Microbial transformation of glycyrrhetinic acid derivatives by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741

Article abstract of DOI:10.1016/j.bmc.2020.115465

Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process, which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them, olean-12-ene-3β,7β,15α,19α,30-pentol (16) and olean-12-ene-3β,7β,15α,27,30-pentol (17) showed significant inhibitory effect with IC₅₀ values of 0.64 and 0.07 μM, respectively.

Full text of DOI:10.1016/j.bmc.2020.115465

Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microbial transformation of glycyrrhetinic acid derivatives by Bacillus
subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741
a,1
a,b,1
a
a
b
a,b,⁎
Pingping Shen , Jian Zhang
b
, Yuyuan Zhu , Wei Wang , Boyang Yu , Weiwei Wang
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, PR
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play
a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized
five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation
and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their
structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the
first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in
the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process,
which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory
effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them,
olean-12-ene-3β,7β,15α,19α,30-pentol (16) and olean-12-ene-3β,7β,15α,27,30-pentol (17) showed significant
inhibitory effect with IC50 values of 0.64 and 0.07 μM, respectively.
Glycyrrhetinic acid derivatives
Pentacyclic triterpene
Biotransformation
Glycosylation
Hydroxylation
1
. Introduction
Biotransformation is an alternative chemical tool for the develop-
ment of sustainable technologies to produce chemicals and drugs of
Licorice (Glycyrrhiza), one of the most widely used herbal medicines,
commercial importance because of the abundant variety of enzymes
10
frequently appearing in various prescriptions for the treatment of dis-
available in microorganisms. In the previous study, more than 100
biotransformation products were obtained from natural pentacyclic
terpenoids (PTs), confirming that microbial transformation is an at-
tractive method to expand the chemical diversity of structurally com-
1
eases ranging from cough to peptic ulcers. The major bioactive in-
gredient in licorice is glycyrrhizin, which is mainly administered in the
2,3
therapy for hepatitis B and C in Japan. Glycyrrhetinic acid (GA, 1), the
aglycone of glycyrrhizin isolated from licorice root, has been reported to
possess a variety of biological effects such as anti-inflammatory, anti-
1
1
plex natural products. With the chemical versatility and a broad
substrate range of cytochromes P450, Bacillus megaterium was widely
served as a biocatalyst for the selective oxidation of various natural
4,5
bacterial, anti-depressant, anti-ulcer, anti-allergen, and anti-malarial.
1
2,13
However, some undesirable physical–chemical properties of GA, in-
cluding high hydrophobicity, low water solubility and membrane per-
meability were observed, which contribute to inadequate bioavailability,
products.
Bacillus subtilis, holding the inherent robust glycosyl-
transferases, could efficiently catalyze a vast variety of compounds for
1
4
the synthesis of high-value glycosylation products. In biosynthetic
pathways of PTs, a series of site-specific oxidations of the skeleton are
involved in the late stages, most probably catalyzed by P450s and fol-
lowed by glycosylations catalyzed through UDP glycosyltransferases
(UGTs). Such modifications are essential for improving their biological
6,7
and seriously restrict its potential clinical applications. To broaden
biological windows, numerous chemical modifications based on the GA
framework have been explored. Deoxoglycyrrhetol dihemiphthalate
(
DGDH) (Fig.1), disodium phthalate of deoxoglycyrrhetol, have been
1
5,16
synthesized and reported to have a remarkable analgesic effect like as-
pirin through suppression of PGE2 production.8
activities and physicochemical features.
Herein, in continuation of
,9
our studies on the biotransformation of bioactive PTs, a series of
⁎
Corresponding author at: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 211198, PR China.
E-mail addresses: shpp1127@163.com (P. Shen), wilsonzhang99@cpu.edu.cn (J. Zhang), yuyuanzhu616@163.com (Y. Zhu),
1
721020352@stu.cpu.edu.cn (W. Wang), weiweiwang@cpu.edu.cn (W. Wang).
