全 文 ：Received 29 Mar. 2004 Accepted 15 Jun. 2004
Supported by the Hi-Tech Research and Development (863) Program of China (2001AA234021, 2002AA2Z343B).
* Author for correspondence. Tel: +86 (0)10 63165197; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 935-939
Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension
Cultures of Catharanthus roseus
LIU Ying1, 2, CHENG Ke-Di1, ZHU Ping1* , FENG Wen-Hua1, MENG Chao1, ZHU Hui-Xin1,
HE Hui-Xia1, MA Xiao-Jun2
(1. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
2. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing 100094, China)
Abstract: Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-
hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures
were established by means of NMR and MS spectral analyses. Nine metabolites were respectively eluci-
dated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-
4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-
dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-
3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above
compounds by biotransformation with Catharanthus roseus cell cultures.
Key words: Catharanthus roseus; cell suspension cultures; biotransformation; 3b-hydroxyandrost-
5-en-17-one
The biochemical potential of plant cell cultures to pro-
duce specific secondary metabolites such as drugs, flavors,
pigments and agrochemicals is of considerable interest in
connection with their biotechnological utilization.
Unfortunately, it has been reported that formation and ac-
cumulation of some secondary metabolites do not normally
occur in the cell cultures of higher plants (Suga and Hirata,
1990). However, much evidence indicated that such cul-
tures retain the ability to specifically transform exogenous
substrates administered to the cultured cells. Therefore,
plant cell culture, as a bioreactor, is considered to be useful
for transforming abundant and interesting compounds into
valuable ones. The compound 3b-hydroxyandrost-5-en-17-
one, also called dehydroepiandrosterone (DHEA), is a C19
antrosterone presented in human adrenal cortex. The de-
scription of DHEA as a neuroactive steroid has raised the
interesting question of whether the steroid itself and/or its
metabolites are active in the treatment of cancer, diabetes
and senescence (Coleman et al., 1982; Daynes et al., 1990;
Ratko et al., 1991). Because of its potential application in
drug research, we select it as the substrate. Classical trans-
formations of DHEA in brain and peripheral tissues include
its conversion to testosterone and estradiol. In the human
brain, the metabolism of DHEA to other metabolites is still
poorly understood. The present study describes the in vitro
transformation of DHEA into 6-hydroxy-DHEA, 14-hy-
droxy-DHEA, 15-hydroxy-DHEA, etc., for the first time in
cell suspension cultures of Catharanthus roseus.
1 Materials and Methods
1.1 Plant tissue and cell culture
Callus cultures of Catharanthus roseus (L.) G. Don
(Apocynaceae) were preserved in the Biosynthesis
Department, Institute of Materia Medica, Chinese Acad-
emy of Medical Sciences and Peking Union Medical
College. The cultures were maintained on MS medium
supplemented with 1.25 mg/L 2,4-dichlorophenoxyacetic
acid (2,4-D), 2 mg/L r-chlorophenoxyacetic acid (CPA), 0.1
mg/L 6-furfurylamino purine (KT) and 5.5 g/L agar. The pH
of the medium was adjusted to 5.8 prior to autoclaving at
121 ℃ for 25 min. The callus cultures grew well at 25 ℃ in
the dark, which was subcultured every four weeks. Three-
week-old friable callus was used to initiate cell suspension
cultures in the same medium. All the cell suspension cul-
tures were maintained on a rotary shaker at 110 r/min at 25
℃ in the dark.
1.2 Biotransformation
The substrate 3b-hydroxyandrost-5-en-17-one (DHEA)
was dissolved in acetone to make a 40.0 mg/mL stock
solution. The cells were cultured in 1 L flasks, each con-
taining 400 mL of MS liquid medium at 25 ℃ on a rotary
shaker. After 10 d of cultivation the stock solution was
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004936
added into the flasks in amount of 1 mL per flask, and the
incubation continued for another 10 d. One mL acetone
was used as the control.
