18α-Glycyrrhetinic acid monoglucuronide as an anti-inflammatory agent through suppression of the NF-κB and MAPK signaling pathway

Article abstract of DOI:10.1039/c7md00210f

Based on the SAR analysis of glycyrrhizin, 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) with strong inhibition against LPS-induced NO and IL-6 production in RAW264.7 cells was discovered. Western blotting and immunofluorescence results showed that 18α-GAMG reduced the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in the LPS-stimulated RAW264.7 cells. Further in vivo results showed that 18α-GAMG could significantly improve the pathological changes of CCl₄-induced hepatic fibrosis.

Full text of DOI:10.1039/c7md00210f

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DOI: 10.1039/C7MD00210F
1
8αꢀGlycyrrhetinic
acid
monoglucuronide
as
antiꢀinflammatory agent through suppression of NFꢀκB and
MAPK signaling pathway†
a
b
a
a
*c
*a
Bo Li, Yongan Yang, Liuzeng Chen, Shichao Chen, Jing Zhang, and Wenjian Tang
a
School of Pharmacy, Anhui Medical University, Hefei 230032, China. E-mail:
ahmupharm@126.com; Fax & Tel: (+86) 551-65161115.
b
Elion Nature Biological Technology Co., Ltd, Nanjing 210038, China.
c
Anhui Prevention and Treatment Center for Occupational Disease, Anhui No. 2
Province People's Hospital, Hefei 230022, China. E-mail: hfzj2552@163.com
†
Electronic supplementary information (ESI) is available: Experimental procedures for
1
13
compound synthesis, characteristic data, H and C NMR spectra, HRMS assays for
Glycyrrhizin analogs. Details for the biological evaluation. Table of cytotoxicity analysis
data. For ESI and other electronic format see DOI:
Bo Li and Yongan Yang contributed equally to this work.

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ABSTRACT: Based on SAR analysis of Glycyrrhizin, 18αꢀglycyrrhetinic acid
monoglucuronide (18αꢀGAMG) with strong inhibition against LPSꢀinduced NO and ILꢀ6
production in RAW264.7 cells was discovered. Western blotting and
immunofluorescence showed that 18αꢀGAMG reduced the expression of iNOS, COXꢀ2,
and MAPKs, as well as activation of NFꢀκB in LPSꢀstimulated RAW264.7 cells. The
further in vivo results showed that 18αꢀGAMG could significantly improve pathological
changes of CCl ꢀinduced hepatic fibrosis.
4
KEYWORDS: Glycyrrhizin, anti-inflammatory activity, structure-activity relationship,
signaling pathway, hepatic fibrosis

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Introduction
Licorice, the roots and rhizomes of Glycyrrhiza species, is a natural sweetener and used
as a traditional herbal medicine for the treatment of inflammation, gastric ulcers, liver
1
‒3
disease, adrenal insufficiency, and tumours. Glycyrrhizin (Glycyrrhizic acid, 18βꢀGA)
4
‒7
is the major bioactive component in licorice with diverse pharmacological activities.
Researches showed that 18βꢀGA can promote maturation of murine dendritic cells (DCs),
regulate interleukin (IL)ꢀ2, ILꢀ10, ILꢀ12, tumor necrosis factorꢀα (TNFꢀα), inducible
8
ꢀ11
nitric oxide synthase (iNOS) and cyclooxygenase (COXꢀ2),
downꢀregulate the
1
2
production of ILꢀ8 and eotaxinꢀ1 in human lung fibroblast cells. Glycyrrhizin also could
prevent enteritis by reducing nuclear factorꢀκB (NFꢀκB) p65 and p38 mitogenꢀactivated
protein kinase (p38MAPK) expression in rat, attenuated neuroinflammation and oxidative
stress in rotenone model of Parkinson's disease, and inflammatory response of
1
3ꢀ15
isofluraneꢀinduced cognitive deficits in neonatal rats.
The antiꢀinflammatory effect of
8βꢀGA may be due to directly binding to highꢀmobility group box 1 protein and thus
1
16,17
inhibiting its chemoattractant and mitogenic activities.
These results indicated that
1
8βꢀGA could be used as immune modulators which precisely regulate the cellular
immunity.
As
a
natural triterpene glycoside, 18βꢀGA contains one molecule of
1
8βꢀHꢀoleananeꢀtype aglycone and two molecules of glucuronic acid (Fig. 1).
8βꢀGlycyrrhetinic acid (18βꢀGCCS), the aglycone of 18βꢀGA, is the active metabolite
1
1
8
by intestinal bacteria after oral administration. 18βꢀGA and 18βꢀGCCS exhibited
similar antiꢀinflammatory effects by inhibiting the production of LPSꢀinduced nitric
oxide (NO), prostaglandin E (PGE ), TNFꢀα, ILꢀ6, ILꢀ1β and intracellular reactive
2
2
oxygen species (ROS), reducing the expression of proꢀinflammatory genes (iNOS and
COXꢀ2), and significantly blocking activation of transcription factors such as NFꢀκB and
1
9,20
PI3K.
8βꢀglycyrrhetinic acid monoꢀglucuronide (18βꢀGAMG).
similar (or stronger) antitumor, antivirus, and antiꢀinflammatory activities to 18βꢀGA.
8αꢀglycyrrhizin (18αꢀGA), a D/Eꢀtransꢀstereoisomer, exhibited antiꢀinflammatory and
anticancer activities by inhibiting the activation of key arachidonic acid (AA) metabolism
18βꢀGA could be metabolized in the liver or be transformed via enzymolysis to
2
1–23
1
18βꢀGAMG showed
2
4–27
1

