5α-Reductase inhibition activity of steroids isolated from marine soft ...

Steroids 69 (2004) 439–444
5-Reductase inhibition activity of steroids isolated
from marine soft corals
P. Radhika a , M. Cabeza b,∗ , E. Bratoeff c , G. García d
a Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India
b Department of Biological Systems Metropolitan University-Xochimilco, Mexico D.F., Mexico
c Department of Pharmacy, Faculty of Chemistry, National University of Mexico, Mexico D.F., Mexico
d Institute of Biology National University of Mexico, Mexico D.F., Mexico
Received 25 November 2003; received in revised form 1 March 2004; accepted 5 April 2004
Abstract
The aim of this study is to determine the 5-reductase inhibitory activity of several new steroidal compounds PR-01–PR-07 by measuring
the conversion of [ 3 H]T to[ 3 H]DHT in Penicillium crustosum broths.
These compounds were obtained from marine soft corals collected on the coasts of Andaman and Nicobar at Hori, Natkal and Kalipur
(Diglipur) Islands and identified as Sinularia grandilobata Verseveldt, Sinularia crassa Tixier- Durivault, Sinularia gravis Tixier- Durivault,
Sinularia sp., Lobophytum sp., Lobophytum crassum and Cladiella sp.
PR-01–PR-04 significantly inhibited the conversion of [ 3 H]T to [ 3 H]DHT (P 0.05) in this model. On the other hand PR-07 stimulated (P

440 P. Radhika et al. / Steroids 69 (2004) 439–444
2.2. Collection of corals (PR-01–PR-07)
The soft corals were collected on the coasts of Andaman
and Nicobar at Hori, Natkal and Kalipur (Diglipur) Islands
during March 1993. The collection was carried out manually
in the inter-tidal rocky region at 1 m depth when exposed
under favorable tide conditions. The animals were collected
in plastic net bags and brought to the shore, washed with
fresh water free from detectable adhering other organisms
like algae, cut into thin slices and preserved in ethanol in a
plastic carboy. A large specimen with clear morphological
features was separately preserved in ethanol in small plastic
container for identification purpose. The organisms were
brought to the laboratory for processing.
2.3. Identification
The soft corals were identified as Sinularia grandilobataVerseveldt,
Sinularia crassa Tixier- Durivault, Sinularia
gravis Tixier- Durivault, Sinularia sp., Lobophytum
sp., Lobophytum crassum and Cladiella sp. through the
courtesy of Dr. V. Jayasree, NIO, Goa, India, Dr. B. Grebnev
(Biologist) and Dr. A. Malyutin (Hydrobiologist), Pacific
Institute of Bio-Organic Chemistry, Vladivostok-22, Russia,
and Dr. Phil Alderslade, Curator of Coelenterates, Northern
Territory Museum of Arts and Science, Darwin, Australia.
The voucher specimens of these soft corals were preserved
in the Department of Organic Chemistry, Andhra University,
Visakhapatnam, India and also in the above museum.
2.4. Extraction, isolation and purification
The extraction of the fragmented organisms (dry weights
in kg and g) S. crassa (900 g), S. gravis (1.2 kg), two Sinularia
sp. (800, 900 g), Cladiella sp. (1.78 kg), L. crassum
(1.2 kg), Lobophytum sp. (2.0 kg) and S. grandilobata
(2.0 kg) was carried out at room temperature with ethanol
for 4 days. The process of extraction was repeated until, it
left negligible residue on removal of the solvent. The solvent
was removed under reduced pressure. The dark colored
residue was then repeatedly reextracted with ethyl acetate.
The ethyl acetate extracts were combined, and the ethyl acetate
soluble portion was washed with water, dried over anhydrous
MgSO 4 and the solvents removed under reduced
pressure.
The crude ethyl acetate extracts of S. crassa (50 g), S.
gravis (40 g), two species of Sinularia (40, 55 g), Cladiella
sp. (40 g), L. crassum (30 g), Lobophytum sp. (30 g) and
S. grandilobata (40 g). were chromatographed over a column
of silica gel (500 g, Acme, 100–200 mesh) using eluants
of increasing polarities of solvent mixtures starting from
petroleum ether (bp, 35–80 ◦ C), petroleum ether–ethyl acetate
(0–100%), and ethyl acetate–methanol (0–100%) to
yield several fractions. Fractions (800 ml each) were collected
and monitored with silica gel G (mesh size 200,
Acme) TLC. The visualization of the spots was carried
out by spraying with anisaldehyde–phosphomolybdic acid
reagent and heating at 105–110 ◦ C. Similar fractions were
combined.
The purification of each fraction was effected by rechromatography
over small silica gel (finer than 200, Acme)
columns using respective solvent polarities of hexane, ethyl
acetate and methanol and recrystallisation of PR-01–PR-07
from 90% chloroform in methanol. The purity of the each
compound was ascertained by homogenity over silica gel
TLC [glass plates (5 cm × 20 cm)], preparative TLC [glass
plates (20 cm × 20 cm)] and also over silver nitrate impregnated
silica gel thin layers [glass plates (20 cm × 20 cm)]
and by including reverse phase high-performance liquid
chromatography (HPLC), using a gradient elution of
acetonitrile–water (0–100%); methanol–water (0–100%) to
afford pure compounds.
2.5. Identification of the steroidal compounds
PR-01–PR-07
The isolated steroidal compounds were identified by a direct
comparison with an authentic specimen and by comparing
the spectral data with those of reported compounds in
the literature [8–25].
2.6. The effect of steroids PR-01–PR-07 in the conversion
of [ 3 H]T to [ 3 H]DHT in P. crustosum broths
The role of steroids PR-01–PR-07 in conversion of
[ 3 H]Tto[ 3 H]DHT was determined in cultures of P. crustosum,
as described earlier [4,7]. The broths were incubated
for 5 days in duplicate with 2 nM [ 3 H]T (specific activity
85–100 Ci/mmol), 0.5 mM NADPH, 8.7 M (in 5 l of
ethanol) of the test compounds in a final volume of 1 ml
at pH 6, and the mixtures were incubated in a water bath
at 25 ◦ C. The optimum growth was observed on the fifth
day and no contamination was noticed under incubation
conditions.
All reactions were stopped by adding 1 ml of dichloromethane,
and the steroids were extracted four times with one
ml of dichloromethane. The mycelia were separated from
the medium, dried at room temperature, and weighed. The
organic phase was separated, evaporated to dryness and redissolved
in 200 l of methanol. The isolation and the purity
of the radioactive DHT were assessed by the reverse-isotope
dilution technique. The isolated steroid was purified by thin
layer chromatographic system using steroidal standards (T
and DHT) on both sides of the plates and then developed in
chloroform: acetone (9:1). The steroid standards were detected
using phosphomolibdic acid reagent and ultraviolet
light (254 nm). The zone corresponding to DHT (R F 0.49)
was cut out from the plate and the fraction was placed into
a counting vial. Radioactivity was detected and its loss was
determined during the procedure in agreement with the re-

