Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites

Article abstract of DOI:10.1016/j.foodchem.2012.11.138

Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis activity. The present research work aimed to study the biotransformation processes of IMP and evaluated the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new derivatives. Ten transformed products were isolated and purified, and their structures were identified accurately based on spectroscopic data. Eight metabolites (2-8 and 10) were novel and previously unreported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of all products (1-10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8 had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future therapeutic regimens for treating osteoporosis.

Full text of DOI:10.1016/j.foodchem.2012.11.138

Food Chemistry 138 (2013) 2260–2266
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis
activities of its metabolites
Xia Lv a,1, Dan Liu b,1, Jie Hou a, Peipei Dong c, Libin Zhan c, Li Wang a, Sa Deng a, Changyuan Wang a,
Jihou Yao a, Xiaohong Shu a, Kexin Liu a, Xiaochi Ma a,c,
⇑
a College of Pharmacy, Dalian Medical University, Dalian 116044, China
b School of Science, Dalian Nationalities University, Dalian 116600, China
c Research Institute of Integrated Traditional and Western Medicine, Dalian Medical University, Dalian 116044, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 October 2012
Received in revised form 26 November 2012
Accepted 28 November 2012
Available online 28 December 2012
Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis
activity. The present research work aimed to study the biotransformation processes of IMP and evaluated
the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi
screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new deriv-
atives. Ten transformed products were isolated and purified, and their structures were identified
accurately based on spectroscopic data. Eight metabolites (2–8 and 10) were novel and previously unre-
ported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and
the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of
all products (1–10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8
had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future
therapeutic regimens for treating osteoporosis.
Keywords:
Penicillium janthinellum
Biotransformation
Coumarin
Imperatorin
Anti-osteoporosis activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
structures. It possesses the advantages of high regio- and stereose-
lectivity, mild reaction conditions, and simple operation
Imperatorin (IMP) is a linear furanocoumarin, with a chemical
structure of 8-isopentenyloxypsoralen (9-(3-methylbut-2-enyl-
oxy)-7H-furo [3,2-g] chromen-7-one). IMP naturally exists in many
Chinese herbal medicines, especially Angelica dahurica, Cnidium
monnieri (L.) Cusson and Imperatoria osthruthium. These have been
widely used as medicines and functional foods for the treatment of
osteoporosis, relief of swelling and pain (He, Zhang, He, & Cao,
2007). The potential pharmacological activities of IMP included
anti-osteoporosis activity (Tang, Yang, Chen, Chen, & Fu, 2008;
Zhang & Qin, 2003), antitumour (Kim, Kim, & Ryu, 2007), anti-
inflammatory (Garcia-Argaez, Ramirez-Apan, Delgado, Velazquez,
& Martinez-Vazquez, 2000) and antimicrobial effects (Rosselli
et al., 2006), b-secretase (BACE1) inhibitory activity (Marumoto &
Miyazawa, 2010a), inhibition of myocardial hypertrophy (Zhang,
Cao, Zhan, Duan, & He, 2010), and effects on drug-metabolising en-
zymes (Shin & Woo, 1986). However, its poor solubility and oral
availability in water limit its use clinically or as a foodstuff (Li, 2006).
Biotransformation has become an economically competitive
technology for modifying chemical compounds with complex
procedures. In recent years, our research group has frequently re-
ported structural modifications of natural functional products to
obtain new chemical entities with better bioactivity and improved
water solubility (Deng et al., 2012; Li et al., 2011; Ma, Cui, Zheng, &
Guo, 2007; Ma, Wu, & Guo, 2006; Ma, Xin, Liu, Han, & Guo, 2008;
Ma, Zheng, & Guo, 2007; Ma et al., 2011).
In the present research, Penicillium janthinellum AS 3.510 exhib-
ited good capabilities to transform IMP. Ten transformed products
of IMP were isolated and identified, including eight novel metabo-
lites 2–8 and 10 (Fig. 1). Regio-specific hydroxylation, hydrolysis,
methylation, hydrogenation and glycosylation reactions of IMP
by suspended-cell cultures of P. janthinellum AS 3.510 were re-
ported. The anti-osteoporosis activities of all transformed products
were evaluated by using MC3T3-E1 cells, and preliminary struc-
ture–activity relationships were concluded. Some new metabolites
(5 and 8) of IMP with potent bioactivity could be used as preferred
candidates for development of new functional foods or drugs.
