α-Glucosidase inhibitory and anti-inflammatory activities of dammarane triterpenoids from the leaves of Cyclocarya paliurus

DOI: 10.1016/j.bioorg.2021.104847

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Bioorganic Chemistry (2021)

Update date:2022-08-11

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Authors:

Deng, Shengping

Hao, Lili

Huang, Xishan

Huang, Yan

Li, Chenguo

Li, Jun Li, Jun

Liang, Cheng-qin

Liu, Wei

Su, Shanshan

Xu, Xia Xu, Xia

Yang, Ruiyun

Zhang, Gaorong

Zhou, Dexiong

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Article abstract of DOI:10.1016/j.bioorg.2021.104847

Diabetes mellitus is caused by chronic inflammation and affects millions of people worldwide. Cyclocarya paliurus leaves have been widely used in traditional folk tea as a remedy for diabetes, but the antidiabetic constituents remain to be further studied. The α-glucosidase inhibitory and anti-inflammatory activities were examined to evaluate their effects on diabetes mellitus, and bioassay-guided separation of C. paliurus leaves led to the identification of twenty dammarane saponins, including eleven new dammarane saponins (1–11). The structures of the isolates were elucidated by spectroscopic methods. Bioactivity assay results showed that compounds 1 and 2 strongly inhibited α-glucosidase activity, with IC₅₀ values ranging from 257.74 μM, 282.23 μM, and strongly inhibited the release of NO, with IC₅₀ values of 9.10 μM, 9.02 μM. Moreover, compound 2 significantly downregulated the mRNA expression of iNOS, COX-2, IL-1β, NF-κB, IL-6 and TNF-α in LPS-mediated RAW 264.7 cells and markedly suppressed the protein expression of iNOS, NF-κB/p65, and COX-2. Dammarane glucoside 2 exhibited the strongest α-glucosidase inhibitory and anti-inflammatory activities. In addition, the structure–activity relationships (SARs) of the dammarane saponins were investigated. In summary, C. paliurus leaves showed marked α-glucosidase inhibitory and anti-inflammatory activities, and dammarane saponins are responsible for regulating α-glucosidase, inflammatory mediators, and mRNA and the protein expression of proinflammatory cytokines, which could be meaningful for discovering new antidiabetic agents.

Full text of DOI:10.1016/j.bioorg.2021.104847

Bioorganic Chemistry 111 (2021) 104847

Contents lists available at ScienceDirect

Bioorganic Chemistry

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

-Glucosidase inhibitory and anti-inﬂammatory activities of dammarane

triterpenoids from the leaves of Cyclocarya paliurus

Chenguo Li^a, Shengping Deng^a, Wei Liu^a, Dexiong Zhou^a, Yan Huang^a, Cheng-qin Liang^c,

Lili Hao^a, Gaorong Zhang^a, Shanshan Su^a, Xia Xu^a, Ruiyun Yang^a, Jun Li^{a *}, Xishan Huang^a

^aState Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Collaborative Innovation Center for Guangxi Ethnic Medicine, School of

Chemistry and Pharmaceutical Science, Guangxi Normal University, Guilin 541004, China

^bInstitute for Sustainable Development and Innovation, Guangxi Normal University, Guilin 541004, China

^cCollege of Pharmacy, Guilin Medical University, Guilin 541004, China

A R T I C L E I N F O

A B S T R A C T

Keywords:

Diabetes mellitus is caused by chronic inﬂammation and affects millions of people worldwide. Cyclocarya paliurus

Cyclocarya paliurus

Diabetes

leaves have been widely used in traditional folk tea as a remedy for diabetes, but the antidiabetic constituents

remain to be further studied. The α-glucosidase inhibitory and anti-inﬂammatory activities were examined to

-glucosidase

evaluate their effects on diabetes mellitus, and bioassay-guided separation of C. paliurus leaves led to the

identiﬁcation of twenty dammarane saponins, including eleven new dammarane saponins (1–11). The structures

of the isolates were elucidated by spectroscopic methods. Bioactivity assay results showed that compounds 1 and

Anti-inﬂammatory

Triterpenoid

2 strongly inhibited

inhibited the release of NO, with IC₅₀values of 9.10

regulated the mRNA expression of iNOS, COX-2, IL-1β, NF-κB, IL-6 and TNF-

and markedly suppressed the protein expression of iNOS, NF-κB/p65, and COX-2. Dammarane glucoside 2

exhibited the strongest -glucosidase inhibitory and anti-inﬂammatory activities. In addition, the structur-

e–activity relationships (SARs) of the dammarane saponins were investigated. In summary, C. paliurus leaves

showed marked -glucosidase inhibitory and anti-inﬂammatory activities, and dammarane saponins are

responsible for regulating

-glucosidase activity, with IC₅₀values ranging from 257.74

M, 282.23

M. Moreover, compound 2 signiﬁcantly down-

in LPS-mediated RAW 264.7 cells

μM, and strongly

M, 9.02

-glucosidase, inﬂammatory mediators, and mRNA and the protein expression of

proinﬂammatory cytokines, which could be meaningful for discovering new antidiabetic agents.

1. Introduction

digestive enzyme, is an essential target for the regulation of postprandial

serum glucose in diabetic patients [3,4]. Additionally, inﬂammation

In recent years, with an increase in the intake of high-energy-content

foods, nutritional balance has been overlooked, and the incidence of

diabetes is increasing at an alarming rate [1]. Diabetes is associated with

a high-carbohydrate diet, and starches are one of the main sources of

carbohydrates. Inhibiting carbohydrate digestive enzymes by delaying

glucose absorption is one of the most effective ways of overcoming

plays a signiﬁcant role in the progression of diabetes. Higher concen-

trations of inﬂammatory mediators, such as nitric oxide (NO), and

proinﬂammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6

(IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase

(iNOS), nuclear factor kappa-B (NF-κB/p65), and tumor necrosis factor-

alpha (TNF-

α) are present in diabetic patients than in healthy patients

postprandial hyperglycemia [2].

-Glucosidase,

carbohydrate

[5,6]. These proinﬂammatory cytokines cause damage in pancreatic

Abbreviations: PNPG, 4-nitrophenyl-α-D-glucopyranoside; IL-1β, interleukin-1β; IL-6, interleukin-6; COX-2, cyclooxygenase-2; iNOS, inducible nitric-oxide syn-

thase; NF-κB/p65, nuclear factor kappa-B; mRNA, messenger RNA; cDNA, complementary DNA; HRESIMS, high resolution electrospray ionization mass spectroscopy;

NMR, nuclear magnetic resonance; HMBC, heteronuclear multiple-bond correlation; H–H COSY, H–H correlated spectroscopy; ROESY, rotating frame Overhauser

effect spectroscopy; DMEM, Dulbecco’s modiﬁed eagle medium; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPR, surface plasmon

resonance; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; PVDF, poly (vinylidene diﬂuoride).

* Corresponding authors at: State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Collaborative Innovation Center for Guangxi

Ethnic Medicine, School of Chemistry and Pharmaceutical Science, Guangxi Normal University, Guilin 541004, China.

E-mail addresses: lijun9593@gxnu.edu.cn (J. Li), huangxishan13@foxmail.com (X. Huang).

https://doi.org/10.1016/j.bioorg.2021.104847

Received 5 August 2020; Received in revised form 14 January 2021; Accepted 18 March 2021

Available online 22 March 2021

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Fig. 1. Structures of compunds 1–20 isolated from C. paliurus.

tissue, adipose tissue and the vasculature. In particular, NF-κB plays an

important role in initiating the inﬂammatory response during the pro-

duction of proinﬂammatory cytokines and to the mRNA expression and

were used as bioassay-guided evaluation models to verify the antidia-

betic effects of C. paliurus leaves and identify the active constituents. The

active portion was separated to obtain twenty dammarane saponins

(1–20), including eleven new dammarane saponins (1–11) (Fig. 1).

protein expression of iNOS, COX-2, IL-1β, NF-κB, IL-6 and TNF-α [7,8].

Activation of the NF-κB signaling pathway and excessive amounts of

inﬂammatory cytokines are considered to be possible mechanisms that

lead to diabetic diseases [9,10].

Furthermore, the inhibition of α-glucosidase and NO by the extracts and

the isolates was evaluated. In addition, the effects of the most active

anti-inﬂammatory compound on the mRNA expression of the inﬂam-

Cyclocarya paliurus, an endemic species that grows in southern

China, has been widely used as a traditional sweet tea to prevent dia-

betes, inﬂammation, hypertension, and heart disease and as a traditional

medicine for the treatment of diabetes mellitus, chronic inﬂammatory

diseases, hyperlipidemia, and hypertension [11,12]. Dammarane tri-

terpenoids, of which compounds were ﬁrstly isolated in 1992 [13,14],

are considered characteristic indicators of the plant and the key func-

tional and active constituents of C. paliurus. The extract of C. paliurus

leaves has shown signiﬁcant antidiabetic effects in many reports

[15–21]. The C. paliurus antidiabetic prescription reduces blood glucose

and improves glucose tolerance [22,23]. The C. paliurus antidiabetic

mechanism may be related to antioxidative and anti-inﬂammatory ef-

fects [12]. However, the possible antidiabetic compounds in C. paliurus

leaves and their corresponding mechanisms remain unclear.

matory cytokines iNOS, NF-κB, COX-2, IL-6, IL-1β and TNF-α and the

protein expression of iNOS, COX-2 and NF-κB/p65 were tested using

LPS-mediated RAW 264.7 macrophages. Herein, the isolation, puriﬁ-

cation and determination of these isolates, the assays used to determine

the antidiabetic and anti-inﬂammatory activities of the constituents, the

effects of the most active compound on mRNA and the protein expres-

sion of some inﬂammatory cytokines, and the structure–activity re-

lationships (SARs) are described.

