An insight into the curdione biotransformation pathway by Aspergillus niger

Article abstract of DOI:10.1080/14786419.2013.873434

Curdione (1), a sesquiterpene with a germacrane skeleton from rhizomes of Curcuma wenyujin, has attracted attention due to its important pharmacological properties. Herein, we investigated the chemo-biotransformation of curdione (1) systematically using Aspergillus niger AS 3.739. Regio- and stereoselective hydroxylation of curdione with filamentous fungus A. niger AS 3.739 led to seven metabolites including four new compounds 3α-hydroxycurcumalactone, 2β-hydroxycurcumalactone, (10S)-9,10-dihydroxy-curcumalactone and (10R)-9,10-dihydroxy-curcumalactone. Their structures were determined by spectroscopic techniques including two-dimensional NMR and TOF-MS (Time of Flight Mass Spectrometry). Based upon the analysis of biological and chemical conversions of curdione, a tentative metabolic pathway via chemo-bio cascade reactions is proposed in A. niger system, which provides an insight into the corresponding metabolism of curdione in animal systems. In addition, experiments with selected monooxygenase inhibitors suggest that cytochrome P450 monooxygenase played a crucial role in the hydroxylation of curdione.

Full text of DOI:10.1080/14786419.2013.873434

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An insight into the curdione
biotransformation pathway by
Aspergillus niger
Yinan Chena, Lang Zhangab, Bin Qina, Xin Zhanga, Xian Jiaab,
Xiaoying Wanga, Danni Jina & Song Youa
a School of Life Science and Biopharmaceutics, Shenyang
Pharmaceutical University, Shenyang 110016, P.R. China
b Department of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Shenyang, P.R. China
Published online: 23 Jan 2014.
To cite this article: Yinan Chen, Lang Zhang, Bin Qin, Xin Zhang, Xian Jia, Xiaoying Wang, Danni
Jin & Song You (2014) An insight into the curdione biotransformation pathway by Aspergillus
niger, Natural Product Research: Formerly Natural Product Letters, 28:7, 454-460, DOI:
10.1080/14786419.2013.873434
To link to this article: http://dx.doi.org/10.1080/14786419.2013.873434
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Natural Product Research, 2014
Vol. 28, No. 7, 454–460, http://dx.doi.org/10.1080/14786419.2013.873434
An insight into the curdione biotransformation pathway by Aspergillus
niger
Yinan Chena, Lang Zhanga, Bin Qina, Xin Zhanga, Xian Jiab, Xiaoying Wanga, Danni Jina and
Song Youa*
aSchool of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016,
P.R. China; bDepartment of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang, P.R. China
(Received 13 September 2013; final version received 1 December 2013)
Curdione (1), a sesquiterpene with a germacrane skeleton from rhizomes of Curcuma
wenyujin, has attracted attention due to its important pharmacological properties.
Herein, we investigated the chemo-biotransformation of curdione (1) systematically
using Aspergillus niger AS 3.739. Regio- and stereoselective hydroxylation of
curdione with filamentous fungus A. niger AS 3.739 led to seven metabolites including
four new compounds 3a-hydroxycurcumalactone, 2b-hydroxycurcumalactone,
(10S)-9,10-dihydroxy-curcumalactone and (10R)-9,10-dihydroxy-curcumalactone.
Their structures were determined by spectroscopic techniques including two-
dimensional NMR and TOF-MS (Time of Flight Mass Spectrometry). Based upon
the analysis of biological and chemical conversions of curdione, a tentative metabolic
pathway via chemo-bio cascade reactions is proposed in A. niger system, which
provides an insight into the corresponding metabolism of curdione in animal systems.
In addition, experiments with selected monooxygenase inhibitors suggest that
cytochrome P450 monooxygenase played a crucial role in the hydroxylation of
curdione.
Keywords: curdione; Aspergillus niger AS 3.739; biotransformation; hydroxylation;
cytochrome P450
1. Introduction
Ezhu, one of commonly used traditional Chinese medicines, is derived from rhizomes of three
species of Curcuma, namely Curcuma phaeocaulis, Curcuma kwangsiensis and Curcuma
wenyujin. The essential oil of Ezhu is currently embodied in the Chinese Pharmacopoeia
(Chinese Pharmacopoeia Commission 2010) and used for the intravenous injection as an
antivirus in clinical practice (Chinese State drug No. H33022542). Curdione (1), a sesquiterpene
with a germacrane skeleton, is a main ingredient of Ezhu essential oil and is reported to possess
wide pharmacological activities including anti-inflammatory, anticancer (Oh et al. 2007; Hou
et al. 2011), hepatoprotective (Matsuda et al. 2001) and antithrombotic activities (Xia et al.
2012). Therefore, relative compounds have been paid more attention in pharmaceutical fields.
Microbial transformation is a promising strategy for preparing target products which are
difficult to produce by conventional chemical methods (Arantes & Hanson 2007). Curdione (1)
has been reported to be transformed into corresponding saturated ketones, alcohols and
epoxides by various microorganisms, such as Aspergillus niger (Asakawa et al. 1991),
Lonicera japonica (Horiike et al. 1997) and Mucor polymorphosporus (Ma et al. 2007). In our
*Corresponding author. Email: yousong207@163.com
q 2014 Taylor & Francis