2. Materials and methods
2.1. Apparatus
⇑
Corresponding author at: College of Pharmacy, Dalian Medical University,
Lvshun South Road No. 9, Dalian 116044, China. Tel./fax: +86 411 86110419.
1D and 2D NMR spectra were determined in dimethylsulfoxide-
d6 (DMSO-d6) by a Bruker ARX 600 NMR spectrometer with
E-mail address: maxc1978@163.com (X. Ma).
These authors contributed equally to this work.
1
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.11.138

X. Lv et al. / Food Chemistry 138 (2013) 2260–2266
2261
OH
OCH3
O
O
O
O
O
O
O
O
O
O
O
O
O
9
OH
O
OH
HO
HO
OH
10
5
8
4
6
7
10
9
3
2'
3'
2
COOCH3
O
O
COOH
O
O
O
1
O
OH
O
O
OH
O
1'
O
O
O
OH
1a
2a
OH
CH2OH
CH2OH
5a
4a
6
5
IMP
1
10
6
2'
3'
3
4
5
8
COOCH3
COOH
O
OH
OH
O
1'
OH
COOH
7
2
O
OH
O
O
O
1a
OH
2a
5a
4a
OH
OH
CH2OH
7
2
3
COOH
COOH
O
OH
OH
O
O
O
O
OH
4
8
Fig. 1. Biotransformation products of IMP by P. janthinellum AS 3.510.
tetramethylsilane (TMS) as internal standard. Electrospray ionisa-
tion mass spectrometry (ESI-MS) was performed using an
API3200 mass spectrometer (AB SCIEX, Framingham, MA). HRMS
data were obtained using a Bruker APEXII Fourier transform ion
cyclotron resonance (FT-ICR) mass spectrometer. HPLC analyses
were performed on an UltiMate 3000 instrument (Thermo Scien-
tific Dionex) equipped with a diode array detector. Melting points
were measured on an XT4A apparatus (Dianguang Corp., Shanghai,
China) and were uncorrected. Optical rotations were measured
using a Perkin-Elmer 243B polarimeter. Ultraviolet (UV) spectra
were detected on a TU-1091 UV–visible spectrophotometer. Infra-
red (IR) spectra were obtained on an Avatar 360 FT-IR spectropho-
tometer (Thermo Nicolet).
was purchased from Qingdao Marine Chemical Corporation, China.
Octadecylsilane (ODS) was purchased from YMC Co., Ltd. (Kyoto,
Japan). IMP was isolated from C. monnieri (L.) Cusson using HPLC
analysis by the author (X. Lv), with >98% purity. Icariside with
98% purity was purchased from the National Institutes for Food
and Drug Control, Beijing, China.
2.3. Microorganisms
Alternaria alternata AS 3.577, Alternaria alternata AS 3.4578,
Aspergillus niger AS 3.795, Aspergillus candidus CICC 2360, Cunning-
hamella blakesleeana Lendner AS 3.970, Cunninghamella elegans AS
3.1207, Cunninghamella elegans AS 3.2028, Fusarium solani AS
3.1829, Mucor spinosus AS 3.3450, Mucor spinosus AS 3.2450, Mucor
spinosus AS 3.3447, Mucor subtilissimus AS 3.2454, Mucor subtilissi-
mus AS 3.2456, Mucor polymorphosporus AS 3.3443, Penicillium
janthinellum AS 3.510, Penicillium aurantigriseum AS 3.4512, Rhizo-
pus stolonifer AS 3.2050 and Trichoderma viride AS 3.2942 were pur-
chased from the Chinese General Microbiological Culture
Collection Center, Beijing, China.
2.2. Reagents
All organic solvents were of analytic grade and were obtained
from Tianjing Chemical Company (China). For HPLC experiments,
chromatographic grade acetonitrile and methanol (Merck, Darms-
tadt, Germany) were used. Thin-layer chromatography (TLC) anal-
yses were performed on silica gel GF254 (300–400 mesh), which

2262
X. Lv et al. / Food Chemistry 138 (2013) 2260–2266
2.4. Culture medium
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) see Tables 1 and 2.
HRESIMS [MꢀH]ꢀ m/z 337.1290 (calcd. for C17H21O7, 337.1287).
All culture and biotransformation experiments were performed
in liquid potato medium. Minced and husked potato (200 g) was
boiled in water for 1 h, and the solution was filtered. The filtrate
was added to 1 L of water after adding 20 g of glucose.
