Microbial transformation of 20(R)-panaxadiol by Absidia corymbifera AS 3.3387

Article abstract of DOI:10.1016/j.molcatb.2015.11.015

The microbial transformation of 20(R)-panaxadiol (1) by Absidia corymbifera AS 3.3387 was investigated. Seven oxidized and hydroxylated products 20(R),25-epoxy-12β-hydroxydammaran- 3-one (2), 20(R),25-epoxy-7β,12β,24α-trihydroxydammaran-3-one (3), 20(R),25-epoxy- 7β,12β,24β-trihydroxydammaran-3-one (4), 20(R),25-epoxy-12β,15α,24α-trihydroxydammaran- 3-one (5), 20(R),25-epoxy-12β,15α,24β-trihydroxydammaran-3-one (6), 20(R),25-epoxy- 7β,12β,24β-trihydroxydammar-15-en-3-one (7), and 20(R),25-epoxy-7β,12β,24β-trihydroxy- 15,30-cyclodammaran-3-one (8) were obtained. Six metabolites 3-8 are new compounds and are being reported here for the first time. Structures of the new metabolites were elucidated by 1-D (¹H, ¹³C), 2-D NMR (HSQC, HMBC, and ROESY) and HR-MS analyses. In addition, the cytotoxicity of substrate and all transformed products was evaluated by MTT assay using a panel of four human tumor cell lines (Du-145, Hela, HepG2, and MCF-7 cells) and one normal cell line Vero.

Full text of DOI:10.1016/j.molcatb.2015.11.015

Journal of Molecular Catalysis B: Enzymatic 123 (2016) 154–159
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Microbial transformation of 20(R)-panaxadiol by Absidia corymbifera
AS 3.3387
Guangtong Chen, Hongjuan Ge, Jie Li, Jianlin Li∗, Xuguang Zhai, Juanjuan Wu, Yan Song∗
School of Pharmacy, Nantong University, 19 Qixiu Road, Nantong 226001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2015
Received in revised form 22 October 2015
Accepted 11 November 2015
Available online 1 December 2015
The microbial transformation of 20(R)-panaxadiol (1) by Absidia corymbifera AS 3.3387 was inves-
tigated. Seven oxidized and hydroxylated products 20(R),25-epoxy-12␤-hydroxydammaran-
3-one
7␤,12␤,24␤-trihydroxydammaran-3-one (4), 20(R),25-epoxy-12␤,15␣,24␣-trihydroxydammaran-
3-one (5), 20(R),25-epoxy-12␤,15␣,24␤-trihydroxydammaran-3-one (6), 20(R),25-epoxy-
(2),
20(R),25-epoxy-7␤,12␤,24␣-trihydroxydammaran-3-one
(3),
20(R),25-epoxy-
7␤,12␤,24␤-trihydroxydammar-15-en-3-one (7), and 20(R),25-epoxy-7␤,12␤,24␤-trihydroxy- 15,30-
cyclodammaran-3-one (8) were obtained. Six metabolites 3–8 are new compounds and are being
reported here for the first time. Structures of the new metabolites were elucidated by 1-D (1H, 13C), 2-D
NMR (HSQC, HMBC, and ROESY) and HR-MS analyses. In addition, the cytotoxicity of substrate and all
transformed products was evaluated by MTT assay using a panel of four human tumor cell lines (Du-145,
Hela, HepG2, and MCF-7 cells) and one normal cell line Vero.
Keywords:
Microbial transformation
20(R)-Panaxadiol
Absidia corymbifera
Cytotoxic activity
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
relationship studies. Some studies on the structural modification
of 20(R)-panaxadiol by chemical and biological methods were
Ginseng, the root of Panax ginseng C.A. Mey (Araliaceae), has
been widely used in traditional Chinese medicine [1,2]. Ginseno-
sides are the main chemical constituents responsible for the
anticancer and neuroprotective activities of ginseng [3–5]. More
than 40 ginsenosides have been found from ginseng [6,7]. It has
been reported that ginsenosides were poorly absorbed in the
gastrointestinal tract [8], and could be hydrolyzed by intestinal bac-
teria to produce secondary glycosides and triterpene sapogenins
[9]. In recent past, the structure–activity relationship studies of
triterpene sapogenins as anti-cancer agents are carried out for
finding the semi-synthetic analogues with the better bioactivity
[10,11].
