Biotransformation of oleanolic acid by Alternaria longipes and Penicillium adametzi

Article abstract of DOI:10.1080/10286020.2010.547028

Microbial transformation of oleanolic acid (1) was carried out. Six transformed products (2-7) from 1 by Alternaria longipes and three transformed products (8-10) from 1 by Penicillium adametzi were isolated. Their structures were elucidated as 2,3,19-trihydroxy-ursolic acid-28-O - d-glucopyranoside (2), 2,3,19-trihydroxy-ursolic acid-28-O - d-glucopyranoside (3), oleanolic acid 28-O - d-glucopyranosyl ester (4), oleanolic acid-3-O - d-glucopyranoside (5), 3-O-(-d-glucopyranosyl)-oleanolic acid-28-O - d-glucopyranoside (6), 2,3,19a-trihydroxy-oleanolic acid-28-O - d-glucopyranoside (7), 21-hydroxyl oleanolic acid-28-O - d-glucopyranoside (8), 21-hydroxyl oleanolic acid (9), and 7,21-dihydroxyl oleanolic acid (10) based on the extensive NMR studies. Among them, 10 was a new compound and compounds 5 and 8-10 had stronger cytotoxic activities against Hela cell lines than the substrate. At the same time, it was reported for the first time in this paper that the skeletons of compounds 2 and 3 were changed from oleanane to uranane and seven glycosidation products were obtained by biotransformation.

Full text of DOI:10.1080/10286020.2010.547028

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Biotransformation of oleanolic acid
by Alternaria longipes and Penicillium
adametzi
Dai-Lin Liu a b , Ying Liu b , Feng Qiu c , Ying Gao b & Jing-Ze
Zhang b
a Tianjin Key Laboratory for Biomarkers of Occupational and
Environmental Hazard, Tianjin, 300162, China
b Department of Pharmacognosy, Medical College of Chinese
People's Armed Police Forces, Tianjin, 300162, China
c Department of Natural Products Chemistry, Shenyang
Pharmaceutical University, Shenyang, 110016, China
Version of record first published: 28 Jan 2011.
To cite this article: Dai-Lin Liu, Ying Liu, Feng Qiu, Ying Gao & Jing-Ze Zhang (2011):
Biotransformation of oleanolic acid by Alternaria longipes and Penicillium adametzi , Journal of
Asian Natural Products Research, 13:02, 160-167
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Journal of Asian Natural Products Research
Vol. 13, No. 2, February 2011, 160–167
Biotransformation of oleanolic acid by Alternaria longipes and
Penicillium adametzi
Dai-Lin Liuab*, Ying Liub, Feng Qiuc, Ying Gaob and Jing-Ze Zhangb
aTianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard, Tianjin
300162, China; Department of Pharmacognosy, Medical College of Chinese People’s Armed
b
c
Police Forces, Tianjin 300162, China; Department of Natural Products Chemistry, Shenyang
Pharmaceutical University, Shenyang 110016, China
(Received 10 September 2010; final version received 7 November 2010)
Microbial transformation of oleanolic acid (1) was carried out. Six transformed products
(2–7) from 1 by Alternaria longipes and three transformed products (8–10) from 1 by
Penicillium adametzi were isolated. Their structures were elucidated as 2a,3a,19a-
trihydroxy-ursolic acid-28-O-b-D-glucopyranoside (2), 2a,3b,19a-trihydroxy-ursolic
acid-28-O-b-D-glucopyranoside (3), oleanolic acid 28-O-b-D-glucopyranosyl ester (4),
oleanolic acid-3-O-b-D-glucopyranoside (5), 3-O-(b-D-glucopyranosyl)-oleanolic
acid-28-O-b-D-glucopyranoside (6), 2a,3b,19a-trihydroxy-oleanolic acid-28-O-b-D-
glucopyranoside (7), 21b-hydroxyl oleanolic acid-28-O-b-D-glucopyranoside (8), 21b-
hydroxyl oleanolic acid (9), and 7a,21b-dihydroxyl oleanolic acid (10) based on the
extensive NMR studies. Among them, 10 was a new compound and compounds 5 and 8–
10 had stronger cytotoxic activities against Hela cell lines than the substrate. At the same
time, it was reported for the first time in this paper that the skeletons of compounds 2 and
3 were changed from oleanane to uranane and seven glycosidation products were
obtained by biotransformation.