2.6.3. 6,7-Furano-8-(2a-hydroxy-3a,9-
epoxyprenyloxy)hydrocoumaric acid (4)
22
Yellow powder, m.p. 197–198 °C, [
a
]
D
ꢀ10.4 (c = 0.2, MeOH);
UV kmax (MeOH): 212, 253 and 280 nm. 1H NMR (DMSO-d6,
500 MHz) and 13C NMR (DMSO-d6, 125 MHz) see Tables 1 and 2.
HRESIMS [MꢀH]ꢀ m/z 305.1017 (calcd. for C16H18O6, 305.1025).
2.5. Culture and biotransformation procedures
Screening scale biotransformation of IMP was carried out in
250-mL Erlenmeyer flasks containing 100 mL of liquid medium.
The flasks were placed on a rotary shaker operating at 180 rpm
and 27 °C. The substrates were dissolved in acetone to reach a final
concentration of 10 mg/mL. After 36 h of pre-culture, 2 mg of IMP
were added into each flask. The incubation was allowed to con-
tinue for 5 days. Culture controls consisted of fermentation blanks
with microorganisms grown under identical non-substrate condi-
tions. Substrate controls were composed of sterile medium with
substrate, and they were incubated without microorganisms.
Preparative scale biotransformation of IMP by P. janthinellum AS
3.510 was carried out in a 1-L Erlenmeyer flask. The substrate
(10 mg) dissolved in acetone (0.5 mL) was added to pre-cultured
medium (400 mL) and was incubated for an additional 5 days. In
total, 800 mg of substrate were used for the preparative scale bio-
transformation. Other procedures were similar to the screening
scale biotransformation.
2.6.4. 6,7-Furano-8-(5a-hydroxymethylprenyloxy)hydrocoumaric acid
(5)
22
Yellow powder, m.p. 227–228 °C, [
a]
D
ꢀ2.5 (c = 0.3, MeOH);
UV kmax (MeOH): 205, 252 and 286 nm. 1H NMR (DMSO-d6,
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) see Tables 1 and 2.
HRESIMS [MꢀH]ꢀ m/z 305.1021 (calcd. for C16H18O6, 305.1025).
2.6.5. 6,7-Furano-8-(5a-hydroxymethylprenyloxy)hydrocoumaric acid
methyl ester (6)
22
Yellow powder, m.p. 198–199 °C, [
a]
D
ꢀ1.5 (c = 0.1, MeOH);
UV kmax (MeOH): 206, 252 and 286 nm. 1H NMR (DMSO-d6,
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) see Tables 1 and 2.
HR-ESIMS [MꢀH]ꢀ m/z 305.1021 (calcd. for C16H18O6, 305.1025).
2.6.6. 6,7-Furano-8-(5a-hydroxymethyl-2a,3a-
dihydroprenyloxy)hydrocoumaric acid (7)
22
Yellow powder, m.p. 205–206 °C, [
a
]
ꢀ9.5 (c = 0.1, MeOH);
D
UV kmax (MeOH): 210, 253 and 285 nm. 1H NMR (DMSO-d6,
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) see Tables 1 and 2.
HRESIMS [MꢀH]ꢀ m/z 307.1185 (calcd. for C16H20O6, 307.1182).
2.6. Extraction, purification and identification of major biotransformed
products
The culture was filtered and the filtrate was extracted four
times with the same volume of ethyl acetate (EtOAc). The organic
phase was collected and concentrated under reduced pressure at
40 °C. The brown residues obtained (1.4 g) were applied to an octa-
decylsilane (ODS) column eluting with MeOH–H2O (in a gradient
manner from 10:90 to 90:10) to give 50 fractions. Based on the re-
sults of HPLC analysis, the biotransformed metabolites of IMP were
detected in Fr 17–39. Then, Fr 17–28 were successively purified by
semi-preparative HPLC (mobile phase: 45% acetonitrile in 0.03%
aqueous acetic acid; flow rate: 1.5 mL/min) to afford compound 1
(25 mg, 3.1%) and six small fractions, which were further subjected
to semi-preparative HPLC (mobile phase: 40% acetonitrile in 0.03%
aqueous acetic acid; flow rate: 2.0 mL/min) to give compounds 2
(8.0 mg, 1.0%), 3 (4.0 mg, 0.5%), 4 (5.0 mg, 0.63%), 5 (6.0 mg,
0.75%), 6 (3.5 mg, 0.4%) and 7 (3.0 mg, 0.37%). Successful purifica-
tion of Fr 29–39 by semi-preparative HPLC with 50% acetonitrile
in 0.03% aqueous acetic acid as mobile phase and flow rate of
1.5 mL/min yielded compounds 8 (3.0 mg), 9 (5.0 mg, 0.63%) and
10 (4.5 mg, 0.56%). A large quantity of substrates was detected in
the mycelium rather than the culture liquid. About 230 mg of sub-
strates were obtained from the mycelium of P. janthinellum AS
3.510. All the products were identified based on their spectral data.
The 1H and 13C NMR spectral data are given in Tables 1 and 2.
