Microbial transformation of Norkurarinone by Cunninghamella blakesleana AS 3.970

Article abstract of DOI:10.1080/10286020.2012.702759

In this paper, microbial transformation of norkurarinone (1) by Cunninghamella blakesleana AS 3.970 was investigated and seven transformed products were isolated and characterized as kurarinone (2), 4,5- dihydroxykurarinone (3), 6-hydroxyl-2-methoxyl-nork

Full text of DOI:10.1080/10286020.2012.702759

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Microbial transformation of
Norkurarinone by Cunninghamella
blakesleana AS 3.970
Yan-Qiu Shi a , Xiu-Lan Xin b , Hu-Cheng Zhang b , Bao-Jing Zhang c
, Chang-Yuan Wang c , Jie Hou c , Qi-Peng Yuan a , Sa Deng c , Yan
Tian c & Xiao-Chi Ma c
a State Key Laboratory of Chemical Resource Engineering, College
of Life Science and Technology, Beijing University of Chemical
Technology , Beijing , 100029 , China
b College of Bioengineering, Beijing Polytechnic , Beijing ,
100029 , China
c School of Pharmacy, Dalian Medical University , Dalian , 116044 ,
China
Published online: 28 Aug 2012.
To cite this article: Yan-Qiu Shi , Xiu-Lan Xin , Hu-Cheng Zhang , Bao-Jing Zhang , Chang-Yuan
Wang , Jie Hou , Qi-Peng Yuan , Sa Deng , Yan Tian & Xiao-Chi Ma (2012) Microbial transformation of
Norkurarinone by Cunninghamella blakesleana AS 3.970, Journal of Asian Natural Products Research,
14:9, 906-912, DOI: 10.1080/10286020.2012.702759
To link to this article: http://dx.doi.org/10.1080/10286020.2012.702759
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Journal of Asian Natural Products Research
Vol. 14, No. 9, September 2012, 906–912
Microbial transformation of Norkurarinone
by Cunninghamella blakesleana AS 3.970
Yan-Qiu Shia, Xiu-Lan Xinb, Hu-Cheng Zhangb, Bao-Jing Zhangc, Chang-Yuan Wangc,
Jie Houc, Qi-Peng Yuana*, Sa Dengc, Yan Tianc and Xiao-Chi Mac*
aState Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology,
Beijing University of Chemical Technology, Beijing 100029, China; College of Bioengineering,
b
c
Beijing Polytechnic, Beijing 100029, China; School of Pharmacy, Dalian Medical University,
Dalian 116044, China
(Received 23 March 2012; final version received 20 May 2012)
In this paper, microbial transformation of norkurarinone (1) by Cunninghamella
blakesleana AS 3.970 was investigated and seven transformed products were isolated and
characterizedaskurarinone(2),400,500-dihydroxykurarinone(3),600-hydroxyl-20-methoxyl-
norkurarinone 7-O-b-D-glucoside (4), 600-hydroxyl-norkurarinone 40-O-b-D-glucoside
(5), 400,500-dihydroxynorkurarinone (6), 7-methoxyl-norkurarinone (7), and 7-methoxyl-
400,500-dihydroxynorkurarinone (8), respectively. Among them, 3–5 are new compounds,
and the glycosylation reaction in microbial transformation process was reported rarely.
In addition, the cytotoxicities of transformed products (1–8) were also investigated.
Keywords: biotransformation; norkurarinone; Cunninghamella blakesleana;
lavandulated flavonoid
1. Introduction
Microbial transformation is an useful
biotechnique to modify chemical structures
of natural products. And a large number of
new natural products by microbial trans-
formation have been reported for improving
thebioactivitiesorstudyingtherelationships
of structures and activities in our previous
works [7–11]. Meantime, microbial trans-
formationcouldbeusedastheinvitromodel
to simulate the metabolism of natural
products in vivo [12,13].