20(R)-panaxadiol (1) is an important sapogenin of ginsenosides,
can be obtained by acid hydrolysis [12]. It has been reported to
have strong anticancer activities, and the mechanism involved
in inducing apoptosis [13]. It can also enhance inhibitory effects
of 5-fluorouracil [14,15]. Therefore, the structure modification
of 20(R)-panaxadiol may be valuable in obtaining new chemical
derivatives for pharmacological screening and structure-activity
reported [16–18].
In the present study, biotransformation of 1 by Absidia corymb-
ifera AS 3.3387 is reported. Seven metabolites were obtained,
among which six were identified as new compounds (Fig. 1). These
may supply novel bioactive anticancer agents and precursors for
synthesis of other new bioactive anticancer drugs.
2. Experimental
2.1. General experimental procedures
The 1H NMR (nuclear magnetic resonance) and 13C NMR spec-
tra were carried out on a Bruker AM-500 spectrometer at 500
and 125 MHz in CDCl3 (chloroform-d), respectively. Chemical shifts
were reported in ppm (ı) relative to tetramethylsilane (TMS) as an
internal standard, and J values are in hertz. High-resolution mass
spectra (HRMS) were recorded on a Finnigan LCQDECA instrument.
2.2. Microorganism
A. corymbifera AS 3.3387 was purchased from Chinese General
Microbiological Culture Collection Center in Beijing, China. The
strain was maintained on potato agar slants prepared from a potato
infusion. The potato infusion was prepared by boiling 200 g of sliced
∗
Corresponding author. Fax: +86 513 85051892.
E-mail addresses: jianlinli1980@163.com (J. Li), violet200599@ntu.edu.cn
(Y. Song).
http://dx.doi.org/10.1016/j.molcatb.2015.11.015
1381-1177/© 2015 Elsevier B.V. All rights reserved.

G. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 154–159
155
Fig. 1. Biotransformation of 20(R)-panaxadiol by Absidia corymbifera AS 3.3387.
potatoes in 1000 mL of distilled water for 30 min and then decant-
ing the broth through cheesecloth. Glucose 15 g and agar powder
15 g were then added, and the medium was sterilized by autoclav-
ing it for 30 min.
Each fraction was evaporated to dryness under reduced pressure
and analyzed by TLC (CHCl3/MeOH 15:1). Fraction B was further
isolated by semi-preparative RP-HPLC with acetonitrile-water as
mobile phase and flow rate at 2.0 mL/min to obtain the metabo-
lites 2 (33.8 mg), 3 (11.7 mg), 4 (15.2 mg), 5 (22.1 mg), 6 (17.5 mg),
7 (15.8 mg), and 8 (11.3 mg).
2.3. Microbial transformation procedures
The biotransformation process was conducted at two scales:
analytical and preparative. Analytical scale biotransformation was
carried out in 250 mL Erlenmeyer flasks containing 100 mL of potato
dextrose medium. The flasks were placed on a rotary shaker operat-
ing at 160 rpm at 26 ◦C. A standard two-stage fermentation protocol
was employed in all experiments. After 2 days of incubation, the
substrates 2 mg (dissolved in 0.5 mL ethanol) were added into each
flask. These flasks were maintained under fermentation conditions
for 7 days, and then the cultures were pooled and filtered. The fil-
trates were extracted three times with equal volumes of EtOAc, and
the extractions were evaporated under reduced pressure and ana-
lyzed by TLC and HPLC. Culture controls consisted of fermentation
blanks in which fungi were grown without substrate but fed with
the same amount of ethanol.
The preparative scale biotransformation of 20(R)-panaxadiol
(1) by A. corymbifera was carried out in twenty 1000 mL Erlen-
meyer flasks each containing 400 mL of sterilized potato medium.
The flasks were placed on rotary shaker operating at 160 rpm at
26 ◦C. 20(R)-panaxadiol were dissolved in ethanol. After 48 h of
pre-culture, 20 mg of substrates in 1 mL ethanol were added into
each flask. After 7 days incubation, the culture was pooled and fil-
tered. The filtrate was extracted three times with an equal volume
of EtOAc and concentrated under reduced pressure to dryness.