2.6.7. 6,7-Furano-8-(2a-hydroxy-3a-en-prenyloxy)hydrocoumaric
acid (8)
22
Yellow powder, m.p. 221–222 °C, [
a]
ꢀ12.5 (c = 0.1, MeOH);
D
UV kmax (MeOH): 209, 253 and 286 nm. 1H NMR (DMSO-d6,
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) see Tables 1 and 2.
HRESIMS [MꢀH]ꢀ m/z 305.1028 (calcd. for C16H18O6, 305.1025).
2.6.8. 6,7-Furano-5-isopentylcoumarin 8-O-b- -glucoside (10)
D
22
Yellow powder, [
a]
+35.5 (c = 0.1, MeOH); UV kmax (MeOH):
D
222, 252 and 311 nm. 1H NMR (DMSO-d6, 600 MHz) and 13C NMR
(DMSO-d6, 150 MHz) see Tables 1 and 2. HRESIMS [MꢀH]ꢀ m/z
455.1314 (calcd. for C22H25O9, 455.1318).
2.7. Analysis methods
The samples were analysed on an Ultimate 3000 HPLC equipped
with a Dionex C-18 column (inner diameter: 4.6 mm, length:
250 mm, and particle size: 5 lm), and DAD at 285 nm. The eluting
procedure was performed as follows: initial elution for 0–5 min
with MeOH:H2O (40:60, v/v), linear change from MeOH:H2O
(40:60, v/v) to (55:45, v/v) for 5–25 min, isocratic elution (55:45,
v/v) for next 25–35 min, and a linear change to MeOH:H2O
(90:10, v/v). The flow rate was 0.8 mL/min and column tempera-
ture was 30 °C.
2.6.1. 6,7-Furano-8-(2a,3a-dihydroxyprenyloxy)hydrocoumaric acid
(2)
2.8. Acid hydrolysis
22
Yellow powder, m.p. 215–217 °C, [
a
]
ꢀ18.7 (c = 0.2, MeOH);
D
UV kmax (MeOH): 210, 252 and 286 nm. 1H NMR (DMSO-d6,
600 MHz) and 13C NMR (DMSO-d6, 150 MHz) see Tables 1 and 2.
HRESIMS [MꢀH]ꢀ m/z 323.1135 (calcd. for C16H19O7, 323.1131).
Acid hydrolysis of the glycosides was carried out by refluxing
1.0 mg of glycoside 10 in 5 mL of 6% hydrochloric acid in MeOH
for 4 h. The reaction mixture was partitioned by EtOAc (20 ml).
The aqueous layer was evaporated and developed with CHCl3:-
MeOH:H2O (15:7:2, v/v/v). Sugars were detected using thymol in
H2SO4 (0.5 g thymol in 95 mL of EtOH and 5 mL of H2SO4) followed
by heating the plates at 120 °C for 10 min, and were identified as
sugar moieties by co-chromatography with reference samples.
2.6.2. 6,7-Furano-8-(2a,3a-dihydroxyprenyloxy)hydrocoumaric acid
methyl ester (3)
22
Yellow powder, m.p. 205–206 °C, [
a
]
ꢀ23.5 (c = 0.2, MeOH);
D
UV kmax (MeOH): 210, 252 and 286 nm. 1H NMR (DMSO-d6,

X. Lv et al. / Food Chemistry 138 (2013) 2260–2266
2263
Table 1
1H NMR spectral data for IMP and 1–10 (DMSO-d6, 600 MHz, d in ppm, J in Hz).