2. Materials and methods

2.1. General experimental procedures

Silica gel (100–200 mesh, Qingdao Marine Chemical Co. Ltd.),

To further investigate potential constituents to treat diabetes based

reversed-phase C₁₈(50 μm, Merck), and Sephadex LH-20 (Amersham

-glucosidase inhibitory and anti-inﬂammatory activities, the p-

Pharmacia Biotech) columns were used for chromatographic separa-

tions. ^L-Arabinose, D-arabinose, L-glucose, D-glucose, L-quinovose and D-

nitrophenol-a-^D-glucopyranoside (PNPG) method and RAW 264.7 cells

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Table 1

¹H NMR Spectral Data of Compounds 1–6 (500 MHz and 600 MHz, Pyridine‑d₅, δ in ppm).

position

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

δ_H, mult (J in Hz)^a

δ_H, mult (J in Hz)^b

δ_H, mult (J in Hz)^a

δ_H, mult (J in Hz)^b

3.08, td, (9.9, 2.7);

2.12, m

2.99, m; 1.80, m

3.04, m; 1.84, m

3.01, m; 1.83, m

3.02, m; 1.83, m

3.06, td, (9.0, 3.8);

1.83, m

2.16, m; 1.75, m

3.62, m

1.79, m; 1.65, m

3.54, m

1.75, m; 1.66, m

3.56, m

1.81, m; 1.69, m

3.61, br s

1.81, m; 1.70, m

3.60, m

1.73, m; 1.61, m

3.55, m

1.80, m

1.54, m

1.55, m

1.56, m

1.55, m

1.54, m; 1.47, m

1.63, m; 1.22, dt,

(13.0, 3.0)

1.53, m; 1.47, m

1.53, m; 1.19, m

1.54, m; 1.45, m

1.52, m; 1.16, m

1.47, m; 1.41, m

1.52, m; 1.12,

overlapped

1.48, m; 1.45, m

1.54, m; 1.18,

overlapped

1.90, m

1.48, m; 1.46, m

1.53, m; 1.16, m

2.04, m

1.88, m

1.90, m

1.85, m

1.90, m

4.48, td, (10.5, 5.0)

2.95, m; 1.71, m

2.16, m

4.45, td, (10.6, 4.9)

2.97, m; 1.59, m

2.02, dd, (10.2, 2.5)

1.42, m; 1.01, dt,

(11.0, 7.6)

1.75, m; 1.44, m

1.89, m

4.51, m

4.46, dt, (10.6, 5.4)

2.58, m; 1.55, m

2.21, m

4.47, td, (10.6, 4.9)

2.99, t (4.5); 1.67, m

2.12, m

4.48, td, (10.8, 5.1)

3.04, t (3.9); 1.66, m

2.12, m

2.99, m; 1.68, m

2.12, m

1.53, m; 1.06, t (10.4)

1.50, m; 1.03, t, (9.5)

1.48, m; 1.05, m

1.48, m; 1.03, dt,

(12.5, 8.2)

1.48, m, 1.04, t, (8.6)

2.14, m; 1.74, m

1.80, m

1.88, m; 1.80, m

1.90, m; 1.70, m

2.55, m

1.75, m; 1.45, m

1.85, m

1.73, m; 1.45, m

1.86, m

1.96, m

1.11, s

1.08, s

1.00, s

1.02, s

1.07, s

1.03, s

1.40, s

1.33, s

1.34, s

1.35, s

1.32, s

1.44, s

1.25, s

1.42, s

2.07, s

1.43, s

1.47, s

2.12, m; 1.96, m

2.29, td, (12.5, 4.0);

1.75, m

2.65, dd, (13.5, 6.6); 2.50, m

2.14, m; 1.67, m

2.18, m; 1.66, m

1^′

2.10, m; 2.02, m

4.42, m

1.90, m; 1.78, m

3.68, d

6.11, d, (7.7)

6.41, d, (15.5)

5.05, s, 4.94, s

1.83, s

4.39, m

4.42, td, (9.0, 4.5)

5.21, m

5.15, dt, (9.0, 1.3)

1.69, s

5.30, s; 4.98, s

1.91, s

1.50, s

1.72, s

1.29, s

1.68, s

1.71, s

1.00, s

0.93, s

0.88, s

0.95, s

0.91, s

1.26, s

1.24, s

1.25, s

1.29, s

1.28, s

1.25, s

0.87, s

0.76, s

0.70, s

0.79, s

0.77, s

5.49, m

5.52, br s

4.86, m

5.56, br s

4.87, m

5.54, d, (1.3)

4.86, m

5.49, m

2^′

4.84, d, (2.8)

4.61, d, (3.4)

4.72, td, (6.9, 3.1)

4.79, dd, (11.6, 3.0);

4.59, m

4.84, m

3^′

4.82, m

4.84, m

4.82, m

4.83, m

4^′

4.73, m

4.75, m

4.72, d, (3.4)

4.36, m; 4.26, dd,

(11.9, 4.8)

4.95, d, (7.7)

3.95, t, (8.5)

4.09, t, (8.8)

3.66, t, (8.8)

3.63, m

4.71, m

5^′

4.38, m; 4.27, d, (4.6)

4.39, m; 4.28, dd,

(11.7, 4.4)

4.87, dd, (15.9)

3.94, t, (8.4)

4.11, t, (8.8)

3.69, t, (8.9)

3.63, m

4.35, m; 4.26, m

1^′′

2^′′

3^′′

4^′′

5^′′

4.93, d, (7.6)

3.98, t, (8.3)

4.10, t, (8.7)

3.66, t, (8.8)

3.60, d, (5.9)

5.03, d, (7.7)

3.94, t, (8.4)

4.13, t, (8.8)

3.68, d, (8.9)

3.74, m

5.06, br s

4.81, d, (3.9)

4.34, d, (7.4)

4.05, dd, (9.2, 3.7)

4.20, m

3.98, t, (8.2)

4.20, t, (8.5)

3.87, m

4.12, t, (8.8)

4.28, dd, (10.3, 2.2);

3.62, m

6^′′

1.61, d, (5.8)

1.55, d, (5.9)

4.52, dd, (11.2, 2.8); 4.33, dd,

(11.2, 5.3)

1.62, d, (5.9)

1.60, d, (5.9)

–

CH₃COO

1.96, s

–

OCH₃

3.19, s

3.21, s

Recorded at 600 MHz for ¹H NMR in pyridine‑d₅, J values (Hz) were determined from ¹H- decoupling experiments.

Recorded at 500 MHz for ¹H NMR in pyridine‑d₅, J values (Hz) were determined from ¹H- decoupling experiments.

quinovose were used as sugar standards (Sigma, Munich, Germany). All

solvents were of HPLC or analytical grade. The instruments used in the

experiment are shown in the Supporting Information (SI, Text S1).

measuring the inhibition degree of NO production in LPS-mediated RAW

264.7 cells.

The active fraction (300 g) was chromatographed on a silica gel

column eluted with a gradient of increasing methanol amounts (0 →

100%) in a mixture with dichloromethane to yield 8 fractions (Fr.I to Fr.

2.2. Plant material

VIII). Fr.Ⅴ (40 g) with active

α-glucosidase inhibitory and anti-

inﬂammatory activities was subjected to MCI gel column chromatog-

raphy (CHP20P, MeOH/H₂O 50:50 → 90:10, 1000 mL each) to provide

six fractions (Fr.Ⅴ-1 to Fr. ꢀ 6). Fr.Ⅴ-3 (10 g) was fractioned by an ODS

column (MeOH-H₂O, 60:40 → 100:0) to obtain six subfractions (Fr.Ⅴ-3-1

to Fr.Ⅴ-3-6). Fr.Ⅴ-3-2 (800 mg) was isolated with a Sephadex LH-20

column, which was followed by semipreparative HPLC (MeCN-H₂O,

50:50, v/v, 3.0 mL/min, λ ₌205 nm) to yield 2 (t_R13.86 min, 10.7 mg),

4 (t_R19.12 min, 6.2 mg), 10 (t_R15.17 min, 7.1 mg), 12 (t_R23.16 min,

4.0 mg), and 13 (t_R22.06 min, 5.8 mg). Fr.Ⅴ-3-3 was puriﬁed by pre-

parative MPLC (MeCN-H₂O, 50:50, v/v, 8.0 mL/min, λ ₌205 nm) and

semipreparative HPLC (MeOH-H₂O, 78:22, v/v, 3.0 mL/min, λ 205

nm) to obtain 5 (t_R16.98 min, 6.2 mg), 6 (t_R16.16 min, 5.0 mg)⁼, 9 (t_R

16.53 min, 4.7 mg), 11 (t_R16.04 min, 7.5 mg), and 14 (t_R27.44 min, 8.1

mg). Compounds 7 (t_R14.16 min, 6.6 mg), 8 (t_R17.98 min, 4.1 mg), and

15 (t_R35.41 min, 4.7 mg) were isolated from Fr.Ⅴ-3-4 using preparative

Dried leaves (10 kg) of C. paliurus were obtained from Xiushui

County, Jiangxi, China, in July 2017 and identiﬁed by Associate Pro-

fessor Qiang Xie (Guangxi Normal University). A voucher specimen (No.

ID-20170705) was deposited at the State Key Laboratory of Guangxi

Normal University (GXNU), China.

