Microbial transformation of curcumin by Rhizopus chinensis

Article abstract of DOI:10.3109/10242422.2010.532870

Curcumin (1) is a potent antioxidant and antitumor natural product. In spite of its efficacy and safety, its clinical use is hindered mainly by poor water solubility and bioavailability. Structural modification to introduce hydrophilic functions is a promising approach to resolve this problem. In the present study we first found that curcumin could be efficiently converted into glucosides by filamentous fungi including Rhizopus chinensis IFFI 03043, Absidia coerulea AS 3.3389 and Cunninghamella elegans AS 3.1207. Curcumin 4′-O-β-d-glucoside (2), together with hexahydrocurcumin (3), was isolated from a preparative-scale biotransformation with R. chinensis IFFI 03043 and characterized fully by NMR and MS. A time-course study revealed that curcumin could be efficiently converted into curcumin 4′-O-β-d- glucoside within 8 h when administered at 0.05 mmol L^-1 and the productivity was 57%. Additionally, the biotransformation products of curcumin by different fungal strains were analyzed by LC/MS. At least 15 metabolites were detected, and the predominant biotransformation reaction was glucosylation. This study provides a simple, efficient and less expensive approach for the preparation of curcumin glucosides. The introduction of the glucosyl function might be able to enhance the bioavailability of curcumin.

Full text of DOI:10.3109/10242422.2010.532870

Biocatalysis and Biotransformation, September–December 2010; 28(5–6): 380–386
ORIGINAL ARTICLE
Microbial transformation of curcumin by Rhizopus chinensis
XING ZHANG1,2, MINYE1, RUI LI1, JUNYIN2 & DE-AN GUO1
1The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Beijing, People’s Republic of China and 2School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, Shenyang, People's Republic of China
Abstract
Curcumin (1) is a potent antioxidant and antitumor natural product. In spite of its efficacy and safety, its clinical use is
hindered mainly by poor water solubility and bioavailability. Structural modification to introduce hydrophilic functions is
a promising approach to resolve this problem. In the present study we first found that curcumin could be efficiently con-
verted into glucosides by filamentous fungi including Rhizopus chinensis IFFI 03043, Absidia coerulea AS 3.3389 and Cun-
ninghamella elegans AS 3.1207. Curcumin 4′-O-β-D-glucoside (2), together with hexahydrocurcumin (3), was isolated from
a preparative-scale biotransformation with R.chinensis IFFI 03043 and characterized fully by NMR and MS. A time-course
study revealed that curcumin could be efficiently converted into curcumin 4′-O-β-D-glucoside within 8 h when administered
at 0.05 mmol L–1 and the productivity was 57%. Additionally, the biotransformation products of curcumin by different
fungal strains were analyzed by LC/MS. At least 15 metabolites were detected, and the predominant biotransformation
reaction was glucosylation. This study provides a simple, efficient and less expensive approach for the preparation of cur-
cumin glucosides. The introduction of the glucosyl function might be able to enhance the bioavailability of curcumin.
Keywords: Curcumin, biotransformation, glucosylation, fungi, Rhizopus chinensis
Introduction
and, as a result, poor absorption in the gastrointes-
Curcumin is a hydrophobic polyphenolic compound
derived from the rhizomes of a medicinal plant, Cur-
cuma longa L. (turmeric). It has a unique 1,7-diaryl-
heptanoid basic skeleton. Pharmacological studies
have proved that curcumin exhibits antioxidant, anti-
tumor and anti-inflammatory activities (Anand et al.
2008). Most importantly, curcumin is used as dietary
pigment and is safe for human use, even at very high
doses. Therefore, curcumin has been evaluated in
several phase I clinical trials and may be developed
into a novel chemoprevention drug (Johnson &
Mukhtar 2007; Itokawa et al. 2008). In spite of its
efficacy and safety, the clinical use of curcumin as a
drug is hindered by its poor bioavailability (Anand
et al. 2007; Shishodia et al. 2007). Extremely low
serum levels were observed when curcumin was
administered orally to human subjects or animals at
a dose of 2 g kg–1. The major reason for the low
bioavailability of curcumin is its poor water solubility
tinal tract. Therefore, structural modification, par-
ticularly introducing hydrophilic functions into the
molecular structure, may be a promising approach
to improve the bioavailability of curcumin.
