Synthesis, anti-microbial and anti-inflammatory activities of 18β-glycyrrhetinic acid derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.103985

Thirteen 18β-glycyrrhetinic acid (GA) derivatives were obtained by reduction at C-11 position, oxidation at C-3 position and condensation at C-2 position of GA. Anti-microbial activity evaluation indicated that compounds 04, 05, 10, 13 and 14 showed more

Full text of DOI:10.1016/j.bioorg.2020.103985

Journal Pre-proofs
Synthesis, anti-microbial and anti-inflammatory activities of 18β-glycyrrhe-
tinic acid derivatives
Yang Yang, Qiuyan Zhu, Yingying Zhong, Xiping Cui, Zhengyun Jiang,
Panpan Wu, Xi Zheng, Kun Zhang, Suqing Zhao
PII:
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DOI:
https://doi.org/10.1016/j.bioorg.2020.103985
YBIOO 103985
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Bioorganic Chemistry
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29 May 2020
Please cite this article as: Y. Yang, Q. Zhu, Y. Zhong, X. Cui, Z. Jiang, P. Wu, X. Zheng, K. Zhang, S. Zhao,
Synthesis, anti-microbial and anti-inflammatory activities of 18β-glycyrrhetinic acid derivatives, Bioorganic
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Synthesis, anti-microbial and anti-inflammatory
activities of 18β-glycyrrhetinic acid derivatives
Yang Yang1, Qiuyan Zhu1, Yingying Zhong1, Xiping Cui1, Zhengyun Jiang1, Panpan
Wu2, Xi Zheng1, *, Kun Zhang1, 2, *, Suqing Zhao1, *
1Faculty of Pharmaceutical Engineering, School of Biomedical and Pharmaceutical
Sciences, Guangdong University of Technology, Guangzhou 510006, People’s Republic
of China.
2Faculty of Biotechnology and Health, Wuyi University, Jiangmen 529020, People’s
Republic of China.
Corresponding Authors
*E-mail:
xizhengch2006@163.com,
*E-mail:
kzhang@gdut.edu.cn;
*E-mail:
sqzhao@gdut.edu.cn.

Abstract: Thirteen 18β-glycyrrhetinic acid (GA) derivatives were obtained by reduction
at C-11 position, oxidation at C-3 position and condensation at C-2 position of GA.
Anti-microbial activity evaluation indicated that compounds 04, 05, 10, 13 and 14
showed more potent inhibitory activity against Staphylococcus aureus subsp. aureus,
Staphylococcus epidermidis, Staphylococcus aureus than GA, especially compound 10,
the inhibitory activity against Staphylococcus epidermidis was equaled with Ampicillin.
Moreover, in vivo experiments exhibited that compound 10 also has anti-inflammatory
effect, which could decrease about 59.69% TPA-induced ear edema of mice with the
gavage treatment of 40.0 mg/mL. Immunohistochemistry results revealed that the effect
of inhibition was related to inhibition of TPA-induced upregulation of the
pro-inflammatory cytokines TNF-α and IL-1β. Furthermore, compound 10 also
significantly decreased the expression level of p65 in NF-κB signaling pathway. In
general, compound 10, both with antibacterial and anti-inflammatory activities, was
expected to become a promising bio-functional agent.
Keyword:
18β-glycyrrhetinic
acid;
derivatives;
synthesis;
anti-microbial;
anti-inflammatory

1. Introduction
18β-glycyrrhetinic acid (GA, Figure 1) is a natural product extracted from licorice
root,1-3
with wide range of pharmacological effects such as
a
antibacterial,4-5anti-inflammatory,6-7 antitumor,8 antiviral,9 antioxidant,10 as well as
anticancer activities.11-12 According to the structure of GA, the hydroxyl group at C-3 is
important sites for structural modification and participation in various biological
activities.13 Some researchers have reported that oxidation of the hydroxyl group at C-3
could increase the biological activity of GA.14 The effect of the reduction at C-11
carbonyl group on the structure-activity relationship of GA was significant.15 Moreover,
modified the C-2 position of pentacyclic triterpenoid via condensation with various
aldehydes could improve their biological activity.16-17 Furthermore, it has been reported
that GA exhibited narrow-spectrum antibacterial activity against some Gram-negative
organisms, but had a good inhibition activity against Gram-positive bacteria.18 At present,
there are few studies on the antibacterial activity of GA derivatives, so it is necessary to
explore the effect of structural modification on the antibacterial effect of GA.
Additionally, in some special skin infections, patients were not only suffered from
pathogenic microbial infections but also accompanied with inflammatory response.19-21
clinical manifestations include local atrophy, thinning of the epidermis, vasodilation, and
repeated inflammation.22 Ibuprofen, a well known anti-inflammatory drug, was acted via
inhibiting the production of inflammatory cytokines in vivo. However, long-term use of
ibuprofen had a certain effect on the physiological organization and function of the rat

liver, and the side effects on the kidney were also obvious. Therefore, it is very important
to develop a drug that has both anti-inflammatory and anti-bacterial activity.
Therefore, a series of 18β-glycyrrhetinic acid derivatives were designed and
synthesized. The in vitro antibacterial ability of GA and its derivatives were measured
against three Gram positive bacteria, including Staphylococcus aureus subsp. aureus (S.
aureus subsp. aureus), Staphylococcus epidermidis (S. epidermidis), Staphylococcus
aureus (S. aureus) and two Gram-negative bacteria, including Escherichia coli (E. coli),
Staphylococcus typhimurium (S. typhimurium). In vivo, the effects of GA and compound
10 on 12-O-tetradecylphorbol-13-acetate (TPA) induced skin inflammation were
investigated,such as TPA-induced ear edema, pro-inflammatory cytokine expression as
well as TPA-induced NF-κB activation.
2. Results and discussion
2.1 Synthesis of 18β-Glycyrrhetinic Acid Derivatives 04~16
The structural modification of GA was made at the C-2, 3-OH and C-11 positions to
obtain a series of novel glycyrrhetic acid derivatives (Scheme 1). Firstly, according to the
Clemensen reduction reaction, the carboxyl group at position C-11 was reduced to a
methylene group with zinc under refluxing of concentrated hydrochloric acid to obtain
11-deoxo-18β-glycyrrhetinic acid (GA-H ） .Then, 3-ketone compound GA-O was
obtained using Jones' reagent with GA-H. Finally, the target compounds 4~16 were
obtained through Aldol Reaction. All compounds were purified by column