1
Contributed equally to this work.
https://doi.org/10.1016/j.bmc.2020.115465
Received 14 January 2020; Received in revised form 18 March 2020; Accepted 22 March 2020
0968-0896/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Pingping Shen, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2020.115465

P. Shen, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
transformation of GA (1, 200 mg) with B. subtilis resulted in two known
glycosylation products 7 and 8. Compound 7 was obtained as a white
+
powder, possessing a [M+H] ion at m/z 633.3966 (calcd for
C
36
H
57
O , 633.3997), indicating a 162 amu mass increase to that of
9
compound 1. Compound 8 was isolated as a white powder, giving a [M
−
+
Cl] ion at m/z 829.4158 (calcd for C42
66
H O14Cl, 829.4147) in the
HR-ESI-MS, indicating a 162 amu mass increase to that of 7. By com-
2
2
parison of the spectroscopic data in the literature, their structures
were elucidated as 30-O-β-D-glucopyranosyl glycyrrhetinic acid and 3-
O-(β-D-glucopyranosyl) glycyrrhetinic acid-30-O-β-D-glucopyranoside
Fig. 1. Structure of deoxoglycyrrhetol dihemiphthalate (DGDH).
(
Fig. 3).
Incubation of glycyrrhaldehyde (2, 200 mg) with B. subtilis led to
synthetic compounds as starting aglycon, we prepared five glycosylated
derivatives by Bacillus subtilis ATCC 6633 and eight oxidized products
by Bacillus megaterium CGMCC 1.1741. Among them, nine metabolites
were isolated for the first time. All the compounds were evaluated for
their anti-inflammatory activity upon nitric oxide (NO) production in
lipopolysaccharide (LPS)-induced RAW 264.7 cells.
the production of compound 9. It was isolated as a white powder, gave
−
a [M+Cl] ion at m/z 651.4124 (calcd for C36
H
56
8
O Cl, 651.3669) in
the HR-ESI-MS, indicating a 162 amu mass increase to that of 2. By
1
contrast with the D NMR spectrum data of 2, compound 9 was eluci-
dated as 30-O-β-D-glucopyranosyl glycyrrhaldehyde (Fig. 3).
Fermentation of glycyrrhetol (3, 200 mg) with B. subtilis resulted in
one new glycosylated product 10. It was obtained as a white powder,
2
. Results and discussion
+
gave a [M+H] ion at m/z 619.4232 (calcd for C36
H
59
O , 619.4204) in
8
the HR-ESI-MS, indicating a 162 amu mass increase to that of 3. By
2.1. Substrates preparation
1
3
further contrast with the C NMR spectrum of 3, compound 10 was
elucidated as 3-O-(β-D-glucopyranosyl) glycyrrhetol (Fig. 3).
GA and oleanlic acid were obtained commercially and served as a
Treating 11-oxo-oleanolic acid (4, 200 mg) with B. subtilis obtained
one known mono-glycosylated metabolite 11. Compound 11, isolated
precursor in the chemical synthesis of five products 2–6 according to
the previous procedures (Fig. 2). Firstly, GA was reduced with LiAlH
4
+
as a white powder, gave a [M+H] ion at m/z 633.4012 (calcd for
and subsequently oxidated through MnO , to give principal product
2
1
7,18
C
36
H
57
9
O , 633.3997) in the HR-ESI-MS, indicating a 162 amu mass
13
glycyrrhetol and a small quantity of by-product glycyrrhaldehyde.
increase to that of compound 4. By comparing its C NMR spectrum
with those reported in the literature, compound 11 was elucidated as
11-oxo-oleanolic acid-28-O-β-D-glucopyranoside (Fig. 3).
Secondly, oleanlic acid was used as a lead for the preparation of 11-oxo-
oleanolic acid by stepwise chemical reactions (C-3 protection, C-11
1
9,20
oxidation and C-3 deprotection) following the previous study.
Incubation of 11-deoxoglycyrrhetinic acid (5, 200 mg) with B.
megaterium obtained one new hydroxylated product 12. Its structure
was elucidated as 3β,7β,27-trihydroxy-olean-12-ene-30-oic acid on the
basis of HR-ESI-MS and NMR spectrum (Fig. 4).