Keywords: biotransformation; oleanolic acid; Alternaria longipes; Penicillium adametzi
1. Introduction
Microbial transformation is defined as
an enzymatic reaction by micro-organisms
with metabolic activities to modify the
structures of bioactive substrates. It has
such advantages as high stereo- or region-
selectivities, as well as mild reaction
conditions over chemical synthesis [5].
Some reactions fulfilled with difficulty in
the process of chemical synthesis are facile
by microbial transformation. Microbial
transformation is a useful technique to
modify the structures of biologically active
compounds. But there have been a few
reports on biotransformation of oleanolic
acid in recent years [6–9]. In our previous
study, we had screened the biotransform-
ation of oleanolic acid by 25 kinds of fungi
Oleanolic acid, a natural pentacyclic
triterpenoid found in many plant species,
has attracted attention due to its important
pharmacological properties, such as anti-
inflammation, antitumor, and antidiabetes
activities [1,2]. In recent years, to find safe,
effective, stable, and an innovative new
drug target molecules or lead compounds,
the structural modification of oleanolic
acid and the structure–activity relationship
of its derivatives were reported [3,4], but
the reaction sites were mainly at C-3 and C-
28, and the reaction procedures including
hydroxylation, oxidation, and acetylation
focused on the introduction of heteroatom
into structure were reviewed.
*Corresponding author. Email: liudailinlch@hotmail.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2010.547028
http://www.informaworld.com

Journal of Asian Natural Products Research
161
and determined the transformation effi-
ciency [10].
powder and its ESI-MS analysis gave a
1
molecular formula of C36H58O10. The H
In this paper, the biotransformation of
oleanolic acid (1) by Alternaria longipes
and Penicillium adametzi was performed
to find out new chemical entities and with
the aim of improving its activities and
solubility. Nine products were obtained,
and their structures were elucidated based
on the extensive spectral data, including
2D NMR. Among them, compound 10 was
new and compounds 5 and 8–10 had
stronger cytotoxic activities in vitro
against Hela cell lines than the substrate.
NMR spectra showed six quaternary
methyl groups and one tertiary methyl
group at d 1.05 (3H, d, H-29), which
indicated that the skeleton of mother
nucleus was changed from oleanane to
ursane. Compared with the literature
[14], the structure of compound 2 was
determined as 2a,3a,19a-trihydroxy-
ursolic acid-28-Ob-D-glucopyranoside.
13C NMR spectral data of compound 3 are
similar to those of compound 2 except that
an oxygenated signal at d 83.8 (CH, by
DEPT) was present instead of a signal at d
79.3 due to C-3. On the same analysis
methods with compound 2, the structure of
compound 3, the other skeleton-rearranged
metabolite, was determined as 2a,3b,19a-
trihydroxyl ursolic acid-28-O-b-D-gluco-
pyranoside.
2. Results and discussion
The structures of unknown transformed
products were elucidated on the basis of
1
their H, 13C NMR, HSQC, and HMBC
spectral data and the known compounds
were identified by comparison of their
NMR spectral data with those reported in
literature [11–15]. Incubation of oleanolic
acid (1) with A. longipes for 7 days yielded
six compounds (2–7) and using the same
methods of microbiologic culture, three
transformed products (8–10) were obtained
from P. adametzi. Their structures were con
-firmed as 2a,3a,19a-trihydroxy-ursolic
acid-28-O-b-D-glucopyranoside (2), 2a,3b,
19a-trihydroxy-ursolic acid-28-O-b-D-glu-
copyranoside (3), oleanolic acid 28-O-b-D-
glucopyranosyl ester (4), oleanolic acid-3-
O-b-D-glucopyranoside (5), 3-O-(b-D-glu-
copyranosyl)-oleanolic acid-28-O-b-D-glu-
copyranoside (6), 2a,3b,19a-trihydroxy-
oleanolic acid-28-O-b-D-glucopyranoside
(7), 21b-hydroxyl oleanolic acid-28-O-b-
D-glucopyranoside (8), 21b-hydroxyl olea-
nolic acid (9), and 7a,21b-dihydroxyl
oleanolic acid (10). Compound 10 was an
unreported product. Skeleton-rearranged
metabolites, compounds 2 and 3, were
obtained from this biotransformation invol-
ving a methyl migration, whose skeletons
were changed from oleanane to ursane.