chromatography, and the yields of compounds were between 30% ~ 80%. The structures
of all target compounds were identified by 1H NMR, 13C NMR and HRMS.
2.2 In Vitro Antibacterial Activity.
The antibacterial activity of GA and all of its prepared derivatives was tested against
five bacterial strains, namely S. aureus (ATCC 6538), S. aureus subsp. aureus (ATCC
29213), S. epidermidis (ATCC 12228), E. coli (CMCC 44102) and S. typhimurium
(ATCC 14028). Ampicillin was selected as a positive control and a microtiter plate
dilution method was performed to determine the MIC. Each sample concentration was
tested four times for each microorganism. DMSO was set as blank control group in order
to demonstrate that it had no effect on bacterial growth.
As shown in Table 1, it could be found that the antibacterial effect of GA and its
derivatives against the Gram-positive bacterial strains (S. aureus, S. aureus subsp. Aureus
and S. epidermidis) was much higher than the Gram-negative bacterial strains (E. coli
and S. murine). Moreover, due to poor solubility, the intermediates GA-H and GA-O
showed less inhibitory activity against Gram-positive bacterial strains than GA while
most of target compounds exhibited higher inhibition activities, (Table 1). The above
findings suggested that condensation modification at C-2 position could improve the
inhibitory activity of 18β-glycyrrhetinic acid against Gram-positive bacteria. Inaddition,
compounds 04, 05, 10, 13, 14 displayed super inhibitory activity against Gram-positive
bacteria indicating that the replacement of the phenyl ring with thienyl ring, pyridyl ring,
pyrazine ring or imidazolyl at C-2 position had a significant effect on the inhibitory

activity of Gram-positive bacteria. However, compounds 06, 07, 08 and 12 exhibited poor
inhibitory activity against S. epidermidis (ATCC 12228), which implied that the phenyl
ring at C-2 position exhibited weak activity. Among the derivatives, compound 10, which
had pyrazine ring at C-2 position, showed the highest activity against S.epidermidis with
MIC value was equivalent to the positive control. Besides, the inhibition activity of
derivative 06 was worse than derivative 09, but better than derivative 07. The results
showed that the stronger the electron-withdrawing ability of the substituent introduced in
the benzene ring, the better the antibacterial activity.
2.3 Time-Kill Assays
According to Table 1, GA and compound 10, with the lowest MIC values, were
selected for time kill assay, It could be found from Figure 2 that, GA and compound 10
showed a dose-dependent inhibitory activity against growth of Staphylococcus aureus
(ATCC 6538), Staphylococcus aureus subsp. aureus (ATCC 29213) and Staphylococcus
epidermidis (ATCC 12228). When the test concentration was 0.5×MIC, the compound
showed a limited inhibitory effect on the bacteria. However, at the concentration of
1×MIC and 2×MIC, the compounds had a very strong inhibitory effect on the bacteria,
and the survival rate of the bacteria was extremely low. Moreover, it could be seen from
Figure 2 that there was no difference in the killing of bacteria by the two compounds at a
high concentration.
2.4 Inhibitory Effects of GA and 10 on TPA-Induced Edema in a Mouse Ear Model
Usually, mice ear edema induced by 12-O-tetradecylphorbol-13-acetate (TPA) has

been used as a model for testing anti-inflammatory activity in animals.23-24 In vivo
anti-inflammatory activity of GA and compound 10 was evaluated using a TPA-induced
ear edema mice model. TPA was commonly used to induce inflammation in the skin as a
model group. Compared with the model group, the change in the average weight of the
ear punches was an important indicator evaluating the efficacy of the compounds. As
shown in Figure 3, compared with the acetone-treated blank group, the model group in
which 10 μL of TPA (50 μg /mL) was applied to both sides of the ear without
administered, the average weight of the ear punches was significantly increased.
Excitingly, both GA groups and compound 10 groups could inhibit the swelling of mouse
ears to some extent. Compared with TPA, the ear swelling inhibition rates were 12.41,
17.90 and 25.14% for GA groups (10.0, 20.0 and 40.0 mg/mL), and 26.73, 40.21 and
59.69% for compound 10 groups (10.0, 20.0 and 40.0 mg/mL). In addition, the ear
swelling inhibition rates of of Ibuprofen (40 mg/mL) was 46%. The results showed that
the inhibitory effect of compound 10 at 40.0 mg/mL was better than that of the positive
control group, which indicated that it had a good effect of inhibiting swelling of mouse
ears.
2.5 Inhibitory Effects of GA and Compound 10 on the Histological Appearance of Mouse
Ears
To investigate the role of GA and compound 10 in the histological appearance of the
TPA-induced mouse ear model, mice were subjected to continuous intragastric
administration of GA and compound 10 for 7 days, ibuprofen was as the positive control

group GA and Compound 10 were evaluated by continuous intragastric administration. In
this model, mice were continuously administered intragastrically for 7 days in the drug
group, as did the ibuprofen-positive control group. After the end of the gavage, the right
ears of blank group were applied with 20 μL of acetone, and the other groups were
treated with 20 μL of TPA (50 μg/mL). Then, the ear tissues were stained by H&E, as
shown in Figure 4, the histological appearances of the ear sections showed that there was
no significant change in the blank group, the thickness of the auricle was normal, and the
stratum corneum was clear in the section. However, the tissue morphology of the TPA
group showed significant swelling, which was consistented with the results of the ear
punch weight in Figure 3. In addition, GA and compound 10 showed varying degrees of
inhibition of inflammation, such as decreased thickness of the ear tissue and weakened
inflammatory cell infiltration.
2.6 Inhibition of TPA-Induced Expression of TNF-α, IL-1β and p65
In this model, skin edema was associated with the increased levels of
pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β
(IL-1β), and interleukin-6 (IL-6).25-27 To investigate the molecular mechanism by which
GA and compound 10 curbed TPA-induced skin inflammation, the effect of GA and
compound 10 on the expression levels of pro-inflammatory cytokines in mouse ears by
immuno-histochemistry, such as TNF-α and IL-1β was observed, but IL-6 have not been
detected. As shown in Figure 5, the protein levels of the pro-inflammatory cytokines
TNF-α and IL-1βα in the ears of the TPA group were increased by 15.93-fold and