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previous study (Zhou et al. 2012), epoxidation of curdione was done yielding (1S,10S)- and
(1R,10R)-1,10-epoxycurdione by Cunninghamella elegans. However, the transformation
pathways of curdione in fungal systems have not been sufficiently investigated, which will be
helpful to understand the mechanism and provide the basis for further study of the
corresponding metabolism in animal systems (Srisilam & Veeresham 2003).
Special attention has been paid to the filamentous fungi containing monooxygenases,
because they are capable of catalysing regio- and stereoselective hydroxylation of a variety of
non-functionalised hydrocarbon centres of a great variety of substrates (Lehman & Stewart
2001). Among them, A. niger has been proved to be an important microorganism for the
hydroxylation of many natural products (Lamm et al. 2009; Yadav et al. 2011). Herein, this
paper reports the chemo-biotransformation of curdione (1) systematically, with the goals of
elucidating the transformation pathway of curdione (1) as well as preparing a series of
metabolites with new chiral centres.
2. Results and discussion
2.1. Identification of curdione metabolites by chemo-biotransformation
Curdione (1), obtained in large quantities from the essential oil of C. wenyujin, was transformed
to six products (2–5, 7 and 8) by growing cells of A. niger AS 3.739. Biotransformation of
curdione (1) by resting cells of A. niger for 48 h, followed by acid-catalysed reaction for 24 h
afforded another new compound 6. Their structures (Figure 1) were determined as
3a-hydroxycurdione (2), 2b-hydroxycurdione (3), curcumalactone (4), 3a-hydroxycurcuma-
Figure 1. Proposed transformation pathways of curdione (1) via chemo-bio cascade reactions.