Furthermore, GA and glycyrrhetol as substrates, through the Clem-
menson reduction reaction, two C-11 decarbonylation products 5 and 6
were prepared, respectively.21
−
Compound 12, isolated as a white powder, gave a [M+Cl] ion at
2.2. Biotransformation
m/z 523.3179 (calcd for C30
H
48
5
O Cl, 523.3196) in the HR-ESI-MS, in-
13
dicating a 32 amu mass increase to that of compound 5. The C NMR
Preparative scale biotransformation, isolation and structure eluci-
spectrum showed the presence of one new methine carbon signal at δ
dation of purified products were investigated. GA with a free hydroxyl
group on C-3 and a carboxyl group on C-30 on the skeleton is easily
glycosylated, particularly, C-30 carboxyl group. Microbial
7
4.3 ppm. And the new proton signal δ 4.66 (1H, dd, J = 10.9, 4.6 Hz)
1 13
ppm showed the direct H– C correlation with δ 74.3 ppm in HSQC
Fig. 2. Synthetic routes of compounds 2–6.
2

P. Shen, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 3. Biotransformation of 1–4 by Bacillus subtilis ATCC 6633.
which was first discovered in Euonymus mupinensis.23
Compound 14 was obtained as a white powder, possessed a [M
+
+
H] ion at m/z 475.3241 (calcd for C30
H
51
O
4
, 475.3782), indicating
1
a 32 amu mass increase to that of compound 6. In H NMR spectrum,
the new proton signal appeared at δ 4.66 (d, J = 8.3 Hz, 1H) ppm,
which showed a direct correlation with δ 67.0 ppm in HSQC and
1
13
showed long-range H– C correlations with δ 21.2 ppm (C-27), δ
3
7.5 ppm (C-10), δ 47.2 ppm (C-8) in HMBC, confirming δ 67.0 ppm
Fig. 4. Biotransformation of 11-deoxoglycyrrhetinic acid (5) by Bacillus mega-
terium CGMCC 1.1741.
was assigned to C-15. The new proton signal δ 4.66 ppm had NOE
enhancements with δ 1.33 ppm (s, 3H, H-26), indicating that 15-OH
should be located in the α-configuration. According to the similar
analysis of the NMR spectrum with 12, the new carbon signal at δ
and showed the long-range correlations with δ 30.7 ppm (C-6), δ
4
6.9 ppm (C-8), δ 49.7 ppm (C-14) and δ 12.4 ppm (C-26), confirming
7
2.4 ppm could be attributed to C-7. Therefore, compound 14 was
C-7 of the site of hydroxylation. The new proton signal δ 4.66 ppm had
NOE enhancements with δ 1.23 ppm (1H, H-5), indicating that 7-OH
should be located in the β-configuration. Another new carbon signal
appeared at δ 65.1 ppm was assigned to C-27 since the new proton
signal δ 4.40 (d, J = 12.3 Hz, 1H), 4.14 (d, J = 12.3 Hz, 1H) ppm
showed direct correlations with δ 65.1 ppm in HSQC and showed long-
identified as olean-12-ene-3β,7β,15α,30-tetraol.
Compound 15 was obtained as a white powder and HR-ESI-MS
−
showed an [M+COOH]
ion at m/z 535.3617 (calcd for
C
30
H
50
O COOH, 535.3640), which indicated an increment of 48 amu as
13
5
compared to 6. In the C NMR, there were three new carbon signals at
δ 72.7 ppm, δ 67.2 ppm, δ 66.8 ppm. The new carbon signal at δ
1
13
range H– C correlations with δ 140.1 ppm (C-13), δ 46.9 ppm (C-8),
and δ 49.7 ppm (C-14) in HMBC (Fig. 6). Thus, metabolite 12 was
identified as: 3β,7β,27-trihydroxy-olean-12-ene-30-oic acid.
