Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger and the cytotoxicity of the resulting metabolites

Article abstract of DOI:10.1016/j.phytol.2014.11.006

The microbial transformation of 20(S)-protopanaxatriol by cell suspension cultures of Aspergillus niger AS 3.1858 yielded metabolites 1-13. The chemical structures of these transformed products were elucidated based on various spectroscopic analyses, incl

Full text of DOI:10.1016/j.phytol.2014.11.006

G Model
PHYTOL 825 1–4
Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
1
2
3
Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger
and the cytotoxicity of the resulting metabolites
Q1 Guangtong Chen a,1, Yan Song a,1, Hongjuan Ge a, Jie Ren b, Xue Yang a, Jianlin Li a,
*
4
5
6
a School of Pharmacy, Nantong University, Nantong 226001, China
b School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou 213164, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The microbial transformation of 20(S)-protopanaxatriol by cell suspension cultures of Aspergillus niger
AS 3.1858 yielded metabolites 1–13. The chemical structures of these transformed products were
elucidated based on various spectroscopic analyses, including 1D and 2D NMR and HRESIMS.
Metabolites 3, 11, and 13 are new compounds. Furthermore, metabolite 3 exhibited relative better
activity profile toward the tested seven cancer cell lines (Du-145, Hela, K562, K562/ADR, SH-SY5Y,
HepG2, and MCF-7) than substrate and preliminary structure-activity relationships were concluded.
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Received 4 September 2014
Received in revised form 14 November 2014
Accepted 17 November 2014
Available online xxx
Keywords:
20(S)-Protopanaxatriol
Microbial transformation
Aspergillus niger AS 3.1858
Cytotoxicity
7
8
1. Introduction
as stereo- or regioselectivity, mild reaction conditions, avoiding 29
complex protection, and deprotection steps over chemical synthesis 30
9
Ginseng, the root of Panax ginseng (Araliaceae), is a valuable
traditional medicine and functional food. The beneficial effects
of ginseng on diabetes, cardiovascular disease remedy as well as
prevention cancer are well documented (Shibata, 2001; Surh et al.,
2001). It is generally believed that ginsenosides and their
metabolites are mainly responsible for the pharmacological
activities of ginseng (Hsu et al., 2013; Jia et al., 2009). 20(S)-
Protopanaxatriol (PPT) is one of the aglycone of ginsenosides
such as ginsenoside Rg1, Re and Rf, and exhibits strong cytotoxic
activity against human leukemia THP-1 cells, Int-407 cells and
Caco-2 cells (Hasegawa et al., 2002; Popovichand Kitts, 2002, 2004).
Therefore, structural modification of 20(S)-protopanaxatriol may be
of valuable to obtain new chemical derivatives for pharmacological
screening. However, it seems very difficult to modify this compound
with dammarane skeleton by conventional chemical method.
Biotransformation is regarded as an effective and useful
technology in modification of natural products for finding the
new chemical derivatives with the potent bioactivities and different
physical–chemical characteristics (Bhatti and Khera, 2014; Muffler
et al., 2011; Parra et al., 2009). It exhibited many advantages such
(Deng et al., 2012; Li et al., 2011; Lv et al., 2013). In recent years, 31
our research group has frequently reported structural modifications 32
of natural functional products to obtain some new chemical entities 33
for improving the solubilities and biological activities (Chen et al., 34
2007, 2008, 2013a, 2013b, 2013c). In present work, biotransforma- 35
tion of 20(S)-protopanaxatriol by Aspergillus niger AS 3.1858 was 36
carried out. Thirteen metabolites, including three new transformed 37
products, were isolated and identified (Fig. 1). In addition, the 38
cytotoxicities of all transformed products were evaluated by 39
using Du-145, Hela, K562, K562/ADR, SH-SY5Y, HepG2, MCF-7, 40
and Vero cells and preliminary structure-activity relationships were 41
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
concluded.