2.3. Extraction and bioactivity-guided separation of dammarane saponins

The leaves of C. paliurus (sweet tea, 10 kg) were extracted 3 times

with a 75% ethanol-H₂O mixture (3:1, v/v, 20 L) under reﬂux and

concentrated under vacuum to remove the ethanol. The remaining H₂O

portion was further subjected to macroporous resin column chroma-

tography (D101, H₂O/MeOH 10:30 → 10:90, 10000 mL each) to provide

six fractions. The

α-glucosidase inhibitory activity was tested by the

PNPG method, and the anti-inﬂammatory effects were assayed by

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Table 2

¹H NMR Spectral Data of Compounds 7–10 (500 MHz and 600 MHz, Pyridine‑d₅, δ in ppm).

position

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

δ_H, mult (J in Hz)^a

δ_H, mult (J in Hz)^b

δ_H, mult (J in Hz)^a

3.03, td, (10.8, 3.5); 1.84, m

1.83, m, 1.70, m

3.61, m

3.07, m; 1.83, m

1.82, m; 1.70, m

3.55, m

3.02, td, (8.71, 4.5); 1.82, m

1.80, m; 1.69, m

3.57, m

3.06, m; 1.85, m

1.74, m; 1.67, m

3.58, br s

3.06, m; 1.83, m

1.74, m; 1.64, m

3.54, br s

1.56, m

1.54, m

1.56, m

1.55, m

1.50, m; 1.47, m

1.58, m; 1.21, dt, (13.0, 3.0)

1.90, m

1.51, m; 1.44, m

1.55, m; 1.18, overlapped

1.90, m

1.50, m; 1.47, m

1.57, m; 1.21, dt, (13.0, 3.0)

1.90, m

1.55, m; 1.44, m

1.52, m; 1.15, m

1.89, m

1.53 m; 1.40, m

1.51, m; 1.17, d, (12.4)

1.88, m

4.47, td, (10.6, 4.9)

2.98, m; 1.67, m

4.47, td, (10.6, 5.0)

3.02, m; 1.67, m

4.47, td, (10.8, 4.5)

2.98, m; 1.67, m

4.48, td, (10.5, 5.0)

2.97, d, (12.5); 1.57,

4.48, m

2.97, d, (12.3); 1.59, m

2.18, m

2.17, m

1.81, m

1.55, m; 1.07, t, (10.6)

1.53, m; 1.08, t, (9.3)

1.90, m; 1.83, m

1.96, m

1.55, m; 1.06, t, (10.1)

1.39, m; 0.99, t, (9.9)

1.77, m; 1.45, m

1.90, m

1.39, m; 1.00, t, (9.3)

1.79, m; 1.43, m

1.90, m

1.92, m, 1.84, m

1.91, m; 1.83, m

1.96, m

1.95, m

1.09, s

1.05, s

1.09, s

1.02, s

1.35, s

1.32, s

1.34, s

1.36, s

1.35, s

1.44, s

1.47, s

1.44, s

1.18, s

1.19, s

2.61, dd, (13.5, 6.2); 2.43, dd,

2.62, dd, (13.6, 6.0); 2.46, dd,

(13.6, 8.6)

2.61, dd, (13.4, 6.2); 2.43, dd,

1.75, m; 1.56, m

1.77, m; 1.57, m

(13.5, 8.7)

(13.4, 8.5)

1^′

5.98, ddd, (15.8, 8.7, 6.2)

6.00, ddd, (15.9, 8.6, 6.0)

5.67, d, (15.9)

1.31, s

5.97, ddd, (15.9, 8.5, 6.2)

2.04, m; 1.95, m

3.96, m

2.03, m; 1.95, m

3.96, t

5.65, d, (15.8)

5.64, d, (15.9)

1.31, s

1.30, s

1.47, s

1.48, s

1.31, s

1.30, s

1.41, s

1.42, s

0.96, s

0.90, s

0.96, s

0.89, s

0.90, s

1.29, s

1.26, s

1.27, s

0.80, s

0.78, s

0.81, s

0.72, s

0.73, s

5.51, br s

5.48, br s

5.51, br s

5.53, br s

4.87, m

5.50, br s

2^′

4.88, m

4.84, m

4.86, m

4.87, m

3^′

4.84, m

4.81, m

4.63, m

4.83, m

4.63, dt

4^′

4.75, m

4.72, ddd, (6.3, 4.7, 3.5)

4.34, m; 4.25, dd, (11.5, 4.7)

4.73, m

4.74, m

4.74, td, (5.7)

4.81, d, (11.5,

3.0);4.61, m

5.16, d, (7.7)

3.99, t, (8.3)

4.30, t, (8.8)

4.03, m

5^′

4.38, m; 4.28, dd, (11.8, 4.6)

4.81, dd, (11.6, 3.0); 4.62, m

4.37, m; 4.28, m

1^′′

2^′′

3^′′

4^′′

5^′′

6^′′

4.94, d, (7.7)

3.96, t, (8.3)

4.11, t, (8.8)

3.69, t, (8.9)

3.64, m

4.83, br s

4.93, d, (7.7)

3.94, m

5.16, d, (7.7)

4.00, d, (8.3)

4.29, m

4.36, m

4.06, dd, (9.2, 3.6)

4.21, m

4.09, t, (8.7)

3.69, m

4.03, dd, (8.1, 4.2)

4.17, t, (9.0)

4.54, d, (11.1); 4.39,

4.30, m; 3.64, m

3.64, m

4.17, t, (9.1)

4.54, d; 4.36, d, (10.6)

1.62, d, (5.8)

1.61, d, (5.8)

–

OCH₃

3.20, s

3.21, s

3.20, s

1.97, s

–

CH₃COO

1.97, s

Recorded at 600 MHz for ¹H NMR in pyridine‑d₅, J values (Hz) were determined from ¹H- decoupling experiments.

Recorded at 500 MHz for ¹H NMR in pyridine‑d₅, J values (Hz) were determined from ¹H- decoupling experiments.

MPLC (MeCN-H₂O, 50:50, v/v, 8.0 mL/min, λ 210 nm) and semi-

HRESIMS m/z 813.4999 [Mꢀ H]^ꢀ, (calculated for C₄₃H₇₃O_14,

813.5000); for the ¹H (pyridine‑d₅, 500 MHz) and ¹³C NMR (pyr-

idine‑d₅, 125 MHz) data, see Tables 1 and 3.

preparative HPLC (MeOH-H₂O, 78:22, v/v, 3.0 mL/min, λ ₌210 nm).

Fr.Ⅴ-3-5 was chromatographed on a silica gel column by eluting with

CH₂Cl₂-MeOH and puriﬁed by preparative MPLC (C₂H₃N-H₂O, 55:45, v/

v, 8.0 mL/min, λ 205 nm) and semipreparative HPLC (MeOH-H₂O,

2.3.3. Cyclocarioside Z₁₁(3)

80:20, v/v, 3.0 mL/min, λ ₌205 nm) to obtain 1 (t_R33.18 min, 14.3 mg),

3 (t_R16.83 min, 7.4 mg), and 16 (t_R21.44 min, 9.0 mg). Fr.Ⅴ-4 was

fractioned with an ODS column (MeOH-H₂O, 50:50 → 100:0) to obtain

ﬁve subfractions (Fr.Ⅴ-4-1 to Fr.Ⅴ-45). Fr.Ⅴ-4-4 was puriﬁed by pre-

parative MPLC (MeCN-H₂O, 50:50, v/v, 8.0 mL/min, λ ₌205 nm) and

semipreparative HPLC (MeOH-H₂O, 80:20, v/v, 3.0 mL/min, λ 205

nm) to give 17 (t_R14.16 min, 6.0 mg), 18 (t_R17.53 min, 5.2 mg), ⁼19 (t_R

15.08 min, 3.7 mg), and 20 (t_R40.15 min, 4.4 mg).

The separation and puriﬁcation method afforded 7.4 mg of a white

amorphous powder; [α _D²⁵

ꢀ 25.27 (c 0.23, MeOH); HRESIMS m/z

]

775.4613 [M+Na]⁺(calculated for C₄₁H₆₈O₁₂Na, 775.4608); for the ¹H

(pyridine‑d₅, 600 MHz) and ¹³C NMR (pyridine‑d₅, 150 MHz) data, see

Tables 1 and 3.

2.3.4. Cyclocarioside Z₁₂(4)

The separation and puriﬁcation method afforded 6.2 mg of a white

amorphous powder; [α _D²⁵

ꢀ 20.47 (c 0.19, MeOH); HRESIMS m/z

]

2.3.1. Cyclocarioside Z₉(1)

677.3880 [M+Na]⁺(calculated for C₃₅H₅₈O₁₁Na, 677.3877); for the ¹H

(pyridine‑d₅, 600 MHz) and ¹³C NMR (pyridine‑d₅, 150 MHz) data, see

Tables 1 and 3.

The chromatographic separation and puriﬁcation method afforded

14.3 mg of a white amorphous powder; [α _D²⁵

+ 1.10 (c 0.17, MeOH);

]

HRESIMS m/z 645.4342 [M+Na]⁺(calculated for C₃₆H₆₂O₈Na,

645.4341); for the ¹H (pyridine‑d₅, 600 MHz) and ¹³C NMR (pyr-

idine‑d₅, 150 MHz) data, see Tables 1 and 3.

2.3.5. Cyclocarioside Z₁₃(5)

The separation and puriﬁcation method afforded 6.2 mg of a white

amorphous powder; [α _D²⁵

ꢀ 7.26 (c 0.31, MeOH); HRESIMS m/z

]

2.3.2. Cyclocarioside Z₁₀(2)

791.4924 [M+Na]⁺(calculated for C₄₂H₇₂O₁₂Na, 791.4921); for the ¹H

(pyridine‑d₅, 500 MHz) and ¹³C NMR (pyridine‑d₅, 125 MHz) data, see

Tables 1 and 3.

The chromatographic separation and puriﬁcation method afforded

10.7 mg of a white amorphous powder; [α _D²⁵

ꢀ 17.60 (c 0.27, MeOH);

]

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Table 3

¹³C NMR Spectral Data of Compounds 1–11 (125 MHz and 150 MHz, pyridine‑d₅, δ in ppm).

