Biotransformation of isofraxetin-6-O-β-D-glucopyranoside by Angelica sinensis (Oliv.) Diels callus

Article abstract of DOI:10.1016/j.bmcl.2016.11.069

Isofraxetin-6-O-β-D-glucopyranoside, identified from traditional medicinal herbal Xanthoceras sorbifolia Bunge, has been demonstrated to be a natural neuroinflammatory inhibitor. In order to obtain more derivatives with potential anti-neuroinflammatory effects, biotransformation was carried out. According to the characteristics of coumarin skeleton, suspension cultures of Angelica sinensis (Oliv.) Diels callus (A. sinensis callus) were employed because of the presence of diverse phenylpropanoids biosynthetic enzymes. As a result, 15 products were yielded from the suspension cultures, including a new coumarin: 8′-dehydroxymethyl cleomiscosin A (1), together with 14 known compounds. Their structures were elucidated by extensive spectroscopic analysis. Furthermore, the biotransformed pathways were discussed. Among them, compound 13 was transformed from isofraxetin-6-O-β-D-glucopyranoside, while compounds 1–6, 10–12, 14–15 were derived from the culture medium stimulated by the substrate. The biotransformation processes include hydroxylation, oxidation and esterification. Furthermore, their inhibitory effects on lipopolysaccharide (LPS)-activated nitric oxide (NO) production were evaluated in BV2 microglial cells. It is worth noting that, 1, 1′-methanediylbis(4-methoxybenzene) (3), obtucarbamates A (5), 2-nonyl-4-hydroxyquinoline N-oxide (10) and 1H-indole-3-carbaldehyde (11) exhibited significant inhibitory effect against neuroinflammation with IC₅₀values at 1.22, 10.57, 1.02 and 0.76?μM respectively, much stronger than that of the positive control minocycline (IC₅₀35.82?μM).

Full text of DOI:10.1016/j.bmcl.2016.11.069

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biotransformation of isofraxetin-6-O-b-D-glucopyranoside by Angelica
sinensis (Oliv.) Diels callus
a,
Di Zhou a, Yuhua Zhang b, Zhe Jiang c, Yue Hou d, Kun Jiao d, Chunyan Yan b, , Ning Li
⇑
⇑
a School of Traditional Chinese Materia Media, Shenyang Pharmaceutical University, Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Wenhua Road 103, Shenyang 110016, China
b College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
c Department of Pharmacy, Yanbian University Hospital, Yanji 133000, China
d College of Life and Health Sciences, Northeastern University, Shenyang 110004, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Isofraxetin-6-O-b-D-glucopyranoside, identified from traditional medicinal herbal Xanthoceras sorbifolia
Received 12 October 2016
Revised 4 November 2016
Accepted 22 November 2016
Available online xxxx
Bunge, has been demonstrated to be a natural neuroinflammatory inhibitor. In order to obtain more
derivatives with potential anti-neuroinflammatory effects, biotransformation was carried out.
According to the characteristics of coumarin skeleton, suspension cultures of Angelica sinensis (Oliv.)
Diels callus (A. sinensis callus) were employed because of the presence of diverse phenylpropanoids
biosynthetic enzymes. As a result, 15 products were yielded from the suspension cultures, including a
new coumarin: 80-dehydroxymethyl cleomiscosin A (1), together with 14 known compounds. Their struc-
tures were elucidated by extensive spectroscopic analysis. Furthermore, the biotransformed pathways
Keywords:
Biotransformation
Isofraxetin-6-O-b-D-glucopyranoside
Angelica sinensis (Oliv.) Diels callus
were discussed. Among them, compound 13 was transformed from isofraxetin-6-O-b-D-glucopyranoside,
while compounds 1–6, 10–12, 14–15 were derived from the culture medium stimulated by the substrate.