As one skeleton-rearranged metabolite,
compound 2 was obtained as a white
Compound 10 was previously unre-
ported and was obtained as a white power.
17
It was optically active, with ½aꢀD þ47.3
(c ¼ 0.15, MeOH). The molecular formula
C30H48O5 was established by analysis of
positive HR-EI-MS, which gave a quasi-
molecular ion peak at m/z 511.3297
[M þ Na]þ. The 13C NMR spectrum
displayed resonances for 30 carbons,
which were assigned as seven methyls,
eight methylenes, seven methines, and
eight quaternary carbons by analysis of
DEPT spectrum. The HMQC spectrum
showed two new cross-peaks between the
proton signals at d 3.99 and 4.26 with the
carbon signals at d 72.5 and 72.9 in
comparison with the parent compound,
indicating that compound 10 contained two
oxygenated atoms compared with 1. In the
HMBC spectrum, H-29 and H-30 exhibited
cross-peaks with C-20 (d 36.8), C-19 (d
47.6), and C-21 (d 72.5), and H-26 (d 1.33)
exhibited cross-peak with C-7 (d 72.9). In
the 1H–1H COSY spectrum, the signal at d
3.99 exhibited cross-peaks with H-22 (d
2.36) and the signal at d 4.26 exhibited
cross-peaks with H-6 (d 1.82), indicating
that the two oxygenated carbons at d 72.5

162
D.-L. Liu et al.
and 72.9 were attributed to C-21 and C-7,
respectively. Furthermore, the NOESY
spectrum also showed cross-peaks of H-7
with H-27, and H-21 with H-29. Accord-
ingly, the configuration of hydroxyl group
was determined to be b. On the basis of the
above evidence, the structure of 10 was
determined to be 7a,21b-dihydroxyl olea-
nolic acid.
(Figures 1 and 2), the pathways shown in
Figures 3 and 4 were proposed for the
biotransformation of oleanolic acid cul-
tured with A. longipes and P. adametzi,
respectively. The glycoside products,
compounds 4 and 5 were detected at 24 h
of incubation, while other products 2, 3, 6,
and 7 were obtained at 48 h of incubation,
which were shown in Figure 1. Biogeneti-
cally, the oleanane and ursane skeletons
share a common intermediate, and the
Based on the time course in the
biotransformation of oleanolic acid (1)
Figure 1. Time course of the biotransformation of oleanolic acid (1) by A. longipes.
Note: aYield is expressed as a percentage relative to the total amount of reaction products on a molar
basis.
Figure 2. Time course of the biotransformation of oleanolic acid (1) by P. adametzi.
Note: aYield is expressed as a percentage relative to the total amount of reaction products on a molar
basis.

Journal of Asian Natural Products Research
163
HO
C
C
COOH
O
HO
HO
O
O
OH
O
OH
O
O
OH
OH
HO
HO
OH
OH
OH
OH
4
1
7
HO
C
C
OH
C
O
O
O
O
HO
HO
O
OH
O
O
O
OH
O
OH
OH
OH
OH
OH
OH
OH
O
O
OH
OH
OH
5
6
3
OH
OH
OH
OH
HO
C
O
O
HO
HO
OH
O
OH
OH
OH
2
Figure 3. Possible biotransformation pathways of oleanolic acid (1) by A. longipes.