Position

Compound

δ_C

35.9

26.1

75.7

39.1

51.2

18.9

37.0

50.6

54.8

40.6

77.0

35.3

41.1

42.0

31.9

27.5

50.5

17.7

17.2

74.4

27.2

38.2

31.0

76.3

150.3

110.6

19.0

23.4

30.5

17.3

36.0

21.7

79.9

38.2

51.5

18.6

36.7

51.3

54.4

40.3

77.1

34.9

40.9

41.8

31.8

26.1

50.8

17.4

17.0

76.3

27.0

24.8

70.3

75.0

27.7

29.2

23.2

29.2

17.3

106.8

84.4

80.1

81.8

65.4

101.7

75.8

78.7

77.2

73.1

18.9

171.1

21.0

35.9

21.5

79.5

38.2

51.2

18.6

36.5

50.8

54.2

40.2

77.1

35.0

41.0

41.6

31.5

25.9

50.8

17.3

16.9

74.7

27.6

45.1

128.6

135.6

142.9

115.1

19.1

23.2

30.2

17.0

106.6

84.4

79.7

85.8

63.2

102.2

75.6

78.7

78.0

72.6

63.8

36.1

21.8

79.7

38.4

51.4

18.7

36.9

50.5

54.2

40.5

76.6

33.1

41.9

43.3

31.8

27.1

54.3

17.5

16.4

211.3

30.5

36.0

21.7

79.6

38.3

51.5

18.7

36.7

51.3

54.4

40.3

76.8

35.1

40.8

41.8

31.6

26.0

50.8

17.5

17.0

74.6

27.8

45.1

76.1

127.3

135.8

18.5

26.1

23.3

30.3

17.0

106.8

84.4

79.8

86.0

63.3

101.8

75.9

78.6

77.2

73.0

18.9

36.0

21.6

79.6

38.3

51.6

18.7

36.6

51.3

54.4

40.3

76.9

34.9

40.7

41.8

31.6

26.0

50.7

17.5

17.0

74.6

27.9

45.0

76.1

127.4

135.8

18.6

26.1

23.3

30.3

17.2

106.7

84.3

79.8

86.0

63.3

102.8

73.1

75.1

70.1

67.9

35.9

21.6

79.6

38.3

51.3

18.7

36.7

50.9

54.3

40.3

76.8

35.1

40.9

41.8

31.6

25.8

50.7

17.5

16.9

74.4

27.9

44.8

127.8

138.6

75.3

26.2

26.9

23.3

30.3

17.0

106.7

84.4

79.7

86.0

63.3

101.9

75.9

78.6

77.2

73.0

19.0

36.0

21.6

79.6

38.3

51.3

18.6

36.7

50.9

54.3

40.3

77.0

34.9

41.0

41.8

31.6

25.9

50.8

17.5

17.1

74.5

27.8

44.7

127.4

138.6

75.3

26.3

26.9

23.3

30.3

17.2

106.7

84.3

79.8

86.1

63.3

102.8

73.0

75.1

70.1

67.9

35.9

21.8

80.0

38.3

51.3

18.6

36.7

50.9

54.3

40.3

76.8

35.1

40.9

41.8

31.6

25.8

50.7

17.5

16.9

74.4

27.8

44.8

127.7

138.6

75.3

26.3

26.9

23.3

30.2

17.1

106.9

84.5

80.2

81.9

65.5

101.9

75.9

78.6

77.2

73.0

18.9

171.1

21.0

36.1

21.6

79.6

38.3

51.3

18.6

36.6

50.4

54.4

40.3

77.3

34.5

41.4

41.8

31.9

27.1

49.6

17.3

17.1

86.8

24.9

34.8

26.7

84.6

71.5

26.5

28.0

23.2

30.3

17.2

106.7

84.4

79.7

85.9

63.3

102.1

75.7

79.0

78.1

72.8

63.8

36.0

21.7

80.0

38.2

51.2

18.6

36.6

50.4

54.4

40.3

77.3

34.5

41.4

41.8

31.9

27.1

49.6

17.3

17.1

86.8

24.9

34.8

26.7

84.6

71.5

26.5

28.0

23.2

30.2

17.1

106.9

84.5

80.1

81.8

65.4

102.1

75.7

79.0

78.1

72.8

63.8

171.1

21.0

1^′

23.4

30.4

17.0

106.9

84.6

79.7

86.1

63.4

102.0

76.0

78.8

77.3

73.1

19.1

2^′

3^′

4^′

5^′

1^′’

2^′’

3^′’

4^′’

5^′’

6^′’

CH₃COO

102.1

76.2

78.7

77.3

73.2

19.1

–

23-OCH₃

55.5

–

25 OCH₃

50.5

Recorded at 150 MHz for ¹³C NMR in pyridine‑d₅, chemical shifts given in ppm using TMS as internal reference.

Recorded at 125 MHz for ¹³C NMR in pyridine‑d₅, chemical shifts given in ppm using TMS as internal reference.

2.3.6. Cyclocarioside Z₁₄(6)

Tables 2 and 3.

The separation and puriﬁcation method afforded 5.0 mg of a white

amorphous powder; [α _D²⁵

ꢀ 7.76 (c 0.19, MeOH); HRESIMS m/z

]

2.3.9. Cyclocarioside Z₁₇(9)

777.4768 [M+Na]⁺(calculated for C₄₁H₇₀O₁₂Na, 777.4765); for the ¹H

(pyridine‑d₅, 500 MHz) and ¹³C NMR (pyridine‑d₅, 125 MHz) data, see

Tables 1 and 3.

The separation and puriﬁcation method afforded 4.7 mg of a white

amorphous powder; [α _D²⁵

ꢀ 30.61 (c 0.22, MeOH); HRESIMS m/z

]

833.5028 [M+Na]⁺(calculated for C₄₄H₇₄O₁₃Na, 833.5027); for the ¹H

(pyridine‑d₅, 500 MHz) and ¹³C NMR (pyridine‑d₅, 125 MHz) data, see

Tables 2 and 3.

2.3.7. Cyclocarioside Z₁₅(7)

The separation and puriﬁcation method afforded 6.6 mg of a white

amorphous powder; [α _D²⁵

ꢀ 17.75 (c 0.25, MeOH); HRESIMS m/z

]

2.3.10. Cyclocarioside Z₁₈(10)

791.4917 [M+Na]⁺(calculated for C₄₂H₇₂O₁₂Na, 791.4921); for the ¹H

(pyridine‑d₅, 500 MHz) and ¹³C NMR (pyridine‑d₅, 125 MHz) data, see

Tables 2 and 3.

The separation and puriﬁcation method afforded 7.1 mg of a white

amorphous powder; [α _D²⁵

ꢀ 18.42 (c 0.25, MeOH); HRESIMS m/z

]

793.4707 [M+Na]⁺(calculated for C₄₁H₇₀O₁₃Na, 793.4717); for the ¹H

(pyridine‑d₅, 600 MHz) and ¹³C NMR (pyridine‑d₅, 150 MHz) data, see

Tables 2 and 3.

2.3.8. Cyclocarioside Z₁₆(8)

The separation and puriﬁcation method afforded 4.1 mg of a white

amorphous powder; [α _D²⁵

ꢀ 23.51 (c 0.30, MeOH); HRESIMS m/z

]

2.3.11. Cyclocarioside Z₁₉(11)

777.4769 [M+Na]⁺(calculated for C₄₁H₇₀O₁₂Na, 777.4765); for the ¹H

(pyridine‑d₅, 500 MHz) and ¹³C NMR (pyridine‑d₅, 125 MHz) data, see

The separation and puriﬁcation method afforded 7.5 mg of a white

amorphous powder; [α _D²⁵

ꢀ 20.47 (c 0.22, MeOH); HRESIMS m/z

]

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Fig. 2. Key HMBC and COSY correlations of compounds 1–11.

835.4818 [M+Na]⁺(calculated for C₄₃H₇₂O₁₄Na, 835.4820); for the ¹H

(pyridine‑d₅, 600 MHz) and ¹³C NMR (pyridine‑d₅, 150 MHz) data, see

Tables 2 and 3.

performed according to our previous report, are described in the SI (Text

S4) [26,27].

2.7. Reverse transcription-polymerase chain reaction (RT-PCR) assay for

2.4. Hydrolyses of new compounds to determine the linking sugars

active compound 2 on the mRNA expressions of iNOS, COX-2, NF-κB, IL-

6, IL-1β and TNF-

The hydrolysis, which was performed according to the previously

described method, and HPLC test are shown in the SI (Text S2) [24].

The detailed procedures used by the RT-PCR assay to study the

mRNA expression of iNOS, NF-κB, COX-2, IL-6, IL-1β and TNF-α were

2.5. Assay for the inhibitory effects on

dammarane saponins

α-glucosidase of the fractions and

performed according to a previous report and are described in the SI

(Text S5) [28].

The α-glucosidase test was performed according to the previously

described PNPG method and is shown in the SI (Text S3) [25].

2.8. Western blotting assay for active compound 2 on the protein

expressions of iNOS, NF-κB/p65 and COX-2

2.6. Assay for the inhibitory effects on NO production of the extracts and

dammarane saponins

The protein expression of iNOS, NF-κB/p65 and COX-2 was evalu-

ated by performing western blotting according to our previous report, as

detailed in the SI (Text S6) [29].

The RAW 264.7 cell viability and NO production tests, which were

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Fig. 3. ROESY correlations of compounds 1, 2, 4, 5, and 10.

2.9. Statistical analysis

Table 4

The HPLC retention time of sugar moieties of compounds 1–11.

Each experiment was repeated at least three times in triplicate. The

results are expressed as the mean ± SD. The differences were considered

statistically signiﬁcant when (**) p < 0.01 or (*) p < 0.05.