H
Imperatorin
1
2
3
4
5
6
7
8
9
10
3
4
5
8
6.32d(9.5)
7.71d(9.5)
7.31s
6.41d(9.5) 2.51 m
8.12 d(9.5) 2.84t(7.5,8.0) 2.87t(7.5)
2.60t(8.0,7.5) 2.51 m
2.49 m
2.58t(8.0,7.5) 2.50 m
2.52 m
2.83t (7.5)
7.04s
6.31d (10.0) 6.43d (10.0)
8.19d (10.0) 8.20 d (10.0)
2.88 m
7.10s
–
2.82t(7.5) 2.85t(8.0,7.5) 2.84 m
7.01s
–
7.06s
–
7.03s
–
7.03s
–
7.00s
–
7.01s
–
-
-
–
–
7.33s
–
–
1a
4.37d(7.0)
4.63d(9.5) 4.58d(7.5)
4.36t(9.0) 3.92t(8.5)
3.66d(7.0) 3.62d(6.5)
4.58d(7.0)
3.92t(8.5)
3.62 m
4.23 m
3.84 m
3.73 m
–
4.79d(6.5) 4.79d(6.5)
4.21 m
4.30 m
3.98t(7.5,2.5)
3.77d(7.0)
2a
3a
4a
5a
20
5.58t(7.0)
–
1.74s
5.77t(1.5) 5.76t(5.0,1.5) 1.86 m,1.55 m 4.33 m
–
–
5.13d
–
–
–
–
2.59 m
0.89(3H,s)
3.30 m
7.76s
–
–
–
1.07s
1.14s
8.10s
7.63s
–
1.05(3H,s)
1.15(3H,s)
7.78d(1.5)
6.77d(2.0)
–
1.05(3H,s)
1.15(3H,s)
7.78d(2.0)
6.78d(2.0)
3.57(3H,s)
1.13(3H,s) 1.56(3H,s) 1.55(3H,s)
1.40(3H,s) 3.80s 3.80s
7.84d(2.0) 7.77d(2.0) 7.78d(2.0)
6.77d(2.0) 6.76d(2.0) 6.76d(2.5)
1.72 (3H,s)
5.50s; 4.88s
7.77d(2.0)
6.77d(2.0)
–
–
–
1.81(3H,s)
1.64 (3H,s)
8.07 d(2.0)
7.14 d(2.5)
–
1.74s
7.64d(2.0)
6.77d(2.0)
–
8.02d(2.5)
7.39d(2.0)
4.26(3H,s)
30
6.76s
–
–
3.57(3H,s)
–
–OCH3
Glc100
Glc200
Glc300
Glc400
Glc500
Glc600
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.54d(7.5)
3.35 m
3.20 m
3.20 m
3.30 m
3.52 m
3.38 m
Table 2
13C NMR spectral data for IMP and 1–10 (DMSO-d6, 150 MHz, d in ppm, J in Hz).
C
Imperatorin
1
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
9
10
1a
2a
3a
4a
5a
20
30
160.2
114.2
144.3
113.2
125.7
148.3
131.2
143.5
116.2
69.8
119.7
139.2
17.8
25.5
146.4
106.6
159.8
114.1
145.2
113.6
125.8
147.7
131.6
142.5
116.4
75.4
76.7
70.8
24.5
27.2
147.1
107.0
174.0
33.9
25.8
114.6
120.2
145.3
131.4
144.1
124.5
75.1
75.9
70.6
24.2
27.6
144.3
106.7
–
172.8
33.6
25.8
114.7
120.3
145.3
131.4
144.1
124.1
75.1
75.8
70.5
24.2
27.6
144.3
106.7
51.2
173.8
34.2
26.1
114.2
123.5
144.7
136.9
142.8
130.3
71.4
74.1
80.8
19.8
26.7
145.6
106.7
174.1
34.0
26.0
172.9
33.6
25.9
174.0
33.9
26.0
114.4
120.3
145.4
131.4
144.1
125.0
71.1
32.9
33.2
16.9
66.1
144.2
106.6
–
174.1
34.1
25.9
114.8
120.3
145.2
131.2
144.1
124.8
76.3
72.7
144.3
18.6
111.8
144.3
106.7
–
160.0
159.5
112.2
139.4
149.4
112.4
157.7
93.1
152.1
105.6
–
–
–
–
–
113.8
141.9
113.6
125.4
145.9
127.1
142.8
125.9
27.3
114.5
120.2
145.6
131.2
144.4
125.0
68.6
118.9
140.3
13.5
114.5
120.3
145.6
131.2
144.4
124.6
68.6
118.8
140.3
13.5
125.5
131.9
17.9
65.5
65.5
25.3
144.2
106.6
–
144.3
106.6
51.1
145.8
105.6
60.2
147.2
105.9
–
–OCH3
Glc100
Glc200
Glc300
Glc400
Glc500
Glc600
101.9
73.8
77.5
69.6
76.7
60.5
2.9. In vitro anti-osteoporosis activity
3. Results and discussion
Mouse osteoblastic cell line (MC3T3-E1, purchased from the
Chinese Academy of Sciences Cell Bank) was cultured under a
humidified atmosphere of 5% CO2 at 37 °C with Dulbecco’s modi-
fied Eagle’s medium (DMEM) containing 1% penicillin streptomy-
cin (Gibco) and 10% foetal bovine serum. The medium was
refreshed every 2 days. After reaching confluence, the cells were
subcultured using 0.02% EDTA–0.05% trypsin solution. Cell prolifer-
ation was determined by an assay using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT). The cells pre-cultured
in growth media for 24 h at 5% CO2, 37 °C, were treated with differ-
In the present study, IMP was added to the 36-h precultures of P.