Glucosylation is an effective approach to enhance
the water solubility of curcumin (Kaminaga et al.
2003). Although the glucosylation of curcumin has
been reported by chemical synthesis or biotransfor-
mation using cultured suspension cells of Catharan-
thus roseus, the yields were only about 30%. In
addition, the biotransformation reactions were not
specific and a number of analogous glucosides were
produced (Hergenhahn et al. 2002; Kaminaga et al.
2003; Mohri et al. 2003).
In the present paper we report a specific and
efficient glucosylation reaction of curcumin by
filamentous fungi. When curcumin was incubated
with Rhizopus chinensis IFFI 03043 for 8 h, about
60% of the substrate could be converted into
Correspondence: M.Ye and D.-A. Guo,The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
38 Xueyuan Road, Beijing 100191, People's Republic of China. Tel: ꢀ86-10-8280-1516. Fax: ꢀ86-10-8280-2700. E-mail: yemin@bjmu.edu.cn (M. Ye);
gda5958@163.com (D.-A. Guo)
(Received 27 August 2010; accepted 14 October 2010)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2010 Informa UK, Ltd.
DOI: 10.3109/10242422.2010.532870

Biotransformation of curcumin 381
Screening test
curcumin 4′-O-β-D-glucoside. In addition, a series
of minor glucosylated products was observed as
biotransformed products in R. chinensis, Absidia
coerulea and Cunninghamella elegans.
The fungi (3 days old) were inoculated from agar
slants and incubated in 250-mL Erlenmeyer flasks
containing 100 mL liquid medium at 25°C on a
rotary shaker (120 rpm).They were allowed to grow
for 24 h until the exponential phase was reached, at
which time curcumin was added. Unless elsewhere
stated, curcumin was prepared as 2 mg mL–1 solu-
tions in acetone and 2 mg was added to each flask.
After 5 days of incubation, the cultures were pooled,
filtered and extracted with an equal volume of ethyl
acetate. The extract was evaporated to dryness in a
rotary evaporator under reduced pressure, and the
residue was dissolved in 1 mL methanol for TLC
analysis. The TLC plates were developed with chlo-
roform–methanol (50:1, v/v) and were sprayed with
10% (v/v) H2SO4 in ethanol for visualization. Cul-
ture controls consisted of fermentation blanks in
which microorganisms were grown without substrate
but were fed with the same amount of acetone. Sub-
strate controls contained the sterile fermentation
medium with the same amount of substrate, incu-
bated and sterilized as described above. For HPLC
and LC/MS analyses, the solutions were filtered
through 0.45 μm micropore membranes before use
and an aliquot of 10 μL was injected.
Materials and methods
General
HPLC-grade acetonitrile (J.T. Baker, Phillipsburg,
NJ, USA) and ultra-pure water were used for all
HPLC and LC/MS analyses. The other analytical-
grade solvents were from Beijing Chemical Corpo-
ration (Beijing, China). Silica gel for column
chromatography was from Qingdao Haiyang Chem-
ical Incorporation (Qingdao, China). Reversed-
phase preparative HPLC was performed on a TSP
P100 SpectraSeries HPLC system (Thermoquest,
San Jose, CA, USA) connected to a UV detector,
using a Zorbax SB C18 column (250 mm ꢁ 10.0 mm,
5 μm; Agilent Technologies Inc., Santa Clara, CA,
USA) eluted with methanol–water at a flow rate
1
of 2 mL min–1. H and 13C NMR spectra were
recorded on a Bruker AM 400 spectrometer
(Karlsruhe, Germany) in acetone-d6 with TMS as
internal standard. Mass spectra were measured on
an LCQ Advantage ion-trap mass spectrometer
(ThermoFinnigan, San Jose, CA, USA).