6
6.8 ppm was assigned to C-11 since the new proton signal at δ 4.59 (s,
1
H) ppm direct correlation with δ 66.8 ppm in HSQC and long-range
1
13
H– C correlations with δ 129.7 (C-12), 148.4 (C-13), 56.8 (C-9), 39.4
C-10) ppm in HMBC. The signal at δ 4.59 ppm had NOE enhancements
Fermentation of 11-deoxoglycyrrhetol (6, 400 mg) by B. megaterium
yielded a known compound 13 along with six new metabolites 14–19
(
with δ 1.29 ppm (s, 3H, H-25), indicating that 11-OH should be located
in the α-configuration. By contrast with the spectrum data of 14, the
new carbon signals at δ 72.7 ppm and δ 67.2 ppm were attributed to 7β
and 15α, respectively. Therefore, metabolite 15 was characterized as
olean-12-ene-3β,7β,11α,15α,30-pentol.
(
Fig. 5).
Compound 13 was obtained as a white powder and the HR-ESI-MS
+
showed
a
[M+H]
ion at m/z 441.3701 (calcd for
C
30
H
49 2
O ,
1
3
4
41.3727). By comparing its C NMR with literature, compound 13
was identified as olean-3-oxo-12-en-30-ol, also refers to mupinensisone
3

P. Shen, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Fig. 5. Biotransformation of 11-deoxoglycyrrhetol (6) by Bacillus megaterium CGMCC 1.1741.
Fig. 6. Key HMBC and NOESY correlations of compound 12.
−
Compound 16, isolated as a white powder, gave an [M+Cl] ion at
showed the long-range correlations with δ 47.8 ppm (C-8), δ
140.65 ppm (C-13), δ 54.3 ppm (C-14), confirming C-27 of the one site
of hydroxylation. The remaining of new carbon signals at δ 73.5 ppm, δ
68.2 ppm were very similar to those of compound 14 and respectively
assigned to C-7, C-15. Therefore, metabolite 17 was elucidated as olean-
12-ene-3β,7β,15α,27,30-pentol.
1
3
m/z 525.3341 (calcd for C30
H
50
O Cl, 525.3352). In the C NMR, there
5
were three new carbon signals at δ 75.9 ppm, δ 72.5 ppm, δ 67.2 ppm.
The new carbon signal at δ 75.9 ppm was assigned to C-19 since the
new proton signal at δ 3.88 (m, 1H) direct correlation with δ 75.9 ppm
in HSQC and long-range correlations with δ 151.5 (C-13), 39.3 (C-17),
−
5
3.4 (C-18) ppm in HMBC. The NOESY spectrum of 16 showed NOE
Compound 18 was purified as a white powder, gave an [M+Cl]
enhancements between δ 3.88 ppm and δ 1.17 ppm (s, 3H, H-28),
confirming the 19-OH should be α-configuration (Fig. 6). By contrast
with the spectrum data of 14, the remaining of new carbon signals at δ
ion at m/z 523.3178 (calcd for C30
H
48
O Cl, 523.3196), indicating a 48
5
amu mass and one degree of unsaturation increase to that of compound
1
3
6. In the C NMR spectrum, there had two new olefinic carbons, also
1
7
2.5 ppm, δ 67.2 ppm were respectively assigned to C-7, C-15. There-
quaternary carbon, at δ 157.1, 144.9 ppm been observed. In H NMR
fore, metabolite 16 was elucidated as olean-12-ene-3β,7β,15α,19α,30-
spectrum, the new proton signal appeared at δ 5.87 (1H, d, J = 6.1 Hz)
ppm, which showed a direct correlation with δ 125.0 ppm in HSQC and
pentol.
1
13
Compound 17 was purified as a white powder. The HR-ESI-MS
showed long-range H– C correlations with δ 157.1 ppm, δ 46.3 ppm
(C-18), δ 51.5 ppm (C-14) in HMBC, confirming δ 125.0 ppm was as-
signed to C-12. Another new proton signal appeared at δ 5.72 (1H, d,
J = 6.0 Hz) ppm, showed a direct correlation with δ 117.8 ppm in
+
showed a [M+Na] ion at m/z 513.3535 (calcd for C30
H
50
5
O Na,
1
3
5
13.3505). The C NMR spectrum showed the presence of three new
1
carbon signals at δ 73.5, 68.2, 63.4 ppm. In H NMR, the new proton
signal δ 4.71(1H, d, J = 12.6 Hz), 4.39 (1H, d, J = 12.6 Hz) ppm
showed the direct 1H– C correlation with δ 63.4 ppm in HSQC and
1
13
HSQC and showed long-range H– C correlations with δ 144.9 ppm, δ
51.1 ppm (C-8), δ 40.3 ppm (C-10) in HMBC, indicating that δ
13
4

P. Shen, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Table 1
Inhibition on NO production of compounds 1–19 in LPS-induced RAW264.7 cells.