1.3 Extraction and isolation
After 10 d of incubation, the cell cultures were pooled
and filtered. The filtrate was extracted with EtOAc three
times and evaporated under reduced pressure. The extract
(7.8 g) from the total 80 L cultures was chromatographed
over a silica gel (160－ 200 mesh) column, starting with
(100%) petroleum ether and petroleum ether-EtOAc (10:1
to 1:1) gradient elution followed by the elution of EtOAc
(100%) and MeOH (100%). Sterol-containing fractions were
subjected to preparative TLC to give transformed products
(solvent system: CH2Cl2:MeOH=15:1). Structure elucida-
tion of the metabolites was based on spectral data.
1.4 General
IR spectra were obtained on an IMPACT 400 spectro-
photometer (KBr). Optical rotation values were measured
by using a Perkin-Elmer 243B polarimeter. NMR spectra
(1H-NMR, 13C-NMR) were recorded in CDCl3 or MeOH on
Varian INOVA-500 spectrometer (1H-NMR, 500 MHz; 13C-
NMR, 500 MHz). UV spectra were obtained on a GENERAL
Tu-1221 spectrophotometer. All chemicals were obtained
from Beijing Chemical Factory.
2 Results
2.1 Biotransformation of DHEA by cell suspension cul-
tures of Catharanthus roseus
The substrate was administered to 10-day-old cell
cultures, and more than ten metabolites were observed on
TLC after additional 10 d of incubation and none of them
could be detected in control cultures. Nine of the trans-
formed products were isolated from the cultures together
with the substrate. On the basis of spectral data, their struc-
tures were identified as depicted in Table 1 and Fig.1, among
which compounds Ⅱ, Ⅳ, Ⅵ, Ⅶ, Ⅷ also were available as
microbial transformation product (Bell et al., 1975; Crabb
et al., 1975; Holland and Thomas, 1982; Mahato et al., 1984),
and the others were reported by chemical synthesis (Reich
et al., 1969; Hiroshi et al., 1977; Kirk et al., 1990). It is the
first time to obtain all of the compounds by biotransforma-
tion with C. roseus cell cultures.
2.2 Structural identification of the nine products
3b-Hydroxyandrost-5-en-17-one (DHEA) C19H28O2,
molecular weight 288, yellow amorphous powder, mp 221
℃, 1H-NMR (CDCl3, 500 MHz) d: 0.88 (3H, s, H-18), 1.03
(3H, s, H-19), 3.53 (1H, m, H-3), 5.37 (1H, d, H-6).
CompoundⅠ（Androst-4-ene-3 ,17-dione）
C19H26O2, colorless needles, mp 150-155 ℃, [a]25D + 172.6°
(c 0.75, MeOH). UV lmax (MeOH): 239.8 nm; IR nmax (KBr):
2 954, 2 918, 2 852, 1 738, 1 660, 1 614, 1 452, 1 433, 1 381,
1 273, 1 227, 1 196, 1 093, 1 055, 1 016 cm-1; ESI-MS: m/z 287;
1H-NMR (CDCl3, 500 MHz) d: 0.91 (3H, s, H-18)，1.20 (3H,
s, H-19), 5.74 (1H, s, H-4). The structure was identified by
comparison with previous data (Reich et al., 1969).
Compound Ⅱ (6a-hydroxyandrost-4-ene-3,17-dione)
C19H26O3, colorless needles, mp 175-176 ℃, [a]25D +102.3°
(c 0.64, MeOH). UV lmax (MeOH): 234.6 nm; IRnmax (KBr):
3 429, 2 956, 2 924, 2 864, 1 736, 1 660, 1 473, 1 454, 1 404,
1 336, 1 275, 1 230, 1 194, 1 097, 1 030, 1 012 cm-1; ESI-MS:
m/z 303. 1H-NMR (CDCl3, 500 MHz) d: 0.94 (3H, s, H-18),
1.40 (3H, s, H-19), 4.40 (1H, t, J =2 Hz, H-6), 5.82 (1H, s, H-4).
The structure was identified by comparison with previous
data (Bell et al., 1975; Holland and Thomas, 1982; Kirk et
al., 1990).