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enzymes including phospholipase A2 (PLA ), cyclooxygenase (COXs), and
2
5
ꢀlipoxygenase (LOX), and release of AA pathwayꢀgenerated inflammatory lipid
2
8‒30
mediators.
Therefore, all glycyrrhizin analogues (Fig. 1, including metabolite and
isomer) exhibited the antiꢀinflammatory activities, which prompted us to analyze
structureꢀactivity relationships (SAR) of 18βꢀGA.
In this paper, we have synthesized Glycyrrhizin analogs with different glucuronide unit
and/or 18ꢀα/βꢀstereoisomer to investigate the SAR and pharmacological mechanism of
Glycyrrhizin as antiꢀinflammatory agent. Then their antiꢀinflammatory activities in vitro
and in vivo were further evaluated.
O
O
HO
R
O
HO
R
HO
R
O
H
O
H
O
H
H
HOOC
HO
H
O O
HO
H
HOOC
HO
O
O
HO
HO
HO
OH
OH
O
OH
O
COOH
R =
R =
H: 18β-GCCS
H: 18α-GCCS
R =
R =
H: 18β-GAMG
H: 18α-GAMG
R =
R =
H: 18β-GA
H: 18α-GA
Fig. 1 Chemical structures of Glycyrrhizin analogs.
Results and discussion
Glycyrrhizin analogs were synthesized and characterized by NMR and ESIꢀMS, their
details synthetic and structural information are located in the Supporting Information
(Scheme 1).

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O
HO
O
HO
HO
H
H
O
H
(
ii)
(iii)
O
H
O
H
H
H
HOOC
HO
H
O
O
O
HOOC
H
O
O
HO
HO
HO
HO
HO
OH
OH
COOH
OH
O
1
8β-GAMG
1
8β-GCCS
18β-GA
(
i)
(i)
O
O
HO
H
O
HO
H
HO
H
O
H
O
(iii)
O
H
H
HOOC
HO
O
O
O
H
HO
HOOC
HO
HO
O
H
HO
O
H
OH
OH
COOH
HO
O
OH
18α-GCCS
18α-GA
18α-GAMG
Scheme 1 Synthesis of Glycyrrhizin analogs. Reagents and conditions: (i) NaOH
solution (5.0 M), 90 °C, 12 h. (ii) βꢀglucuroidase; (iii) AcOH, 5 N HCl, 100 °C.
To evaluate the antiꢀinflammatory effect of Glycyrrhizin analogs, Griess reagent was
used to detect the level of LPSꢀinduced NO release in RAW264.7 cells. Excessive release
3
1
of NO is regarded as an important factor in inflammatory responses. As shown in Fig. 2,
after Glycyrrhizin analogs treatment, the increase of LPSꢀinduced NO release was
significantly alleviated in RAW264.7 cells. The SAR analysis showed: (i) the
antiꢀinflammatory activity of 18αꢀepimer of the oleananeꢀtype aglycone was superior to
that of 18βꢀepimer (18αꢀGA > 18βꢀGA, 18αꢀGAMG > 18βꢀGAMG, 18αꢀGCCS >
1
8βꢀGCCS); (ii) the number of glucuronic acid at the Cꢀ3 position had effect on the
antiꢀinflammatory activity (monoꢀglucuronide > aglycone > bisꢀglucuronide, such as
8βꢀGAMG > 18βꢀGCCS > 18βꢀGA, 18αꢀGAMG > 18αꢀGCCS > 18αꢀGA).
Glycyrrhizin analogs displayed preferable antiꢀinflammatory activity, among them,
8αꢀGAMG exhibited the strongest activity, and the NO inhibition rate exceeded 70% at
1
1
the concentration of 40 µM.
The cytotoxicity of Glycyrrhizin analogs was evaluated by MTT assay in RAW264.7