2.5. Biotransformation products
2.5.1. 20,25-epoxy-7ˇ,12ˇ,24˛-trihydroxydammaran-3-one
(3)(R)
20
Colorless powder (MeOH); mp 312–313 ◦C; ␣
+47.2◦ (c 0.1,
[
]
D
MeOH); IR ꢀmax (KBr, cm−1): 3421, 2973, 1713. 1466, 1027; 1H
NMR and 13C NMR (CDCl3) spectroscopic data see Table 1 and
Table 2; HR-ESI–MS m/z 513.3558 [M + Na]+ (calcd for C30H50O5Na,
513.3550).
2.5.2. 20,25-epoxy-7ˇ,12ˇ,24ˇ-trihydroxydammaran-3-one
(4)(R)
20
Colorless powder (MeOH); mp 334–336 ◦C; ␣
+24.7◦ (c 0.1,
[
]
D
MeOH); IR ꢀmax (KBr, cm−1): 3404, 2961, 1704. 1401, 1058; 1H
NMR and 13C NMR (CDCl3) spectroscopic data see Table 1 and
Table 2; HR-ESI–MS m/z 513.3554 [M + Na]+ (calcd for C30H50O5Na,
513.3550).
2.5.3. 20,25-epoxy-12ˇ,15˛,24˛-trihydroxydammaran-3-one
(5)(R)
20
Colorless powder (MeOH); mp 285–287 ◦C; ␣
+18.7◦ (c 0.1,
[
]
D
2.4. Chromatographic conditions
MeOH); IR ꢀmax (KBr, cm−1): 3389, 2929, 1721. 1439, 1024; 1H
NMR and 13C NMR (CDCl3) spectroscopic data see Table 1 and
Table 2; HR-ESI–MS m/z 513.3560 [M + Na]+ (calcd for C30H50O5Na,
513.3550).
The residue (0.97 g) was subjected to a reversion phase silica
gel Rp-18 column and eluted with 20% MeOH-H2O, 70% MeOH-
H2O, and MeOH to give the Fractions A, B, and C, respectively.

156
G. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 154–159
Table 1
1H NMR spectroscopic data for metabolites 3–8 (CDCl3, 500 MHz, ı in ppm, J in Hz).
No.
3
4
5
6
7
8
1
2
3
1.94, 1.44 m
2.47 m
–
1.96, 1.44 m
2.48 m
–
1.98, 1.49 m
2.47 m
–
1.96, 1.45 m
2.45 m
–
1.92, 1.41 m
2.47 m
–
1.94, 1.43 m
2.44 m
–
4
–
–
–
–
–
–
5
6
7
8
1.48 m
1.64 m
3.82 m
–
1.49 m
1.64 m
3.82 m
–
1.42 m
1.52 m
1.63 m
–
1.39 m
1.49, 1.60 m
1.59 m
–
1.50 m
1.67 m
3.49 br d (8.3)
–
1.48 m
1.64 m
3.42 m
–
9
1.63 m
–
1.63 m
–
1.52 m
–
1.50 m
–
1.42 m
–
1.61 m
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.88 m
3.49 m
1.43 m
–
1.72, 1.32 m
1.24 m
1.99 m
1.01 s
0.97 s
–
1.22 s
1.43 m
1.31 m
3.54 dd (10.3, 5.3)
–
1.33 s
1.27 s
1.09 s
1.05 s
0.97 s
1.39, 1.90 m
3.53 td (4.9, 10.0)
1.44 m
–
1.72, 1.33 m
1.24, 1.86 m
1.89 m
1.02 s
0.96 s
–
1.21 s
1.74 m
1.76 m
3.45 br t (8.5)
–
1.29 s
1.27 s
1.29, 1.91 m
3.66 td (5.0, 9.8)
1.71 m
–
4.16 br t (7.2)
1.65 m
2.10 m
1.13 s
1.00 s
–
1.25 s
1.45, 1.68 m
1.77, 2.12 m
3.52 br s
–
1.28 s
1.33 s
1.89 m
3.62 td (5.1, 10.3)
1.68 m
–
4.14 br t (9.5)
1.64 m
1.96 m
1.10 s
1.00 s
–
1.21 s
1.46, 1.69 m
1.76 m
3.43 br t (7.4)
–
1.29 s
1.27 s
1.37, 1.95 m
3.73 td (4.6, 10.1)
1.97 m
–
5.35 d (6.0)
6.33 dd (6.0, 2.5)
2.54 m
1.04 s
0.94 s
–
1.19 s
1.63 m
1.81 m
3.78 m
–
1.31 s
1.28 s
1.97 m
3.39 td (5.2, 10.6)
2.07 t (9.6)
–
1.16 m
1.69 m
1.35 m
1.18 s
0.94 s
–
1.21 s
1.58 m
1.74 m
3.34 m
–
1.28 s
1.27 s
1.08 s
1.04 s
1.09 s
1.05 s
0.97 s
1.10 s
1.06 s
1.00 s
1.08 s
1.04 s
0.97 s
1.10 s
1.06 s
0.95 s
0.23 dd (5.3, 3.6)0.60 dd (8.0, 5.3)
Table 2; HR-ESI–MS m/z 513.3553 [M + Na]+ (calcd for C30H50O5Na,
513.3550).