Preparative-scale biotransformation of curcumin by
R. chinensis IFFI 3043
Chemicals
The mycelia from agar slants were aseptically trans-
ferred to 250-mL Erlenmeyer flasks each containing
80 mL liquid potato medium. The flasks were incu-
bated at 25°C on a rotary shaker (180 rpm) in the
dark for 24 h to make stock inoculums, 5 mL of
which were added to 1-L flasks containing 350 mL
potato medium. After 12 h incubation, 900 mg cur-
cumin dissolved in 45 mL acetone was distributed
equally among the 45 flasks.The incubation contin-
ued for 6 days on the shaker.The fermentations were
then pooled and filtered. The filtrate was extracted
three times with 15 L ethyl acetate.The ethyl acetate
extract was evaporated under vacuum to yield 3.2 g
of brown residue.The residue was subjected to silica
gel column chromatography (100–200 mesh, 130 g
silica gel) and eluted with chloroform–methanol
(100:1, 50:1 and 10:1, v/v) to yield compound 3 (56 mg,
6.2% yield). The 5:1 (v/v) fraction was subjected to
preparative reversed-phase LC and eluted with
methanol–water (50:50, v/v) to obtain compound
2 (376 mg, 41.8% yield).
Curcumin (1) was isolated as a bright yellow powder
from C. longa L. by the authors. Its chemical struc-
ture was identified unambiguously by comparison
with literature data (Kiuchi et al. 1993). The purity
was above 95% as determined by HPLC/DAD
analyses.
Microorganisms and culture medium
The fungal strains were purchased from China
General Microbiological Culture Collection Center
(Beijing, China). A total of 11 strains were used for
the screening test, including Gibberella fujikuroi var.
fujikuroi AS 3.4748, Saccharomyces cerevisiae ACCC
2168, Alternaria alternata AS 3.4578, Chaetomium
globosum IFFI 2445, Crebrothecium ashbyii ACCC
2114, Penicillium melinii AS 3.4474, A. coerulea AS
3.3389, Aspergillus niger AS 3.795, Syncephalastrum
racemosum AS 3.264, R. chinensis IFFI 3043 and
C. elegans AS 3.1207. Fermentations were carried
out in potato medium consisting of 100 g potato
extract, 20 g glucose and 1000 mL distilled water.
The media were sterilized in an autoclave at 121°C and
1.06 kg cm–2 for 30 min.
Curcumin 4′-O-β-D-glucoside (2): orange amor-
1
phous powder. UV (MeOH), λmax (nm): 425. H-
NMR (acetone-d6, 400 MHz), δ (ppm): 7.62 (1H, d,
Jꢂ16.0 Hz, H-1); 7.61 (1H, d, Jꢂ16.0 Hz, H-7);

382 X. Zhang et al.
mobile phase consisted of acetonitrile (A) and water
(B). The following gradient elution program was
used (v/v): 20–40% A/80–60% B for 0–15 min,
40–55%A/60–45%Bfor15–25min,55–95%/45–5%
B A for 25–38 min, and 95% A/5% B for 38–45
min. The flow rate was 1.0 mL min–1 and the col-
umn temperature was 30°C. The detection wave-
length was 425 nm and 350 nm for compounds 2
and 3, respectively. For LC/MS analysis, the HPLC
instrument was connected to an LCQ Advantage
ion-trap mass spectrometer (ThermoFinnigan) via
an electrospray ionization (ESI) interface. The LC
effluent was introduced into the ESI source in a
post-column splitting ratio of 5:1. High-purity
nitrogen was used as the nebulizing gas (including
sheath gas and auxiliary gas) and ultra-high purity
helium as the collision gas. The ESI source was
monitored in the negative-ion polarity mode. The
optimized detection parameters were as follows: ion
spray voltage, 4.5 kV; sheath gas (N2), 50 arbitrary
units; auxiliary gas (N2), 10 units; capillary tem-
perature, 325°C; capillary voltage, –4 V; tube lens
offset voltage, –45 V. For tandem mass spectrome-
try (MSn) analysis, the collision energy for CID
(collision-induced dissociation) was adjusted to
35% of maximum and the isolation width of pre-
cursor ions was 2.0 Th. The LC/MS system was
controlled by Xcalibur 1.3 software.