The biotransformation processes include hydroxylation, oxidation and esterification. Furthermore, their
inhibitory effects on lipopolysaccharide (LPS)-activated nitric oxide (NO) production were evaluated in
BV2 microglial cells. It is worth noting that, 1, 10-methanediylbis(4-methoxybenzene) (3), obtucarba-
mates A (5), 2-nonyl-4-hydroxyquinoline N-oxide (10) and 1H-indole-3-carbaldehyde (11) exhibited sig-
Anti-neuroinflammation
nificant inhibitory effect against neuroinflammation with IC50 values at 1.22, 10.57, 1.02 and 0.76
respectively, much stronger than that of the positive control minocycline (IC50 35.82 M).
lM
l
Ó 2016 Elsevier Ltd. All rights reserved.
Isofraxetin-6-O-b-
D
-glucopyranoside, a coumarin glycoside, is
than 160 compounds from A. sinensis, mainly including phthalides,
phenylpropanoids, terpenoids and essential oils.8 Thus, phenyl-
propanoids biosynthesis enzymes are abundant in A. sinensis. In
order to get more bioactive derivatives of the bioactive molecule,
the major active constituent of the testa of Xanthoceras sorbifolia
Bunge (X. sorbifolia).1–4 We previously found that isofraxetin-6-O-
b-D-glucopyranoside could exhibit significant inhibitory effect of
NO production in over-activated microglia cells, which suggested
its potency to be used as natural neuroinflammatory inhibitor.1
Biotransformation is an efficient approach for structural modifi-
cation of complicated natural products.5,6 The plant cultured cells
have abilities of the regio- and stereoselective hydroxylation,
oxido-reduction, hydrogenation, glycosylation, and hydrolysis for
various organic compounds as well as microorganisms under mild
reaction conditions.7 Over the past decades, phytochemical inves-
tigations have revealed the isolation and characterization of more
isofraxetin-6-O-b-
A. sinensis callus.
However, there is no report about biotransformation of
coumarin glycosides using A. sinensis callus up to now. Our inves-
tigation is to find out bioactive derived products of isofraxetin-6-O-
D
-glucopyranoside
was
transformed
by
b-D-glucopyranoside by A. sinensis callus. As a result, 15 products
were identified, including one new coumarin and 14 known ones,
from the suspension cultures of A. sinensis callus (Fig. 1). The pos-
tulated biosynthetic pathways of the transformed products were
deduced as shown in Figs. 3 and 4. Furthermore, the anti-neuroin-
flammatory effects of the transformed products were evaluated in
lipopolysaccharide (LPS)-induced BV2 microglial cells (Fig. 5).
NMR spectra were measured on Bruker AVANCE III HD 600 MHz
instrument (Bruker, Switzerland), Bruker ARX-400 or ARX-600
⇑
Corresponding authors at: School of Traditional Chinese Materia Medica 49#,
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharma-
ceutical University), Ministry of Education, Wenhua Road 103, Shenyang 110016,
China (N. Li).
E-mail addresses: ycybridge@163.com (C. Yan), liningsypharm@163.com (N. Li).
http://dx.doi.org/10.1016/j.bmcl.2016.11.069
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.069

2
D. Zhou et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
OH
OH
O
HO
HO
O
OH
H3CO
O
O
Substrate: isofraxetin-6-O- -D-glucopyranoside
β
transformed products
4
5
4
3
5
H3CO
O
H3CO
O
3
OCH3
6
6
O
O
O
O
2
2
7
7
8
8
6'
6'
O
O
8'
5'
4'
HO
5'
4'
HO
OCH3
H3CO
OCH3
7'
7'
1'
2'
1'
8'
9'
2'
OH
3'
3'
OCH3
OCH3
3
2
4
1
H
N
O
OCH3
OH
O
O
H
H
N
NH2
H3CO
N
OCH3
O
HN
NH
O
O
O
HN
OCH3
OCH3
OCH3
O
N
H
8
O
7
9
6
8
10
5
OCH3
O
OH
O
H3CO
H3CO
O
H3CO
HO
H3CO
HO
H3CO
H3CO
H3CO
N
H3CO
O
O
O
O
OCH3
H
H3CO
H3CO
OH
H3CO
13
11
12
15
14
Fig. 1. Structures of compounds 1–15.