42
2. Results and discussion
43
The biotransformation of 20(S)-protopanaxatriol (PPT) with A. 44
niger AS 3.1858 for five days yielded thirteen metabolites. Their 45
chemical structures were identified as 12-oxo-20(S)-protopanax- 46
atriol (1), 29-hydroxy-20(S)-protopanaxatriol (2), 24-methylene- 47
20(S)-protopanaxatriol (3), (20S,24R)-epoxy-dammaran-3
,25-tetrol (4), (20S,24S)-epoxy-dammaran-3 ,6 ,12 ,25-tetrol 49
(5), 12-oxo-23 -hydroxy -20(S)-protopanaxatriol (6), 12-oxo- 50
26-hydroxy-20(S)-protopanaxatriol (7), 12-oxo-27-hydroxy-20 51
(S)-protopanaxatriol (8), 12-oxo-15 -hydroxy-20(S)-protopanax- 52
atriol (9), 12-oxo-11 -hydroxy-20(S)-protopanaxatriol (10), 53
b,6a,12 48
b
b
a
b
b
*
Corresponding author at: School of Pharmacy, Nantong University, Qixiu Road
No. 19, Nantong 226001, China. Tel.: +86 513 85051749; fax: +86 513 85051749.
a
E-mail address: jianlinli1980@163.com (J. Li).
1
These two authors contributed equally to this work.
b
http://dx.doi.org/10.1016/j.phytol.2014.11.006
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Chen, G., et al., Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger and the cytotoxicity of
the resulting metabolites. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.11.006

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PHYTOL 825 1–4
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G. Chen et al. / Phytochemistry Letters xxx (2014) xxx–xxx
HO
HO
HO
OH
OH
O
HO
HO
HO
1
OH
OH
OH
3
2
OH
HO
HO
O
HO
O
OH
OH
O
HO
O
HO
21
17
HO
OH
12
18
O
HO
HO
HO
5
4
OH
23
OH
26
6
19
5
OH
1
14
30
27
9
3
7
HO
HO
HO
HO
HO
13
OH
OH
29
28
O
PPT
O
O
OH
HO
OH
OH
HO
HO
OH
9
OH
OH
8
7
HO
O
HO
HO
O
O
O
HO
HO
HO
HO
HO
OH
OH
12
OH
11
10
Fig. 1. Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger AS 3.1858.
54
55
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12-oxo-11
epoxy-3
24S)-epoxy-3
a
-hydroxy-20(S)- protopanaxatriol (11), (20S,24R)-
,25-trihydroxy-dammarane-12-one (12), and (20S,
,6 ,25-trihydroxy-dammarane-12-one (13).
was confirmed. In the HMBC spectrum, two methylene protons at
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
b
,6
a
dH 4.77 and 4.71 (H-10) correlated with the methane carbon at dC
b
a
34.2 (C-25), the methyl protons at dH 1.04 (H-26) and 1.05 (H-27)
Among them, 3, 11, and 13 were elucidated as new compounds
by mass spectroscopic analyses. The 1H and 13C NMR signals of all
new compounds were fully assigned based on their HSQC, HMBC,
correlated with the carbons at dC 156.8 (C-24) and 34.2 (C-25)
(Fig. 2). These evidences implied that the methylene group was at
C-24. Consequently, compound 3 was identified as 24-methylene-
20(S)-protopanaxatriol.
Compound 11 was isolated as a colorless powder (MeOH). The
molecular formula was determined to be C30H50O5 on the basis of
HRESI-MS. By comparing with the 13C NMR spectrum of PPT, a
new carbonyl signal at dC 211.7 and a new oxygenated methane
signal at dC 77.5 were observed which indicated that 11 was a
hydroxylated and oxidized product of PPT. In the HMBC spectrum,
carbonyl signal showed long-range correlations with H-11 (dH
60 Q2 and NOESY spectra, as listed in Tables 1 and 2.
61
2.1. Identification of metabolites 3, 11, and 13
62
63
64
65
66
Compound 3 was obtained as white amorphous powder
(MeOH). Its molecular formula was determined to be C31H54O4
on the basis of HRESI-MS. Compared to the 1H NMR spectrum of
PPT, the specific signal of H-24 at about 5.17 ppm was disappear-
ing, along with two new methylene protons at
d
H 4.77 (1H, s) and
3.91, t, J = 10.5 Hz), H-13 (dH 2.87, d, J = 9.5 Hz) and H-17 (dH 2.31)
67 Q3 4.71 (1H, s) that were observed. According to 13C NMR, DEPT and
which suggested that the position of the carbonyl group was at C-
12. Analysis of the HMBC data showed that hydroxylation had
taken place at C-11, because its corresponding proton signal (dH
68
69
HSQC spectra of 3, the presence of two methylene carbon signals at
dC 106.9 and 156.8, and a new methane carbon signal at dc 34.2,
HO
HO
O
O
HO
OH
O
HO
HMBC
NOESY
HO
HO
HO
13
OH
3
OH
OH
11
Fig. 2. Key 2D NMR correlations of metabolites 3, 11, and 13.