Table 2
13C NMR spectroscopic data for metabolites 3–8 (CDCl3, 125 MHz; ı in ppm).
Position
3
4
5
6
7
8
2.5.5. 20,25-epoxy-7ˇ,12ˇ,24ˇ-trihydroxydammar-15-en-3-one
1
2
3
4
5
6
7
8
39.3
33.9
39.3
33.9
39.8
34.1
39.8
34.1
38.9
33.9
38.5
34.0
(7)(R)
20
Colorless powder (MeOH); mp 292–294 ◦C; ␣
+26.4◦ (c 0.1,
217.2
47.0
52.7
29.4
74.3
45.2
50.0
36.6
30.7
69.4
48.7
50.9
34.4
25.5
53.3
9.5
15.9
75.7
19.8
28.7
23.2
69.6
76.9
27.3
27.4
26.8
21.0
16.8
217.3
47.0
52.7
29.4
74.3
45.2
50.0
36.6
30.7
69.4
48.7
50.9
34.4
25.8
53.3
9.5
15.9
76.7
19.2
36.4
25.2
74.6
77.2
29.6
21.2
26.8
21.0
16.8
217.8
47.3
55.0
19.6
34.9
40.2
49.2
36.8
30.8
69.4
47.5
51.9
72.9
35.0
52.0
15.4
16.1
76.0
20.0
28.6
23.1
69.5
76.5
27.1
27.3
26.7
21.0
10.0
217.7
47.3
55.0
19.6
34.9
40.2
49.3
36.8
30.8
69.4
47.4
51.9
72.9
25.1
52.0
15.4
16.1
76.1
19.3
36.3
25.1
74.6
77.2
29.5
21.1
26.6
21.0
10.0
217.0
47.0
53.1
29.5
74.6
43.7
49.0
36.8
31.8
67.8
52.3
56.8
129.4
144.8
59.2
13.6
15.5
75.3
20.9
36.9
25.2
74.8
77.2
29.5
21.0
26.7
21.1
15.5
217.0
47.0
52.8
29.7
74.6
40.8
53.3
36.7
30.8
72.2
46.5
40.7
19.4
31.5
51.0
15.5
15.3
75.9
19.7
36.2
25.3
74.7
77.2
29.7
21.3
26.7
21.1
[
]
D
MeOH); IR ꢀmax (KBr, cm−1): 3403, 2946, 1732. 1461, 1019; 1H
NMR and 13C NMR (CDCl3) spectroscopic data see Table 1 and
Table 2; HR-ESI–MS m/z 511.3405 [M + Na]+ (calcd for C30H48O5Na,
511.3394).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.5.6. 20,25-epoxy-7ˇ,12ˇ,24ˇ-trihydroxy-15,30-
cyclodammaran-3-one
(8)(R)
20
Colorless powder (MeOH); mp 352–353 ◦C; ␣
+30.3◦ (c 0.1,
[
]
D
MeOH); IR ꢀmax (KBr, cm−1): 3391, 2962, 1714. 1438, 1034; 1H
NMR and 13C NMR (CDCl3) spectroscopic data see Table 1 and
Table 2; HR-ESI–MS m/z 511.3401 [M + Na]+ (calcd for C30H48O5Na,
511.3394).