janthinellum AS 3.510 followed by a further incubation of 5 days. A
blank culture control and substrate control were used as described
above. A total amount of 800 mg of substrate was used for the pre-
parative-scale biotransformation. Ten products were isolated by
the preparative chromatographic methods. Based on the 1H NMR,
13C NMR and 2D-NMR spectra, their chemical structures were
elucidated as 2a,3a-dihydroxyimperatorin (1), 6,7-furano-8-(2a,
3a-dihydroxyprenyloxy)hydrocoumaric acid (2), 6,7-furano-8-
(2a,3a-dihydroxyprenyloxy)hydrocoumaric acid methyl ester (3),
6,7-furano-8-(2a-hydroxy-3a,9-epoxyprenyloxy)hydrocoumaric
acid (4), 6,7-furano-8-(4a-hydroxymethylprenyloxy)hydrocoumaric
acid (5), 6,7-furano-8- (4a-hydroxymethylprenyloxy)hydrocouma-
ric acid methyl ester (6), 6,7-furano-8- (4a-hydroxymethyl-2a,
3a-dihydroprenyloxy)hydrocoumaric acid (7), 6,7-furano-8- (2a-
hydroxy-3a-en-prenyloxy)hydrocoumaric acid (8), bergapten (9),
ent concentrations (1, 10 and 100 lM) of the target compounds for
24 h. Then, MTT (5 mg/mL) was added to the cell cultures, and the
samples were incubated for 4 h at 37 °C. In addition, the cells in
normal culture medium were treated with the same volume of
DMSO, similar to the test concentration of purified metabolites
(1–10) as blank. Finally, the absorbance was measured in an optical
96-well microplate reader at a wavelength of 570 nm (Tai et al.,
2010). Icariside was used as the positive control.
and 6,7-furano- 5-isopentylcoumarin 8-O-b-
D-glucoside (10).
Among them, eight metabolites (2–8 and 10) were novel (Fig. 1).

2264
X. Lv et al. / Food Chemistry 138 (2013) 2260–2266
Compounds 1 and 9 were identified as 2a,3a-dihydroxyimpera-
additional methoxyl group at d 51.1. In the HMBC spectrum, the
proton signal of d 3.57 was found to be correlated with C-2
(d 172.9), suggesting that a methoxyl group was located at C-2.
Hence, compound 6 was identified as 6,7-furano-8-(5a-hydroxy-
methyl-prenyloxy) hydrocoumaric acid methyl ester.
Compound 7 was obtained as yellow powder (MeOH). HRMS
data suggested a molecular formula of C16H20O6. When compared
with 5, the 13C NMR spectrum of 7 showed the absence of olefinic
carbon signals of d 118.9 and d 140.3, suggesting the reduction of
the side chain double bond. In the HMBC spectrum, H-5a had
long-range correlations with C-3a (d 33.2) and C-4a (d 66.1). These
evidences implied that the double bond of C-2a and C-3a was re-
duced. Consequently, compound 7 was identified as 6,7-furano-
torin and bergapten, respectively. Their 1H NMR and 13C NMR spec-
tral data were in agreement with those reported in the literature
(Harkar, Razdan, & Waight, 1984; Liu, Feng, Sun, & Kong, 2004).