Please cite this article in press as: Chen, G., et al., Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger and the cytotoxicity of
the resulting metabolites. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.11.006

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3
88
89
90
3.91) had correlations with C-8 (
d
C 44.9), C-9 (
d
C 61.4), and C-13 (dC
3. Experimental
150
151
50.1) (Fig. 2). The NOE enhancement of H-11 with H-18 (dH 1.34)
and H-19 ( H 1.03) indicated the -configuration for 11-OH. From
d
a
3.1. General experimental produces
91
the above results, 11 was determined as 12-oxo-11
a-hydroxy-
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110
111
112
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114
20(S)-protopanaxatriol.
1D and 2D NMR spectra were carried out on a Bruker DRX-500 152
spectrometer operating at 500 MHz (for 1H) and 125 MHz (for 13C) 153
using CDCl3 as solvent and internal reference. HRESI-MS was 154
recorded on a Finnigan LCQDECA instrument (Thermo Finnigan, San 155
Jose, CA, USA). Optical rotations in CH3OH were measured on a 156
Perkin-Elmer 341 polarimeter. Reversed-phase preparative HPLC 157
was performed on a Shimadzu LC-20A instrument with a Thermo 158
Compound 13 was isolated as a colorless powder (MeOH).
HRESIMS suggested a molecular formula of C30H50O5. Compared
with PPT, 13C NMR spectrum of 13 exhibited an additional
carbonyl signal at
signal showed long-range correlation with H-17 (
In addition, the chemical shift of C-11 ( C 40.0) and C-13 (d
d
C 210.9. In the HMBC experiment, this carbonyl
H 2.53) ( Fig. 2).
C 57.2)
d
d
enabled it to be assigned to C-12. Further compared 13C NMR data
with PPT showed that significant differences were in the carbon
signals from C-20 to C-27. The side-chain double bond (C24–C25)
in PPT was replaced with a new oxygenated quaternary resonance
C18 column (250 mm ꢀ 10 mm, i.d. 5
mm) and a Shimadzu SPD- 159
20A detector. Melting points were measured on an XT4A apparatus 160
(Dianguang Corp., Shanghai, China) and were uncorrected. All 161
organic solvents were of analytic grade and were obtained from 162
Sinopharm Chemical Reagent Co., (China). For HPLC experiments, 163
chromatographic grade acetonitrile and methanol (Merck, Darm- 164
stadt, Germany) were used. Thin-layer chromatography (TLC) 165
analyses were carried out on pre-coated silica gel GF-254 plates 166
(0.25 mm thick), which was purchased from Qingdao Marine 167
Chemical Corporation, China. Octadecylsilane (ODS) was pur- 168
(
d
C 70.3) and a new oxygenated tertiary signal (
resonated at a much lower field ( C 85.5) and NOE enhancements
were observed between 21-CH3 dH 1.03) and H-26 (dH 1.09),
dC 88.0). The C-20
d
(
and H-27 (dH 1.19), indicating cyclization of the side-chain. The
structure and stereochemistry of the side-chain was established
by comparison of the 1H NMR and 13C NMR spectra of 13
with data reported in the literature (Zhou et al., 2013).
Comparison of the 13C NMR spectrum of 13 with that of
chased from YMC Co., Ltd. (Kyoto, Japan).
169
(20S,24S)-epoxy-3
b
, 25-dihydroxy-dammaran-12-one showed
3.2. Substrate
170
a close similarity between the C-20 and C-27 data. Thus, the
structure of 13 was determined as (20S,24S)-epoxy-3
trihydroxy-dammarane-12-one.
b
,6
a
,25-
20(S)-protopanaxatriol (500 mg) was purchased from Shanghai 171
Tauto Biotech Co., Ltd in China. Its purity was determined to be 98% 172
by HPLC analysis.