Compounds

Retention times (min)

Types of the monosaccharide

8.551

D-Qui

7.861, 8.554

7.597, 7.844

7.840, 8.537

7.845, 8.519

7.862

L-Ara, D-Qui

L-Ara, D-Glc

L-Ara, D-Qui

L-Ara

3. Results and discussion

3.1. Elucidating the structures of the new dammarane saponins

7.832, 8.498

7.876

L-Ara, D-Qui

L-Ara

Cyclocarioside Z₉(1) was determined to have the molecular formula

7.833, 8.505

7.531, 7.828

7.512, 7.824

7.537

L-Ara, D-Qui

L-Ara, D-Glc

₃₆H₆₂O₈by a positive sodium ion HRESIMS peak at 645.4342

D-Glc

L-Ara

D-Qui

[M+Na]⁺(calcd 645.4342 for C₃₆H₆₂O₈Na) (Fig. S1). The ¹H NMR data

(pyridine‑d₅, Table 1) of 1 exhibited eight methyl signals at δ_H0.87 (s,

30-CH₃), 1.00 (s, 28-CH₃), 1.26 (s, 29-CH₃), 1.91 (s, 27-CH₃), 1.11 (s,

18-CH₃), 1.44 (s, 21-CH₃), 1.40 (s, 19-CH₃), and 1.61 (d, 6^′-CH₃); one

pair of oleﬁnic protons at δ_H5.30 (br s, H-26a) and 4.98 (br s, H-26b);

and one sugar anomeric proton at δ_H4.93 (d, J = 7.6 Hz, H-1^′). The ¹³C

NMR (Fig. S3, pyridine‑d₅, Table 1) and DEPT (distortionless enhance-

ment by polarization transfer, pyridine‑d₅) data (Fig. S4) of 1 displayed

the existence of 36 carbon signals, in which there was one terminal

7.843

8.530

–

CH₂(16)CH₂(15) , which was conﬁrmed from the H– H correlations of

1, shows that one hydroxyl group was connected at position 11 instead

of position 12. Moreover, quinovopyranose was identiﬁed in the acid

hydrolysis solution of 1 by comparing with the authentic sample by

double bond signal at δ_C150.3 (C-25) and 110.6 (C-26) and one sugar

performing a HPLC assay (Table 4). The HMBC correlations between δ

anomeric carbon signal at δ 102.1 (C-1^′). In the H–¹H COSY (corre-

4.93 (td, J = 7.6 Hz, H-1^′) and δ 77.0 (C-11) indicated that the qui-

lation spectroscopy) spectrum of 1, the correlations of H-1/H-2/H-3, H-

novopyranosyl group was located at C-11 (Fig. 2). To determine the

absolute conﬁguration for chiral centers C-3/11/17/20/24 of 1, chem-

ical shift comparison for chiral centers (C-3/11/17/20/24) were

collected for both R- and S-enantiomer conﬁgurations. For analogous

dammaranes, the absolute conﬁgurations at C-3/11/17/20 were deter-

5/H-6/H-7, H-9/H-11/H-12/H-13/H-17/H-16/H-15, and H-22/H-23/

–

H-24 substantiated the existence of four fragments of CH₂CH₂CH

–

– –

–

CHCH₂CH₂

CHCHCH₂CHCHCH₂CH₂

and

CH₂CH₂CH

(Figs. 2 and S6). In the HMBC spectrum (Fig. 2 and S7, pyridine‑d₅), the

cross peaks from H-26 (δ 5.30, 4.98, br s) to C-24 (δ 76.3) and C-27 (δ

–

mined to be R with the signals at δ 82.4–82.5 (C-3), 76.3 (C-11), 45.0

–

19.0) suggested that the terminal double bond (

CH ) was con-

(C-17), 70.2 (C-20) and be S with the signals at δ_C73.2–73.3 (C-3), 72.8

(C-11), 51.0 (C-17), and 76.2 (C-20) [20,36]. The carbon signals of

compound 1 at δ_C75.7 (C-3), 77.0 (C-11), 50.5 (C-17), 74.4 (C-20)

suggested its chiral centers to be 3S, 11R, 17S, and 20S. Moreover,

25,26-en-24(R)-hydroxyl-20(S)-protopanaxadiol and 25,26-en-24(S)-

hydroxyl-20(S)-protopanaxadiol [30], whose structures, together with

their absolute conﬁgurations, had been identiﬁed by analyzing the

spectroscopic data as well as chemical methods, have the same chiral

centers at C-24 as 1 and could be used as model compounds to assign the

absolute conﬁguration of 1 by comparing their C-24 chemical shift

values. The ¹³C NMR datum at δ_C76.3 (C-24) of 1 more closely resem-

bled that at δ 76.4 (C-24) of 25,26-en-24(R)-hydroxyl-20(S)-proto-

panaxadiol tha^Cn that at δ_C76.0 (C-24) of 25,26-en-24(S)-hydroxyl-20

nected to C-25. The HMBC correlations from H-21-CH₃(δ 1.44, s) to C-

17 (δ_C50.5) substantiated that the side chain was locate^Hd at C-17. The

ROESY correlations of H-5/H-9/H-17/H-CH₃-21/H-CH₃-30 suggested

that H-5, H-9, H-17, CH₃-21, CH₃-24, and CH₃-30 are α-oriented. The

–

correlations of H-3/H-13/H-CH₃18/H-CH₃-19 in the ROESY spectrum

indicated that H-3, H-13, H-CH₃-18, and H-CH₃-19 are in the β-orien-

tation (Figs. 3 and S8). Detailed analyses of the abovementioned NMR

data showed that the data of 1 was highly similar data to those of 25,26-

en-24(R)-hydroxyl-20(S)-protopanaxadiol, suggesting that 1 includes

the basic protopanaxadiol triterpenoid skeleton, differing from 25,26-

en-24(R)-hydroxyl- 20(S)-protopanaxadiol only in the signals at posi-

tions 11 and 12 and possessing an extra quinovopyranose (Tables 1 and

–

3) [30]. The fragment of

CH(9)CH(11)CH₂(12) CH(13)CH(17)

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

(S)-protopanaxadiol [30]. Consequently, the structure of 1 was deduced

as (20S,24R)-dammarane-24-hydroxyl-11-O-β-^D-quinovopyranosyl and

named cyclocarioside Z₉.

β-oriented. The ROESY cross peaks of H-5/H-9/H-17/H-CH₃-21/H-CH₃-

30 suggested that H-5, H-9, H-17, CH₃-21, CH₃-24, and CH₃-30 are

-oriented (Fig. 3). Furthermore, to determine the absolute conﬁgura-

Cyclocarioside Z₁₀(2) was established to have the molecular formula

C₄₃H₇₄O₁₄by the negative ion HRESIMS peak at m/z 813.4999 [Mꢀ H]^ꢀ

(calcd 813.5000 for C₄₃H₇₃O₁₄) (Fig. S9). The ¹H NMR data (pyr-

idine‑d₅, Table 1, Fig. S10) of 2 showed eight methyl singlets at δ_H0.76

(s, 30-CH₃), 0.93 (s, 28-CH₃), 1.08 (s, 18-CH₃), 1.24 (s, 29-CH₃), 1.33 (s,

19-CH₃), 1.25 (s, 21-CH₃), 1.29 (s, 27-CH₃), and 1.50 (s, 26-CH₃), a

doublet methyl signal at δ 1.55 (d, J = 5.9 Hz, 6^′’-CH₃) and two

glycosyl anomeric protons a^Ht δ_H5.49 (m, H-1^′) and 5.03 (d, J = 7.7 Hz,

H-1^′’). The ¹³C NMR (pyridine‑d₅, Table 3, Fig. S11) and DEPT (dis-

tortionless enhancement by polarization transfer, pyridine‑d₅) spectra

displayed the existence of eight methyl carbon signals at δ 17.4 (C-18),

17.0 (C-19), 27.0 (C-21), 27.7 (C-26), 29.2 (C-27), 23.2 (C^C-28), 29.2 (C-

tion for chiral centers C-3/11/17/20 of 3, the comparisons of the carbon

signals of compound 3 at δ_C79.5 (C-3), 77.1 (C-11), 50.8 (C-17), 74.7 (C-

20) to those of analogous dammaranes demonstrated its chiral centers to

be 3R, 11R, 17S, and 20S [20,36]. Thus, the structure of 3 was deduced

(20S,23E)-dammarane-11-O-β-^D-glucopyranosyl-3-O-α-L-arabino-

furanoside and named cyclocarioside Z₁₁

Cyclocarioside Z₁₂(4) was separated as a white amorphous powder.

The molecular formula of C₃₅H₅₈O₁₁of 4 was determined by a positive

sodium ion HRESIMS peak at m/z 677.3880 [M+Na]⁺(calcd 677.3877

for C₃₅H₅₈O₁₁Na) (Fig. S25). The ¹H- and ¹³C NMR data of 4 (pyr-

idine‑d₅, Tables 1 and 3) were similar to those of cyclocarioside P [34],

with the exception of the resonances of the side chain and connecting

positions of the glycosyl units, suggesting that 4 also possessed a

dammarane-type triterpenoid skeleton. The side chain is cleaved be-

tween C-20 and C-22, which is followed by the formation of a keto group

at C-20, as conﬁrmed by the HMBC correlations of H-21 (δ_H2.07) with

29) and 17.3 (C-30) and two glycosyl anomeric carbon signals δ 106.8

(C-1^′) and 101.7 (C-1^′’). A dammarane-type triterpenoid glycoside

structure was established for compound 2, which is similar to cyclo-

carioside H, except for the signals of the side chain and connecting po-

sition of the glycosyl unit [31]. In the ¹H–¹H COSY spectrum of 2, the

correlations of H-9/H-11/H-12/H-13/H-17 and H-22/H-23/H-24 sub-

C-17 (δ 54.2) and C-20 (δ 211.3). The ¹H–¹H correlation spectroscopy

(COSY)^Cresults of H-9/H-11/H-12/H-13/H-17/H-16/H-15 in 4 demon-

–

stantiated the existence of the two fragments CHCHCH₂CHCH and

strated the presence of one

CHCHCH₂CHCHCH₂CH₂fragment

–

CH₂CH₂CH (Figs. 2 and S14). The HMBC peak from H-21-CH₃(δ_H

(Figs. 2 and S30). Furthermore, ^L-arabinose and D-quinovose were

conﬁrmed in the acid hydrolysis solution of 4 by comparing with the

authentic sugars in the HPLC assay (Table 4). The HMBC correlations

from the anomeric protons H-1^′(δ 5.56, br s) to C-3 (δ 79.7) and H-1^′’