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Y. Chen et al.
lactone (5), 2b-hydroxycurcumalactone (6), (10S)-9,10-dihydroxy-curcumalactone (7) and
(10R)-9,10-dihydroxy-curcumalactone (8). The structures of compounds 5–8 are new and
previously unreported (see Supplementary material).
The absolute configuration of the C-10 hydroxyl group of 7 and 8 was characterised by the
Snatzke’s method (Bari et al. 2001, 2004). Compounds 7 and 8 (1.1 mg) were dissolved in 1 mL
DMSO containing Mo2(OAc)4 (1.3 mg), respectively. The induced CD spectra were recorded at
30 min after mixing. Following Snatzke’s nomenclature, we referred to the special band as band
IV (310 nm, positive Cotton effect), indicating that the O–C–C–O dihedral angle was positive
for 7 and negative for 8. Therefore, the absolute configuration of C-10 was S for compound 7,
which was characterised as (10S)-9,10-dihydroxy-curcumalactone; R for compound 8 was
confirmed as (10R)-9,10-dihydroxy-curcumalactone (Figure S2). The optical rotation value and
the NMR spectral data of the 9,10-dihydroxy-curcumalactone were reported in previous study
(Asakawa et al. 1991), but the absolute configuration was not specified. The NMR spectral data
of compounds 7 and 8 were significantly different and the absolute configuration of 7 and 8 was
determined in this study. Therefore, 7 and 8 were identified as novel compounds.
2.2. Biotransformation pathway in the A. niger system
In order to gain a full profile of the metabolism, the time course of curdione (1)
biotransformation by growing cells of A. niger AS 3.739 was initially recorded, and the
Figure 2. Degradation and accumulation patterns of curdione and biotransformation metabolites. Results
are the mean ^ SD, n ¼ 3.

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variations in the contents of seven compounds in the cultures are summarised in Figure 2. The
quantity of the starting material 1 decreased dramatically at the beginning of the culture and was
undetected after 48 h, which indicated that 1 was completely consumed and catalysed to other
metabolites. The quantities of compounds 2 and 4 increased rapidly at the first 36 h, and then
decreased from 36 to 84 h, suggesting that they were intermediate products. Since compounds 3,
5 and 7 increased slowly during the overall incubation, it was deduced that these three
compounds might be stable products.
Regio- and stereoselective hydroxylation of curdione (1) in A. niger cultures gave two
hydroxylated products, 2 and 3. Curdione (1) was reported to be capable of being converted to a
spirolactone skeleton via intracellular ene reaction, which was supposed to be an acid-catalysed
rearrangement (Inayama et al. 1985). During the biotransformation process, a significant decline
in pH value (final pH value of 1.7, Figure S4) was observed, which was in the agreement with the
fact that A. niger grown in nutrient medium secreted large amounts of acid (Clark and Lubs
1917). Interestingly, the results (Figure S5) showed that curdione (1) and 3a-hydroxycurdione
(2) could be transformed to the corresponding spirolactones 4 and 5, respectively, whereas 2b-
hydroxycurcumalactone (3) could not be converted. To obtain initial information regarding the
mechanism of this process, the influence of acid was evaluated. The conversion of 1 using
resting cell of A. niger at pH 7.0 gave the production of only hydroxylated curdiones 2 and 3
without further reactions (Figure S6). However, acid-catalysed intracellular ene reaction from
compounds 1–3 occurred at pH 2.0 to generate the corresponding spirolactones 4–6,
respectively (Figure S7), with 2b-hydroxycurcumalactone (6) as a new compound. These results
suggested that the intracellular ene reactions of curdiones were accomplished mainly by the acid
produced by A. niger. It was noted that compound 3 either failed to be converted into 6 by
A. niger growing cells, or gave 6 with low yield in acidic solutions. As described by Inayama
et al. (1985), the conversion of curdione to the curcumalactones involves the steric course. The
difficulty in the intracellular ene reaction from compounds 3 to 6 may be due to the steric
hindrance at 2-C by hydroxylation.
Following hydroxylation and acid-mediated intracellular ene reaction, dihydroxylation in
the generated olefinic compounds by A. niger occurred. There are two steps involved: the
epoxidation of the olefinic linkage and the subsequent hydrolysis of the epoxide (Fourneron et al.
1989). By HPLC analysis, two dihydroxylation products 7 and 8 appeared, whereas the
intermediate epoxides could not be detected. This may be due to the large amounts of acid
excreted by A. niger which efficiently catalysed hydration of the epoxides to the corresponding
dihydroxylation products 7 and 8. A tentative metabolic pathway for the transformation of
curdione (1) via chemo-bio cascade reactions is proposed in Figure 1. Regio- and stereoselective
Figure 3. Effect of monooxygenase inhibitors on the biotransformation by resting cells of A. niger. Results
are the mean ^ SD, n ¼ 3. *P , 0.05, means significantly differ from the control at the same level.