2.6. Cytotoxicity assay
Four cancer cell lines, Du-145, Hela, HepG2, MCF-7 and one nor-
mal cell line Vero were kindly provided by Shanghai Jiao Tong
University. All cells were maintained in RPMI 1640 medium or
DMEM, supplemented with 10% (v/v) neonatal bovine serum or
10% fetal bovine serum. The culture was maintained at 37 ◦C, 5% CO2
and grown in 96-well microliter plates for the assay. All media were
supplement with 100 U/mL penicillin and 100 ␮g/mL streptomycin.
The cytotoxic activity in vitro was measured using the MTT
assay. The MTT solution (10.0 mL/well) was added in culture media
after cells were treated with various concentrations of compounds
for 72 h, and cells were incubated for further 4 h at 37 ◦C. The
purple formazan crystals were dissolved in 100 mL DMSO. After
10 min, the plates were read on an automated microplate spec-
trophotometer (Bio-Tek Instruments, Winooski, VT) at 570 nm and
6.99
2.5.4. 20,25-epoxy-12ˇ,15˛,24ˇ-trihydroxydammaran-3-one
(6)(R)
20
Colorless powder (MeOH); mp 311–313 ◦C; ␣
+21.2◦ (c 0.1,
[
]
D
MeOH); IR ꢀmax (KBr, cm−1): 3415, 2936, 1718. 1446, 1061; 1H
NMR and 13C NMR (CDCl3) spectroscopic data see Table 1 and

G. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 154–159
157
Fig. 2. Key HMBC correlations for compounds 3, 6, and 8.
Fig. 3. Key NOE correlations for compounds 3 and 6.
630 nm. Assays were performed in triplicate on three independent
experiments. The concentration required for 50% inhibition of cell
viability (IC50) was calculated using the software “Dose-Effect Anal-
ysis with Microcomputers”. The tumor cell lines panel consisted of
Du-145, Hela, HepG2, and MCF-7. In all of these experiments, three
replicate wells were used to determine each point.
27) (Fig. 3). Therefore, the structure of 3 was elucidated as 20(R),
25-epoxy-7␤,12␤,24␣-trihydroxydammaran-3-one. Its 1H and 13
C
NMR spectral data were shown in Table 1 and Table 2.
Metabolite 4 was obtained as colorless powder (methanol).
HRMS gave a quasi-molecular ion [M + Na]+ at m/z 513.3554 sug-
gesting the molecular formula of C30H50O5. By comparing with the
13C NMR spectrum of 3, the presence of a carbonyl carbon signal at
ıC 217.3 and two new bearing-oxygen carbons at ıC 74.3 and 74.6
were observed. This suggested metabolite 4 was very similar to 3.
The only different is the relative configuration of 24-OH, and 24-
OH was deduced as ␤ by ROESY correction between ıH 3.45 (H-24)
and ıH 1.29 (H-26). Thus, the structure of 4 was elucidated as 20(R),
3. Results and discussion
The biotransformation of 20(R)-panaxadiol (1) by A. corymbifera
AS 3.3387 yielded one known metabolite and six new metabolites
(Fig. 1). The known metabolite was identified as 20(R), 25-epoxy-
12␤-hydroxydammaran-3-one (2) by the combined analyses of IR,
MS and NMR spectroscopic data [18]. The structures of six new
metabolites were determined on the basis of extensive spectro-
scopic analyses.
Metabolite 3 was obtained as colorless powder (methanol).