OH
C
O
OH
HO
OH
10
OH
OH
COOH
C
O
HO
OH
O
HO
1
9
C
O
OH
O
HO
OH
OH
8
OH
Figure 4. Possible biotransformation pathways of oleanolic acid (1) by P. adametzi.
ursane nucleus is biosynthesized from an
oleanane precursor via a methyl migration
from C-20 to C-19 [16]. To investigate the
mechanism of this biotransformation and
to study the biotransformation pathway, a
conversion experiment was performed on
utilizing compound 7 as substrate. Com-
pound 7 was found to be converted to
compound 3 when incubated with A.
longipes under the same conditions for
2 days. On the other hand, no appreciable
reaction could be detected for compound 2
after extended incubation for 4 days. This
implies that oleanolic acid 1 is converted

164
D.-L. Liu et al.
stepwise to the final product compound 3
via one intermediate, compound 7, which
is further converted to compound 2
through extended incubation (Figures 3
and 4). Otherwise, it was reported for the
first time in this paper that the skeletons of
compounds 2 and 3 were changed from
oleanane to uranane by biotransformation
approach, which was formed by the
participation of ‘retro-biosynthetic’ methyl
migration from C-20 to C-19.
experiment indicated that hydroxylation
at C-21 (9), C-7 (10), and C-3 (5)
glycosylation could increase the cytotoxi-
cities, but the glycosylation at C-28 (8)
would significantly reduce the activities.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a digital
polarimeter in methanol on a Jasco P-1020
digital polarimeter (model AUTOPOL,
Japan). The mass spectra were recorded
on an ion trap mass spectrometer. The IR
spectrum was obtained on an Avatar 360
At the same time, seven glycosidation
products are reported in this paper. Glyco-
sylationisacharacteristicbiotransformation
reaction in plant cells, because glycosyl-
transferases are widespread in plants [17].
But glycosidation is not common in the
microbial transformation in aquatic con-
ditions. Recently, the synthesis mechanism
of oligosaccharides was reported by a
chemoenzymatic method using cyclodex-
trin glucanotransferase [18]. Microbial
transformation, which is also an enzymatic
reaction with good region- and stereo-
selectivity, can overcome the limitation of
chemical synthesis and satisfy the demands.
So, the capacity of A. longipes and P.
adametzi for the glycosylation of oleanolic
acid should be useful for the preparation of
even more highly water-soluble oleanolic
glycosides. This procedure overcomes the
time-consuming protection and deprotec-
tion steps necessary in chemical synthesis.
Thus, the ability and regioselectivity of
A. longipes and P. adametzi for the
glycosylation and methyl migration of
oleanolic acid are elucidated for the first
time in this paper.
1
FT-IR spectrophotometer. The H NMR
spectra were recorded on a Bruker ARX-
1
400 spectrometer (400 MHz for H NMR
and 100 MHz for 13C NMR) in pyridine-d5
as solvent. The HR-ESI-MS were recorded
on a Jeol HX 110 mass spectrometer.
Chromatography was performed with
Diaion HP20 (Mitsubishi Chemical Cor-
poration, Tokyo, Japan), silica gel (200–
300 mesh, Qingdao Haiyang Chemical
Corporation, Qingdao, China). Preparative
HPLC was performed in Thermo Scientific
Spectra SYSTEM P1000 LC (Thermo
Fisher Scientific, Waltham, MA, USA).
Pump type: Spectra SYSTEM P1000,
detector: Spectra SERIES UV100, col-
umn: RP-18 (5 mm, 250 £ 10 mm) (YMC-
Pack ODA-A, YMC Company Ltd.,
Kyoto, Japan, flow rate: 4.5 ml/min,
elution with MeOH–H2O). TLC plates
(silica gel) were used to check the purity of
compounds and all spots were detected by
spraying with 10% H2SO4, followed by
heating. All reagents used were of
analytical grades. Oleanolic acid (1) was
supplied by JF-natural (Tianjin Jianfeng
Natural Product R&D Ltd. Co., Tianjin,
China). The purity of 1 was above 98%
determined by HPLC.