Compound
IC50/μM a
Cell viability (%)
Compound
IC50/μM
Cell viability (%)
b
1
2
3
4
5
6
7
8
9
NA
4.035 ± 0.31
89.64 ± 2.38
56.57 ± 1.25
111.25 ± 2.69
83.41 ± 1.55
101.89 ± 3.01
94.79 ± 2.19
120.54 ± 7.55
7.33 ± 0.59
11
12
13
14
15
16
17
18
19
11.05 ± 4.30
NA
82.73 ± 2.40
113.75 ± 3.22
109.80 ± 0.73
119.87 ± 4.61
111.84 ± 1.16
124.59 ± 0.91
106.62 ± 2.56
129.85 ± 0.72
130.09 ± 1.66
106.20 ± 2.20
47.17 ± 4.97
50.28 ± 7.28
38.34 ± 0.70
NA
NA
25.61 ± 0.78
NA
48.19 ± 2.12
0.64 ± 0.31
0.07 ± 0.87
68.87 ± 0.93
NA
NA
NA
NA
NA
c
1
0
1.22 ± 0.06
quercetin
4.53 ± 0.81
a: Concentration necessary for 50% inhibition (IC50).
b: NA = no activity; Values are mean ± SD (n = 3).
c: Quercetin was used as a positive control.
1
1
17.8 ppm was assigned to C-11. And, the new olefinic carbons δ
57.1 ppm was assigned to C-9 since the proton signal at δ 1.56 (s, 3H,
addition, it was found that the inhibitory activity disappeared after the
primary alcohol at C-27 was oxidized to aldehyde. As indicated above,
the substituent site of hydroxyl group was even crucial to their anti-
inflammatory activity.
1
13
H-26) and δ 1.25 (s, 3H, H-25) ppm showed long-range H– C corre-
lations with it, so another olefinic carbon δ 144.9 ppm should be lo-
cated at C-13. It is speculated that the new conjugate double bond in
C9-C11 and C12-C13 was formed. Compared to the NMR spectra of 12
and 17, the three new carbon signals at δ 73.6 ppm, δ 69.2 ppm and δ
3. Conclusions
1
3
6
2
9
4.9 ppm in the C NMR spectrum were assigned to C-7β, C-15α and C-
7, respectively. Therefore, the metabolite 18 was identified as: olean-
(11),12(13)-diene-3β,7β,15α,27,30-pentol.
As mentioned in the introduction, glycyrrhizin and especially its
biologically active metabolite GA, involved in a variety of biological
processes, are essential for the treatment of several diseases and dis-
orders. Herein, GA and oleanlic acid were served as leads in the semi-
synthesis of five ring C/E-modified derivatives. Biotransformation of all
the six compounds by B. subtilis and B. megaterium were investigated,
+
Compound 19, isolated as a white powder, possessed an [M+Na]
ion at m/z 511.3379 (calcd for C30
H
48
O Na, 511.3394), indicating one
13
5
degrees of unsaturation increase to that of compound 6. In the C NMR
spectrum, three new carbon signals, respectively, displayed at δ
2
2
08.2 ppm, δ 74.9 ppm, δ 67.4 ppm. The new carbon signal at δ
08.2 ppm was assigned to C-27 since δ 10.4 ppm (s,1H) showed direct
Table 2
1
13
13
C NMR data of compounds 9–19 in C
5
D N(150 MHz).
5
correlations with δ 208.2 ppm in HSQC and showed long-range H–
C
correlations with δ 139.5 (C-13) ppm, δ 60.7 (C-14) ppm, δ 23.6 (C-15)
ppm in HMBC. In contrast to the NMR spectrum of 15, the remaining
new carbon signals at δ 74.9 ppm, δ 67.4 ppm were assigned to C-7 and
C-11 Thus, metabolite 19 was determined as 3β,7β,11α,30-tetra-
hydroxy-olean-12-en- 27- aldehyde.