6.89–7.37 (6H, m, H-Ar); 6.78 (1H, d, Jꢂ16.0 Hz,
H-2); 6.74 (1H, d, Jꢂ16.0 Hz, H-6); 6.01 (1H, s,
H-4); 5.05 (1H, d, Jꢂ7.2 Hz, Glc-H-1); 3.40–4.30
(6H, m, Glc-H); 3.93 (1H, s, MeO-3′); 3.91 (1H, s,
MeO-3″). 13C-NMR (acetone-d6, 100 MHz), δ
(ppm): 184.2 (C-3); 183.6 (C-5); 150.0 (C-4′);
149.6 (C-4″); 149.2 (C-3′); 147.9 (C-3″); 140.8
(C-1); 139.8 (C-7); 129.7 (C-1′); 127.2 (C-1″);
123.0 (C-2); 122.7 (C-6); 122.1 (C-6′); 121.4
(C-6″); 116.3 (C-5′); 115.4 (C-5″); 111.5 (C-2′);
110.7 (C-2″); 101.1 (C-4); 101.0 (glc-1′); 77.1
(glc-2′); 77.0 (glc-3′); 73.8 (glc-4′); 70.4 (glc-5′);
61.7 (glc-6′); 55.6 (MeO-3′); 55.4 (MeO-3″).
ESI-MS: m/z 529.0. These data were in accordance
with the literature (Mohri et al. 2003).
Hexahydrocurcumin (3): yellowish amorphous
powder. UV (MeOH), λmax (nm): 280. 1H-NMR (ace-
tone-d6, 400 MHz), δ (ppm): 6.81 (1H, d, Jꢂ2.0 Hz,
H-2′), 6.80 (1H, d, Jꢂ2.0 Hz, H-2″); 6.72 (1H, d,
Jꢂ8.0 Hz, H-5′); 6.71 (1H, d, Jꢂ8.0 Hz, H-5″); 6.64
(2H, dd, Jꢂ8 and 2 Hz, H-6′ and H-6″); 4.02 (1H,
m, H-5); 3.82 (1H, s, MeO-3′); 3.81 (1H, s, MeO-3″);
2.77 (2H, m, H-1a and H-1b); 2.75 (2H, m, H-2a and
H-2b); 2.69 (2H, m, H-7a and H-7b); 2.59 (1H, dd,
Jꢂ16.8 and 6.0 Hz, H-4b); 2.55 (1H, dd, Jꢂ16.8 and
6.0 Hz, H-4a); 1.70 (1H, dt, Jꢂ13.6 and 5.2 Hz,
H-6a); 1.68 (1H, dt, Jꢂ13.6 and 5.2 Hz, H-6b). 13C-
NMR (acetone-d6, 100 MHz), δ (ppm): 209.9 (C-3);
148.2 (C-3′ and C-3″); 145.6 (C-4′); 145.5 (C-4″);
134.5 (C-1′);133.7 (C-1″);121.5 (C-6′);121.4 (C-6″);
115.6 (C-5′ and C-5″); 112.8 (C-2′ and C-2″); 56.4
(MeO-3′ and MeO-3″); 56.2 (C-5); 50.9 (C-4); 40.3
(C-6); 32.1 (C-1); 30.2 (C-7). ESI-MS: m/z 373.1
([M-H]–). These data were in accordance with the
literature (Itokawa et al. 1985).
Results and discussion
Microbial screening
A total of 11 strains of filamentous fungi were
screened for the biotransformation of curcumin (1).
According to theTLC results, curcumin was metab-
olized by six strains, which included G. fujikuroi var.
fujikuroi AS 3.4748, A. coerulea AS 3.3389, A. niger
AS 3.795, R. chinensis IFFI 3043, C. elegans AS
3.1207 and S. cerevisiae ACCC 2168. Among them,
R. chinensis IFFI 3043 was the most potent strain
and was selected for preparative-scale biotransfor-
mation to isolate the products.
HPLC and LC/MS analyses
Samples were analyzed on an Agilent 1100 series
HPLC instrument equipped with a quaternary
pump, a diode-array detector, an autosampler and
a column compartment. An Agilent Zorbax SB C18
column (4.6 mm ꢁ 250 mm, 5 μm) was used. The
Figure 1.The biotransformation pathway of curcumin by Rhizopus chinensis IFFI 3043.