The MTT bioassay showed that all the
transformed products had cytotoxicities
against Hela (human cervical carcinoma)
cells. Compounds 5 and 8–10 had
significant inhibitory effects against Hela
cells with IC50 values of 7.4, 25.0, 7.6, and
0.78 mM, and the IC50 values of substrate
and other products were all greater than
25.0 mM, respectively. Compounds 5 and
8–10 had more potent inhibitory effects
than the substrate 1. The pharmaco-
3.2 Fungi
A. longipes AS 3.2875 and P. adametzi AS
3.447 were purchased from the China
General Microbiological Culture Collec-

Journal of Asian Natural Products Research
165
tion Center in Beijing, China. All cultures
were maintained on potato dextrose agar
slants and stored in a refrigerator at 58C
prior to use.
tion Q-4 (4.6 g) was separated by a silica
gel open column (F 35 cm £ 3.7 cm) with
CHCl3–MeOH gradient elution to yield
six subfractions (Fractions Q-41–Q-46).
Crystallization of Fraction Q-44 yielded
compound 4 (294 mg, 4.54% yield). The
purification of Fraction Q-45 (395 mg) on
PHPLC with MeOH–H2O (3:2) afforded
metabolites 2 (13 mg, 0.2% yield), 3 (15 mg,
0.23% yield), and 5 (20 mg, 0.31% yield).
Using the same method, Fractions Q-46
(384 mg) were purified by PHPLC
with MeOH–H2O (1:1) to furnish 6
(6 mg, 0.10% yield) and 7 (65 mg, 1.03%
yield).
3.3 Biotransformation and isolation
Stock cultures of fungi were stored on
potato dextrose agar slants at 58C. Seed
cultures of each fungus were obtained by
transferring fungi from stock cultures to a
liquid medium containing (g L21) potato
(20%) and glucose (2%). The experiments
were conducted in conical flasks (500 ml),
containing the same liquid medium
(200 ml) inoculated with the fungi. The
flasks were shaken at 288C and 160 rpm for
48 h. The substrates (oleanolic acid, 1),
previously dissolved in a minimum
volume (15 mg ml21) of dehydrated alco-
hol, were evenly distributed among the
flasks (15 mg per flask of substrate), except
one that was kept as a control, and the
reaction was allowed to proceed for 4 days.
The mycelium was then removed by
filtration, and the broth was dealt in an
ultrasonic bath for 30 min and extracted
with n-BuOH for three times (n-BuOH
was used in the same volume with broth
every time) and all extracts were combined
and dried. After filtration, the solvents
were evaporated under reduced pressure,
and the crude extract was fractionated by
chromatography. Controls were carried
out in order to verify the action of the
medium (without fungus) on the substrates
and the presence of similar metabolites on
the fungi cultures (without substrates).
3.3.2 Biotransformation of oleanolic
acid (1) by P. adametzi
The crude extract obtained from the
biotransformation of oleanolic acid (1)
(14.6 g) was chromatographed on Diaion
HP20 (F 45 cm £ 3.7 cm) eluted stepwise
by H2O, 30% EtOH, 50% EtOH, 70%
EtOH, and 95% EtOH to give five
corresponding fractions (Qi-1–Qi-5). Fr.
Qi-4 (1.8 g) was separated by a silica gel
open column (F 25 cm £ 2.8 cm) with
CHCl3–MeOH gradient elution to yield
six subfractions (Qi-41–Qi-46) and crys-
tallization of Fraction Qi-42 yielded
compound 9 (203 mg, 3.13% yield). Fr.
Qi-3 (1.7 g) was separated by an ODS open
column with MeOH–H2O gradient elution
to yield six subfractions (Qi-31–Qi-36).
The purification of Fraction Qi-33
(577 mg) on PHPLC with MeOH–H2O
(85:15) afforded metabolite 10 (185 mg,
2.90% yield). Using the same method,
Fractions Qi-36 (237 mg) was purified by
PHPLC with MeOH–H2O (7:3) to furnish
8 (13 mg, 0.20% yield).