Compound Ⅲ (6a,17b-dihydroxyandrost-4-en-3-one)
C19H28O3, white amorphous powder , mp 174-175 ℃, [a]25D
+11.9°(c 0.42, MeOH). UV lmax (MeOH): 237.2 nm; IRnmax
(KBr): 3 369, 2 935, 2 868, 2 827, 1 660, 1 471, 1 448, 1 377,
1 325, 1 279, 1 188, 1 124, 1 057, 1 043 cm-1; ESI-MS: m/z 305.
1H-NMR (CDCl3, 500 MHz) d: 0.82 (3H, s, H-18), 1.39 (3H, s,
H-19), 3.66 (1H, t, J = 9 Hz, H-17), 4.35 (1H, t, J = 3 Hz, H-6),
5.82 (1H, s, H-4). The structure was identified by compari-
son with previous data (Kirk et al., 1990).
Compound Ⅳ (6b-hydroxyandrost-4-ene-3,17-dione)
C19H26O3, colorless needles, mp 194-195 ℃, [a]25D +115°
(c 0.70, CHCl3). UV lmax (MeOH): 234.6 nm; IRnmax (KBr):
3 504, 2 951, 2 852, 1 738, 1 655, 1 645, 1 610, 1 448, 1 377,
Table 1 The yields of the nine products biotransformed from 3b-hydroxyandrost-5-en-17-one (DHEA)
Compound Entry name of the products Molecular formula Molecular weight Yield (mg) Yield (%)
Ⅰ Androst-4-ene-3,17-dione C19H26O2 286 2 210.4 27.63
Ⅱ 6a-Hydroxyandrost-4-ene-3,17-dione C19H26O3 302 110.2 1.38
Ⅲ 6,17-Dihydroxyandrost-4-en-3-one C19H28O3 304 55.6 0.70
Ⅳ 6b-Hydroxyandrost-4-ene-3,17-dione C19H26O3 302 139.7 1.75
Ⅴ 17-Hydroxyandrost-4-en-3-one C19H28O2 288 600 7.50
Ⅵ 15a,17-Dihydroxyandrost-4-en-3-one C19H28O3 304 17.5 0.22
Ⅶ 15b ,17-Dihydroxyandrost-4-en-3-one C19H28O3 304 34.2 0.43
Ⅷ 14-Hydroxyandrost-4-ene-3,17-dione C19H26O3 302 33.5 0.42
Ⅸ 17-Hydroxyandrost-4-ene-3,16-dione C19H26O3 302 17.2 0.22
LIU Ying et al.: Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus 937
1 336, 1 273, 1 230, 1 138, 1 092, 1 012 cm-1; ESI-MS: m/z 303;
1H-NMR (CDCl3, 500 MHz) d: 0.91 (3H, s, H-18), 1.20 (3H, s,
H-19), 4.38 (1H, dd, J1=5.5 Hz, J2=12 Hz, H-6), 6.20 (1H, s, H-
4). The structure was identified by comparison with previ-
ous data (Reich et al., 1969; Holland and Thomas, 1982).
Compound Ⅴ (17b-hydroxyandrost-4-en-3-one)
C19H28O2, colorless needles, mp 162-164 ℃, [a]25D +100.1°
(c 0.85, MeOH). UV lmax (MeOH): 241.2 nm; IRnmax (KBr):
3 400, 2 952, 2 933, 2 850, 2 821, 1 658, 1 612, 1 450, 1 346,
1 331, 1 273, 1 228, 1 190, 1 070, 1 059 cm-1; ESI-MS: m/z 289;
1H-NMR (CDCl3, 500 MHz) d: 0.78 (3H, s, H-18), 1.18 (3H, s,
H-19), 3.62 (1H, t, J = 9 Hz, H-17), 5.72 (1H, s, H-4). Therefore,
the structure of compound Ⅴ was identified as 17b-
hydroxyandrost-4-en-3-one. This conclusion is in line with
previously report (Reich et al., 1969).