30.91-fold compared with the blank group, which indicated that TPA could significantly
increase the expression levels of the pro-inflammatory cytokines IL-1β and TNF-α in the
rat ears. However, the GA groups and the compound 10 groups that administered
continuously for seven days were effectively and dose-dependently reduced TPA-induced
of TNF-α and IL-1β expression. Compared with the TPA group, the compound 10 (20
mg/kg) delayed TPA-induced overexpression of TNF-α was better than the ibuprofen (40
mg/kg). For IL-1β, the compound 10 at 40 mg/kg was preferable to the ibuprofen at 40
mg/kg (Figure 6). The up-regulated expression of pro-inflammatory cytokines was
consistent with the in vitro results in Figure 3 and Figure 4. Therefore, the results
indicated that compound 10 could significantly inhibit the overexpression of
proinflammatory cytokines.
The activation of NF-κB signaling pathway was the key to regulate the expression of
genes involved in TFA-induced inflammation, such as the increased expression of p65
and I-κB.28-33 To further investigate the in vivo anti-inflammatory mechanisms of GA and
compound 10. The effects of GA and compound 10 on NF-κB signaling pathway were
investigated by immunohistochemistry. The activation of NF-κB was significant for the
regulation of TNF-α and IL-1β, overexpression in the TPA-induced inflammatory model.
When TPA induced activation of the NF-kB signaling pathway, the expression of related
inflammatory factors in the pathway was significantly increased, such as p65, but I-κB
have not been tested .As shown in Figure 5, The TPA group caused a significant increase
in the protein levels of p65 (10.91-fold), compared to the control-acetone group. This

result revealed that TPA could effectively activate the NF-κB signaling pathway.
Compared with the TPA group, the delayed TPA-induced overexpression of p65 for
compound 10 at 40 mg/kg was higher than 40 mg/kg of GA (Figure 6), which showed
that compound 10 had good anti-inflammatory activity by inhibiting the expression of
p65 in the NF-kB pathway.
2.7 Conclusion
Thirteen 18β-glycyrrhetinic acid derivatives were synthesized through reduction
reaction, oxidation reaction and condensation reaction, and were biologically evaluated as
agents with antibacterial and anti-inflammatory activities. Among them, compounds 04,
05, 10, 13 and 14 displayed great inhibitory effect on Gram-positive bacteria than GA. In
particular, compound 10 exhibited the most effective biological activity, and the MIC
value was similar to the positive control group, ampicillin.
In the TPA-induced skin inflammation model, compound 10 and GA could inhibit
TPA-induced skin inflammation in mouse ears by intragastric administration. However,
compound 10 had much stronger inhibitory effect than GA. The anti-inflammatory
effects by compound 10 were related with the suppression of TPA-stimulated production
of TNF-α and IL-1β and the reduction of p65 expression in NF-κB signaling pathway.
The results of study indicated that the derivatives of 18β-glycyrrhetinic acid could be
used as a good antibacterial agent. In conclusion, compound 10 was expected to be
developed as a bifunctional agent having anti-microbial and anti-inflammatory activity.
3. Material and Methods

3.1 General Experimental Procedures
GA was bought from Macklin Co., Ltd. (Shanghai, China), and the aldehydes were
obtained from Macklin Co., Ltd. (Shanghai, China), the purity of all compounds are
higher than 98%. Ampicillin was purchased from Aladdin Chemical (Shanghai, China).
TPA was offered by Sigma-Aldrich (St. Louis, MO, USA). Ibuprofen was provided by
Beijing Lnoke Technology Co., (Ltd). Sodium carboxymethylcellulose was obtained
from Beijing Lnoke Technology Co., (Ltd). The antibodies against TNF-α, IL-1β and p65
were bought from Rebiosci Biotech Co., Ltd (Shanghai, China). Thin Layer
chromatography (TLC) was performed on silica gel 60 F254 glass plates (10 × 20 cm)
purchased from Tsingtao Marine Chemistry Co., (Ltd). Mueller Hinton broth (MHB)
was obtained from Guangdong Huankai Microbial Sci. & Tech.Co., Ltd. (Guangdong,
China). The rest of the chemicals and reagents were high purity and analytical grade.
The anti-microbial activity was tested by using a Multimode Plate Reader (Infinite
200, TECAN, Guangzhou, Guangdong, China). 1H NMR, 13C NMR and 19F NMR spectra
were determined by Bruker AVANCE NMR spectrometer, High-resolution mass spectra
were gained on BRUKER microOTOF-Q in the ESI mode (HR-ESI-MS).
The S.aureus subsp. aureus (ATCC 29213), S. epidermidis (ATCC 12228), S.
aureus (ATCC 6538), E. coli (CMCC 44102) and S. typhimurium (ATCC 14028) were
obtained from Guangdong Huankai Microbial Sci. & Tech.Co., Ltd. (Guangdong, China).
All bacteria were cultured in MHB on a shaker at 37 °C. After incubating for 24 h, a
suitable amount of bacterial solution was taken from MHB for turbidimetry assays.

Healthy and clean Kunming female mice (body weight 18~22 g) were taken from
Guangdong Animal Experimental Center. The room temperature was kept at around
26 °C and the relative humidity was kept at 50%. The mice have sufficient light time and
are free to get food and water. All experimental procedures involving animals and their
care were conformed to the National Care and Use of Laboratory Animals by the
National Animal Research Authority (China).
3.2 General Procedure for the Preparation of Analogues (04~16).
Firstly, GA (10.0 mmol) were dissolved in the solution of 1,4-dioxane (100 mL),
and then zinc powder (30 mg), HgCl2 (1.2 mg) and 5% HCl (50 mL) were added to
initiate the reaction, after removing the oxides on zinc powder surface by the dilute
hydrochloric acid, 5 mL of concentrated HCl was slowly added at 0 °C. After stirring for
6 h, the reaction mixture was filtered with a Buchner funnel to remove excess solid. Next,
the reaction mixture was concentrated under reduced pressure, extracted with ethyl
acetate and water, filtered and dried by anhydrous magnesium sulfate. A white solid
GA-H (4.11 mg, 90%) was obtained by the column chromatography [petroleum
ether-ethyl acetate-acetic acid (40:10:1)]. Secondly, After GA-H (2.28 g, 5.0 mmol) was
dissolved in 100 mL acetone and 2.0 mL the prepared Jones reagent was added in an
ice-salt bath, the mixture was stirred for 0.5 h and then extracted with ethyl acetate and
water, filtered and dried by anhydrous magnesium sulfate. Finally, a white solid GA-O
(2.15 mg, 90%) was obtained by the column chromatography [petroleum ether-ethyl
acetate (3:1)]. In the end, to a solution of GA-O (1.19 mmol) and potassium hydroxide