The Chinese crude drug ‘Kushen’, derived
from the dry roots of Sophora flavescens, is
a commonly used traditional Chinese
medicine with gastric disturbance, antifeb-
rile, and anthelmintic activities [1]. A large
amount of lavandulated flavonoids were
isolated and purified from this plant by
phytochemical methods in recent years
[2–4]. These flavonoids such as kurarinone
and norkurarinone are the major constitu-
ents of S. flavescens with widely various
biological activities including cytotoxicity
[5] and glycosidase inhibition [6]. So more
and more pharmacological researches of the
lavandulyl flavonoids from S. flavescens
were reported for the future development of
new drug. However, the poor water
solubility would limit its wide medicine use.
In this work, microbial transformation of
norkurarinone (1) by Cunninghamella bla-
kesleana AS 3.970 was studied to obtain the
novel bioactive flavonoid derivatives with
the lavandulyl groups. Totally seven trans-
formed products of 1 were isolated and
identified by widely spectral methods
including NMR and MS. Among them,
products 3–5are new. And compounds 4and
*Corresponding authors. Email: yuanqp@mail.buct.edu.cn; maxc1978@sohu.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.702759
http://www.tandfonline.com

Journal of Asian Natural Products Research
907
HO
OH
OH
HO
OH
OH
MeO
O
OH
HO
O
HO
GluO
O
HO
MeO
O
HO
O
MeO
O
4
2
3
HO
OH
HO
7"
6"
4"
2"
OH
OGlu
OH
HO
9"
HO
OH
4'
1"
O
HO
8"
2'
O
O
8
HO
HO
7
2
1'
6'
5
4
OH O
10
O
6
HO
HO
O
5
1
HO
OH
OH
HO
HO
OH
O
MeO
O
MeO
O
HO
HO
O
7
8
Figure 1. A possible biotransformation pathway of 1 by C. blakesleana AS 3.970.
5 were the glycoside derivatives of 1, which
was the rare microtransformation reaction.
The biotransformation reactions of 1 by C.
blakesleana AS 3.970 were hydroxylation,
methylation, and glycosylation. In addition,
the cytotoxicities of transformed products
(2–8) against Hela and A375 cells were also
investigated (Figure 1).
their structures were elucidated as kurar-
inone (2), 400,500-dihydroxyl-kurarinone (3),
600-hydroxyl-20-methoxyl-norkurarinone 7-
O-b-D-glucoside (4), 600-hydroxyl-norkur-
arinone 40-O-b-D-glucoside (5), 400,500-dihy-
droxy-norkurarinone (6), 7-methoxyl-
norkurarinone (7), and 7-methoxyl-400,500-
dihydroxynorkurarinone (8), respectively.
Among them, 3–5 are new products.
The possible biotransformation pathway of
1 by C. blakesleana AS 3.970 was proposed.
The biotransformation reactions such as
demethylation, glycosylation, and hydroxy-
lation were observed. And glycosylation
was a rare reaction in the microbial
transformation process.
2. Results and discussion
Inthisstudy,compound1wasaddedintothe
36-h-old cultures of C. blakesleana AS
3.970 and continued to incubate for 5 days.
And a blank culture control and substrate
control were performed as described above.
A total amount of 1.0 g of substrate was
addedtothecultureliquidinthepreparative-
scale biotransformation. After 5 days of
incubation, seven products were isolated by
the preparative chromatographic methods.
Based on the extensive spectra of 1H NMR,
13C NMR, and 2D NMR techniques,
Compounds 2 and 6–8 were identified
as kurarinone, 400,500-dihydroxy-norkurari-
none, 7-methoxyl-norkurarinone, and 7-
methoxyl-400,500-dihydroxy-nor-kurarinone,
respectively. Their 1H NMR and 13C NMR

908
Y.-Q. Shi et al.
spectral data were in agreement with those
reported in literatures [14–16].
the carbonsignalat d 165.4 (C-7), indicating
that the glucopyranosyl moiety should be at
C-7 position. The proton signal at d 3.85 had
HMBC correlations with C-700 and C-400,
suggestingthatthehydroxylgroupshouldbe
located at C-600. A positive absorption at
315 nm and a negative absorption at 285 nm
in the CD spectrum of 4 indicated the
absolute configuration of C-2 as S. On the
basis of above analysis, compound 4 was
characterized as 600-hydroxyl-20-methoxyl-
norkurarinone 7-O-b-D-glucoside. All the
1H NMR and 13C NMR spectral data were
unambiguously assigned by 2D NMR
spectra (Table 1).