Position
9
10
12
14
15
16
17
18
19
1
38.9
27.0
89.0
40.0
55.7
18.0
33.2
43.8
62.5
37.6
39.9
28.6
88.9
40.3
55.7
18.0
33.3
45.9
62.4
37.6
39.4
28.6
78.2
39.5
53.5
30.7
74.3
46.9
49.0
38.1
39.5
28.9
78.2
39.4
53.1
29.5
72.4
47.2
48.6
37.5
24.4
41.5
28.9
78.2
39.9
53.3
29.6
72.7
49.6
56.8
39.4
66.8
43.0
29.1
78.0
39.8
53.0
29.5
72.5
49.4
48.2
33.2
21.2
38.1
29.0
78.1
39.5
53.1
29.6
73.5
47.8
48.4
39.3
24.8
38.7
28.5
77.7
39.8
49.5
29.8
73.6
51.1
41.6
28.9
77.8
39.9
53.0
31.0
74.9
51.9
2
3
4
5
6
2.3. Anti-inflammatory activity of all the compounds
7
8
Compounds 1–19 were submitted to the bioassay of NO inhibitory
9
157.1 57.6
40.3 40.0
117.8 67.4
effect to evaluate possible improvement after structural modifications
in LPS-induced RAW 264.7 cells. Discovery of new potential com-
pounds against NO production may be the key to the treatment of in-
flammation-related diseases, including arthritis, diabetes, metabolic
syndrome and tumor.
10
1
1
1
1
1
2
3
4
199.8 199.8 24.5
129.2 128.9 128.6 124.5 129.7 124.1 129.5 125.0 134.0
168.9 170.3 140.1 146.5 148.4 151.5 140.6 144.9 139.5
47.3
24.2
26.6
32.4
48.3
40.3
45.9
28.9
37.8
27.0
17.1
17.4
19.1
27.3
28.6
28.7
47.6
28.6
23.9
41.2
47.6
44.0
30.3
32.8
36.3
29.0
17.4
17.1
19.2
27.3
28.6
27.0
49.7
26.6
28.5
39.3
50.2
42.9
44.7
32.2
32.8
29.0
17.1
16.8
12.5
65.1
29.2
29.5
49.6
67.0
37.1
37.9
48.8
42.6
36.5
30.5
33.3
29.0
17.0
16.1
11.4
21.2
29.2
28.5
50.5
67.2
37.3
37.1
48.0
42.5
36.4
30.4
33.2
29.2
17.0
17.5
13.4
21.6
29.6
28.9
65.8
50.2
67.2
37.1
39.3
53.4
75.9
36.4
30.4
40.0
28.9
16.9
13.8
29.7
21.2
17.7
28.9
65.9
54.3
68.2
37.7
37.1
48.6
41.4
36.5
30.1
33.1
28.9
17.0
16.5
12.7
63.4
29.5
28.6
66.1
51.5
69.2
38.5
37.0
46.3
42.7
36.5
30.3
60.7
23.6
27.9
36.7
48.0
36.2
39.8
30.1
15
1
1
1
1
6
7
8
9
As shown in Table 1, among all the substrates, two C-11 dec-
arbonylation compounds 5 and 6 possessed a higher inhibitory effect on
NO production and lower cytotoxic activity than the corresponding
parent compounds. Secondly, glycosylation did not enhance the in-
hibitory activity of metabolites except for compound 11. Furthermore,
B. megaterium catalyzed highly efficient regio-selective oxidation on a
non-activated C-H bond of 11-deoxoglycyrrhetol and yielded one
known and six new compounds. Among the series of oxidated products,
compounds 13 and 18 exhibited moderate NO inhibition effects with
IC50 values of 38.34–68.87 μM. Compounds 16 and 17 showed sig-
nificant inhibitory activity against NO production than the positive
control, with IC50 values of 0.64, 0.07 μM, respectively, which is in-
creased by 75 and 688 times compared to the mother compound. A
simple structure–activity analysis revealed that compounds 14 and 15
with hydroxyl groups at C-7, C-11 and C-15, exhibited lower NO in-
hibition activity in contrast to the lead compound 6. However, com-
pounds 16 and 17, with hydroxyl groups at C-19 and C-27 respectively,
strongly enhanced inhibitory activity over that of quercetin. In
20
21
2
2
2
2
2
3
4
5
33.44 32.6
29.1
17.0
26.4
15.9
64.9
29.3
28.8
66.1
28.9
17.0
17.8
13.1
208.2
28.8
28.7
65.8
26
2
2
2
3
7
8
9
0
206.4 65.8
107.3 107.2
180.1 65.9
Glu-1′
2′
76.2
79.2
72.6
78.7
63.4
76.1
79.1
72.2
78.6
63.3
3
4
5
6
′
′
′
′
5

P. Shen, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
Table 3
1
H NMR data of compounds 12–19 in C
5
5
D N(600 MHz).