1.25, s) to C-17 (δ 51.3) suggested that the side chain was connected to

C-17. The connect^Cions of the two hydroxyl groups at C-24 and C-25 were

determined by the key HMBC correlations of H-26 (δ_H1.50, s) to C-24

(δ 70.3), C-25 (δ 75.0), C-27 (δ 29.2) and H-27 (δ 1.29, s) to C-25 (δ

(δ_H4.87, dd) to C-11 (δ 76.6) afﬁrmed that the two glycosyl moieties

75.0) and C-26 (δ 27.7). Furthermore, L-arabinose and D-quinovose

are linked at C-3 and C-11, respectively (Fig. 2). The β-orientations of H-

3, H-11, H-13, H-CH₃-19, and H-CH₃-29 were then veriﬁed by the

ROESY peaks of H-13/H-CH₃-18/H-CH₃-19. Similarly, the diagnostic

ROESY peaks of H-17 and H-30 demonstrated that H-17 and H-30 are

were identiﬁed in the acid hydrolysis solution of 2 by comparing with

authentic sugar samples in the HPLC assay (Table 4). The HMBC cor-

relations from the anomeric protons H-1^′(δ_H5.49, m) to C-3 (δ 79.9)

and H-1^′’ (δ 5.03, d) to C-11 (δ 77.3) suggested that L-arabinose and D-

α-oriented (Figs. 3 and S32). In addition, to determine the absolute

quinovose were situated at posi^Ctions 3 and 11, respectively. The HMBC

conﬁguration for chiral centers C-3/11/17/20 of 4, the comparisons of

the carbon signals of compound 4 at δ_C79.7 (C-3), 76.6 (C-11), 50.8 (C-

17) to those of analogous dammaranes suggested its chiral centers to be

3R, 11R, and 17S [20,36]. Thus, compound 4 was elucidated as

peak from H-5 -CH₃COO (δ_H1.96, s) to C-5^′(δ_C65.5) demonstrated

′

–

′

–

that CH₃COO was linked to position 5 (Fig. 2). Moreover, the diag-

nostic ROESY correlations of H-3/H-11/CH₃-19/CH₃-29 suggested that

H-3, H-11, CH₃-19, and CH₃-29 are β-oriented. The correlations of H-5/

(3α,11α)-11-O-β-D-quinovopyranosyl-3-O-α-L-arabinofuranoside-

–

H-9/H-17/H-CH₃21/H-CH₃-30 in the ROESY spectrum indicated that

23,24,25,26,27-hexanor-dammarane-20-one and named cyclocarioside

H-5, H-9, H-17, CH₃-21, CH₃-24, and CH₃-30 are

-oriented (Fig. 3).

Z₁₂

Moreover, to determine the absolute conﬁguration for chiral centers C-

3/11/17/20/24 of 2, the comparisons of the carbon signals of com-

pound 2 at δ_C79.9 (C-3), 77.1 (C-11), 50.8 (C-17), 76.3 (C-20) to those

of analogous dammaranes indicated its chiral centers to be 3R, 11R, 17S,

and 20S [20,36]. In addition, the ¹³C NMR datum at δ_C70.3 (C-24) of 2

The molecular formula of cyclocarioside Z₁₃(5) was deduced to be

₄₂H₇₂O₁₂due to the positive sodium ion HRESIMS peak at m/z

791.4917 [M+Na]⁺(calcd 791.4921 for C₄₂H₇₂O₁₂Na) (Fig. S33). The

¹H NMR data (pyridine‑d₅, Table 1) of 5 showed one double bond

hydrogen signal at δ_H5.15 (dt, J = 9.0, 1.3 Hz, 24-H). The detailed

analysis of the NMR data showed that 5 was similar to compound 12,

except for the number and positions of the double bonds and the sub-

stitution of the methoxy group on the side chain, indicating that 5 also

possesses a dammarane skeleton. Compound 5 possessed a double bond

between positions 24 and 25 and a methoxy group linking to position 23

in the side chain. ^L-arabinose and D-quinovose were conﬁrmed in the

acid hydrolysis solution of 5 by comparing with the authentic sugars in

the HPLC assay (Table 4). The ¹H–¹H COSY correlations of H-9/H-11/H-

more closely resembled that at δ 70.8 (C-24) of 24R,25-dihydroxyl

protopanaxadiol than that at δ_C77.1 (C-24) of cyclofoetoside B [32,33].

Thus, compound 2 was deduced as dammarane-(20S,24R,25)-penthy-

droxyl-11-O-β-^D-quinovopyranosyl-3-O-(5^′-O-acetyl)-

α-L-arabinofurano-

side and named cyclocarioside Z₁₀

Cyclocarioside Z₁₁(3) was determined to have the molecular for-

mula C₄₁H₆₈O₁₂by a positive sodium ion HRESIMS peak at m/z

775.4613 [M+Na]⁺(calcd 775.4608 for C₄₁H₆₈O₁₂Na) (Fig. S17).

Detailed analyses of the H- and ¹³C NMR data (pyridine‑d₅, Tables 1

12/H-13/H-17/H-16/H-15 and H-22 (δ 2.14, m, 1.67, m)/H-23 (δ

and 3) demonstrated that 3 showed features that were closely similar to

4.39, m)/H-24 (δ_H5.15, dt, 9.0, 1.3) demonstrated the presence of th^He

–

– –

–

those of cyclocarioside P, except the quinovopyranosyl moiety was

fragments CHCHCH₂CHCHCH₂CH₂and CH₂CHCH (Figs. 2 and

S38). The connection of the side chain to position 17 was determined by

the key HMBC correlations of δ 1.43 (s, H-21) to δ 50.8 (C-17), δ 74.6

replaced by a glucopyranoside moiety [34]. α-L-Arabinopyranose and

β-^D-glucopyranoside were conﬁrmed in the hydrolysate of 3 by

comparing with the authentic samples in the HPLC assay (Table 4). The

¹H–¹H COSY correlations of H-9/H-11/H-12/H-13/H-17 and H-22/H-

23/H-24 of 3 demonstrated the presence of the two fragments

(C-20), and δ_C45.1 (C-22), those of δ_H1.69 (s, H-26) to δ_C127.3 (C-24),

δ 135.8 (C-25), and δ 26.1 (C-27), and the HMBC correlation of δ 3.19

(s, 23-OCH₃) to δ_C76^C.1 (C-23) (Fig. 2). The diagnostic ROESY correla-

–

– –

–

CHCHCH₂CHCH and CH₂CHCH (Figs. 2 and S22). The HMBC

tion of H-23 and H-21 suggested that CH₃-21 and H-23 are α-oriented,

correlations from the anomeric protons H-1^′(δ 5.52, br s) to C-3 (δ

indicating that the methoxy group at position 23 in the side chain is

β-oriented (Fig. 3). In addition, compound 5 and cycloarta-24-ene-

3β,23-diol possess almost the same fragments on the side chain.

79.5) and H-1^′’ (δ_H5.06, br s) to C-11 (δ 77.1)^Hsuggested that the two

sugar units were situated at positions 3^Cand 11, respectively (Fig. 2).

Furthermore, in the diagnostic ROESY of 3, the correlations of H-3/H-

11/CH₃-19/CH₃-29 indicated that H-3, H-11, CH₃-19, and CH₃-29 are

Considering their ¹³C NMR data, the ¹³C NMR data of δ 127.3 (C-24)

and δ 135.8 (C-25) of 5 more closely resembled those of δ 128.4 (C-24)

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

and δ_C135.6 (C-25) in 23β-cycloarta-24-ene-3β,23-diol than those of δ_C

quinovopyranosyl-3-O-

Z₁₅

-L-arabinofuranoside and named cyclocarioside

129.1 (C-24) and δ 133.8 (C-25) in 23

-cycloarta-24-ene-3β,23-diol

[35]. Moreover, to^Cdetermine the absolute conﬁguration for chiral

centers C-3/11/17/20/23 of 5, the comparisons of the carbon signals of

compound 5 at δ_C79.6 (C-3), 76.8 (C-11), 50.8 (C-17), 74.6 (C-20) to

those of analogous dammaranes demonstrated its chiral centers to be 3R,

11R, 17S, and 20S [20,36]. Correspondingly, the absolute conﬁguration

of position 23 of 5 was unambiguously conﬁrmed to be R. Therefore, the

The molecular formula of cyclocarioside Z₁₆(8) was determined to

be C₄₁H₇₀O₁₂by the positive sodium ion HRESIMS peak at m/z

777.4769 [M+Na]⁺(calcd 777.4765 for C₄₁H₇₀O₁₂Na) (Fig. S57),

which is 14 Da less than that of 7. The detailed analysis of the ¹H- and

¹³C NMR (pyridine‑d₅, Tables 2 and 3) data showed that 8 was very

similar to 7, except the quinovopyranosyl moiety was replaced by an

arabinopyranosyl moiety at C-11 in 8. Moreover, the hydrolysate of

compound 8 obtains ^L-arabinose, which was determined by comparing

with the authentic sample in the HPLC test (Table 4). Accordingly, the

structure of 5 was deduced as (20S,23R)-dammarane-(3

,11

)-23-

and

methoxy-11-O-β-^D-quinovopyranosyl-3-O- -L-arabinofuranoside

named cyclocarioside Z₁₃

The molecular formula of cyclocarioside Z₁₄(6) was deduced to be

₄₁H₇₀O₁₂due to the positive sodium ion HRESIMS peak at m/z

structure of 8 was deduced as (23E,20S)-dammarane-(3

droxyl-11-O- -L-arabinopyranosyl-3-O- -L-arabinofuranoside

named cyclocarioside Z₁₆

,11

)-25-hy-

and

777.4768 [M+Na]⁺(calcd 777.4765 for C₄₁H₇₀O₁₂Na) (Fig. S41),

which is 14 Da less than that of 5. The ¹H- and ¹³C NMR (pyridine‑d₅,

Tables 2 and 3) data of 6 are similar to those of 5, but 6 differed from 5

due to the absence of a methyl group (ꢀ CH₃) at δ_H1.60 (d, J = 5.9 Hz).