Biotransformation of curcumin 383
Preparative biotransformation of curcumin by
R. chinensis IFFI 3043
Curcumin (1) could be effectively metabolized by
R. chinensis IFFI 3043. After 6 days of incubation,
the culture supernatants were extracted with ethyl
acetate.The extract was separated by silica gel chro-
matography and semi-preparative HPLC to yield
compound 2 (376 mg, 41.8% yield) and compound
3 (56 mg, 6.2% yield) (Figure 1). On the basis of
NMR and MS analyses, the structures of compounds
2 and 3 were identified as curcumin 4′-O-β-D-gluco-
side and hexahydrocurcumin (5-hydroxy-1,7-bis(4-
hydroxy-3-methoxyphenyl)-3-heptanone),
respectively. Interestingly, compound 3 has been
identified as a mammalian metabolite of curcumin
(Ireson et al. 2001).
Figure 2. Biotransformation kinetics of curcumin by Rhizopus
chinensis IFFI 3043: 1, curcumin; 2, curcumin 4′-O-β-D-glucoside;
3, hexahydrocurcumin.The detection wavelength was 425 nm for
1 and 2, and 280 nm for 3. Concentrations are the average of
duplicate tests.
Biotransformation kinetics of curcumin by
R. chinensis IFFI 3043
curcumin, including A. coerulea and C. elegans. After
1 day of incubation, the majority of administered
curcumin was metabolized and curcumin 4′-O-β-D-
glucoside (2) was the predominant product (Figure
3). When the HPLC chromatogram was monitored
at 425 nm, only a few minor products were observed.
More products could be observed at 280 nm or 360
nm. However, their peak areas were much lower
than that of curcumin 4′-O-β-D-glucoside. There-
fore, the glucosylation of curcumin by filamentous
fungi appears to be a specific and efficient biotrans-
formation reaction.
The biotransformation time course of curcumin in
R. chinensis for 24 h was studied.We found that cur-
cumin could be rapidly metabolized within only 8 h of
incubation (Figure 2). Curcumin 4′-O-β-D-glucoside
(2) was the predominant product and its productiv-
ity was 57%, calculated on the basis of peak area. In
this period, a small amount of product 3 was gener-
ated, which was detected at 350 nm. As the incuba-
tion continued, product 3 accumulated.
Kaminaga et al. (2003) reported the glucosylation
of curcumin by cell suspension cultures of C. roseus.
At least five major glucosides, including monogluco-
sides and diglucosides, were produced when admin-
istered at 0.5 mmol L–1 and the productivity of total
glucosides was 32%. The microbial transformation
reaction reported in the present study was more effi-
cient and more specific than previous reports.
We also tried to further improve the biotransfor-
mation productivity of curcumin 4′-O-β-D-glucoside
(2) by R. chinensis. Previous studies reported that
β-cyclodextrin might induce biocatalytic reactions
(Dai et al. 2008). We added β-cyclodextrin into the
cultures, but unfortunately this decreased the pro-
ductivity. In addition, we examined the substrate
concentration tolerance of the reaction. When the
concentration of curcumin was increased from
0.05 mmol L–1 to 0.25 mmol L–1 and 1 mmol L–1,
the productivity of curcumin 4′-O-β-D-glucoside
(2) decreased from 57% to 39% and 20%, respec-
tively (data not shown).The low productivity at high
substrate concentration may be due to the poor
water solubility of curcumin, as yellow precipitates
were observed even after several days of incubation.