3.3.1 Biotransformation of oleanolic
acid (1) by A. longipes
The crude extract obtained from the
biotransformation of oleanolic acid (1)
(17.4 g) was chromatographed on Diaion
HP20 (F 45 cm £ 3.7 cm) eluted stepwise
by H2O, 30% EtOH, 50% EtOH, 70%
EtOH, and 95% EtOH to give five
corresponding fractions (Q-1–Q-5). Frac-
3.3.3 7a,21b-dihydroxyl oleanolic acid
(10)
White powder; Liebermann–Burchard
17
reaction was positive; ½aꢀD 47.3 (c ¼ 0.15,
MeOH). IR (KBr) nmax: 3422, 3130, 2962,

166
D.-L. Liu et al.
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR spectral data of 10 in pyridine-d5 (d in ppm,
J in Hz).
No.
1H
13C
No.
1H
13C
1
2
3
4
5
6
7
8
1.62 m, 1.06 o
1.85 m
3.47 m
38.9 t
28.2 t
77.9 d
39.1 s
53.1 d
30.5 t
72.9 d
45.6 s
48.3 d
37.6 s
24.0 t
123.1 d
144.0 s
43.9 s
32.3 t
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.31 m, 2.22 m
25.7 t
48.8 s
42.6 d
47.6 t
36.8 s
72.5 d
41.8 t
28.6 q
16.6 q
15.6 q
10.8 q
26.0 q
179.4 s
29.8 q
17.7 q
3.55 m
2.15 m, 1.53 m
1.09 o
2.03 m, 1.82 m
4.26 m
3.99 dd, J ¼ 11.1, 5.2 Hz
2.36 m
1.23 s
1.03 s
0.98 s
1.33 s
1.52 s
9
1.75 m
2.06 m
5.67 t, J ¼ 3.3 Hz
10
11
12
13
14
15
1.29 s
1.30 s
2.70 td, J ¼ 14.5, 4.4 Hz, 2.19 m
Notes: Overlapped signals are indicated by ‘o’. The carbon and proton signals were unambiguously assigned
through the HMQC spectrum and HMBC spectrum.
2873, 1696, 1399, 1073, and 860 cm21. For
1H and 13C NMR spectral data see Table 1.
HR-ESI-MS m/z: 511.3357 [M þ Na]þ
(calcd for C30H48O5Na, 511.3399).
3.5 Bioassay
The cell line was maintained in an RPMI
1640 medium (GIBCO/BRL, MD, USA)
supplemented with 10% (v/v) of fetal
bovine serum, 100 IU/ml of penicillin and
100 mg/ml of streptomycin at 378C, and
5% CO2, and grown in 96-well microtiter
plates for the assay. All compounds were
dissolved in DMSO. After 48 h of incu-
bation, compounds underwent serial
dilution to give final concentrations of
1027–1024 mol/L. The cell growth was
evaluated by the MTT assay. The activities
were measured as IC50 values, which are a
concentration of the test compound (mM)
that inhibited cell growth by 50%.
3.4 Time course and HPLC analysis
Time course experiments were performed
by a procedure similar to normal trans-
formation experiments except that 5 mg/ml
of substrate was administered to each of
eight flasks (250 ml) containing 60 g of
liquid medium. At a regular time interval
(12 h), one of the flasks was tested to
evaluate the conversion yields of the
products by HPLC methods.
The HPLC analysis was carried out
using an analytical Luna ODS C18 column
(250 £ 4.6 mm i.d., 5 mm; Phenomenex
Inc., Guangzhou, China). The isocratic
mobile phase was MeOH–1% glacial
acetic acid water solution (90:10, v/v).
The mobile phase was filtered through
0.45 mm Millipore filters before use. The
injection volume was 20 ml and the flow
rate was 1.0 ml/min. Peaks were detected at
208 nm, which is the maximum absorbance
of these products. The system was operated
at room temperature (258C).
Acknowledgements
We thank the Foundation (YFJMJC08300) of
Tianjin Natural Science Foundation of China
for financial support.
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