Compound 3 was obtained as yellow
powder (MeOH). It was optically active,
22
with ½aꢀD 2 9.2 (c 0.3, MeOH). Its HR-
ESI-MS provided a pseudo-molecular ion at
m/z 471.1958 [M–H]2, suggesting a mol-
ecular formula of C26H32O8. Comparing
with compound 2, two oxygenated carbon
signals at d 80.0 and 81.5 were observed in
the 13C NMR spectrum of 3, while the CvC
double bond carbons at d 124.4 and 131.6 in
compound 2 disappeared [14]. The HMBC
correlations of the carbon signal at d 81.5
withtheprotonsatd1.06(Me-600 and700)and
3.61 (H-400) were observed, and the 1H–1H-
COSY correlations of H-300 with H-200 and
H-400 implied that two hydroxyl groups were
introduced at C-400 and C-500 of the
lavandulyl group, respectively. In addition,
the circular dichroism (CD) spectrum of 3
showed a positive absorption at 315 nm and
a negative absorption at 285 nm. Thus, the
absoluteconfigurationofC-2wasconfirmed
as S [14]. On the basis of the above analysis,
compound 3 was elucidated as 400,500-
Compound 5 was afforded as yellow
powder (MeOH). Its HR-FAB-MS provided
a quasi-molecular ion at m/z 601.2298 [M–
H]2, suggesting the molecular formula of
C31H38O12. Similar with compound 4, the
carbon signals at d 102.8, 74.2, 77.5, 71.0,
77.5 and 62.4 indicated that a sugar moiety
was introduced in the molecular formula of
5. Acid hydrolysis and thin-layer column-
chromatography with the reference sample
exhibited that the sugar was D-glucose. The
anomeric carbon signal at d 102.8 and the
anomeric proton coupling constant (7.0Hz)
suggested that the configuration of gluco-
pyranosyl should be b. In HMBC spectrum,
the anomeric proton had long-range corre-
lation with the carbon signal at d 155.9,
suggesting that glucopyranosyl moiety
should be at C-40 position. In addition, the
methyl group at d 1.45 had HMBC
correlations with the carbon signals at d
124.2 and 68.4. The proton signal at d 3.80
had long-range correlations with C-500 at d
136.3 and C-700 at d 13.6. In NOESY
spectrum, H-600 (d 3.80) had correlation with
H-400 (d5.00).Alloftheevidencessuggested
that the hydroxyl group should be located at
C-600. Similar to the CD spectrum of 1, a
positive absorption at 320 nm and a negative
absorption at 290 nm implied that the
absolute configuration of C-2 was S. On
the basis of the above analysis, compound 5
was determined as 600-hydroxyl-norkurar-
inone 40-O-b-D-glucoside. All the 1H NMR
1
dihydroxykurarinone. All the H and 13C
NMR spectral data were unambiguously
assigned by 2D NMR spectra (Table 1).
Compound 4 was obtained as yellow
powder (MeOH). Its HR-ESI-MS provided
a pseudo-molecular ion at m/z 615.2418
[M–H]2, suggesting a molecular formula of
C32H39O12. The methoxyl group (d 3.81)
had the HMBC correlation with C-20 (d
159.8). And at the same time, the methoxyl
group had the NOESY correlation with H-30
(d 6.64). These evidences proved that this
methoxyl group should be located at C-20.
In addition, the carbon signals of d 101.8,
75.4, 78.4, 71.8, 79.0 and 63.0 indicated that
a sugar moiety was introduced in the
structure of 4. The sugar moiety was
identified as D-glucose by acid hydrolysis.