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Y. Chen et al.
hydroxylation of curdione (1) catalysed by A. niger yielded 2 and 3, and subsequent intracellular
ene reaction induced by acid yielded lactones 4 and 5. Compound 4 further underwent
epoxydation, as well as acid-catalysed hydrolysis leading to the formation of 7 and 8, which are
novel compounds.
2.3. Effects of monooxygenase inhibitors on the biotransformation of curdione
In a previous study, various monooxygenases in A. niger have been reported (Faber et al. 2001),
and hydroxylation and epoxidation reactions can be catalysed by monooxygenases such as
cytochrome P-450 (CYP450) and flavin-containing monooxygenases (FMOs) (Cirino & Arnold
2002). To further elucidate the responsible enzyme in curdione biotransformation by A. niger,
piperonyl butoxide (PB), chlorpyrifos (CP), 1-aminobenzotriazole (ABT) and methimazole
(MZ) were added to the cultures as PB, CP and ABT are well-known CYP450 inhibitors (Keum
et al. 2009), while MZ is a well-known FMO inhibitor (Nace et al. 1997). PB, CP and ABT
strongly inhibited the hydroxylation of curdione (1), while the treatment of MZ had no effect
(Figure 3). Based on these results, CYP450 rather than FMO might be involved in the
hydroxylation, which suggested an excellent starting point for studying the metabolism of
curdione in human liver microsomal CYP450 systems, as well as directed evolution of CYP450
from A. niger devoted to increase the regio- and stereoselectivty of hydroxylation.
3. Experimental
3.1. General analytical techniques
NMR spectra of curdione derivatives were recorded in CDCl3 (d 7.26 and 77.00 ppm) on a
1
Bruker AV-600 spectrometer (Bruker, Germany, 600 MHz for H NMR and 150 MHz for 13C
NMR) with tetramethylsilane as an internal standard. The signals in the 1H and 13C NMR spectra
were assigned unambiguously using 2D NMR techniques: COSY, HMQC and HMBC. HR-MS
were recorded on a Bruker micrOTOF-Q spectrometer (Bruker, USA). CD spectra were
measured with a Bio-Logic MOS-450 spectrophotometer (Bio-Logic, France) in MeOH. Optical
rotations were measured with a PerkinElmer 241 polarimeter (Perkin-Elmer, USA) in MeOH.
UV spectra were recorded with a Shimadzu UV 2201 UV–vis spectrophotometer (Shimadzu,
Japan). IR spectra were recorded with a Bruker IFS 55 spectrometer (Bruker, Germany).
Analytical thin layer chromatography (TLC) was carried out on precoated silica gel GF254 plate
(Merck, Darmstadt, Germany) using ethyl acetate/petroleum ether (1:1) as the eluent. Spots for
compounds were visualised by spraying with 10% aq. H2SO4 followed by heating. The HPLC
analyses were performed on Jasco HPLC 2000 series with a Jasco UV-2075 detector on a Dikma
Diamonsil C18 column (5 mm, 4.6 £ 200 mm) at 308C. The composition of the eluent was
increased linearly from acetonitrile–H2O (20:80) to acetonitrile–H2O (80:20) in 30 min and
decreased to acetonitrile–H2O (20:80) in 30–35 min. The wavelength of the UV detector was
205 nm with the eluent flow rate of 1 mL min21
.
3.2. Plant material
The essential oil obtained by steam distillation from the rhizomes of Zingiberaceae C. wenyujin
was purchased from Shuinan Yaoyong Baicaoyou Factory (Jishui, Jiangxi, China). The plant
was identified by Dr Y.H. Chen, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College. A voucher specimen (No. 4587) of the rhizomes is
available from the plant collection in the Chinese Virture Herbarium.