Compound Ⅵ (15a,17b-dihydroxyandrost-4-en-3-
one) C19H28O3, colorless needles, mp 102-110 ℃, [a]25D
+136°(c 0.88, MeOH). IRnmax (KBr): 3 500, 3 350, 2 941,
2 846, 1 660, 1 610, 1 452, 1 356, 1 236, 1 130, 1 049 cm-1; ESI-
MS: m/z 305; 1H-NMR (CDCl3) d: 0.80 (3H, s, H-18), 1.20
(3H, s, H-19), 3.89（1H, t, J = 9 Hz, H-17）, 4.11 (1H, ddd,
J1=3.5 Hz, J2=9 Hz, H-15), 5.73 (1H, s, H-4). The structure
was identified by comparison with previous data (Hiroshi
et al., 1977).
Compound Ⅶ (15b,17b-dihydroxyandrost-4-en-3-
one) C19H28O3, colorless needles, mp 220-222 ℃, [a]25D
+57°(c 1.0, EtOH). IRnmax (KBr): 3 450, 2 945, 2 426, 1 655,
1 610, 1 450, 1 333, 1 227, 1 147, 1 068, 1 011 cm-1; ESI-MS:
m/z 305; 1H-NMR (CDCl3) d: 0.98 (3H, s, 18-H), 1.22 (3H, s,
19-H), 3.44 （1H, t, J = 8.5 Hz, 17-H）, 4.10 (1H, ddd,
J1=2.5 Hz, J2 = 6 Hz, J3 = 7.25 Hz, 15-H), 5.67 (1H, s, 4-H). The
structure was identified by comparison with previous data
(Hiroshi et al., 1977; Kirk et al., 1990).
Compound Ⅷ (14a-hydroxyandrost-4-ene-3,17-
dione) C19H26O3, colorless needles, mp 261-263 ℃, [a]25D
+162°(c 2.16, CHCl3). UV lmax (MeOH): 240 nm; IRnmax
(KBr): 3 423, 2 951, 2 864, 1 745, 1 655, 1 608, 1 448, 1 410, 1
360, 1 342, 1 281, 1 234, 1 192, 1 030, 1 012 cm-1; ESI-MS: m/
z 303; 1H-NMR (CDCl3, 500 MHz) d: 1.04 (3H, s, H-18), 1.22
(3H, s, H-19), 5.74 (1H, s, H-4). The structure of compound
Ⅷ was identified as 14a-hydroxyandrost-4-ene-3,17-dione.
This conclusion is in line with previously report (Ryoji et
al., 1972; Crabb et al., 1980; Mahato et al., 1984).
Compound Ⅸ (17b-hydroxyandrost-4-ene-3,16-dione)
C19H26O3, colorless needles, mp 161-162.5 ℃, [a]25D +66°
(c 1.0, CHCl3). UV lmax (MeOH): 234.6 nm; IRnmax (KBr):
3 427, 2 939, 2 856, 1 749, 1 670, 1 614, 1 452, 1 379, 1 230,
1 086, 1 051 cm-1; ESI-MS: m/z 303; 1H-NMR (CDCl3, 500
MHz) d: 0.76 (3H, s, H-18), 1.21 (3H, s, H-19), 3.76 (1H, s, H-
6), 5.74 (1H, s, H-4). The structure was identified by com-
parison with previous data (Reich et al., 1969).