HRMS gave a quasi-molecular ion [M + Na]+ at m/z 513.3558 sug-
gesting the molecular formula of C30H50O5. According to 13C NMR
and DEPT-135 experiments of 3, the presence of a carbonyl carbon
signal at ıC 217.2 and two new bearing-oxygen carbons at ıC 74.3
and 69.6 were observed. These suggested it to be a hydroxylated
and oxidized derivative of 20(R)-panaxadiol. The carbonyl group
was localized at C-3 due to the HMBC correlations between the
resonation at ıH 1.09 (H-28), 1.05 (H-29), 1.48 (H-5) and ıC 217.2
(C-3). The two hydroxyl groups were located at C-7 and C-24 due
to the HMBC correlations between the resonation at ıH 3.87 (H-
7) and ıC 29.4 (C-6), 9.5 (C-18), the resonation at ıH 3.54 (H-24)
and ıC 27.3 (C-26), 27.4 (C-27), 76.9 (C-25) (Fig. 2). Furthermore,
the relative configuration of 7-OH was deduced as ␤ by the ROESY
correlation between ıH 3.87 (H-7) and ıH 0.97 (H-30). The relative
configuration of 24-OH was deduced as ␣ by ROESY correlation
between ıH 3.54 (H-24) and the H-atom resonating at ıH 1.27 (H-
25-epoxy-7␤,12␤,24␤-trihydroxydammaran-3-one. Its 1H and 13
C
NMR spectral data were shown in Table 1 and Table 2.
The HRMS data of metabolite 5 gave a peak at 513.3560
[M + Na]+, suggesting a molecular formula of C30H50O5. Its 13C
NMR spectrum showed an additional carbonyl signal at ıC 217.8
and two new oxygen-bearing carbons at ıC 72.9 and 69.5. These
suggested it to be a hydroxylated and oxidized derivative of 20(R)-
panaxadiol. The new oxygen-bearing methine resonance at ıC 72.9
was assigned to C-15 due to its long-rang correlation with ıH 1.00
(H-30). The ROESY correlation between ıH 4.16 (H-15) and 1.13 (H-
18) suggested an ␣ configuration for the 15-OH group. The ıC 69.5
signal was assigned to C-24 due to its HMBC correlations with ıH
1.28 (H-26) and 1.33 (H-27). The 24-OH group was deduced to be in
the ␣ configuration by the ROESY correlation of ıH 3.52 (H-24) with
1.33 (H-27). Therefore, metabolite 5 was identified as 20(R), 25-
epoxy- 12␤,15␣,24␣-trihydroxydammaran-3-one. Its 1H and 13C
NMR spectral data were shown in Table 1 and Table 2.
The molecular formula of metabolite 6 was established as
C
30H50O5 by HRMS m/z 513.3553 [M + Na]+. Its 13C NMR and DEPT-
135 spectra indicated an additional carbonyl signal at ıC 217.7 and
two new oxygen-bearing methine signals at ıC 72.9 and 74.6. One

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G. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 154–159
Table 3
hydroxyl group should be located at C-15 according to HMBC cor-
relation of ıC 72.9 with ıH 0.97 (H-30) (Fig. 2). The 15-OH was
determined to be in the ␣ configuration by the ROESY correlation of
ıH 4.14 (H-15) with 1.10 (H-18). The other hydroxyl group should
be located at C-24 according to the HMBC corrections of ıC 74.6
with ıH 1.29 (H-26) and 1.27 (H-27) (Fig. 2). In the ROESY spec-
trum, the correction between ıH 3.43 (H-24) with 1.29 (H-26) was
consistent with the ␤ configuration for the 24-OH group (Fig. 3).
Thus, metabolite 6 was identified as 20(R), 25-epoxy-12␤,15␣,24␤-
trihydroxydammaran-3-one. Its 1H and 13C NMR spectral data were
shown in Table 1 and Table 2.
The HRMS spectrum of metabolite 7 suggested a molecular
formula of C30H48O5 ([M + Na]+, m/z 511.3405). Its 13C NMR and
DEPT-135 spectra showed a new carbonyl signal at ıC 217.0, a
new C&9552;C bond signal at ıC 144.8 and 129.4, and two new
oxygen-bearing methine signals at ıC 74.6 and 74.8. The C&9552;C
group was located at C-15, due to the HMBC correlations between
ıH 5.35 (H-15) and 6.33 (H-16) with ıC 52.3 (C-13), 56.8 (C-14),
and 59.2 (C-17), the H-atom resonation at ıH 0.95 (H-30) with
ıC 129.4 (C-15). The ıC 74.6 signal was assigned to C-7 due to
its HMBC correlation with ıH1.04 (H-18). The ROESY correlation
between ıH 3.49 (H-7) and ıH 0.95 (H-30) suggested that 7-OH was
␤-oriented. The ıC 74.8 resonance was assigned to C-24 due to its
HMBC correlations with ıH 1.31 (H-26) and 1.28 (H-27). The ROESY
correlation between ıH 3.78 (H-24) and ıH 0.95 (H-26) indicated
the ␤-configuration of 24-OH. Therefore, the structure of 7 was
identified as 20(R), 25-epoxy-7␤,12␤,24␤-trihydroxydammar-15-
en-3-one. Its 1H and 13C NMR spectral data were shown in Table 1
and Table 2.