173
115
2.2. Biotransformation characteristics
3.3. Microorganisms
174
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127
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In the biotransformation of 20(S)-protopanaxatriol by A. niger
AS 3.1858, it was observed that the dehydrogenation, hydroxyl-
ation, and side-chain oxidation–reduction were the main reaction
Aspergillus niger AS 3.1858 was purchased from the Chinese 175
General Microbiological Culture Collection Center, Beijing, China. 176
All of the culture and biotransformation experiments were 177
performed in liquid potato medium. Minced and husked potato 178
(200 g) was boiled in water for 0.5 h, and the solution was filtered. 179
The filtrate was added to 1 L of water after adding 20 g of glucose. 180
type. The 12
b-hydroxyl groups were selectively dehydrogenated
into carbonyl groups, while the 3
b
-OH and 6 -OH remained
a
intact. These results indicated that enzymes in the Aspergillus
culture could specifically and efficiently catalyze dehydrogenation
of 12b-OH. Moreover, these enzymes could catalyze hydroxylation
of methylene (C-11, C-15 or C-23) and methyl (C-26, C-27 or C-29)
groups. These highly specific reactions may be difficult in chemical
synthesis. In addition, we found a new methylene reaction which
has never been reported in the biotransformation of dammarane-
type compounds.
3.4. Biotransformation procedures
181
Preliminary screening scale biotransformation was carried out 182
in 250 mL Erlenmeyer flasks containing 100 mL of potato dextrose 183
medium. The flasks were placed on a rotary shaker operating at 184
160 rpm at 26 8C. A standard two-stage fermentation protocol was 185
employed in all experiments. After 2 days of incubation, the 186
substrates 2 mg (dissolved in 0.5 mL ethanol) were added into each 187
flask. These flasks were maintained under fermentation conditions 188
for 5 days. Culture controls consisted of fermentation blanks in 189
which fungi were grown without substrate. Substrate controls 190
were composed of sterile medium with substrate, and they were 191
incubated without microorganisms. The preparative scale bio- 192
transformation of PPT was carried out in twenty 1000 mL flasks 193
each containing 400 mL of potato dextrose medium. The fungus 194
was incubated for 2 days before 20 mg of substrate PPT (1 mL, 195
ethanol) was fed to each flask. Incubation conditions and the 196
129
2.3. Cytotoxicity testing
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144
145
146
147
148
149
The cytotoxic activities of thirteen products were evaluated
with seven cancer cell lines Du-145, Hela, K562, K562/ADR, SH-
SY5Y, HepG2, and MCF-7 and one normal cell line Vero. The results
expressed as IC50 values are summarized in Table 3.
As illustrated in Table 3, metabolite 3 exhibited relative better
activity profile toward the tested seven cancer cell lines than 20(S)-
protopanaxatriol. Among the tested cell lines, metabolite 3
displayed the most potent cytotoxicity against Hela, K562,
K562/ADR, and SH-SY5Y cell, with IC50 values of 7.3
mmol/L,
12.6 mol/L, 9.9 mol/L, and 11.4 mol/L. This result indicated
m
m
m
extraction process were the same as described above.
197
that the 24-methylene side-chain would increase the cytotoxi-
cities. The metabolite 10 showed the lower potent inhibitory
effects against tested cells than metabolite 1, which suggested
3.5. Extraction, purification and identification of biotransformed
products
198
199
that the hydroxylation at C-11b could significantly reduce the
cytotoxicities. The results in Table 3 also shown that metabolite 11
had more potent cytotoxicity toward the all tested cell lines than
metabolite 10. This result suggested that the hydroxylation at C-
The culturewas filteredandthefiltrate was extractedthree times 200
with equal volumes of ethyl acetate (EtOAc), and the extractions 201
were evaporated in vacuo and afforded a crude extract (1.3 g). The 202
extract was subjected to column chromatography on a silica gel 203
column (60.0 g), with CH2Cl2-EtOH (30:1–1:1) as solvent, which 204
yielded fractions A-G. Fraction B was purified by semi-preparative 205
11a would increase the activities. The pharmacological signifi-
cance of new biotransformed products obtained in this study will
be further evaluated.
Please cite this article in press as: Chen, G., et al., Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger and the cytotoxicity of
the resulting metabolites. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.11.006

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210
211
212
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HPLC (mobile phase: 55% acetonitrile; flow rate: 1.5 mL/min) to
yield metabolite 1 (8.6 mg). Fraction C was purified using semi-
preparative HPLC (mobile phase: 60% methanol; flow rate: 3.0 mL/
min) to afford metabolite 2 (11.4 mg). Purification of fraction D by
semi-preparative HPLC with 64% acetonitrile as mobile phase and
flow rate of 2.0 mL/minyielded compounds 3 (10.2 mg), 4 (9.8 mg),
and 5 (7.5 mg). Fraction E was purified by semi-preparative HPLC
with 80% methanol as mobile phase and flow rate of 1.5 mL/min led
to 6 (9.3 mg), 7 (8.9 mg), 8 (12.3 mg), and 9 (10.5 mg). Fraction F was
further subjected to semi-preparative HPLC with 68% acetonitrile as
mobile phaseand flow rateof1.5 mL/minyielded to10(14.4 mg), 11
(5.7 mg), 12 (9.3 mg), and 13 (6.8 mg).