3 Discussion
All the results make it clear that the suspension cultures
of Catharanthus roseus have an obvious ability to modify
DHEA. The biotransformation of DHEA involves various
reactions, such as: (1) regiospecific hydroxylation at C6,
C14 and C15; (2) selective oxidation and reduction at C3 and
Table 2 13C-NMR data of biotransformed compounds (CDCl3, 125 MHz)
C DHEA Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ Ⅶ Ⅷ Ⅸ
1 35.8 35.7 37.1 37.1 36.3 35.7 35.7 36.8 35.7 35.5
2 31.4 33.8 34.2 34.2 33.8 33.9 35.3 34.7 33.9 33.9
3 71.6 199.2 200.1 200.3 199.2 199.5 199.5 202.1 199.2 199.2
4 42.2 124.1 126.5 126.4 120.1 123.9 123.8 124.1 124.2 124.2
5 141.0 170.2 167.7 168.2 170.7 171.2 170.9 175.4 169.5 169.9
6 120.9 32.5 72.8 73.0 68.3 32.8 32.7 32.7 30.3 32.5
7 31.5 31.3 37.2 38.0 40.0 31.5 32.2 32.3 24.5 31.8
8 31.5 35.1 29.4 29.8 30.0 35.7 33.9 33.9 37.9 34.5
9 50.2 53.8 53.6 53.7 53.8 53.9 53.9 55.8 46.9 53.7
10 36.6 38.6 38.0 38.0 39.0 38.6 40.0 40.2 38.6 38.7
11 20.3 20.3 20.3 20.6 20.3 20.6 20.5 21.7 19.1 20.3
12 30.8 30.7 31.3 36.4 33.8 36.4 36.6 39.1 25.5 35.4
13 47.5 47.4 47.6 42.9 47.5 42.9 44.3 43.3 52.4 42.4
14 51.7 50.8 50.9 50.5 50.6 50.5 58.5 56.4 80.7 44.5
15 21.9 21.7 21.7 23.3 21.7 23.3 72.5 69.4 32.2 36.2
16 37.1 35.7 35.7 30.5 35.7 30.4 43.6 43.6 33.0 216.4
17 220.1 220.2 220.4 81.7 219.9 81.6 78.7 81.8 217.9 86.1
18 13.5 13.7 13.8 11.1 10.1 11.0 12.6 14.2 17.8 11.3
19 19.4 17.3 19.6 19.5 13.7 17.4 17.5 17.7 17.3 17.4
DHEA, 3b-hydroxyandrost-5-en-17-one.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004938
C17; (3) the double bond shifting from C5-C6 to C4-C5; (4)
selective carbonylation at C16. In other words, there are
several types of enzymes involved in the bioprocess.
Interestingly, the C3 hydroxyl group of DHEA was oxidized
to carbonyl group and the double bond shifted from C5-C6
to C4-C5 for all transformed products identified. A compari-
son of the structures of the substrate and transformed prod-
ucts leads to the proposed biotransformation pathway as
shown in Fig.1.
Biotransformation refers to the technique that converts
various substrates to more useful products using freely
suspended, immobilized plant (or microorganisms) cells or
enzymes derived from those organisms. Biotransformations
employing plant cell cultures cover a wide range of
reactions, such as glycosylation, glycosyl esterification,
hydroxylation, oxido-reductions, methylation and
demethylation, hydrolysis, hydrogenation, etc. To a cer-
tain extent, plant cells act as a poly-enzyme system that can
biocatalyze an exogenous substrate to several products
through various types of reactions. In this paper,
regiospecific hydroxylation (C6, C14, C15) and selective
carbonylation (C16) were observed in the C. roseus cell
line, which showed somewhat reaction diversity. It is de-
duced that the significant different yields of the products
might be due to the activities or amounts of enzymes re-
sponsible for these reactions. Thus, the major biotransfor-
mation of DHEA by C. roseus cells might be considered as
C3 oxidation, C6 hydroxylation, and C17 reduction. Generally,
those compounds were obtained either by chemical syn-
thesis or by microbial conversion. It is the first report that
all of them could be generated by C. roseus cell
transformation. Because of the pharmaceutical activities,
DHEA has drawn a lot of attention. However, this com-
pound has some side-effects in clinical applications, such
Fig.1. Structures of compounds 3b-hydroxyandrost-5-en-17-one (DHEA) and Ⅰ－Ⅸ and their relationships.
LIU Ying et al.: Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus 939
as the dysfunction of human endocrine and metabolism
and even a potential carcinogenicity, etc. On the other hand,
it is difficult to modify the structure of DHEA by chemical
methods. It would be a suitable approach to get the desired
DHEA related compounds through transformation of C.
roseus cells.
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(Managing editor: WANG Wei)