H3CO
O
the chromatographic and analytical grade reagents were obtained
from Tianjin DaMao Chemical Company (Tianjin, China).
Fresh A. sinensis was collected in the South China Botanical
Garden, Guangzhou, China. Petiole I (the portion adjacent to the
leaf) was collected from the cultivated plant for callus induction.9
O
O
O
HO
Isofraxetin-6-O-b-D-glucopyranoside was prepared from the testa
OCH3
of X. sorbifolia and the purity was determined to be 99.8% by HPLC
analysis.
Fig. 2. Key HMBC (
) correalations of compound 1.
We carried out a mass of assays to optimize the induce of A.
sinensis callus and subculture condition through composition of
callus induction media, plant organs and callus type, and obtained
OH
OH
OH
HO
HO
large-scale tissue culture of A. sinensis callus.9 Isofraxetin-6-O-b-
D-
O
O
OH
HO
Deglycosylation
glucopyranoside (859 mg) was distributed into A. sinensis callus
and they were co-incubated for one week under the optimal sub-
culture condition.10 After incubation, mixtures were separated into
the cultures and medium, then they were furthered extracted and
purified by chromatographic and recrystallization methods
repeatedly.11
H3CO
O
O
H3CO
O
O
13
Fig. 3. Possible biosynthesis pathway of compound 13 from isofraxetin-6-O-b-
glucopyranoside in A. sinensis callus.
D-
As a result, fifteen products were yielded, spectra data (1D, 2D
NMR, HR-ESI-MS) revealed the structures of the transformed prod-
ucts, including one new compound 1: 80-dehydroxymethyl
cleomiscosin A (1), cleomiscosin A (2),12 1, 10-methanediylbis (4-
methoxybenzene) (3),13,14 2-methoxyphenyl-(4-methoxyphenyl)
methane (4),13,14 obtucarbamates A (5),15,16 obtucarbamates B
(6),15,16 2-phenylacetamide (7),17 1-(6-hydroxy-2, 3-dimethoxy-
phenyl)ethan-1-one (8),18 uracil (9),19 2-nonyl-4-hydroxyquino-
line N-oxide (10),20 1H-indole-3-carbaldehyde (11),21 isofraxidin
(12),22 5, 6-dihydroxy-7-methoxycoumarin (13),23 (+)-isoschisan-
spectrometers, with TMS as an internal standard. Bruker micro
TOF-Q mass spectrometer was used to collect HR-ESI-MS data
recorded as m/z (rel.%) mode. Optical rotations were detected with
MCP 200 polarimeter produced by Anton Paar GmbH (Graz,
Austria). Silica gel GF254 used for thiner layer chromatography
and column chromatography was purchased from Qingdao Ocean
Chemical Group Co (Qingdao, China). Sephadex LH-20 was pro-
ducted by Pharmacia Company (Uppsala, Switzerland). ODS
drin (14),25 -schisandrin (15).24
c
(50
Co. Ltd (Kyoto, Japan). HPLC separation was carrried out on a
YMC ODS-A C-18 column (5
lm) for column chromatography was purchased from YMC
Compound 1 was initially isolated as white oil, ½a D20
þ 49:8 (c
ꢁ
lm, 250 mm ꢀ 10 mm) equipped with
1.0, CH3OH), the molecular formula C19H16O7 was established by
the presence of a pseudo-molecular-ion peak at m/z 379.0787 [M