The molecular formula of cyclocarioside Z₁₇(9) was determined to

be C₄₄H₇₄O₁₃by the positive sodium ion HRESIMS peak at m/z

833.5028 [M+Na]⁺(calcd 833.5027 for C₄₄H₇₄O₁₃Na) (Fig. S65),

which is 42 Da more than that of 7. Detailed comparisons of the ¹H- and

¹³C NMR (pyridine‑d₅, Tables 2 and 3) data showed that 9 was highly

Comparing the H- and ¹³C NMR (Tables 1 and 3) and HRESIMS data

between 6 and 5 revealed a signiﬁcant difference in the data obtained

for the sugars and the existence of another arabinose instead of a methyl

pentacarbonose in 6, indicating that 6 also possesses a dammarane

skeleton. ^L-Arabinose was conﬁrmed in the acid hydrolysis solution of 6

by comparing with the authentic sugars in the HPLC assay (Table 4). The

arabinopyranoses are linked to positions 3 and 11 in 6, which was

further veriﬁed by the key HMBC peaks from the anomeric proton H-1^′

(δ 5.49, m) to C-3 (δ 79.6) and H-1^′’ (δ 4.81, d) to C-11 (δ 76.9)

analogous to 7, except for the replacement of an acetyl moiety

′

–

(CH₃CO ) at C-5 of L-arabinose linked to C-3 in 9, which was conﬁrmed

by the HMBC correlations of H-5^′(δ 4.81, dd, 11.6, 3.0; δ 4.62, m)

–

with C O (CH CO , δ_C171.1) (Fig. 2). Besides, the glycosyl moieties

were determined by acid hydrolysis and HPLC analysis with ^L-arabinose

and ^D-quinovose standard (Table 4). Accordingly, the structure of 8 was

deduced as (23E,20S)-dammarane-(3α,11α)-25-hydroxyl-11-O-α-L-ara-

(F^Hig. 2). In addition, to determine the absolute conﬁguration for chiral

binopyranosyl-3-O-

Z₁₆

α-L-arabinofuranoside and named cyclocarioside

centers C-3/11/17/20/23 of 6, the comparisons of the carbon signals of

compound 6 at δ 79.6 (C-3), 76.9 (C-11), 50.7 (C-17), 74.6 (C-20) to

Cyclocarioside Z₁₈(10) yielded a positive sodium ion HRESIMS peak

those of analogous dammaranes indicated its chiral centers to be 3R,

11R, 17S, and 20S [20,36]. The absolute conﬁguration of position 23 of

6 was unambiguously conﬁrmed to be R by comparing the ¹³C NMR data

of δ 127.4 (C-24) and δ 135.8 (C-25) of 6 with the ¹³C NMR data of δ

at m/z 793.4717 [M+Na]⁺(calcd 793.4707 for C₄₁H₇₀O₁₃Na)

(Fig. S73), which revealed the molecular formula of C₄₁H₇₀O₁₃

Comparing the ¹H- and ¹³C NMR data (pyridine‑d₅, Tables 2 and 3) of 10

with those of the related compounds in the literature indicated that the

data of 10 were highly similar to those of cyclocarioside I, [34] except

for the signals replacing ^D-glucopyranose with D-quinovose and the

connecting position of ^D-glucopyranose (pyridine‑d₅, Tables 2 and 3).

The ¹H–¹H COSY correlations of H-9/H-11/H-12/H-13 of 10 demon-

127^C.3 (C-24) and δ_C135.8 (C-25) of 5. Accordingly, the structure of 6

was deduced as (20S,23R)-dammarane-(3α,11α)-23- methoxy-11-O-α-L-

arabinopyranosyl-3-O-

-L-arabinofuranoside and named cyclocarioside

Z₁₄

–

C₄₂H₇₂O₁₂was determined to have the molecular formula cyclo-

strated the presence of the fragment CHCHCH₂CH (Figs. 2 and S78).

Furthermore, the diagnostic ROESY correlations of H-3 and H-11 to H-

19 suggested that H-3, H-11 and H-19 are β-oriented. Similarly, the

diagnostic ROESY peaks of H-17 and H-30 indicated that H-17 and H-30

carioside Z₁₅(7) by the positive sodium ion HRESIMS peak at m/z

791.4917 [M+Na]⁺(calcd 791.4921 for C₄₂H₇₂O₁₂Na) (Fig. S49) and

possesses the same formula as 5. Compound 7 had NMR (pyridine‑d₅,

Tables 2 and 3) signals identical to those of 5, as revealed by comparing

their ¹H- and ¹³C NMR data. The sugar units were conﬁrmed in the acid

hydrolysis solution of 5 by comparing with the authentic ^L-arabinose

and ^D-quinovose in the HPLC assay (Table 4). Small differences between

7 and 5 existed in the ¹H- and ¹³C NMR data (pyridine‑d₅) of positions

23, 24, and 25. The ¹H NMR data of H-23 and 24 shifted from δ_H4.39

(m) and 5.15 (dt, 9.0, 1.3) in 5 (pyridine‑d₅, Table 1) to δ_H5.98 (ddd,

15.8, 8.7, 6.2) and 5.65 (d, 15.8) in 7 (pyridine‑d₅, Table 2). Moreover,

the ¹³C NMR data of 23, 24, and 25 changed from δ_C76.1, 127.3, and

135.8 in 5 to δ 127.8, 138.6, and 75.3 in 7, respectively (pyridine‑d₅,

Table 3). The ¹^CH NMR spectrum of 7 showed a pair of coupled oleﬁnic

protons at δ 5.98 and 5.65, whose coupling constant of J = 15.8 Hz

showed that^Hthe conﬁguration of the double bond was E. The ¹H–¹H

are α-oriented (Fig. 3). In addition, α-L-arabinopyranose and β-D-gluco-

pyranose were conﬁrmed by the hydrolysis and HPLC test (Table 4). The

HMBC correlations from the anomeric proton δ_H5.53 (br s, H-1^′) to δ_C

79.6 (C-3) and δ 5.16 (d, H-1^′’) to δ 77.3 (C-11) suggested that

α-L-

arabinopyranose^Hand β-^D-glucopyranoside are connected to C-3 and C-

11, respectively (Fig. 2). In addition, to determine the absolute conﬁg-

uration for chiral centers C-3/11/17/20/24 of 10, the comparisons of

the carbon signals of compound 10 at δ 79.6 (C-3), 77.3 (C-11), 49.6 (C-

17), 86.8 (C-20), 84.6 (C-24) to those of analogous dammaranes

demonstrated its chiral centers to be 3R, 11R, 17S, 20S, and 24S [20,36].

Finally, the structure of 10 was deduced as (20S,24R)-epoxydammara-

α,11α)-25-hydroxyl-11-O-β-D-glucopyranoside-3-O-α-L-arabinofur-

anoside, and 10 was named cyclocarioside Z₁₈

COSY peaks of H-22 (δ 2.61, dd, 13.5, 6.2; δ 2.43, dd, 13.5, 8.7)/H-23

Cyclocarioside Z₁₉(11) yielded a positive sodium ion HRESIMS peak

at m/z 835.4818 [M+Na]⁺(calcd 835.4820 for C₄₃H₇₂O₁₄Na)

(Fig. S81), demonstrated a molecular formula of C₄₃H₇₂O₁₄and is 42 Da

(δ_H5.98, ddd, 15.8, 8.7, 6.2)/H-24 (δ_H5.65,^Hd, 15.8) of 7 demonstrated

–

the presence of the fragment CH₂CHCH (Figs. 2 and S54). In addi-

tion, the HMBC correlations from 25-OCH₃(δ_H3.20, s) to C-25 (δ_C75.3)

more than that of 10. Analysis of the H- and ¹³C NMR (pyridine‑d₅,

and H-26 (δ 1.31, s) to C-24 (δ 138.6), C-25 (δ 75.3) and C-27 (δ

Tables 2 and 3) data showed that 11 was analogous to 10, except for the

replacement of an acetyl group at C-5^′of L-arabinose linking to C-3 in 11,

26.9) (Fig. 2^H) indicated that the methoxy group is linked to position 25.

Furthermore, to determine the absolute conﬁguration for chiral centers

C-3/11/17/20 of 7, the comparisons of the carbon signals of compound

which was conﬁrmed by the HMBC correlations of H-5^′(δ 4.81, dd,

–

11.5, 3.0; δ 4.61 m) with C O (CH CO , δ 171.1) (Fig. 2). In addi-

7 at δ 79.6 (C-3), 76.8 (C-11), 50.7 (C-17), 74.4 (C-20) to those of

tion,

-L-arabinopyranose and β-D-glucopyra^Cnose were conﬁrmed by

analogous dammaranes indicated its chiral centers to be 3R, 11R, 17S,

and 20S [20,36]. Therefore, the structure of 7 was deduced as

acid hydrolysis and HPLC analysis (Table 4). Therefore, the structure of

11 was deduced as (20S,24R)-epoxydammarane-(3

α,11

α)-25-hydroxyl-

(23E,20S)-dammarane(3

,11

α)-25-hydroxyl-11-O-β-D-

11-O-β-^D-glucopyranoside-3-O-(5^′-O-acetyl)-

α-L-arabinofuranoside and

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Fig. 5. Inhibitory effects against NO of the different fractions in LPS-mediated

RAW 264.7 cells.

Fig. 4. Inhibitory effects against α-glucosidase of the different fractions.

Table 6

Table 5

Inhibitory Activities on NO of Compounds 1–20 in LPS-induced RAW 264.7 Cell

Inhibitory activities on

-Glucosidase of Compounds 1-20.^a

Compounds

IC₅₀

(

mol)

Compounds

IC₅₀(μmol)

Compounds

IC₅₀

(

Compounds

IC₅₀(μM)

282.23 ± 1.84

257.74 ± 1.55

290.84 ± 0.99

578.77 ± 0.94

330.29 ± 1.17

369.54 ± 0.71

447.72 ± 0.62

>500

274.77 ± 1.13

287.71 ± 1.08

306.99 ± 0.79

383.50 ± 0.82

>500

9.10 ± 0.11

9.02 ± 0.14

>40

30.76 ± 0.43

23.74 ± 0.12

24.18 ± 0.66

30.19 ± 0.17

>40

13.94 ± 0.17

12.57 ± 0.41

16.41 ± 0.12

15.42 ± 0.75

15.86 ± 0.44

26.47 ± 0.78

27.19 ± 0.51

>500

478.15 ± 1.44

>500

23.17 ± 0.57

30.44 ± 0.19

451.22 ± 1.63

>500

acarbose ^b

359.36 ± 0.65

Dexamethasone ^b

9.17 ± 0.41

>500

All values are means of three independent experiments.

Acarbose, a hypoglycemic drug used as positive control.

All values are means of three independent experiments.