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cells. As shown in Table S1, Glycyrrhizin analogs and LPS showed a low toxicity, and
the relative cell viabilities treated with them were all more than 96%. These results
indicated that Glycyrrhizin analogs did not possess significant cytotoxic effects against
the cells at the used concentrations.
Fig. 2
The inhibitory effects of Glycyrrhizin analogs on NO production in
LPSꢀstimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with Glycyrrhizin
analogs (40 µM) for 2 h, and then in the presence or absence of LPS (1 µg/mL) for 20 h.
The results were showed as means ± SD (n=3) of at least three independent experiments.
#
##
###
*
**
p < 0.05, p < 0.01, p < 0.001 compared with the blank group; p < 0.05, p < 0.01,
***
p < 0.001 compared with LPSꢀstimulated group.
To further evaluate the effects of Glycyrrhizin analogs on LPSꢀinduced ILꢀ6
production, RAW264.7 cells were cultured with LPS (1 µg/mL) in the presence of
Glycyrrhizin analogs for 20 h, and the levels of ILꢀ6 in the supernatant were determined
by ELISA. As shown in Fig. 3, the LPSꢀinduced ILꢀ6 production was decreased after
Glycyrrhizin analogs treatment, and inhibitory effects with the above conclusions were
observed: (i) for inhibition of ILꢀ6 production, 18αꢀepimer was better than 18βꢀepimer
(18αꢀGA > 18βꢀGA, 18αꢀGAMG > 18βꢀGAMG, 18αꢀGCCS ≈ 18βꢀGCCS); (ii) the
glucuronic acid’ number had effect on the antiꢀinflammatory activity (monoꢀglucuronide
>
>
aglycone > bisꢀglucuronide, such as 18βꢀGAMG > 18βꢀGCCS > 18βꢀGA, 18αꢀGAMG
18αꢀGCCS > 18αꢀGA). Thus, combining with the antiꢀinflammatory activity and
cytotoxicity of Glycyrrhizin analogs, 18αꢀGAMG was selected to further explore the
mechanisms of the antiꢀinflammatory effect.

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Fig. 3 The inhibitory effects of Glycyrrhizin analogs on LPSꢀinduced ILꢀ6 production
in RAW264.7 cells. RAW264.7 cells were incubated with Glycyrrhizin analogs (40 µM
and 10 µM) for 2 h, and then in the presence or absence of LPS (1 µg/mL) for 20 h. The
#
results were showed as means ± SD (n = 3) of at least three independent experiments. p
###
*
***
<
0.05, p < 0.001 compared with the control group; p < 0.05,
p < 0.001 compared
with LPSꢀstimulated group.
18αꢀGAMG was the most effective in inhibiting NO and ILꢀ6 production. Thus, it was
3
2,33
used to investigate the expression of inflammationꢀrelated proteins.
The expression of
nitric oxide synthase (iNOS) and COXꢀ2 was examined in LPSꢀstimulated RAW264.7
cells. Western blotting showed that 18αꢀGAMG strongly attenuated the expression of
iNOS and COXꢀ2 in LPSꢀstimulated RAW264.7 cells in a doseꢀdependent manner (Fig.
4
). These preliminary results demonstrated that 18αꢀGAMG may participate in signaling
pathways activated by LPS in macrophages.