Position 12
14
15
16
17
18
19
1
2.76 d (13.9)
1.91, m
1.93, m
2.93, dd (10.7, 3.3)
1
.75, m
2.63, t (12.6)
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1.83, m
3.40, dd (11.3,4.8) 3.48, dd (10.2,5.3)
3.57, d (10.7)
1.25, m
3.51, d (7.1)
1.15, m
3.38, d (8.9)
1.21, m
3.42, m
1.35, m
5.34, m
3.45, dd (11.4, 4.7)
1.05, m
1.23, m
2.06, m
1.07, m
4.66, dd (10.9,4.6) 4.37, d (7.6)
4.41, d (7.4)
2.02, m
4.34, d (8.1)
2.33, m
4.74, m
4.02, dd (10.8,4.4)
2.11, m
2.47(m)
2.02, m
1.66, m
2.44, m
0
1
2
3
4
5
4.59, s
1.70, m
5.72, d (6.0)
5.87, d (6.1)
4.79, dd (8.9,2.5)
6.18, d (2.8)
5.46, m
5.81, d (3.4)
5.73, d (3.5)
5.83, m
2.15, m
4.66, d (8.3)
4.71, d (7.5)
1.33, m
4.70, d (9.2)
4.96, d (8.9)
1.39, m
2
.41, m
1
1
1
1
6
7
8
9
2.18, m
2.60, m
1.92, m
2.25, m
1.93, m
1.68, m
2.2, m
1.93, m
3.88, m
2.34, m
1.99, m
1.88, m
2.34, d (13.8)
1.94, m
2.33, dd (13.4,3.7)
1.85, m
1.67, m
2
.51, m
1.62, m
2
2
0
1
1.44, m
.28, m
2
22
23
24
25
26
27
2.04, m
1.30, s
1.12, s
1.36, s
1.05, s
1.70, s
1.19, s
1.27, s
1.08, s
1.03, s
1.33, s
1.61, s
1.32, s
1.14, s
1.29, s
1.44, s
1.74, s
1.23, s
1.21, s
1.13, s
1.08, s
1.30, s
1.46, s
10.46, s
1.07, s
1.08, s
1.04, s
1.03, s
1.02, s
1.25, s
1.33, s
1.33, s
1.56, s
4.14, d (12.3)
4.71, d (12.6)
4.39, d (12.6)
1.06, s
4.62, d (12.0)
4.39, d (12.0)
0.98, s
4
.40, d (12.3)
28
29
30
1.06, s
1.31, s
1.02, s
1.18, s
1.05, s
1.10, s
1.17, s
0.99, s
1.14, s
1.14, s
1.08, s
0.99, s
3.84, dd (11.7, 11.5) 3.87, d (10.3) 3.70, d
3.90, d (12.3) 3.77, d
(10.2)
3.86, dd (13.1,
9.8)
3.79, dd (18.1,
10.7)
3.79, d (10.6) 3.79, d
(10.6)
(
10.4)
and nine previously undescribed and four known products were iso-
lated. The results showed B. subtilis, holding the robust regio-selective
glycosylation ability, could efficiently catalyze aglycones 1–4 to pre-
pare a series of glycosides. Additionally, the culture displayed its pre-
ference to the free carboxyl group on the framework than the free hy-
droxyl group, as indicated in 7 and 11. B. megaterium was observed to
be capable of regio- and stereo-selective hydroxylation at unactivated
chromatography on silica gel (200–400 mesh). HPTLC was carried out
on precoated silica gel GF254 plates, and the silica gel was bought from
Qingdao Marine Chemical Group Co., PR China.