The anomeric proton coupling constant of
7.5 Hz and the anomeric carbon signal at d
101.8 suggested that the configuration of
glucopyranosyl should be b. The anomeric
proton had the long-range correlation with

Journal of Asian Natural Products Research
909
d
d
d
d
d
d

910
Y.-Q. Shi et al.
Table 2. In vitro cytoxic activities of trans-
formed products.
d
Tumor cell
lines and
IC50 (uM)
Tumor cell
lines and
IC50 (uM)
No.
Hela
A375 No.
Hela
A375
1
2
3
4
30.6
35.8
.100 .100
5.3 21.4
49.8
61.5
5
6
7
8
23.6
68.9
.100 .100
55.7 .100
.100 .100
d
and 13C NMR spectral data were unambigu-
ously assigned by 2D NMR spectra
(Table 1).
The cytotoxic activities of compounds
1–8 against Hela and A375 cells were
tested. Our results indicated that the
cytotoxicities of transformed products of 1
against Hela cells were more sensitive than
A375 cells. The glycosylation in the
skeleton of lavandulyl flavonoid would
increase the cytotoxicities of products 4
and 5. And the glycosylated derivative of
C-7 showed better bioactivity than that of
C-40. While the methoxylation at C-7,
hydroxylation at C-600, and dihydroxylation
at the double bond of C-400 and C-500 would
decrease the cytotoxicities significantly.
In addition, the methoxylation of C-5 did
not have the significant influence on the
cytotoxicities of 1 (Table 2).
d
d
d
3. Experimental
3.1 General experimental procedures
UV spectra were measured by Cary 300
spectrophotometer (Beijing Tongyong
LLC, Beijing, China). IR spectra were
recorded on a NEXUS-470 FT-IR spectro-
photometer in KBr pellets (Thermo Fisher
Scientific, Dubuque, IA, USA). Melting
points were measured by XT4A micromelt-
ing point apparatus and were uncorrected
(Beijing Keyi Company, Beijing, China).
CD spectra were performed on JASCO J-
810 CD instrument (Jasco, Tokyo, Japan).
1H NMR and 13C NMR spectra were
measuredwithanINOVA-500spectrometer
d

Journal of Asian Natural Products Research
911
(500MHzfor1HNMRand125 MHzfor13C
NMR) in Dimethyl Sulfoxide (DMSO)-d6
with TMS (tetramethylsilane) as internal
standard (Varian, Las Vegas, NV, USA).
HR-MS were obtained on a Bruker APEXII
Fourier transform ion cyclotron resonance
mass spectrometer (Bruke, Ettlingen,
Germany). Thin Layer Chromalography
(TLC) analyses were performed on silica
gel G. All chemicals were purchased from
Beijing Chemical Factory (Beijing, China).
Silica gels were supplied by Qingdao
Haiyang Chemical Group Co. (Qingdao,
China). Norkurarinone (1)wasisolatedfrom
S. flavescens by Y.-Q. Shi. The purity was
above 97% which was determined by HPLC
analysis.
180 rpm at 288C. After 36h of pre-culture,
the substrate (10 mg) in 1 ml acetone was
added to 350 ml medium. In total, 1000mg
of substrate was used for biotransformation.
The incubation was continued under the
above conditions for five additional days.
The culture was filtered and the filtrate was
extracted with same volume of EtOAc for
four times. The organic phase was collected
and concentrated to dryness in vacuo.
The crude extract (3.5 g) was subjected to
silica gel column and eluted with chloro-
form–acetone [in a gradient manner from
100:3 (v/v) to 1:1, at a flow rate of
1.5 ml/min] to yield six fractions (I–VI).
