of 26 showed a molecular formula of

10 based on a deprotonated molecular ion at m/z 659.37659 [M

3α

-trihydroxyurs-12-en-23,28-dioic acid 23-methyl ester 28-O-

-dihydroxyurs-12-en-3-one-23,28-dioic

acid 23-methyl ester 28-O-β-D-glucopyranosyl ester, and 19 ,23,24-tri-

hydroxyurs-12-en-3-one-28-O-β-D-glucopyranosyl ester, respectively.

β-D-glucopyranosyl ester, 6β,19

Na] (calcd for C35

bles 3 and 5) of 26 closely resembled those of 3β,19

norurs-12-en-28-oic acid [25]. In the ROESY spectrum, a key correlation

of H -24 (δ 3.92, 3.56) with H -25 (δ 0.79) indicated a 24β-OH

conﬁguration, which supported 26 to be a 23-nortriterpenoid saponin.

Additionally, the ROESY correlations of H-3 (δ 3.81) with H-5 and H-18

2.51) with H -29 (δ 1.20) indicated 3β-OH and 19 -OH conﬁgu-

rations. The 1D NMR spectra of 26 also displayed characteristic reso-

nances assignable to a sugar unit [δ

10Na, 659.37657). The 1D NMR data (Ta-

,24-trihydroxy-23-

Compound 33 had the same molecular formula (C37

12) as 30,

which was deduced from the HRESIMS ion at m/z 717.3819 [M + Na]

(calcd for C37H O12Na, 717.3820). The H and C NMR data (Tables 3

and 5) of 33 displayed high similarities with those of 30, especially for

the resonances assigned to the BCDE ring and one glucopyranose unit,

while the A ring was different. The major difference was the large

(

coupling constant (J = 11.9, 4.2 Hz) between H-3 and H

gesting that 3-OH is β-oriented. The ROESY correlation of H-3 (δ

with H-5 (δ 1.48) also supported the 3β-OH conﬁguration. In addition,

-2 in 33, sug-

5.32 (1H, d, J = 8.2 Hz, H-1ʹ),

.32–3.39 (4H, overlap, H-2ʹ to H-5ʹ, 3.79 (1H, dd, J = 12.0, 2.3 Hz, H-

ʹa), 3.68 (1H, dd, J = 12.0, 4.8 Hz, H-6ʹb); δ 95.9, 74.0, 78.5, 71.3,

8.7, 62.6]. Furthermore, the sugar unit was determined to be linked at

3.90)

β-D-glucopyranose was determined to be present by the large coupling

constant of the anomeric proton and the acid hydrolysis method. Finally,

C-28 by the HMBC correlation of the anomeric proton H-1ʹ with the

carbonyl carbon C-28 (δ 178.7). A large coupling constant of the

anomeric proton (J = 8.2 Hz) indicated the sugar moiety to be

β-conﬁguration. An acid hydrolysis assay of 26 revealed that the sugar

unit is D-conﬁguration. Thus, the structure of 26 was identiﬁed as

the structure of 33 was reﬁned as 3β,6β,19α-trihydroxyurs-12-en-23,28-

dioic acid 23-methyl ester 28-O-β-D-glucopyranosyl ester.

In addition, fourteen known compounds were isolated from

E. chinensis. Their structures were identiﬁed as 3β,6β,19

droxyolean-12-en-28-oic acid (6) [27], oleanolic acid (7) [28], β-amyrin

(8) [29], 19 ,23,24-trihydroxyurs-12-en-3-one-28-oic acid (34) [30],

clethric acid (35) [31], 3β,19 ,23,24-tetrahydroxyurs-12-en-28-oic acid

(36) [32], 4-epi-barbinervic acid (37) [33], rotundic acid (38) [34],

3β,6β,19 ,23-tetrahydroxyurs-12-en-28-oic acid (39) [27], pomolic

acid (40) [35], ursolic acid (41) [36], 19 ,24-dihydroxyurs-12-en-3-

,23-tetrahy-

β,19

,24-trihydroxy-23-norurs-12-en-28-O-β-D-glucopyranosyl ester.

Compound 27 showed an [M + Na] ion at m/z 657.3611 (calcd for

O10Na, 657.3609) in the HRESIMS, which revealed a molecular

formula of C35

10. The 1D NMR data (Tables 3 and 5) of 27 closely

resembled those of ilexpuson B, a 24-nortriterpenoid saponin from Ilex

pubescens [26], except for an additional hydroxy group at C-6. This

assignment was further supported by the fragment [ꢀ CH(5) ꢀ CH(6) ꢀ

one-28-oic acid (42) [37], taraxerol (43) [38], and lupeol (44) by

comparison of their obtained NMR spectra with the reported data [39].

The hepatoprotective effects of compounds 3, 5, 7–9, 12–14, 16–19,

22, 24, 25, 32, 34–41, and 44 were evaluated in an acetaminophen

(7) ꢀ ] in the H– H COSY spectrum. The ROESY corrections of H-6

(

4.12)/H

-23 (δ

1.00) and H-4 (δ

2.83)/H

-25 (δ 1.41) indicated

that H-6 is

-oriented. Additionally, the presence of β-D-glucopyranose

(APAP)-induced damage model of HepG2 cells at 10 M. The results are

was conﬁrmed based on the large coupling constant of the anomeric

shown in Fig. 7, and compounds 8, 9, 12–14, 17, 24, and 44 signiﬁ-

proton and the acid hydrolysis assay. According to the above evidence,

cantly improved the survival rates of HepG2 cells from 54.6% to 73.1%

H.-Q. Wang et al.

Bioorganic Chemistry 111 (2021) 104866

Table 6

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