(1.78 mmol) in 10 mL ethanol, aldehyde (1.78 mmol) was added, the mixture was reacted
for 12 h.34 The pretreatment of the crude product was similar to the intermediates GA-H
and GA-O, the column chromatography was used to purify the crude product for gaining
the derivatives 04~16.
3.2.1 2-Thiophenemethylene-3-oxo-18-olean-12-en-30-oic acid (04). According to the
above general methods, derivative 04 was obtained by Claisen Schmidt condensation of
GA-O with 2-Thiophenecarboxaldehyde and purified by petroleum [Ether-AcOEt (10 : 1,
1
Rf = 0.35)]; Yield: 53%; yellow amorphous powder; H NMR (400 MHz, CDCl3) δ 7.74
(s, 1H), 7.51 (d, J = 4.9 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 7.15 – 7.11 (m, 1H), 5.43 (s,
1H), 3.03 (d, J = 16.9 Hz, 1H), 2.22 (d, J = 16.9 Hz, 1H), 2.15 – 2.01 (m, 3H), 2.01 –
1.89 (m, 3H), 1.88 – 1.75 (m, 2H), 1.70 (t, J = 13.7 Hz, 1H), 1.64 – 1.41 (m, 6H), 1.41 –
1.29 (m, 3H), 1.24 (s, 3H), 1.22 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.98 (s,
1H), 0.94 (s, 3H), 0.90 (s, 1H), 0.86 (d, J = 10.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
207.42, 183.49, 144.33, 139.47, 132.68, 130.78, 130.33, 129.69, 127.56, 122.62, 52.67,
48.24, 45.58, 44.94, 44.83, 44.15, 42.61, 41.83, 39.69, 38.28, 36.06, 32.08, 31.72, 31.11,
29.97, 28.73, 28.25, 27.01, 26.11, 25.77, 23.82, 22.52, 20.52, 16.33, 15.93.
(+)-HRESIMS m/z 571.3221 [M+Na]+ (calcd for C36H49NO3Na, 571.3216).
3.2.2 Pyridinemethylene-3-oxo-18-olean-12-en-30-oic acid (05). According to the above
general methods, derivative 05 was obtained by Claisen Schmidt condensation of GA-O
with 2-Pyridinecarboxaldehyde and purified by petroleum [Ether -AcOEt (15: 1, Rf =
0.35)]; Yield: 80%; yellow amorphous powder; 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J

= 4.6 Hz, 1H), 7.72 – 7.65 (m, 1H), 7.40 (s, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.21 – 7.15 (m,
1H), 5.38 (s, 1H), 3.52 – 3.43 (m, 1H), 2.45 (d, J = 17.9 Hz, 1H), 2.07 – 1.88 (m, 6H),
1.86 – 1.76 (m, 2H), 1.68 (t, J = 13.5 Hz, 1H), 1.60 – 1.47 (m, 4H), 1.46 – 1.26 (m, 6H),
1.22 (s, 3H), 1.20 (s, 6H), 1.14 (s, 3H), 1.06 (s, 1H), 1.02 (s, 3H), 0.90 (s, 3H), 0.83 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 208.86, 183.11, 155.62, 149.60, 144.39, 138.43,
136.28, 134.41, 126.85, 122.85, 122.44, 53.20, 48.36, 45.48, 45.30, 44.77, 44.24, 42.78,
41.99, 39.79, 38.43, 36.09, 32.20, 31.94, 31.28, 29.79, 28.88, 28.37, 27.14, 26.23, 25.89,
23.92, 22.72, 20.56, 16.54, 15.75. (+)-HRESIMS m/z 544.3795 [M+Na]+ (calcd for
C36H49NO3Na, 544.3785)
3.2.3 4-Chlorobenzalmethylene-3-oxo-18-olean-12-en-30-oic acid (06). According to the
above general methods, derivative 06 was obtained by Claisen Schmidt condensation of
GA-O with 4-Chlorobenzaldehyde and purified by petroleum [Ether -AcOEt (18: 1, Rf =
1
0.38)]; Yield: 58%; yellow amorphous powder; H NMR (400 MHz, CDCl3) δ 7.47 (s,
1H), 7.39 – 7.32 (m, 4H), 5.36 (s, 1H), 2.97 (d, J = 16.3 Hz, 1H), 2.26 (d, J = 15.9 Hz,
1H), 2.05 – 1.88 (m, 6H), 1.86 – 1.74 (m, 2H), 1.68 (t, J = 13.5 Hz, 1H), 1.62 – 1.40 (m,
6H), 1.39 – 1.29 (m, 3H), 1.22 (s, 3H), 1.21 (s, 3H), 1.19 (s, 3H), 1.15 (s, 3H), 1.07 (s,
1H), 1.02 (s, 3H), 0.95 (s, 1H), 0.88 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
207.75, 183.31, 144.49, 136.10, 134.45, 134.35, 131.56, 128.70, 128.33, 122.43, 53.03,
48.21, 45.39, 45.25, 44.24, 44.11, 42.58, 41.87, 39.71, 38.23, 36.22, 32.07, 31.81, 31.08,
29.71, 28.71, 28.22, 27.00, 26.08, 25.78, 23.80, 22.69, 20.39, 16.42, 15.46.
(+)-HRESIMS m/z 599.3265 [M+Na]+ (calcd for C37H49ClONa, 599.3262)