+Na]+ (calcd. 379.0788 for C19H16NaO7) in the HR-ESI-MS spec-
trum. 1H NMR spectrum of 1 showed characteristic signals for a
typical 6, 7, 8-O-tri-substituted coumarin at dH 7.85 (1H, d,
a Shimadzu SPD-20A UV detector and a Shimadzu LC-6AD or LC-
20AR series pumping system (Shimadzu Corporation, Kyoto,
Japan). Dimethyl sulfoxide (DMSO), DMSO-d6, CD3OD were
obtained from Sigma-Aldrich Company (St. Louis, MO, USA). All
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D. Zhou et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
H3CO
H3CO
Deglycosylation
OH
H3CO
O
O
OH
HO
HO
O
OCH3
O
HO
O
OCH3
O
O
O
O
Condensation
HO
O
12
Eleutheroside B1
OH
OH
OCH3
2
OH
-CH2OH
O
OH
HO
H3CO
O
OCH3
guaiacylglycerol
O
O
HO
1
OCH3
H3CO
H3CO
Methylation
OCH3
O
H3CO
OCH3
OCH3
COOH
3
+
Condensation
Anisic acid
OCH3
OCH3
4
H
Anisole
N
OCH3
H
N
H
N
H
NH2
CH3COOH
-H2O
OCH3
H3CO
+
N
OCH3
O
ethylation
M
O
O
O
HN
OCH3
NH2
HN
OCH3
5a
O
5
6
m-phenylenediamine
O
O
O
COOH
8
O
Pent-2-enoic acid
NH2
Pyrolysis
Condensation
+
HO
Palmitic acid
N
7
N
H
8
H
10
11
Aniline
HO
HO
COOH
OH Hydroxylation
OH
Reduction
HO
HO
OCH3
Ferulic acid
O
OCH3
OCH3
OCH3
Methylation
H3CO
O
H3CO
H3CO
H3CO
OH
Dehydration
Reduction
H3CO
H3CO
Condensation
H3CO
H3CO
H3CO
H3CO
OH
OCH3
H3CO
14
15
Fig. 4. Possible biosynthesis pathways of compounds 1–6, 10–12, 14–15 in A. sinensis callus stimulated by isofraxetin-6-O-b-D-glucopyranoside.
J = 9.5 Hz, H-4), 6.79 (1H, s, H-5) and 6.26 (1H, d, J = 9.5 Hz, H-3).
Meanwhile, we can also observe resonances of a ABX system of
aromatic ring presented as dH 7.02 (1H, d, J = 2.0 Hz, H-20), 6.81
(1H, d, J = 8.1 Hz, H-50), 6.90 (1H, dd, J = 8.1, 2.0 Hz, H-60). And
the signals for two methoxy groups were exhibited at dH 3.86
(3H, s, 6-OCH3) and 3.85 (3H, s, 30-OCH3). Moreover, the resonances
at dH 4.51 (1H, dd, J = 11.6, 2.1 Hz, H-80a), 4.14 (1H, dd, J = 11.6,
8.6 Hz, H-80b) and dH 5.13 (1H, dd, J = 8.6, 2.1 Hz, H-70) afforded
an oxygenated methylene and an oxygenated methane group
respectively (Table 1). Then in the 13C NMR spectrum, 19 carbon
signals were observed, including 9 carbons for a typical umbellifer-
one skeleton, 6 carbons for one benzene ring and 4 carbons for
high-field regions. The long-range correlations shown in HMBC
spectrum (Fig. 2) between dH 5.13 (H-70), 4.51 (H-80a), 4.14 (H-
80b) and dC 126.7 (C-10) confirmed the connection 6, 7, 8-O-tri-sub-
stituted coumarin and phenethyl alcohol moiety. Thus, compound
1 was determined as 80-dehydroxymethyl cleomiscosin A. There
are similar structure framework in compounds 1 and 2, so the chi-
ral center C-70 in compound 1, the absolute configuration is iden-
tify to the compound 2 according to the biosynthesis pathway.25
Above all, the compound was identified as 80-dehydroxymethyl
cleomiscosin A.