Fraction II (0.2 g) was subjected to prepara-
tive TLC and eluted with chloroform–
acetone (5:1) to yield 2 (7 mg). Fraction IV
(0.11 g) was separated by preparative HPLC
and eluted with MeOH–H2O (85:15, v/v) to
give 7 (12 mg, Rt ¼ 19.0 min). Fraction V
(415 mg) was subjected to preparative
HPLC and eluted with MeOH–H2O
(60:40) to yield 3 (12 mg, Rt ¼ 15.5 min),
6 (16 mg, Rt ¼ 25.0 min), and 8 (7 mg,
Rt ¼ 28.5 min). Fraction VI (460 mg) was
isolated by HPLC with MeOH–H2O (55:45)
to obtain products 4 (7 mg, Rt ¼ 16.5 min)
and 5 (9 mg, Rt ¼ 22.4 min). Purities of all
the transformed products were above 95% by
Rp-HPLC analysis, and the detected wave-
length and the flow rate were set at 285 nm
and 1.5 ml/min, respectively.
3.2 Microorganism
Mucor subtilissimus AS 3.2454, Mucor
spinosus AS 3.2450, M. spinosus AS
3.3447, M. subtilissimus AS 3.2456, Alter-
naria alternata AS 3.577, A. alternata AS
3.4578, Alternaria longipes AS 3.2875,
C. blakesleana lender AS 3.970, Cunning-
hamella echinulata AS 3.3400, Cunning-
hamella elegans AS 3.2028, Curvularia
lunata AS 3.4381, Penicillium melinii AS
3.4474, Penicillium janthinellum AS 3.510,
Rhizopus stolonifer AS 3.3463, R. stolonifer
AS 3.2050, Syncephalastrum racemosum AS
3.264, Trichoderma viride AS 3.2942, and
Rhizopus arrhizus AS 3.2897 were pur-
chased from China General Microbiological
Culture Collection Center in Beijing, China.
3.4.1 400,500-Dihydroxykurarinone (3)
Yellow powder (MeOH). m.p. 185–1868C.
UV (MeOH) lmax: 285, 325 nm; CD
(MeOH): D1315.1 nm þ4.860, D1285.5
212.570. IR (KBr) nmax (cm21): 3380,
3.3 Culture medium
All cultures of filamentous fungi in bio-
transformationexperiments wereperformed
in potato medium, which was made by the
following compositions (liters): 200 g
potato and 20g glucose [17].
2820, 1700, and 990; for 1H NMR and 13
C
NMR spectral data, see Table 1; HR-
ESIMS: m/z 471.1958 [M–H]2 (calcd for
C26H31O8, 471.2019).
3.4 Culture and biotransformation
3.4.2 600-Hydroxyl-20-methoxyl-
norkurarinone 7-O-b-D-glucoside (4)
Preparative-scale transformation of 1 by
C. blakesleana AS 3.970 was performed in a
1000-ml Erlenmeyer flask. The flasks were
placed on the rotary shakers, operating at
Yellow powder (MeOH). m.p. 287–2888C.
UV (MeOH) lmax 288, 320 nm; CD

912
Y.-Q. Shi et al.
[2] P.L. Ding, D.F. Chen, K.F. Bastow,
A.K. Nyarko, X.H. Wang, and K.H. Lee,
Helv. Chim. Acta 87, 2574 (2004).
(MeOH): D1315 þ 5.360, D1286.2 210.190.
IR (KBr) nmax (cm21): 3290, 2850, 1760,
and 1025; for 1H NMR and 13C NMR
spectral data, see Table 1; HR-ESI-MS: m/z
615.2418 [M–H]2 (calcd for C32H38O12,
615.2441).
[3] H.J. Shin, H.J. Kim, J.H. Kwak, J.H. Kim,
H. Park, D.H. Kim, and Y.S. Lee,
Bioorg. Med. Chem. Lett. 12, 2313 (2002).
[4] T.H. Shin, S.J. Jeong, W.G. Ko, N.Y. Kim,
B.H. Lee, M. Inagaki, T. Miyamoto, and
Y.C. Kim, J. Nat. Prod. 63, 680 (2000).
[5] S.K. Hyun, W.H. Lee, M. Jeong, Y. Kim,
and J.S. Choi, Biol. Pharm. Bull. 31, 154
(2008).
[6] J.K. Son, J.S. Park, J.A. Kim, Y.S. Kim,
S.R. Chung, and S.H. Lee, Planta Med.