3.2.4 4-(2-Pyridinyl)benzalmethylene-3-oxo-18-olean-12-en-30-oic acid (07). According
to the above general methods, derivative 07 was obtained by Claisen Schmidt
condensation of GA-O with 4-(2-Pyridinyl)benzaldehyde and purified by petroleum
1
[Ether -AcOEt (15: 1, Rf = 0.4)]; Yield: 64%; yellow amorphous powder; H NMR (400
MHz, CDCl3) δ 8.68 (d, J = 4.6 Hz, 1H), 8.01 (d, J = 5.0 Hz, 1H), 7.75 – 7.68 (m, 2H),
7.57 (s, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.51 (s, 1H), 7.24 – 7.17 (m, 1H), 5.34 (s, 1H),
3.07 (d, J = 16.3 Hz, 1H), 2.33 (d, J = 16.3 Hz, 1H), 2.10 – 1.87 (m, 6H), 1.86 – 1.73 (m,
2H), 1.72 – 1.62 (m, 1H), 1.62 – 1.40 (m, 6H), 1.39 – 1.29 (m, 3H), 1.21 (s, 6H), 1.19 (s,
3H), 1.16 (s, 3H), 1.13 – 1.06 (m, 1H), 1.01 (s, 3H), 0.97 (d, J = 8.7 Hz, 1H), 0.93 (d, J =
9.0 Hz, 1H), 0.89 (s, 3H), 0.82 (s, 3H).13C NMR (100 MHz, CDCl3) δ 207.88, 182.66,
156.60, 149.61, 144.40, 139.09, 136.99, 136.88, 136.57, 134.38, 130.83, 126.98, 122.43,
120.78, 53.05, 48.23, 45.43, 45.27, 44.42, 44.08, 42.63, 41.87, 39.71, 38.29, 36.24, 32.07,
31.83, 31.16, 29.73, 28.71, 28.26, 27.02, 26.10, 25.78, 23.80, 22.71, 20.41, 16.44, 15.50,
15.26. (+)-HRESIMS m/z 642.3923 [M+Na]+ (calcd for C42H53NO3Na, 642.3918)
3.2.5 3-Fluoro-4-methoxybenzalmethylene-3-oxo-18-olean-12-en-30-oic acid (08).
According to the above general methods, derivative 08 was obtained by Claisen Schmidt
condensation of GA-O with 3-Fluoro-4-methoxybenzaldehyde and purified by petroleum
[Ether -AcOEt (20: 1, Rf = 0.35)]; Yield: 48%; yellow amorphous powder; 1H NMR (400
MHz, CDCl3) δ 7.42 (s, 1H), 7.25 – 7.20 (m, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.98 (t, J =
8.8 Hz, 1H), 5.38 (s, 1H), 3.90 (s, 3H), 2.98 (d, J = 16.3 Hz, 1H), 2.27 (d, J = 16.1 Hz,

1H), 2.06 – 1.89 (m, 6H), 1.87 – 1.75 (m, 2H), 1.69 (t, J = 13.7 Hz, 1H), 1.64 – 1.40 (m,
6H), 1.40 – 1.29 (m, 3H), 1.22 (s, 3H), 1.22 (s, 3H), 1.19 (s, 3H), 1.14 (s, 3H), 1.10 –
1.06 (m, 1H), 1.02 (s, 3H), 0.98 – 0.92 (m, 1H), 0.89 (s, 3H), 0.83 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 207.75, 183.44, 153.12 (d, JC-F = 244.40 Hz), 148.02 (d, JC-F = 13.90 Hz),
144.36, 136.11, 132.83, 129.12 (d, JC-F = 6.60 Hz), 127.48 (d, JC-F = 3.10 Hz), 122.52,
117.76 (d, JC-F = 18.60 Hz), 112.98 (d, JC-F = 1.70 Hz), 56.19, 52.89, 48.21, 45.42, 45.11,
44.38, 44.12, 42.55, 41.86, 39.70, 38.26, 36.13, 32.07, 31.79, 31.10, 29.83, 28.71, 28.23,
19
27.00, 26.09, 25.74, 23.82, 22.64, 20.43, 16.41, 15.51. F NMR (376 MHz, CDCl3) δ
−134.74 (s, F), (+)-HRESIMS m/z 613.3673 [M+Na]+ (calcd for C38H51FO4Na,
613.3664).
3.2.6 4-(Trifluoromethyl) benzalmethylene-3-oxo-18-olean-12-en-30-oic acid (09).
According to the above general methods, derivative 09 was obtained by Claisen Schmidt
condensation of GA-O with 4-(Trifluoromethyl) benzaldehyde and purified by petroleum
[Ether -AcOEt (15: 1, Rf = 0.30)]; Yield: 60%; yellow amorphous powder; 1H NMR (400
MHz, CDCl3) δ 7.65 (s, 1H), 7.63 (s, 1H), 7.52 (s, 1H), 7.51 (s, 1H), 7.49 (s, 1H), 5.35 (s,
1H), 2.99 (d, J = 16.4 Hz, 1H), 2.29 (d, J = 16.0 Hz, 1H), 2.05 – 1.88 (m, 6H), 1.87 –
1.74 (m, 2H), 1.67 (t, J = 13.6 Hz, 1H), 1.62 – 1.40 (m, 6H), 1.40 – 1.31 (m, 3H), 1.25 (s,
1H), 1.22 (s, 3H), 1.21 (s, 3H), 1.20 (s, 3H), 1.17 (s, 3H), 1.06 (d, J = 13.0 Hz, 1H), 1.02
13
(s, 3H), 0.97 – 0.92 (m, 1H), 0.89 (s, 3H), 0.82 (s, 3H). C NMR (100 MHz, CDCl3) δ
207.70, 183.52, 144.48, 139.42, 135.89, 135.61, 130.26, 129.90 (q, JC-F = 32.30 Hz),