The possible biosynthesis pathways were supposed on the basis
of the structures. Among them, compounds 1, 2, 12 and 13 were
related to the substrate. Compounds 3–6, 10–11, 14–15 were pro-
duced through substrate or the culture medium stimulated by sub-
strate.19
Firstly,
isofraxetin-6-O-b-
D
-glucopyranoside
was
transformed into compound 13 by deglycosylation as shown in
Fig. 3. Similarly, compound 12 was from eleutheroside B1 existing
in the system of the A. sinensis callus (Fig. 4).25 Then compound 2
was constructed by condensation reaction between compound 12
and guaiacylglycerol, which come from the system of A. sinensis
callus. Next, compound 2 further lost the hydroxymethyl group
at C-80 to yeild compound 1. However, anisic acid from the system
was methylated and obtain methyl 4-methoxybenzoate, which
was condensed with anisole and afforded compounds 3 and 4.26
Compound 5 and compound 6 were provided by methylation of
intermediate product 5a, which was from the acid-base reaction
between acetic acid and benzene-1, 3-diamine.27 Furthermore,
compounds 10 and 11 were derived from the condensation of
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4
D. Zhou et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
A
LPS+10μM
Con
LPS
LPS+1μM
LPS+30μM
LPS+100μM
150
100
50
#
#
#
#
#
#
#
#
#
*
*
*
*
*
*
*
*
*
*
*
*
0
1
2
3
4
5
6
7
8
MINO
B
Con LPS LPS+1μM LPS+10μM LPS+30μM LPS+100μM
150
100
50
#
#
#
#
#
#
#
#
*
*
*
*
*
*
*
*
*
*
0
9
10
11
12
13
14
15
MINO
Fig. 5. Anti-neuroinflammatory activities of the transformed products 1–15 on LPS-induced NO production in BV2 microglial cells. (A: Inhibitory effect of the transformed
products 1–8 and MINO on LPS-induced NO production in BV2 microglial cells; B: Inhibitory effect of the transformed products 9–15 and MINO on LPS-induced NO
production in BV2 microglial cells. Significance: *P < 0.001 compared to LPS groups. #P < 0.001 compared to control groups. NO: nitric oxide, LPS: lipopolysaccharide, MINO:
minocycline).
Table 1
NMR data of compounds 1 and 2 (1H: 600 MHz, 13C: 150 MHz in CD3OD).
Position
1
2
C
H
C
H
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
70
80
90
161.9
112.4
144.9
100.6
145.4
138.3
131.4
113.4
138.2
126.7
110.2
147.2
146.8
114.6
119.4
74.7
163.6
114.2
146.6
102.2
145.3
133.7
132.8
112.4
139.3
125.2
113.3
147.7
149.4
116.5
122.2
76.2
6.26 (1H, d, J = 9.5 Hz)
7.85 (1H, d, J = 9.5 Hz)
6.79 (1H, s)
6.31 (1H, d, J = 9.5 Hz)
7.90 (1H, d, J = 9.5 Hz)
6.82 (1H, s)
7.02 (1H, d, J = 2.0 Hz)
7.04 (1H, d, J = 2.0 Hz)
6.81 (1H, d, J = 8.1 Hz)
6.90 (1H, dd, J = 8.1, 2.0 Hz)
5.13 (1H, dd, J = 8.6, 2.1 Hz)
4.51 (1H, dd, J = 11.6, 2.1 Hz) 4.14 (1H, dd, J = 11.6, 8.6 Hz)
6.86 (1H, d, J = 8.1 Hz)
6.95 (1H, dd, J = 8.1, 2.0 Hz)
5.04 (1H, d, J = 8.0 Hz)
4.33 (1H, m)
68.5
77.4
59.9
3.40 (1H, ddd, J = 11.3, 4.3, 3.2 Hz) Hz).
3.67 (1H, ddd, J = 11.3, 4.3, 3.2 Hz)
3.87 (3H, s)
6-OCH3
30-OCH3
54.9
55.3
3.86 (3H, s)
3.85 (3H, s)
55.7
55.8
3.88 (3H, s)
aniline and pent-2-enoic acid; which was the pyrolysis product of
palmitic acid.28 With analogical pathway, ferulic acid, as one of the
major constituents of A. sinensis callus, could product compounds
14 and 15 through a serise of reactions including reduction,
hydroxylation, methylation and condensation as shown in
Fig. 4.24 Therefore, we briefly concluded the transformation rules
according to the biosynthesis pathway of the identified products.