69, 559 (2003).
[7] F.Y. Li, R.P. Cang, S.S. Huang,
B.J. Zhang, X.L. Xin, J.H. Yao, Q. Zhou,
Y. Tian, S. Deng, and X.C. Ma, J. Mol.
Cata. 68, 187 (2011).
3.4.3 600-Hydroxyl-norkurarinone 40-O-
b-D-glucoside (5)
Yellow powder (MeOH). m.p. 256–2578C.
UV (MeOH) lmax 290, 320 nm; CD
(MeOH): D1320 þ 3.290, D1290 216.810.
IR (KBr) nmax (cm21): 3410, 2780, 1820,
and 1150; for 1H NMR and 13C NMR
spectral data, see Table 1; HR-ESI-MS: m/z
601.2298 [M–H]2 (calcd for C31H37O12,
601.2285).
[8] X.L. Xin, L.B. Zhan, F.Y. Li, X.C. Ma,
K.X. Liu, J. Han, and D.A. Guo, J. Asian
Nat. Prod. Res. 11, 187 (2009).
3.5 Bioassay
[9] S. Deng, B.J. Zhang, C.Y. Wang, Y. Tian,
J.H. Yao, L. An, S.S. Huang, J.Y. Peng,
K.X. Liu, and X.C. Ma, Bioorg. Med.
Chem. Lett. 22, 1615 (2012).
[10] X.L. Xin, L.B. Zhan, F.Y. Li, X.C. Ma,
K.X. Liu, and D.A. Guo, J. Asian Nat.
Prod. Res. 11, 7 (2009).
[11] X.L. Xin, D.H. Su, S. Deng, Y. Tian,
B.J. Zhang, X.C. Ma, L. An, S.S. Huang,
J.H. Yao, and X. Cui, J. Asian Nat. Prod.
Res. 13, 831 (2011).
[12] X.C. Ma, J. Ning, G.B. Ge, S.C. Liang,
X.L. Wang, B.J. Zhang, S.S. Huang,
J.K. Li, and L. Yang, Drug Metab. Dispos.
39, 675 (2011).
[13] J. Ning, T.H. Wu, Y. Tian, C.Y. Wang,
G. Tian, B.J. Zhang, and X.C. Ma,
Xenobiotica 40, 48 (2010).
[14] X.C. Ma, X.L. Xin, B.J. Zhang, F.J. Li,
K.X. Liu, and D.A. Guo, Magn. Reson.
Chem. 46, 903 (2008).
[15] L.J. Wu, T. Miyase, A. Ueno,
M. Kuroyanagi, T. Noro, and S. Fukush-
ima, Chem. Pharm. Bull. 33, 3231 (1985).
[16] X.C. Ma, C.K. Sun, S.S. Huang,
J.K. Wang, B.J. Zhang, F.Y. Li,
G. Wang, S. Deng, and J. Cui,
Sep. Purif. Technol. 76, 140 (2010).
[17] X.C. Ma, X.L. Xin, K.X. Liu, J. Han, and
D.A. Guo, J. Nat. Prod. 71, 1268 (2008).
The cyctoxicities of transformed products
(2–8) were evaluated by 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) method [14]. Hela
and A375 cells were maintained in
RPMI1640 medium with 10% (v/v) fetal
bovine serum and cultured in 96-well
microtiter plates. Appropriate dilutions of
compounds 2–8 were added to the culture.
And then cells were continued to culture at
378C, 5%CO2 for72h. Theconcentrationof
test compound to give 50% inhibition of cell
growth was expressed as IC50 value.
Acknowledgments
We thank LNET (LJQ2011095), Personnel
Department of Liaoning Province, Natural
Science Foundation of China (20976009 and
21176018), and Science and Technology Devel-
opment Common Project (KM2011 10858001)
for financial support.
References
[1] X.C. Ma, X.L. Xin, K.X. Liu, J. Han,
B.R. Wang, and D.A. Guo, Chromatogra-
phia 68, 471 (2008).