4.2. Microorganism and culture medium
Bacillus subtilis ATCC 6633 were obtained from courtesy of Prof.
CH
2
/CH on the skeleton of 5 and 6, covering C-7, 11, 15, 19, 27 sites,
3
John P.N. Rosazza of the University of Iowa, USA. Bacillus megaterium
CGMCC 1.1741 was purchased from China Center of Industrial Culture
Collection (CICC). Cultures were cultivated in 50 mL of soybean meal
glucose medium by a two-stage procedure held in 250 mL culture
flasks. The soybean meal glucose medium contained 20 g glucose, 5 g
which greatly expand the chemical diversity of structurally complex
PTs. Moreover, the substitution sites at C-19 and C-27 of such PTs was
found for the first time by biotransformation, but seen in plant-derived
2
4,25
triterpenoids.
The anti-inflammatory activity results indicated that
metabolite 16, 17 with hydroxyl groups at C-19, C-27, respectively,
significantly inhibited NO release even better than that of positive
control quercetin with IC50 values of 0.64, 0.07 μM. In summary, we
enriched the structural diversity of PTs by biotransformation and their
anti-inflammatory activity at low micromolar concentrations may be
worthy of further studies.
soybean meal, 5 g yeast extract, 5 g NaCl and 5 g K
2
HPO in one-liter
4
distilled water and was adjusted to pH 7.0 with 6 N HCl before being
autoclaved at 121 °C for 20 min. GA was purchased from Nanjing Plant
Origin Biological Technology Co., Ltd.
4.3. Fermentation, extraction and isolation
4
. Materials and methods
Cultures were incubated on a rotary shaker at 28°C and 180 rpm. A
1
0% inoculum derived from the 24-h-old stage I culture was used to
4.1. General experimental procedures
initiate the stage II culture, which was incubated for 24 h, and 1 mL of
acetone with 10 mg of the substrates was sequentially added. Cultures
were incubated for 4 days and extracted with equal volumes of EtOAc
three times. The organic layer was concentrated and isolated on silica
All the chemicals and solvents used for extraction and isolation were
of analytical grade. NMR spectra were recorded on a Bruker AV-600
spectrometer in C
5
D
5
N solution, with TMS as the internal standard. The
gel column chromatography and gradient eluted with CH
2
Cl
2
/CH OH
3
HR-ESI-MS experiments were performed on Agilent 1100 Series MSD
Trap mass spectrometer. Preparative reversed-phase HPLC was per-
formed on Agilent 1100 instrument equipped with Alltech 3300 ELSD
detector. Separation and purification were performed by column
(100:1–2:1, v/v). The samples were spotted on silica gel HPTLC plates,
and the results were visualized by spraying with H
2
SO
4
(10% in
ethanol). According to high-performance thin layer chromatography
(HPTLC) analysis, similar fractions were combined and further
6

P. Shen, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxxx
subjected to preparative HPLC with CH
3
CN/H
2
O as the mobile phase.
Appendix A. Supplementary material
The compounds were isolated at 30%-40% acetonitrile in water and
were further characterized by spectrometry.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115465.
1
3
0-O-β-D-glucopyranosyl glycyrrhaldehyde (9): white powder;
H
1
3
NMR and C NMR (pyridine‑d
5
), see Tables 2 and 3, respectively; ne-
−
gative HR-ESI-MS m/z 651.4124 [M+Cl] (calcd for C36
H
56
O
8
Cl,
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3
β,7β,27-Trihydroxy-olean-12-ene-30-oic acid (12): white solid;
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Declaration of Competing Interest
2. Hayashi H, Yamada K, Fukui H, Tabata M. Metabolism of exogenous 18β-glycyr-
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Acknowledgements
This Project was supported by Jiangsu Planned Projects for
Postdoctoral Research Funds (Grant No. 2018K085B) and the
Fundamental Research Funds for the Central Universities (Grant No.
2
632018PY14).
7