It was found that glycosidic bond in the substrate was easily
hydrolzed, such as compounds 12 and 13. And, condensation,
observed in the biosynthesis of compounds 3, 4, 14, 15, was a pre-
ponderant reaction in A. sinensis callus culture system. Further-
more, many products were afforded by hydroxylation and
methylation.
The anti-neuroinflammatory activities of the total extract and
transformed products were assayed in BV2 microglial cells by
monitoring LPS-induced NO production.29 Moreover, the cytotoxic
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D. Zhou et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
5
Table 2
References
Effects of transformed products from the suspension cultures on NO production by
LPS-activated microglia cells (Mean SEM).
1. Xiao W, Wang Y, Zhang P, et al. Eur J Med Chem. 2013;60:263.
2. Li Z, Li X, Li D, Gao L, Xu J, Wang Y. Fitoterapia. 2007;78:605.
3. Wang Y, Pan Y, Xing YC, Tang YZ, Li N, Jiang S. Chin J Med Chem. 2013;23:397.
4. Li ZL, Li X, Li DY, Gao LS, Xu J, Wang Y. Phyto Commun. 2007;78:605.
5. Loughlin WA. Bioresour Technol. 2000;74:49.
6. Rather MY, Mishra S, Verma V, Chand S. Bioresour Technol. 2012;107:28.
7. Ishihara K, Hamada H, Hirata T, Nakajima N. J Mol Catal B Enzym. 2003;23:1459.
8. Yi LZ, Liang YZ, Wu H, Yuan DL. J Chromatogra A. 2009;1216:1999.
9. Huang B, Han LJ, Li SM, Yan CY. Pharmacogn Mag. 2015;13:574.
10. PetioleⅠof A. sinensis was placed running tap water for 30 min to wash dust and
other particles, then surface-sterilized for 1 min by soaking in 75% (v/v)
ethanol, after that overall-sterilized in 0.1% (w/v) aqueous solution of mercuric
chloride for 8–10 min, finally washed 5 times in sterilized water. Furthermore,
it was cut into pieces of 1.5 cm in size, and induced on MS (Murashige and
a
a
Sample name
IC50
Sample name
IC50
Extract
38.58 3.15
﹥100
﹥100
1.22 2.30
﹥100
10.57 3.05
﹥100
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Minocyclineb
﹥100
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
1.02 2.16
0.76 2.52
﹥100
﹥100
﹥100
﹥100
35.82 3.60
﹥100
﹥100
a
IC50
(l
g/mL for extracts and
lM for compounds).
Skoog) medium, supplemented with 5 mg/L NAA (a-naphthaleneacetic acid),
b
Positive control.
0.5 mg/L BA (6-benzyladenine), 0.7 mg/L 2, 4-D (2, 4-dichlorophenoxy acetic
acid), 30 g/L sucrose and 7.5 g/L agar, kept in darkness at 25 °C 1 °C, the
medium pH was adjusted to 5.75 before autoclaving for 20 min at 121 °C. After
15 days of pre-culturing on a gyratory platform shaker at 100 rpm, then the
primary calli was subcultured in 250 mL conical flasks containing B5 medium
with 1.0 mg/L 2, 4-D, 0.2 mg/L BA and 30 g/L sucrose at 25 °C 1 °C in darkness
every three weeks. B5 liquid medium (15 g/L sucrose) containing 1.0 mg/L NAA,
0.5 mg/L 2, 4-D and 0.05 mg/L KT (kinetin) was the medium for culture of fresh
A. sinensis callus. And the pH value of the medium was adjusted to 5.75 before
autoclaving for 20 min at 121 °C. 6 g callus cells were inoculated into a 250 mL
Erlenmever flask with 120 mL B5 medium, which were kept in the dark at
25 °C 1 °C on a rotary shaker at 110 rpm for 20 days.