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Fig. 4 Effects of 18αꢀGAMG on LPSꢀinduced iNOS and COXꢀ2 gene expression in
RAW264.7 cells. The cells were treated with different concentrations of 18αꢀGAMG, and
then in the presence or absence of LPS (1 ꢁg/mL) for 20 h. Bay 11ꢀ7082 is the NFꢀκB
inhibitors (20 µM). The results were showed as means ± SD (n = 3) of at least three
#
##
###
independent experiments. p < 0.05, p < 0.01, p < 0.001 compared with the control
*
**
***
group; p < 0.05, p < 0.01, p < 0.001 compared with LPSꢀstimulated group.
NFꢀκB is a wellꢀknown transcription factor that positively regulates inflammatory
3
4
genes such as iNOS, COXꢀ2, and ILꢀ6 in response to inflammatory stimuli. NFꢀκB
activation is controlled by phosphorylation and degradation of IκBꢀα, a cognate
3
5
regulatory subunit of NFꢀκB. Therefore, western blotting was used to examine the
effects of 18αꢀGAMG on NFꢀκB pathways in LPSꢀstimulated RAW 264.7 cells. As
shown in Fig. 5A, LPS significantly increased the levels of phosphorylated NFꢀκB p65,
IκBα and 18αꢀGAMG treatment could attenuate activation of these proteins in varying
degrees.Furthermore, the nuclear translocation of NFꢀκB was examined in
LPSꢀstimulated RAW264.7 cells. Immunofluorescence analysis showed that 18αꢀGAMG
clearly inhibited NFꢀκB p65 nuclear translocation from the cytosol to the nucleus (Fig.
5
B).These results further confirmed that 18αꢀGAMG might regulate the expression of
proꢀinflammatory proteins through inhibition of NFꢀκB signaling pathways.
A

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B
Fig. 5 A: 18αꢀGAMG suppressed LPSꢀinduced activation of NFꢀκB signaling pathway
in RAW 264.7 cells. RAW264.7 cells were treated with 18αꢀGAMG (10 µM – 30 µM)
and LPS (1 µg/mL) for 30 min. The levels of NFꢀκB p65, IκB, and their phosphorylated
forms were analyzed using western blotting. Bay 11ꢀ7082 is the NFꢀκB inhibitors (20
µM). The results were showed as means ± SD (n = 3) of at least three independent
#
###
###
*
experiments. p < 0.05, p < 0.01, p < 0.001 compared with the control group; p <
**
***
0
.05, p < 0.01, p < 0.001 compared with LPSꢀstimulated group.
B: 18αꢀGAMG clearly inhibited NFꢀκB p65 nuclear translocation. RAW264.7 were
pretreated with 18αꢀGAMG for 1 h and then stimulated with LPS (1 µg/mL) for 3 h.
The mitogenꢀactivated protein kinases (MAPK) transduction pathway is activated by
3
6,37
NFꢀκB in mammalian cells.
Inhibiting the activation of MAPK downꢀregulates the
expression of inflammatory mediators and thus improve the outcome of experimental
3
8,39
inflammatory diseases.
To determine the role of 18αꢀGAMG in modulating MAPK
activation in LPSꢀstimulated RAW264.7 cells, the expression of ERK, JNK, and p38 was
examined. As expected (Fig. 6), levels of phosphorylation of p38, JNK, and ERK were
increased after LPSꢀstimulated for 30 min. 18αꢀGAMG doseꢀdependently (10, 20, and 30
µM) inhibited LPSꢀinduced phosphorylation of ERK, but had little effect on
phosphorylation of JNK or p38 in RAW264.7 cells. These results suggested that the