Metabolite 8 was obtained as colorless powder (methanol).
Its molecular formula was stablished as C30H48O5 by HRMS m/z
511.3401 [M + Na]+. The 1H NMR spectrum indicated seven methyl
groups. The 13C NMR and DEPT-135 spectra showed a new car-
bonyl signal at ıC 217.0, a new methene signal at ıC 6.99, and
two new oxygen-bearing methine signals at ıC 74.6 and 74.7. The
special metene signal at ıC 6.99 was assigned at C-30 due to the
HMBC correlations between H-30 (ıH 0.23 and 0.60) with C-8 (ıC
40.8), C-13 (ıC 46.5), C-14 (ıC 40.7), C-15 (ıC 19.4), and C-16 (ıC
31.5) (Fig. 2). Furthermore, the relative configuration of C-15 was
deduced as ␣ by the ROESY correlations between H-30 (ıH 0.23
and 0.60) and H-7 (ıH 3.42), H-12 (ıH 3.39), and H-17 (ıH 1.35).
The hydroxyl group should be located at C-7 according to HMBC
correlation of ıC 74.6 with ıH 1.18 (H-18). The ROESY correlation
between ıH 3.42 (H-7) and ıH 0.23 (H-30) suggested that 7-OH
was ␤-configuration. The ıC 74.7 resonance was assigned to C-24
due to its HMBC correlations with ıH 1.28 (H-26) and 1.27 (H-
27). The ROESY correlation between ıH 3.34 (H-24) and ıH 1.28
(H-26) indicated the ␤-configuration of 24-OH. Thus, metabolite 8
was identified as 20(R), 25-epoxy-7␤,12␤,24␤-trihydroxy-15,30-
cyclodammaran-3-one. Its 1H and 13C NMR spectral data were
shown in Table 1 and Table 2.
The cytotoxicity of compounds 1–8 against DU-145, Hela, HepG2, MCF-7, and Vero
cell lines.
Compound
IC50 value (␮M)
DU-145
Hela
HepG2
MCF-7
Vero
1
2
3
4
5
6
7
8
46.2
44.1
31.4
44.3
38.7
6.5
37.8
58.2
52.1
32.1
61.7
21.2
10.3
18.0
29.6
34.7
36.5
58.1
35.4
12.2
20.3
14.2
42.1
63.9
31.7
21.4
29.5
11.0
18.8
13.4.
87.5
>100
78.6
>100
>100
69.3
>100
>100
4.8
13.1
side-chain oxidation-reduction, hydroxylation and ketonization
were the main reaction types [21].
In this work, we found a new cyclization reaction which was
never been reported on the biotransformation of dammarane-
type compounds. In the transformation of 20(R)-panaxadiol by
A. corymbifera AS 3.3387, it was also observed that the 3␤-
hydroxyl group was selectively dehydrogenated into carbonyl
group, while the 12␤-OH remained intact. The hydroxylation
was another main reaction type. The specific 7␤-hydroxylated
product, 24␤-hydroxylated product, 24␣-hydroxylated product,
and 15␣-hydroxylated product yielded from this bio-process. The
dehydration to form double bond at C-15 was also observed.
These results indicate that enzymes in the Absidia culture could
specifically and efficiently catalyze dehydrogenation of 3␤-OH,
hydroxylation of methylene groups (C-7, C-15, and C-24), and the
dehydration at C-15. These highly specific reactions may be difficult
in chemical synthesis. Thus, biotransformation is a potent approach
to diversifying the structures of natural products and preparing a
variety of derivatives for the search of new lead compounds.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (81102327, 81400634), Natural Science Foun-
dation of Jiangsu Province (BK20140436), and Innovation Project
of Jiangsu Graduate Education (YKC14059). Guangtong Chen
gratefully acknowledges the Jiangsu Government Scholarship for
overseas Studies (JS-2013-238).
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.molcatb.2015.11.
015.
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