Dexamethasone, an anti-inﬂammatory agent used as positive control.

named cyclocarioside Z₁₉

values of the positive control acarbose. Dammarane saponins 4, 7, 9, and

18 had moderate effects, with IC₅₀values ranging from 451.22 M to

478.15 M. Dammarane saponins 8, 10–11, 16–17 and 19–20 exhibited

By comparing the NMR (¹H and ¹³C) and MS data to those reported in

the literature, the known dammarane saponins were identiﬁed as

cyclocarioside P (12) [34], cyclocarioside O (13) [34], cyclocarioside L

(14) [36], cyclocarioside M (15) [36], cyclocarioside H (16) [34],

cyclocarioside B (17) [37], cyclocarioside J (18) [37], cyclocarioside I

(19) [14], and octyl ursolate (20) [38].

no inhibition against α-glucosidase. The results showed that there are

abundant dammarane saponins in C. paliurus eaves, showing α-glucosi-

dase inhibition.

3.3. The extracts and dammarane saponins reduce the production of NO

in LPS-mediated RAW 264.7 cells

3.2. The extracts and dammarane saponins inhibit the activity of

-glucosidase

The RAW 264.7 cell viability assay showed that the extracts and all of

The inhibitory activities towards

-glucosidase of the PE, EtOAc, n-

the dammarane saponins were not cytotoxic at a concentration of 40

and the cells had a survival rate up to 90%.

μM,

butanol, and H₂O fractions obtained from the 75% EtOH/H₂O extract

are shown in Fig. 4. The results show that the EtOAc fraction (Fr. EtOAc)

The inhibitory effects on NO production of the 75% EtOH/H₂O

extract (crude Ext) and the PE fraction (Fr. PE), Fr. EtOAc, Fr. n-butanol,

and Fr⋅H₂O from the crude Ext are shown in Fig. 5. Fr. EtOAc strongly

inhibited the production of NO in LPS-mediated RAW 264.7 cells and

exhibited an inhibitory effect equal to that of dexamethasone (positive

control). The active anti-inﬂammatory Fr. EtOAc was further isolated to

obtain 20 dammarane saponins. The inhibitory effects of dammarane

saponins 1–20 against LPS-mediated NO production in RAW 264.7 cells

are listed in Table 6. The results show that dammarane saponins 1 and 2

obtained from the 75% EtOH/H₂O extract strongly inhibited α-glucosi-

dase, in a manner comparable to the inhibition effect of the positive

control acarbose.

The inhibitory effects of dammarane saponins 1–20 isolated from the

active EtOAc fraction on α-glucosidase are shown in Table 5. Dammar-

ane saponins 1–3, 5, and 12–14 showed a strong inhibitory activity

against

-glucosidase, with IC₅₀values ranging from 257.74

M. These values are superior to those of the positive control,

M). Dammarane saponin 2 revealed the strongest

-glucosidase, with an IC₅₀value of 257.74

M. Dammarane saponins 6 and 15 had good effects, with IC₅₀values of

369.54 M and 383.50 M, respectively, which are equal to the IC₅₀

μM to

290.84

acarbose (359.36

strongly suppressed NO production, with IC₅₀values of 9.10

9.02 M, respectively, and 1 and 2 were better than dexamethasone,

with an IC₅₀value of 9.17 M. Dammarane saponins 5–9 had moderate

effects on inhibiting NO production, with IC₅₀values ranging from

μM and

inhibitory activity against

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Fig. 6. Effect of compound 2 on LPS-mediated mRNA expression of IL-6, IL-1β, iNOS, NF-κB, COX-2 and TNF-

. RAW264.7 cells were pre-incubated with various

concentrations of compound 2 (5, 10 and 20 µM) for 1 h followed by stimulation with LPS (1 µg/mL) for 24 h. The mRNA expression of iNOS (6A), NF-κB (6B), COX-2

(6C), IL-6 (6D), IL-1β (6E) and TNF-

α (6F) was analyzed using Real-time RT-PCR. The data are presented as mean ± SDs (n = 3), **p < 0.01 represent signiﬁcance

when compared to LPS-only treated cells.

12.57

inhibited NO production, with IC₅₀values ranging from 23.17

30.76 M. Dammarane saponins 3–4, 12, 17 and 20 did not inhibit NO

M to 16.41

M. Dammarane saponins 10–11, 13–16 and 18–19

For example, 4, which has a side chain that is cleaved between C-20 and

C-22, showed a lower inhibitory activity against -glucosidase than

M to

dammarane saponins 5–9 and 13–16 and exhibited no activity against

NO production. In addition, compounds with many double bonds on the

side chains more weakly inhibited NO production than did compounds

with few double bonds. For example, 3, 12, and 13 with two double

bonds on the side chains exhibited weak or no activity against NO

production, with IC₅₀values above 40 μM. The results demonstrated

that there are abundant dammarane saponins in C. paliurus leaves that

inhibit NO production.

Although dammarane saponin 1 has a hydroxyl group linked to po-

sition 3, all other dammarane saponins, 2–20, have arabinose or its

derivatives linked to position 3. The differences between these com-

pounds are mainly in the sugars linked to position 11 and the changes in

the side chains. Comparing the structures of these dammarane saponins

production. In summary, the

α-glucosidase inhibitory and anti-

inﬂammatory activities of these dammarane saponins are related to

the kind of glycosyl unit substituted at position 11, the number of hy-

droxyl groups, the epoxy ring, the cleavage of the side chain, and the

number of double bonds on the side chain.

with their inhibitory activities towards α-glucosidase and NO production

revealed that the number of hydroxyl groups in the isolate side chains

affects their inhibitory activity. Dammarane saponins 1 and 2, which

have many hydroxyl groups in their side chains, showed a better activity

than the compounds with fewer hydroxyl groups. Moreover, an epoxy

3.4. Active dammarane saponin 2 inhibits the mRNA expression of iNOS,

COX-2, NF-κB, IL-6, IL-1β and TNF-

ring on the side chain seemed to be related to

-glucosidase and NO

-glucosidase and

LPS alone markedly increased the mRNA expression of iNOS

production potency. For example, the inhibition of

(Fig. 6A), NF-κB (Fig. 6B), COX-2 (Fig. 6C), IL-6 (Fig. 6D), IL-1β (Fig. 6E)

NO production of 1, 2, 5, 6, 7, 12, and 13 with no ring in the side chain

were greater than those of 10, 11, 16, 17, and 19 with epoxy rings on the

side chains. Furthermore, the kinds of glycosyl units signiﬁcantly

and TNF-α (Fig. 6F) in LPS-mediated RAW 264.7 cells, as shown in

Fig. 6. However, secretions of these inﬂammatory cytokines all

decreased after pretreatment with compound 2 at concentrations of 20,

inﬂuenced the inhibitory effects on

α-glucosidase and NO production.

10, and 5 μM. Generally, the results suggested that compound 2 can

Dammarane saponins 6, 8, 13, and 18, with an arabinopyranosyl unit at

position 11, more strongly inhibited NO production than their corre-

sponding dammarane saponins 5, 7, 12, and 17 with a quinovopyr-

anosyl unit at position 11. In addition, the inhibition of NO production

seems to be related to whether there is a full side chain at position 17.

inhibit the mRNA expression of iNOS, NF-κB, COX-2, IL-6, IL-1β and

TNF- in a concentration-dependent manner in LPS-mediated RAW

264.7 cells.

C. Li et al.

Bioorganic Chemistry 111 (2021) 104847

Fig. 7. Effects of compound 2 at concentrations of 5, 10, and 20

M on iNOS, COX-2, and NF-κB/p65 expression in LPS-mediated RAW 264.7 cells. β-Actin served as

the loading control. ** p < 0.01 compared with cells treated with LPS, ^###p < 0.001 compared with control that was activated without LPS.

3.5. Active dammarane saponin 2 inhibits the protein expression of iNOS,

number of double bonds in the side chain. These ﬁndings demonstrated

that C. paliurus leaves contain abundant dammarane saponins with

NF-κB/p65 and COX-2

-glucosidase inhibitory and anti-inﬂammatory activities, which could

Mediated by LPS, the protein expression levels of iNOS, NF-κB/p65,

and COX-2 were signiﬁcantly higher than the levels of the control cells

(Fig. 7). After treatment with 2, the protein expression levels of iNOS,

NF-κB/p65, and COX-2 were signiﬁcantly suppressed. Compound 2

inhibited the protein expression levels of iNOS, NF-κB/p65, and COX-2

in LPS-mediated RAW 264.7 cells in a dose-dependent manner.

be meaningful for discovering new antidiabetic agents.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

4. Conclusions

Diabetes mellitus is caused by chronic inﬂammation and affects

millions of people worldwide. C. paliurus leaves have been widely used

in traditional folk tea as a remedy for diabetes. An extract of C. paliur-

us leaves showed signiﬁcantly reduced blood glucose, an improved

glucose tolerance, and attenuated damage to pancreatic islets. However,

the possible antidiabetic constituents and mechanisms remain unclear.

Acknowledgments

Authors (J. Li. and X. S. Huang) acknowledge the following grants for

funding this project: National Natural Science Foundation of China

(32060097); The Natural Science Fund of Guangxi Province

(2018GXNSFDA050007,

2018GXNSFAA050042,

and

2018GXN

The α-glucosidase inhibitory- and anti-inﬂammatory-guided separation

SFAA281102);TheOpenResearchFundprogramoftheKeyLaboratoryfor

the Chemistry and Molecular Engineering of Medicinal Resources

(CMEMR2020-A04); The Fund of Guilin Scientiﬁc Research and Technol-

ogy Development Program (20190205).

of the C. paliurus leaves led to the identiﬁcation of 20 dammarane sa-

ponins, including eleven new dammarane saponins (1–11). Bioassays

demonstrated that dammarane saponins 1–3, 5, and 12–14 showed

potent inhibitory effects against α-glucosidase and NO production in

LPS-mediated RAW 264.7 cells. The most active compound (2) signiﬁ-

cantly downregulated the mRNA expression of iNOS, COX-2, IL-1β, NF-

Appendix A. Supplementary material

κB, IL-6 and TNF-α and markedly suppressed the protein expression of

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.104847.

iNOS, NF-κB/p65, and COX-2 in LPS-mediated RAW 264.7 cells, sug-

gesting that compound 2 produced -glucosidase inhibitory and anti-

inﬂammatory activities by inhibiting NF-κB signaling pathways. Dam-

marane glucoside 2 exhibited the strongest -glucosidase inhibitory and

anti-inﬂammatory activities. In addition, the structure–activity rela-

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