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3.3. Microorganisms and cultures
A. niger AS 3.739, purchased from China General Microbiological Culture Collection Center
(Beijing, China), was cultured in the modified culture medium (15.0 g dextrose, 15.0 g sucrose,
5.0 g peptone, 1.0 g KH2PO4, 0.5 g MgSO4, 0.5 g KCl, 0.01 g FeSO4 and 1000 mL H2O, pH 7.0)
at 288C and 210 rpm for 48 h.
3.4. Isolation and characterisation of curdione and its metabolites
Isolation and characterisation of curdione was performed as previously described (Zhou et al.
2012). A stock solution of curdione (1) was prepared in methanol. Further dilutions were made
immediately prior to each experiment. After 36 h-incubation of A. niger 3.739 growing cells
supplemented with curdione (250 mg), the culture was filtered to remove mycelium (1 L), further
subjected to macroporous resin D101 chromatography (110 mL, 2.5 £ 35 cm) using H2O
(100 mL), 30% ethanol (200 mL) and 98% ethanol (300 mL). The fraction of 98% ethanol was
collected, concentrated (239 mg) and separated on a silica gel HG/T2354-92 column (10 g,
38–48 mm, 1 £ 30 cm, flow rate 1.5 mL min–1, fraction size 5 mL). A step gradient of petroleum
ether–EtOAc 5:1 (300 mL), 4:1 (700 mL) and 3:1 (500 mL) was applied. All fractions eluted
were monitored by TLC, and fractions with the same Rf were combined affording compound 4
(tR 30–50 mL, Rf 0.85, 11.5 mg), compound 5 (tR 165–200 mL, Rf 0.29, 5.3 mg), compound 2
(tR 570–800 mL, Rf 0.23, 33.2 mg) and compound 3 (tR 850–1100 mL, Rf 0.15, 30.5 mg),
compound 8 (tR 1100–1200 mL, Rf 0.14, 3.4 mg), compound 7 (tR 1300–1500 mL, Rf 0.12,
8.2 mg) with purity .98%. Their structures were identified by means of spectral analyses (UV,
IR, TOF-MS, NMR, CD and elemental analysis). To accumulate the amount of compounds 5, 7
and 8, a 72 h-biotransformation was performed.
After biotransformation of curdione (0.3 mg mL21) by resting cells of A. niger for 48 h,
followed by acid-catalysed reaction for 24 h, the mixture was evaporated under reduced pressure
to give a crude product (50 mg) which was chromatographed on a silica gel HG/T2354-92
column (10 g, 38–48 mm, 1 £ 30 cm, flow rate 1.5 mL min–1, fraction size 5 mL) using
petroleum ether–EtOAc (30:1) to yield 6 (Rf 0.48, 4 mg) with purity .98%. The structure was
determined by spectral analyses.
4. Conclusions
Microbial transformation with A. niger AS 3.739 afforded seven metabolites and four of them
are novel. The research work also provided a proof of evidence that transformation of curdione
(1) by A. niger AS 3.739 followed the chemo-bio cascade reactions. The involvement of
CYP450 in curdione biotransformation suggested an excellent starting point for studying the
metabolism of curdione in human liver microsomal CYP450 systems, as well as directed-
evolution of CYP450 from A. niger devoted to increase the regio- and stereoselectivty of
hydroxylation.
Supplementary material
Supplementary material relating to this article is available online, alongside Tables S1–S3 and
Figures S1–S8.
Acknowledgement
This work is dedicated to Prof Dr Xinsheng Yao on the occasion of his 80th birthday.

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Y. Chen et al.
Funding
This work was financially supported by the National Scientific Major Program (No. 2010ZX09301-012)
and Shenyang Municipal Scientific and Technology Research Fund (No. F11-243-1-00). Support from
Liaoning Province Education Administration (No. L2011173) is also appreciated.
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