Aside from R. chinensis, a number of fungal
strains were able to catalyze the glucosylation of
LC/MS analysis of curcumin metabolites by different
fungal strains
In order to fully understand the metabolic proper-
ties of filamentous fungi on curcumin, minor prod-
ucts from the three most capable fungal strains,
R.chinensis, A.coerulea and C.elegans, were analyzed
by LC/MS. A total of 15 curcumin derivatives (1a–
1m, 2, 3) were identified by analyzing their UV and
MS spectral data. Their proposed structures are
given in Table I. Among them, three compounds
(1a, 1c, 1g) are reported for the first time. Struc-
tural elucidation of the products was based mainly
on their tandem mass spectrometry and by refer-
ring to the fragmentation pathway of curcuminoids
proposed by Jiang et al. (2006a,b). UV spectra were
also used as supplementary information as the
absorption maxima of curcuminoids were corre-
lated with the unsaturation degree of the heptane
chain. Here, we take 1j (dihydrocurcumin O-gluco-
side) as an example to illustrate the structural elu-
cidation process (Figure 4).
Compound 1j showed a UV absorption maxi-
mum at 364 nm. In comparison, curcumin showed

384 X. Zhang et al.
Figure 3. Glucosylation of curcumin (1) by different fungal strains, showing the HPLC chromatograms of curcumin (a) and its
biotransformed products in Rhizopus chinensis IFFI 3043 (b), Cunninghamella elegans AS 3.1207 (c) and Absidia coerulea AS 3.3389 (d).
an absorption maximum at 425 nm, due to the
multiple double-bond conjugation system of the hep-
tane chain. The wavelength shift of 1j suggested
hydrogenation of the double bond. The ESI-MS
spectrum showed an [MꢃH]ꢃ ion at m/z 531, which
produced m/z 371 by collision-induced dissociation.
The loss of 162 Da could be attributed to a glucosyl
residue. The mass of m/z 371 was two mass units
Figure 4. Identification of the biotransformed product 1j (dihydrocurcumin O-glucoside) of curcumin by Rhizopus chinensis IFFI 3043
by means of HPLC/DAD/ESI-MSn. The UV spectrum and the MS2 and MS3 spectra of the ion at m/z 531 are shown.

Biotransformation of curcumin 385
Table I. HPLC/DAD/ESI-MS characterization of the biotransformation products of curcumin by Rhizopus chinensis IFFI 3043, Absidia
coerulea AS 3.3389 and Cunninghamella elegans AS 3.1207.
UV λmax
No. tR (min)
(nm) [MꢃH]ꢃ
MS/MS
MS2 [537]: 375 (100)
Identification
3043 3.3389 3.1207
1a
7.37
–
537
Octahydrocurcumin
O-glucoside
ꢃ
ꢃ
ꢀ
MS3 [537→375]: 357 (20), 329 (30), 250 (15),
191 (20), 179 (22), 152 (25), 135 (100)
MS2 [533]: 371 (100)
1b
1c
8.37
8.88
360
–
533
535
Tetrahydrocurcumin
O-glucoside
Hexahydrocurcumin
O-glucoside
ꢀ
ꢀ
ꢃ
ꢃ
ꢃ
ꢀ
MS3 [533→371]: 235 (78), 191 (100), 178 (74)
MS2 [535]: 373 (100), 371 (60), 191 (18)
MS3 [535→373]: 249 (15), 235 (15), 193 (20),
191 (100), 179 (60), 165 (12)
1d
1e
1f
9.19
11.36
12.83
420
360
360
691
533
371
MS2 [691]: 673 (20), 571 (8), 529 (100),
487 (5), 378 (5), 367 (5), 217 (5)
MS3 [691→529]: 497 (10), 217 (100), 173 (10)
MS2 [533]: 371 (100)
Curcumin di-O-glucoside
ꢀ
ꢀ
ꢀ
ꢃ
ꢀ
ꢀ
ꢃ
ꢀ
ꢃ
Tetrahydrocurcumin
O-glucoside
MS3 [533→371]: 249 (100), 235 (90), 207 (15),
193 (8), 175 (5)
MS2 [371]: 353 (8), 249 (100), 235 (90), 207
(20), 193 (8)
Tetrahydrocurcumin
MS3 [371→207]: 192 (100), 149 (50)
MS2 [515]: 427 (15), 353 (100)
1g
1h
1i
13.45
13.77
14.77
420
–
515
375
371
Demethylcurcumin
O-glucoside
ꢀ
ꢃ
ꢃ
ꢃ
ꢃ
ꢀ
ꢃ
ꢀ
ꢃ
MS3 [515→353]: 217 (70), 173 (85), 161 (100)