Compound 2 was obtained as yellow powder (MeOH). Its
molecular formula of C16H20O7 was established from high-resolu-
tion electrospray ionisation mass spectrometry (HRESIMS); a qua-
si-molecular ion [M–H]ꢀ was observed at m/z 323.1135. Compared
with IMP, 13C NMR spectrum of 2 exhibited the disappearance of
olefinic carbons at d 144.3 (C-4), d 114.2 (C-3), d 119.7 (C-2a),
and d 139.2 (C-3a), while the additional oxygenated methylenes
at d 75.9 and d 75.1, and the aliphatic carbon signals of d 25.8
and d 33.9 were observed. It was also found that C-2 (d 174.0)
and C-10 (d 124.6) shifted downfield
D
+13.8 ppm and
D
8-(4a-hydroxymethyl-3a,4a-dihydroprenyloxy)
acid.
hydrocoumaric
+8.3 ppm. All these evidences implied hydroxylation of the double
bond, and hydrolysis of the lactone ring. In HMBC spectrum, CH3-
4a (d 1.05) had long-range correlations with C-5a (d 27.6), C-3a
(d 70.6) and C-2a (d 75.9), and H-1a (d 3.92) had HMBC correlations
with C-2a (d 75.9) and C-8 (d 131.4). This indicated that two hydro-
xyl groups were located at C-2a and C-3a, respectively. In addition,
the HMBC cross-peaks of H-3 (d 2.51) with C-10 (d 124.5) and C-4
(d 25.8) were observed, and H-4 (d 2.84) had HMBC correlations
with C-3 (d 33.9), C-5 (d 114.6), C-9 (d 144.1) and C-10 (d 124.5),
indicating the reduction of C-3,4 double bond and the hydrolysis
of a lactone ring. The proton signal of d 8.78 (OH) had HMBC cor-
relations with C-10 (d 124.5), C-8 (d 131.4) and C-9 (d 144.1), sug-
gesting a hydroxyl group was located at C-9. Based on the above
Compound 8 was obtained as yellow powder (MeOH). HRMS
suggested a molecular formula of C16H18O6. Similarly to IMP, the
oxo group of C-2 (d 160.2) shifted downfield to d 174.1 in the 13C
NMR spectrum. An additional carbon signal of d 72.7 was observed.
In the HMBC spectrum, H-4 (d 2.83) had long-range correlations
with C-3 (d 34.1), C-5 (d 114.8), C-9 (d 144.1), C-10 (d 124.8), and
C-2 (d 174.1). These observations suggested that the lactone ring
was hydrolysed. In addition, H-4a (d 1.72) showed HMBC correla-
tions with C-2a (d 72.7), C-3a (d 111.8) and C-5a (d 144.3), indicat-
ing rearrangement of the double bond at C-3a and C-5a. In
addition, H-5a (d 5.50 and d 4.88) had HMBC correlations with
C-4a (d 18.6) and C-2a (d 72.7), while H-1a (d 3.98) was correlated
with C-2a (d 72.7) and C-8 (d 131.2). All this evidence suggested
that the hydroxyl group was located at C-2a. Therefore, compound
8 was identified as 6,7-furano-8-(2a-hydroxy-3a-en-prenyloxy)
hydrocoumaric acid.
analysis, compound
2 was identified as 6,7-furano-8-(2a,3a-
dihydroxyprenyloxy)hydrocoumaric acid. All the 1H and 13C NMR
spectral data were unequivocally assigned by 2D NMR spectra
(see Tables 1 and 2).
Compound 3 was isolated as a yellow powder (MeOH). Its
molecular formula was determined as C17H22O7 by HRMS. Com-
pared to metabolite 2, only an additional carbon signal of methoxyl
group at d 51.2 was observed in 13C NMR spectrum of 3. In the
HMBC experiment, this methoxyl signal of d 3.57 showed long-
range correlations with C-2 (d 172.8), suggesting that a methoxyl
group was substituted at C-2. Therefore, compound 3 was identi-
fied as 6,7-furano-8(2a,3a-dihydroxyprenyloxy)hydrocoumaric
acid methyl ester. All the 1H and 13C NMR spectral data were
unambiguously assigned by 2D NMR spectra (see Tables 1 and 2).
Compound 4 was isolated as yellow powder (MeOH). HRMS
suggested a molecular formula of C16H18O6. Compared to 2, the
characteristic proton signal of d 8.78 disappeared in 1H NMR spec-
trum of 4. Meantime, the 13C NMR spectrum of 4 exhibited the oxy-
gen-bearing carbon signals of d 80.8 and d 74.1. In the HMBC
spectrum, correlations of the proton signal of d 5.57 with C-1a
(d 71.4), C-2a (d 74.1) and C-3a (d 80.8) were observed. These data
indicated the dehydration of two hydroxyl groups at C-9 and C-3a
to form a new six-membered ring. On the basis of the above
analysis, compound 4 was identified as 6,7-furano-8-(2a-hydro-
xy-3a,9-epoxyprenyloxy)hydrocoumaric acid.