activities were assayed to avoid possible effects of reduced viabil-
ity on NO release (Fig. S9).30 Results showed that the total extract
inhibited NO production remarkably with IC50 value of 38.58 lg/
mL. Among isolated compounds, 3, 5, 10 and 11 exhibited signifi-
cant inhibition on NO production in a concentration-dependent
manner with their IC50 values of 1.22 2.30, 10.57 3.05,
1.02 2.16
and
0.76 2.52
l
M
respectively
(Table
2,
11. Extraction and isolation: Isofraxetin-6-O-b-D-glucopyranoside (859 mg) was
Fig. 5A and B), comparing with positive control (IC50 35.82
lM),
dissolved in CH3OH (86.0 mL) and distributed into 59 bottles of the suspension
cultures of A. sinensis callus equally. The mixture was co-cultured for 7 days,
then the cells and medium were separated by filtration. The medium was
concentrated to 200 mL and partitioned by the same volume of EtOAc (ethyl
acetate) and n-BuOH (n-butyl alcohol) successively for three times. Next, the
organic phase was collected and concentrated under the reduced pressure to
yield EtOAc extract (Fr. 1, 0.25 g) and n-BuOH extract (Fr. 2, 1.20 g)
respectively. The cells were dried under 50 °C, and then were extracted in an
ultrasonic bath with CH3OH for 30 min. After that, the extract was further
partitioned by EtOAc and n-BuOH respectively to afford EtOAc extract (Fr. 3,
0.62 g) and n-BuOH extract (Fr. 4, 1.46 g). Compared with the experimental
and they are no cytotoxicity on the effective concentration except
that compound 10 are slender toxic to the cells at the concentra-
tions of 10 lM (Fig. S9). So, compounds 3, 5, 10 and 11 might be
a promising therapeutical approach for inflammatory diseases.
In our research, it’s the first time to biotransform coumarin gly-
cosides using the A. sinensis callus. Isofraxetin-6-O-b-
D-glucopyra-
noside, natural coumarin, was biotransformed through
a
deglycosylating. Moreover, a new compound were produced by
the system with the stimulation of substrate and the biosynthesis
pathway was also suggested. The extract and isolated compounds
showed anti-inflammation activities in LPS-induced BV2 microglial
cells. Moreover, compounds 3, 5, 10 and 11 exhibited significant
inhibition on NO production with their IC50 values of 1.22 2.30,
group, cultures and medium without isofraxetin-6-O-b-D-glucopyranoside act
as control group and was processed to get Fr. 5–8 by the same procedures. The
combination of Fr. 1 and Fr. 3 (EE, 0.87 g) was subjected on ODS column
chromatography (CC) with gradient elution CH3OH/H2O (from 0:100 to 100:0)
to get five subfractions (Fr. 1a–5a). Next, Fr. 1a was separated by HPLC with
ODS column (250 mm ꢀ 10 mm, flow rate 3 mL/min) eluted with CH3OH/H2O
(49: 51) to afford compounds 5 (2.1 mg, tR = 14 min), 6 (2.3 mg, tR = 18 min)
10.57 3.05, 1.02 2.16 and 0.76 2.52 lM, respectively. Based
and
3 (5.9 mg, tR = 39 min). Fr. 2a was firstly fractioned by silica gel CC
on structures of transformed products, it suggested that com-
pounds were prone to be condensation, deglycosylation, hydroxy-
lation and methylation. Polymerase, hydrolase, hydroxylase and
methylase are mainly systhesis enzymes, which are existed in A.
sinensis callus. Our continuing efforts to discover more biotransfor-
mation regulars in A. sinensis callus.