125.37, 125.33, 125.30, 125.26, 122.37, 53.13, 48.19, 45.37, 44.11, 42.56, 41.87, 39.71,
38.22, 36.28, 32.06, 31.81, 31.06, 29.72, 29.63, 28.70, 28.21, 26.99, 26.07, 25.76, 23.77,
22.72, 20.37, 16.42, 15.45. 19F NMR (376 MHz, CDCl3) δ −62.74 (s, 3F), (+)-HRESIMS
m/z 633.3530 [M+Na]+ (calcd for C38H48F3O3Na, 633.3526).
3.2.7 2-Pyrazinecarbalmethylene-3-oxo-18-olean-12-en-30-oic acid (10). According to
the above general methods, derivative 10 was obtained by Claisen Schmidt condensation
of GA-O with Pyrazine-2-carbaldehyde and purified by petroleum [Ether -AcOEt (25: 1,
Rf = 0.30)]; Yield: 55%; yellow amorphous powder; 1H NMR (400 MHz, CDCl3) δ 8.69 –
8.61 (m, 2H), 8.43 (s, 1H), 7.37 (s, 1H), 5.39 (s, 1H), 5.29 (s, 1H), 3.54 (d, J = 18.4 Hz,
1H), 2.48 (d, J = 18.2 Hz, 1H), 2.08 – 1.88 (m, 6H), 1.87 – 1.74 (m, 2H), 1.68 (t, J = 13.6
Hz, 1H), 1.64 – 1.41 (m, 6H), 1.40 – 1.31 (m, 3H), 1.22 (s, 3H), 1.21 (s, 6H), 1.14 (s, 3H),
1.02 (s, 3H), 0.96 (s, 1H), 0.91 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
208.38, 183.01, 151.67, 147.52, 144.33, 144.16, 142.41, 141.48, 129.87, 122.65, 53.44,
48.25, 45.38, 45.19, 45.09, 44.12, 42.65, 41.87, 39.66, 38.27, 35.92, 32.08, 31.77, 31.12,
29.67, 28.74, 28.24, 27.00, 26.09, 25.77, 23.81, 22.54, 20.42, 16.39, 15.76.
(+)-HRESIMS m/z 567.3564 [M+Na]+ (calcd for C35H47N2O3Na, 567.3557).
3.2.8 5-Fluoro-2-methoxynicotinalmethylene-3-oxo-18-olean-12-en-30-oic acid (11).
According to the above general methods, derivative 11 was obtained by Claisen Schmidt
condensation of GA-O with 5-Fluoro-2-methoxynicotinaldehyde and purified by
petroleum [Ether -AcOEt (15: 1, Rf = 0.44)]; Yield: 45%; yellow amorphous powder; 1H

NMR (400 MHz, CDCl3) δ 7.98 (d, J = 2.8 Hz, 1H), 7.58 (s, 1H), 7.36 – 7.31 (m, 1H),
5.34 (s, 1H), 3.95 (s, 3H), 2.87 (d, J = 16.2 Hz, 1H), 2.19 (d, J = 16.0 Hz, 1H), 2.02 –
1.86 (m, 6H), 1.85 – 1.73 (m, 2H), 1.69 (t, 1H), 1.61 – 1.41 (m, 6H), 1.39 – 1.33 (m, 3H),
1.21 (s, 3H), 1.20 (s, 3H), 1.18 (s, 3H), 1.16 (s, 3H), 1.09 (d, J = 14.4 Hz, 1H), 1.01 (s,
3H), 0.94 (d, J = 7.4 Hz, 1H), 0.90 (s, 3H), 0.82 (s, 3H).13C NMR (100 MHz, CDCl3) δ
207.08, 183.13, 158.48, 153.41 (d, JC-F = 244.60 Hz), 144.39, 136.16, 133.09 (d, JC-F
=
25.60 Hz), 130.05, 125.62 (d, JC-F = 21.40 Hz), 122.37, 119.93 (d, JC-F = 4.30 Hz), 54.10,
53.17, 48.19, 45.38, 45.34, 44.09, 43.89, 42.53, 41.85, 39.72, 38.25, 36.38, 32.05, 31.82,
31.11, 29.54, 28.69, 28.22, 26.98, 26.08, 25.73, 23.73, 22.80, 20.32, 16.45, 15.48. 19F
NMR (376 MHz, CDCl3) δ −138.90 (s, F), (+)-HRESIMS m/z 614.3622 [M+Na]+ (calcd
for C37H50FNO4Na, 614.3616).
3.2.9
4-Bromo-2-fluorobenzalmethylene-3-oxo-18-olean-12-en-30-oic
acid
(12).
According to the above general methods, derivative 12 was obtained by Claisen Schmidt
condensation of GA-O with 4-Bromo-2-fluorobenzaldehyde and purified by petroleum
[Ether -AcOEt (18: 1, Rf = 0.37)]; Yield: 55%; yellow amorphous powder; 1H NMR (400
MHz, CDCl3) δ 7.50 (s, 1H), 7.33 – 7.30 (m, 1H), 7.29 – 7.26 (m, 1H), 7.20 (t, J = 8.1
Hz, 1H), 5.33 (s, 1H), 2.82 (d, J = 16.2 Hz, 1H), 2.16 (d, J = 15.9 Hz, 1H), 2.03 – 1.87
(m, 6H), 1.84 – 1.72 (m, 2H), 1.66 (t, J = 13.8 Hz, 1H), 1.61 – 1.41 (m, 6H), 1.40 – 1.32
(m, 3H), 1.30 – 1.24 (m, 1H), 1.21 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H), 1.17 (s, 3H), 1.09
(d, J = 16.8 Hz, 1H), 1.00 (d, J = 6.7 Hz, 3H), 0.96 – 0.92 (m, 1H), 0.88 (s, 3H), 0.82 (s,