Compound 5 was obtained as yellow powder (MeOH). HRMS
suggested a molecular formula of C16H18O6. When compared with
2, additional carbon signals of d 118.9 and d 140.3, and only one
oxygen-bearing carbon of d 65.5 were observed. A methyl carbon
signal at d 25.5 disappeared in the 13C NMR spectrum of 5. In HMBC
spectrum, H-4a (d 3.80) was correlated with C-2a (d 118.9), C-3a
(d 140.3) and C-5a (d 13.5), while H-1a (d 4.79) showed HMBC
correlations with C-8 (d 131.2), C-3a (d 140.3) and C-2a (d 118.9).
These findings suggested that a hydroxyl group was substituted
at C-4a. Thus compound 5 was identified as 6,7-furano-8-(5a-hy-
droxyl-methyl-prenyloxy)hydrocoumaric acid.
Compound 10 was isolated as yellow powder (MeOH), with a
suggested molecular formula of C22H25O9. In the 13C NMR spec-
trum the additional carbon signals of d 60.5, d 69.6, d 73.8, d
76.7, d 77.5 and d 101.9 indicated that a sugar moiety was intro-
duced in the chemical structure of 10. The sugar moiety was iden-
tified as D-glucose by chemical hydrolysis. The anomeric proton
coupling constant of 7.5 Hz and anomeric carbon signal of d
101.9 (C-1) suggested that the configuration of glucopyranosyl
should be b. In the HMBC spectrum, the proton signal of d 5.54
was observed to be correlated with C-8 (d 127.1), indicating that
the glucopyranosyl moiety should be at C-8. In the HMBC spec-
trum, H-4a (d 1.81) was found to be correlated with C-5a
(d 25.3), C-2a (d 125.5) and C-3a (d 131.9), while H-1a (d 3.77)
had HMBC correlations with C-5 (d 113.6), C-6 (d 125.4), C-2a
(d 125.5), C-3a (d 131.9) and C-10 (d 125.9). The findings suggested
that an isopentane group was located at C-5. On the basis of above
analysis, the chemical structure of compound 10 was identified as
6,7-furano-5-isopentyl-coumarin 8-O-b-D
-glucoside. All the 1H and
13C NMR spectral data were assigned by 2D NMR spectra (see
Tables 1 and 2).
A previous report showed that Glomerello cingulata could
metabolise some coumarins such as isopimpinellin, bergapten,
xanthotoxin and isoimperatorin, to the corresponding reduced acid
(Marumoto and Miyazawa, 2010b, 2010c, 2011a, 2011b), while
IMP, as a specific coumarin with a prenyloxy side-chain, could be
transformed by G. cingulata to yield the dealkylated metabolite
xanthotoxol in high yield (81%), rather than the corresponding re-
duced acid derivatives. In addition, IMP could also be metabolised
by Aspergillus flavus to form products specifically oxidised at C-4a
and C-5a positions with xanthotoxol as a minor metabolite, pro-
ducing cleavage at the prenyloxy side-chain (Teng, Huang, Huang,
Chung, & Chen, 2004).
Compound 6 was isolated as yellow powder (MeOH). HRMS
suggested a molecular formula of C17H20O6. The spectral data of
6 were found to be very similar to those of 5, except for an
In our present work, P. janthinellum exhibited a great capability
to transform IMP into a series of novel transformed products, most
of which were the corresponding derivatives of hydrocoumaric

X. Lv et al. / Food Chemistry 138 (2013) 2260–2266
2265
at a,b-unsaturated lactone ring, hydroxylation, methylation, dehy-
dration and glycosylation, which were observed for the first time in
microbial transformation processes of IMP. Some novel metabo-
lites (5 and 8) showed potent bioactivity in increasing MC3T3-E1
cell growth. Hence, these metabolites could be used as potent can-
didates for the further development of anti-osteoporosis drugs or
functional foods.
Acknowledgements
We thank the National Natural Science Foundation of China
(No. 81274047), Personnel Department of Liaoning province and
the Program for Liaoning Excellent Talents (LJQ2011095) in the
University, the Education Department of Liaoning Province
(LS2010059) for funding towards this research.
Fig. 2. HPLC chromatograms of the blank of P. janthinellum AS 3.510 (A) and
administrating IMP for 5 days (B). All UV spectra were similar to that of metabolite
1.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.foodchem.
2012.11.138.
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