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antiꢀinflammatory activity of 18αꢀGAMG might be associated with its negative effects on
ERK activation.
Fig. 6 18αꢀGAMG suppressed LPSꢀinduced activation of MAPK signaling pathway in
RAW 264.7 cells. RAW264.7 cells were treated with 18αꢀGAMG (10 µM – 30 µM) and
LPS (1 µg/mL) for 30 min. The levels of JNK, ERK1/2, and p38 MAPK proteins, and
their phosphorylated forms were analyzed using western blotting. The results were
#
###
showed as means ± SD (n = 3) of at least three independent experiments. p < 0.05,
p
#
##
*
**
***
<
0.01, p < 0.001 compared with the control group; p < 0.05, p < 0.01, p < 0.001
compared with LPSꢀstimulated group.
In order to further verify the antiꢀinflammatory activity of 18αꢀGAMG in vivo, mice
model of CCl ꢀinduced hepatic fibrosis was established in this study to investigate its
4
4
0
effect on hepatic fibrosis. Healthy C57BL6 mice (SPF, male, 20 ± 2 g) were purchased
from the experimental animal center of Anhui Medical University. Animals were housed
in a temperature (22 ± 2 °C) and relatively humidity (50%)ꢀcontrolled room on a 12 h
light/dark cycle, given free access to food and water, and acclimatized for at least one
week prior to use. All the animal experiments were performed in accordance with the
Regulations of the Experimental Animal Administration issued by the State Committee of
Science and Technology of China. Efforts were made to minimize the number of animals
used and their suffering. Animals were maintained in accordance with the Guides of

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Center for Developmental Biology, Anhui Medical University for the Care and Use of
Laboratory Animals and all experiments used protocols approved by the institutions’
subcommittees on animal care.
As shown in Fig. 7, in the control group, the structure of the liver was clear, and the
size of hepatocytes was constant. The hepatic lobule was intact, without denaturation or
necrosis (Fig. 7A, 7F). In the model group, the amount of blue collagen fibers was
obviously increased. The fatty degeneration was apparent and ballooning degeneration of
hepatocyte can be seen in model group (Fig. 7B, 7G). The extent of inflammatory cell
infiltration, blue collagen fibers and fibrosis of liver in colchicine group (0.1 mg/kg),
highꢀdose 18αꢀGAMG group and lowꢀdose 18αꢀGAMG were significant decreased, and
highꢀdose group was better than lowꢀdose group (Fig. 7C, 7D, 7E, 7H, 7I, 7J). These
results showed that 18αꢀGAMG could significantly improve pathological changes of
CCl ꢀinduced hepatic fibrosis.
4
Fig. 7 Typical light photomicrographs of liver tissue showing influence of 18αꢀGAMG
on histological changes (H&E and Masson staining). (7Aꢀ7E) represented H&E staining of
the control group, model group, colchicine group (0.1 mg/kg), lowꢀdose 18αꢀGAMG
group (100 mg/kg), and highꢀdose 18αꢀGAMG group (200 mg/kg), respectively. (7Fꢀ7J)
represented Masson staining of the control group, model group, colchicine group (0.1
mg/kg), lowꢀdose 18αꢀGAMG group (100 mg/kg), and highꢀdose 18αꢀGAMG group (200
mg/kg), respectively.

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Conclusions
In conclusion, the clear structureꢀactivity relationships of glycyrrhizin with
antiꢀinflammatory activity was explained, among them, glucuronide unit and
1
8ꢀα/βꢀstereoisomer are important factors. In these compounds, 18αꢀglycyrrhetinic acid
monoglucuronide (18αꢀGAMG) with the strongest inhibition was found. Western blotting
and immunofluorescence showed that 18αꢀGAMG decreased the expression of iNOS,
COXꢀ2, and MAPKs, as well as activation of NFꢀκB in LPSꢀstimulated RAW264.7 cells.
Taken together, 18αꢀGAMG exerted its antiꢀinflammatory activity through inhibition of
NO generation as a result of inhibiting NFꢀκB and MAPKsꢀrelated inflammatory
signaling pathways. In addition, the in vivo results showed that 18αꢀGAMG could
significantly improve pathological changes of CCl ꢀinduced hepatic fibrosis. Therefore,
4
1
8αꢀGAMG may be clinically useful for the reduction of inflammation in the future.
Acknowledgements
Financial support was provided by the National Natural Science Funding of China
(21572003), Anhui University Natural Science Research Project (KJ2016A339), and
University Project of Introduction and Cultivation of Leading Talents (gxfxZD2016044).
Conflict of Interest
The authors declare no competing interests.
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MedChemComm
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DOI: 10.1039/C7MD00210F

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Page 16 of 16
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DOI: 10.1039/C7MD00210F
1
8αꢀGAMG exhibited strong antiꢀinflammatory activity through inhibiting the
expression of iNOS, COXꢀ2, and MAPKs, as well as activation of NFꢀκB.