3H).13C NMR (100 MHz, CDCl3) δ 207.17, 183.37, 159.33 (d, JC-F = 263.90 Hz), 144.47,
136.47, 131.30 (d, JC-F = 3.30 Hz), 128.83, 127.24 (d, JC-F = 3.60 Hz), 123.09 (d, JC-F
=
13.60 Hz), 122.72 (d, JC-F = 9.60 Hz), 122.36, 119.64 (d, JC-F = 25.20 Hz), 53.25, 48.18,
45.47, 45.34, 44.10, 43.91, 42.55, 41.86, 39.71, 38.22, 36.37, 32.06, 31.84, 31.06, 29.44,
28.70, 28.21, 26.98, 26.07, 25.77, 23.72, 22.80, 20.31, 16.45, 15.43. 19F NMR (376 MHz,
CDCl3) δ −109.29 (s, F) (+)-HRESIMS m/z 661.2671 [M+Na]+ (calcd for
C37H48BrFO3Na, 661.2663).
3.2.10 1-Methyl-1H-imidazole-2-carbalmethylene-3-oxo-18-olean-12-en-30-oic acid
(13). According to the above general methods, derivative 13 was obtained by Claisen
Schmidt condensation of GA-O with 1-Methyl-1H-imidazole-2-carbaldehyde and
purified by petroleum [Ether -AcOEt (25: 1, Rf = 0.36)]; Yield: 73%; yellow amorphous
1
powder; H NMR (400 MHz, CDCl3) δ 7.34 (s, 1H), 7.24 (s, 1H), 6.95 (s, 1H), 5.41 (s,
1H), 3.73 (s, 3H), 2.38 (d, J = 18.0 Hz, 1H), 2.26 – 2.18 (m, 1H), 2.16 – 2.08 (m, 1H),
2.03 – 1.77 (m, 6H), 1.67 – 1.58 (m, 2H), 1.54 (d, J = 13.8 Hz, 1H), 1.50 – 1.31 (m, 6H),
1.30 – 1.23 (m, 3H), 1.21 (s, 3H), 1.19 (s, 3H), 1.18 (s, 3H), 1.13 (s, 3H), 1.02 (s, 3H),
0.97 – 0.92 (m, 1H), 0.89 (s, 3H), 0.83 (s, 3H).13C NMR (100 MHz, CDCl3) δ 207.93,
181.73, 143.91, 143.65, 136.51, 129.94, 123.19, 122.62, 119.63, 53.06, 47.94, 45.12,
44.93, 44.34, 43.98, 42.66, 41.86, 39.61, 38.45, 35.84, 33.30, 32.05, 31.84, 31.45, 29.83,
28.76, 28.40, 27.12, 26.13, 25.71, 23.77, 22.82, 20.51, 16.50, 15.72. (+)-HRESIMS m/z
547.3906 [M+Na]+ (calcd for C35H50N2O3Na, 547.3894).

3.2.11 5-Bromo-2-furalmethylene-3-oxo-18-olean-12-en-30-oic acid (14). According to
the above general methods, derivative 14 was obtained by Claisen Schmidt condensation
of GA-O with 5-Bromo-2-furaldehyde and purified by petroleum [Ether -AcOEt (25: 1,
1
Rf = 0.42)]; Yield: 66%; yellow amorphous powder; H NMR (400 MHz, CDCl3) δ 7.22
(s, 1H), 6.53 (d, J = 3.5 Hz, 1H), 6.42 (d, J = 3.4 Hz, 1H), 5.40 (s, 1H), 3.08 (d, J = 17.5
Hz, 1H), 2.20 (d, J = 17.1 Hz, 1H), 2.08 – 1.91 (m, 6H), 1.87 – 1.75 (m, 2H), 1.69 (t, J =
13.6 Hz, 1H), 1.64 – 1.41 (m, 6H), 1.41 – 1.32 (m, 3H), 1.23 (s, 3H), 1.21 (s, 3H), 1.18
(s, 3H), 1.10 (s, 3H), 1.03 (s, 3H), 1.01 – 0.95 (m, 1H), 0.93 (s, 3H), 0.90 (d, J = 11.3 Hz,
1H), 0.84 (s, 3H).13C NMR (100 MHz, CDCl3) δ 207.07, 183.60, 154.53, 144.24, 131.45,
124.86, 123.05, 122.66, 117.51, 114.08, 52.78, 48.22, 45.40, 44.95, 44.29, 44.15, 42.57,
41.80, 39.67, 38.29, 35.74, 32.07, 31.73, 31.10, 29.85, 28.75, 28.24, 26.98, 26.12, 25.76,
23.70, 22.59, 20.46, 16.35, 15.86. (+)-HRESIMS m/z 633.2551 [M+Na]+ (calcd for
C35H47BrO4Na, 633.2550).
3.2.12 5-Methyl-2-furalmethylene-3-oxo-18-olean-12-en-30-oic acid (15). According to
the above general methods, derivative 15 was obtained by Claisen Schmidt condensation
of GA-O with 5-Methyl-2-furaldehyde and purified by petroleum [Ether -AcOEt (20: 1,
1
Rf = 0.34)]; Yield: 70%; yellow amorphous powder; H NMR (400 MHz, CDCl3) δ 7.30
(s, 1H), 6.51 (d, J = 3.2 Hz, 1H), 6.11 (d, J = 2.9 Hz, 1H), 5.41 (s, 1H), 3.11 (d, J = 17.3
Hz, 1H), 2.37 (s, 3H), 2.19 (d, J = 17.3 Hz, 1H), 2.10 – 1.90 (m, 6H), 1.87 – 1.76 (m,
2H), 1.69 (t, J = 13.6 Hz, 1H), 1.62 – 1.41 (m, 6H), 1.40 – 1.31 (m, 3H), 1.23 (s, 3H),
1.21 (s, 3H), 1.18 (s, 3H), 1.11 (s, 3H), 1.04 (s, 3H), 1.02 – 0.95 (m, 1H), 0.93 (s, 3H),

0.91 – 0.86 (m, 1H), 0.85 (s, 3H).13C NMR (100 MHz, CDCl3) δ 207.16, 183.73, 155.01,
151.23, 144.30, 129.19, 124.55, 122.71, 117.26, 108.85, 52.79, 48.24, 45.42, 44.83,
44.34, 44.16, 42.60, 41.81, 39.68, 38.29, 35.69, 32.07, 31.78, 31.09, 29.92, 28.75, 28.24,
26.99, 26.11, 25.77, 23.73, 22.60, 20.47, 16.37, 15.82, 14.06. (+)-HRESIMS m/z
569.3613 [M+Na]+ (calcd for C36H50O4Na, 569.3601).
3.2.13 3-Methylthiophene-2-carboxalmethylene-3-oxo-18-olean-12-en-30-oic acid (16).
According to the above general methods, derivative 16 was obtained by Claisen Schmidt
condensation of GA-O with 3-Methylthiophene-2-carboxaldehyde and purified by
petroleum [Ether -AcOEt (18: 1, Rf = 0.41)]; Yield: 42%; yellow amorphous powder; 1H
NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.42 (d, J = 5.0 Hz, 1H), 6.96 (d, J = 5.1 Hz,
1H), 5.43 (s, 1H), 3.06 (d, J = 16.8 Hz, 1H), 2.40 (s, 3H), 2.19 (d, J = 17.1 Hz, 1H), 2.10
– 1.89 (m, 6H), 1.88 – 1.75 (m, 2H), 1.69 (t, J = 13.6 Hz, 1H), 1.63 – 1.42 (m, 6H), 1.41
– 1.33 (m, 3H), 1.23 (s, 3H), 1.21 (s, 3H), 1.21 (s, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.99 (d,
J = 14.3 Hz, 1H), 0.94 (s, 3H), 0.91 – 0.87 (m, 1H), 0.83 (d, J = 9.5 Hz, 3H).13C NMR
(100 MHz, CDCl3) δ 207.50, 183.04, 144.24, 142.11, 133.07, 130.45, 129.52, 128.65,
128.14, 122.70, 52.69, 48.25, 45.62, 44.89, 44.74, 44.12, 42.64, 41.83, 39.71, 38.28,
36.12, 32.07, 31.74, 31.12, 30.02, 28.70, 28.24, 27.03, 26.12, 25.76, 23.81, 22.57, 20.53,
16.34, 15.88, 14.69. (+)-HRESIMS m/z 585.3384 [M+Na]+ (calcd for C36H50O3SNa,
585.3373).
3.3 Evaluation of the Antibacterial Activity in Vitro
The values of minimum inhibitory concentration (MIC) were determined by