Acknowledgements
257
We thank the National Natural Science Foundation of China (No. Q5 258
81102327) and Innovation Project of Jiangsu Graduate Education
(YKC14059) for funding toward this research.
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3.5.1. 24-Methylene-20(S)-protopanaxatriol (3)
White amorphous powder, m.p. 152–154 8C,
½
a 2D2
+48.38
ꢁ
(c = 0.1, MeOH). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,
125 MHz) see Tables 1 and 2. HRESIMS [M+Na]+ m/z 513.3927
(calcd. for C31H54O4Na, 513.3920).
223
224
225
226
227
3.5.2. 12-oxo-11a-hydroxy-20(S)-protopanaxatriol (11)
Colorless powder, m.p. 137–139 8C, ½a D22
ꢁ
+46.58 (c = 0.1, MeOH).
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) see
Tables 1 and 2. HRESIMS [M+Na]+ m/z 513.3558 (calcd. for
C30H50O5Na, 513.3556).
228
229
230
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233
3.5.3. (20S,24S)-epoxy-3b,6a,25-trihydroxy-dammarane-12-one
(13)
Colorless powder, m.p. 127–129 8C, ½a D22
ꢁ
+38.68 (c = 0.1, MeOH).
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) see
Tables 1 and 2. HRESIMS [M+Na]+ m/z 513.3553 (calcd. for
C30H50O5Na, 513.3556).
234
3.6. Bioassay
235
236
237
238
239
240
241
242
243
244
245
Seven cancer cell lines, Du-145, Hela, K562, K562/ADR, SH-SY5Y,
HepG2, MCF-7 and one normal cell line Vero, were maintained in
RPMI1640mediumorDMEM,supplementedwith10%(v/v)neonatal
bovine serum or 10% fetal bovine serum. The culture was maintained
at 37 8C, 5% CO2 and grown in 96-wellmicrotiter plates for the assay.
All media weresupplemented with 100 U/mL penicillin and 100
mg/
mL streptomycin. The survival rates of the cancer cells were
evaluated by the MTT method. Cytotoxicities were determined as
IC50 values, namely, the concentrationof test compounds required to
provide 50% inhibition of cell growth. The results were expressed as
the mean value of triplicate determinations.
246
4. Conclusions
247
248
249
250
251
252
253
254
The incubation of PPT with A. niger AS 3.1858 yielded thirteen
products in total, including three new compounds. The enzymatic
reactions included dehydrogenation, hydroxylation, and side-
chain. In addition, we found a new methylene reaction which has
never been reported in the biotransformation of dammarane-type
compounds. Thus, biotransformation is a potent approach to
diversify the structures of natural products and preparing a variety
of derivatives for the search of new lead compounds.
Tian, Y., Guo, H.-Z., Han, J., Guo, D.-A., 2005. Microbial transformation 20(S)-
protopanaxatriol by Mucor spinosus. J. Nat. Prod. 68, 678–680.
Zhou, Z.-W., Ma, C., Zhang, H.-Y., Bi, Y., Chen, X., Tian, H., Xie, X.-N., Meng, Q.-G., Lewis,
P.-J., Xu, J.-Y., 2013. Synthesis and biological evaluation of novel ocotillol-type
triterpenoid derivatives as antibacterial agents. Eur. J. Med. Chem. 68, 444–453.
Zhang, J., Guo,H.-Z.,Tian, Y., Liu,P., Li, N., Zhou,J.-P.,Guo,D.-A.,2007. Biotransformation
of 20(S)-protopanaxatriol by Mucor spinosus and the cytotoxic structure activity
relationships of the transformed products. Phytochemistry 68, 2523–2530.
255 Q4 Uncited references
256 He et al. (2014), Tian et al. (2005) and Zhang et al. (2007).
Please cite this article in press as: Chen, G., et al., Biotransformation of 20(S)-protopanaxatriol by Aspergillus niger and the cytotoxicity of
the resulting metabolites. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.11.006