(3 ꢀ 50 cm) with gradient elution petroleum/acetone (0:100–100:0) to get
substraction 2a-1 and 2a-2. Then Fr. 2a-1 was purified by HPLC with ODS
column (250 mm ꢀ 10 mm, flow rate 3 mL/min) with CH3CN/H2O (37: 63) to
provide compounds 8 (1.0 mg, tR = 22 min), 10 (1.1 mg, tR = 27 min) and 14
(1.2 mg, tR = 35 min). While compound 15 (0.9 mg, tR = 25 min) was purified
from substration Fr. 2a-2, using HPLC with ODS column (250 mm ꢀ 10 mm,
flow rate 3 mL/min) eluted with CH3CN/H2O (39: 61). And Fr. 3a was
chromatographied on HPLC with ODS column (250 mm ꢀ 10 mm, flow rate
3 mL/min) to yield compounds
9 (1.1 mg, tR = 40 min) and 11 (0.9 mg,
tR = 56 min) using CH3OH/H2O (35: 65) as elute. However, compounds 12
(0.8 mg, tR = 37 min) and 13 (1.3 mg, tR = 49 min) were obtained from Fr. 4a by
HPLC with ODS column (250 mm ꢀ 10 mm, flow rate 3 mL/min) using CH3CN/
H2O (8:92) as eluting solvent. Equally, Fr. 2 and Fr. 4 was combined as BE
(2.66 g) to subject on ODS CC with CH3OH/H2O (from 0:100 to 100:0) to get
seven fractions (Fr. 1b–7b). Fr. 1b was isolated using HPLC with ODS column
(250 mm ꢀ 10 mm, flow rate 3 mL/min) eluted by CH3OH/H2O (20: 80) and
then purified by Sephadex LH-20 chromatography eluted with CH3OH to afford
compounds 1 (1.1 mg) and 2 (1.7 mg). Furthermore, compound 4 (2.0 mg) was
obtained from Fr. 3b by recrystallization in CH3OH. Next, Fr. 4b was subjected
on silica gel CC (3 ꢀ 50 cm) with gradient elution CH2Cl2/CH3OH (0:100-100:0)
to get substraction Fr. 4b-1 and Fr. 4b-2. Then Fr. 4b-1 was purified by HPLC
with ODS column (250 mm ꢀ 10 mm, flow rate 3 mL/min) with CH3CN/H2O
Acknowledgements
The work was supported partially by the National Natural
Science Foundation of China (Grant No. U1403102, 81173531,
81473330, 81673323), the Shenyang Science and Technology
Research Project, Liaoning, China (F15-199-1-26), the Research
Project for Key Laboratory of Liaoning Educational committee,
Liaoning, China (Grant No. LZ2015067), the Natural Science
Foundation of Liaoning Province, China (Grant No. 2015020732),
the Innovation Team Project of Liaoning Province, China (Grant
No. LT2015027), the Fund for Long-term Training of Young
Teachers in Shenyang Pharmaceutical University, Liaoning, China
(ZCJJ2013409).
(13: 87) to provide compounds
R = 39 min).
7 (1.1 mg, tR = 19 min) and 16 (1.8 mg,
t
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.11.
069.
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.069

6
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control group. Fifty microliter culture supernatant fluids were mixed with an
equal volume of Griess reagent (50 L) at room temperature for 15 min. Then,
the absorbance of the mixture was determined at 540 nm with a plate reader
(Bio-Tek).
l
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30. Briefly, cells were plated on 96-well microtiter plates at a concentration of
3 ꢀ 104 cells/well and pretreated with the extract at concentrations of 100
lg/
mL, 30
l
g/mL, 10
lg/mL,
1
l
g/mL,
0 lg/mL and purified compounds at
concentrations of 100
lM, 30
lM, 10 lM, 1 lM, 0 lM in the presence of LPS
29. Cells were cultured into 96-well microtiter plates at a density of 3 ꢀ 104 cells/
(100 ng/mL) for 24 h. Compared with the experimental group, cells without LPS
act as a control group. And then the cells were incubated with MTT (0.25 mg/
mL) for 4 h at 37 °C. The supernatant of the cells was removed and the
formazan formed was dissolved in DMSO. Finally, the optical density were
measured at 490 nm using a plate reader (Bio-Tek, USA).
well and treated with the extract at concentrations of 100 lg/mL, 30 lg/mL,
10
100
for 24 h. Compared with the experimental group, cells without LPS act as a
l
l
g/mL, 1
l
g/mL, 0
l
g/mL; and purified compounds at concentrations of
M, 30
l
M, 10 M, 1
l
l
M, 0 M in the presence of LPS (100 ng/mL) at 37 °C
l
Please cite this article in press as: Zhou D., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.069