turbidimetry.35 A series of diluted test compounds were obtained by two-fold serial
dilution using DMSO as the solvent. The bacterial culture was inoculated at a
concentration of 1 × 105 CFU / mL in a 96-well microplate. Each well contained 5 μL of
specific concentration of compound and 195 μL of bacterial solution. In addition, a blank
group (DMSO and bacteria-free medium), and a negative group (DMSO and
microorganisms) were set. After incubating in a bacteria oven at 37 °C for 24 h, the
absorbance of each well was measured with a Multimode Plate Reader (Infinite 200), 600
nm. The lowest concentration that did not cause significant bacterial growth compared to
the blank control was defined as the MIC.
The killing kinetic of compounds 05, 10 against Gram-positive strains, including
S.aureus (ATCC 6538), S. aureus subsp. aureus (ATCC 29213) and S.epidermidis
(ATCC 12228), was determined in 96-well microplates.36-37 The microplate was
incubated at 37 ° C for 24 hours. The absorbance was measured every 2 hour by using a
multi-plate reader (infinite 200) at 600 nm to monitor bacterial growth,
3.4 TPA-Induced Skin Inflammation in Mice
Experimental mice were randomly divided into nine groups (six animals each),
including blank group, TPA group, ibuprofen (40 mg/kg) group, GA (10mg/kg, 20 mg/kg,
40 mg/kg) groups and compound 10 (10 mg/kg, 20 mg/kg, 40 mg/kg) groups. All
compounds, suspended in 1.0% sodium carboxymethylcellulose, were prepared fresh
immediately prior to the assay. The drug was injected into the mice for seven consecutive
days by gavage, while the blank group and TPA group were injected with equal doses of

sodium carboxymethylcellulose per day. After 1 hour at the end of the last gavage, TPA
(0.125 μg) dissolved in 20 μL of acetone was coated to the inner and outer surfaces of the
mice ears, and the blank group was smeared with an equal dose of acetone. All mice were
killed in 6 hours and ear punches (diameter 6mm) were quickly taken out, weighed, and
stored at low temperature.
3.5 Histopathological Analyses: Hematoxylin-Eosin (HE) Staining
For morphological assessment of cutaneous inflammation, biopsies from control and
treated ears of mice in each treatment group were collected.38 The ear samples were fixed
in 4% paraformaldehyde, decalcified in EDTA buffer, subjected to a series progression of
dehydration. The processed tissues were embedded in paraffin, and cut into a thickness of
4 μm using a paraffin placement machine and stained with hematoxylin and eosin. A
representative area was selected for qualitative light microscopic (AXIO Scan. Z1)
analysis, cellular infiltration and epidermal hyperplasia were evaluated using
magnifications of 200×. To minimize a source of bias, the investigator did not know the
group that he was analyzing.
According to the commonly used tissue section H&E staining criteria, the epidermal
stratum corneum hyperplasia and leukocyte infiltration of each section were scored.39 The
higher the score, the deeper the staining intensity ： 0, indicating no staining; 1+,
indicating weak; 2+, indicating mild; 3+, indicating strong. 1+, 2+, and 3+ were
expressed as 1, 2, and 3 points, respectively. The evaluation of the extent of staining was
similar：0, no staining; 1+, ≤ indicating 25% of positive cells; 2+, indicating 26–50%

of positive cells; and 3+, ≥ indicating 51% of positive cells.
3.6 Immunohistochemical Assays for TNF-α, IL-1β and p65
The immunohistochemical study was performed as previously reported with minor
modifications.40 Firstly, the tissue sections were heated at 100 °C for 20 min in 10 mM
EDTA buffer with 0.05% Tween-20 (pH 6.0) for antigen retrieval. After natural cooling,
it was washed with PBS buffer (pH = 7.4), and the endogenous peroxidase activity was
quenched after incubation in 3% H2O2. For blocking buffer, 10% normal goat serum was
used for 30 min. Then, the sections were incubated with the TNF-α, IL-1β and p65
antibodies overnight (4 °C). After diluting in PBS, the sections were incubated with the
anti-goat IgG for 50 min and stained with 3, 3-diaminobenzidine-peroxide (DAB)
cromophore and Harris hematoxylin. Finally, the average optical density values of TNF-α,
IL-1β and p65 were detected by Image-Pro Plus (version 6.0) software.
3.7 Statistical Analysis
The results were expressed at the mean ± SD. Statistical comparisons among groups
were performed using the Dunnett’s multiple test. The significance level was set at *P <
0.05, **P < 0.01 compared with the controls. P < 0.05 indicated a significant difference
between the two sets of data.
Supporting Information
Brief descriptions in nonsentence format listing the contents of the files supplied as
Supporting Information.

Copies of the NMR and HRMS spectra of all of the new compounds (Compounds 04 ~
16)
Acknowledgement
This work was financially supported by the Guangzhou Science and Technology
Foundation (2016201604030025,201902010430100011), Guangdong Science and
Technology Foundation (2017A050501034).
Competing financial interests
The authors declare no competing financial interest.
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