MS2 [375]: 359 (40), 339 (8), 325 (5), 223 (15), Octahydrocurcumin
209 (20), 179 (40), 135 (100), 109 (15)
360
MS2 [371]: 249 (100), 235 (75), 207 (18), 193 Tetrahydrocurcumin
(5), 177 (8), 121 (2)
MS3 [371→249]: 234 (100), 207 (80), 191 (25),
125 (5), 119 (4)
3a
1j
15.01
16.02
–
373
531
MS2 [373]: 355 (20), 193 (45), 179 (100),
165 (28), 121 (15)
Hexahydrocurcumin
ꢀ
ꢀ
ꢃ
ꢃ
ꢀ
ꢀ
364
MS2 [531]: 369 (100)
Dihydrocurcumin
O-glucoside
MS3 [531→369]: 233 (50), 219 (100), 175 (30),
149 (60)
2a
16.33
16.45
420
360
529
369
MS2 [529]: 367 (100), 217 (68), 173 (18),
157 (8)
Curcumin 4′-O-β-D-
glucoside
ꢀ
ꢃ
ꢀ
ꢀ
ꢀ
ꢃ
MS3 [529→367]: 352 (20), 217 (100), 173 (72),
175 (50)
1k
MS2 [369]: 233 (100), 219 (70), 191 (10),
175 (50), 159 (8), 149 (45), 131 (5)
MS3 [369→219]: 204 (25), 173 (15), 149 (100)
MS2 [371]: 249 (50), 235 (100), 233 (15),
219 (15)
Dihydrocurcumin
1l
19.77
26.29
360
360
371
371
Tetrahydrocurcumin
Tetrahydrocurcumin
ꢀ
ꢀ
ꢃ
ꢃ
ꢃ
ꢃ
1m
MS2 [371]: 343 (8), 329 (8), 249 (100),
135 (90), 193 (8)
MS3 [371→249]: 235 (90), 207 (100), 191 (30),
111 (20)
aCompounds were identified unambiguously by comparison with reference standards.
greater than curcumin [MꢃH]ꢃ (m/z 367), suggest-
ing the saturation of one double bond.The MS3 spec-
trum of m/z 371 further yielded fragment ions at m/z
223, 219, 175 and 149.These fragments were in good
accordance with the reports of Jiang et al. (2006a,b),
and should be due to the cleavage of the heptane
chain. Particularly, the production of m/z 233 indi-
cated the cleavage of C1–C2 (Fig. 4). Therefore, the
C1–C2 double bond of curcumin should have been
saturated. Based on the above evidence, the structure
of metabolite 1j was tentatively characterized as dihy-
drocurcumin O-glucoside.
The other metabolites were characterized in a
very similar manner to 1j. For all metabolites, three
biotransformation reactions were observed, includ-
ing glucosylation, reduction (hydrogenation) and
demethylation. Some identified metabolites were
obtained via only a single type of reaction, like
compound 2 (glucosylation); some others were
obtained after more complicated reactions, like 1a
(reduction and glucosylation) and 1g (demethyla-
tion and glucosylation). Obviously, the major
biotransformation reaction was glucosylation. Out
of the 15 identified products, eight were glucosides.

386 X. Zhang et al.
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Conclusions
We have shown herein that fungal strains can
efficiently metabolize curcumin into its glucosides.
The predominant product was curcumin 4′-O-β-D-
glucoside and for R. chinensis IFFI 3043, the yield
was 57% after 8 h of incubation. In addition, a
number of minor glucosylation, reduction and
demethylation products of curcumin were observed
by LC/MS. This study revealed that filamentous
fungi could be efficient catalysts for the production
ofcurcuminglucosides,whichsignificantlyimproved
the water solubility of curcumin. Given its high pro-
ductivity and specificity, and low cost, this biotrans-
formation reaction might be valuable in industrial
application.
Declaration of interest: This work was supported
by Beijing Natural Science Foundation (No.
7102103) and the Beijing NOVA Program (No.
2009B02). The authors report no conflicts of inter-
est.The authors alone are responsible for the content
and writing of the paper.
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