P. Radhika et al. / Steroids 69 (2004) 439–444 441
PR - 01
OH
PR - 02
OH
HO
OH
O
O
OH
OH
HO
OH
O
O
OH
PR - 03
PR - 04
OH
HO
OH
OH
OH
HO
OH
OH
PR - 05
PR - 06
OH
OH
OAc
HO
HO
OH
OH
PR - 07
O
HO
Fig. 1. Steroidal structures extracted from soft corals.

442 P. Radhika et al. / Steroids 69 (2004) 439–444
35
30
fmol of DHT /mg of mycellium
25
20
15
10
5
0
T PR01 PR02 PR03 PR04 PR05 PR06 PR07
TREATMENT
Fig. 2. Effect of steroidal structures on T conversion to DHT in P. crustosum broths.
sults obtained from the control experiment without the fungi.
The formation of DHT was calculated and expressed in fmol
of DHT/mg of mycelium. A duplicate fraction of DHT was
eluted from the chromatogram and recrystallized to constant
specific activity in order to know the radiochemical purity
of the isolated compound.
3. Results
3.1. Obtained compounds
The pure compounds were obtained from different marine
soft corals as follows:
PR-01 Sinularia crassa Tixier–Durivault, Sinularia gravis Tixier–Durivault, and Sinularia sp.
[Molecular formula: C 34 H 56 O 7 ]: 24-Methylenecholest-5-ene-3,7, 16-triol-3-O--l-fucopyranoside (30 mg)
TLC: (R F 0.36, chloroform: methanol 9:1)
PR-02 Two Sinularia sp. and Cladiella sp.
[Molecular formula: C 34 H 56 O 6 ]: 24-Methylenecholest-5-ene-3,16- diol-3-O--l-fucopyranoside (60 mg)
TLC: (R F 0.43, chloroform: methanol 9:1)
PR-03 Lobophytum crassum
[Molecular formula: C 28 H 50 O 4 ]: (24S)-24-Methylcholestane- 3,5,6,7-tetrol (150 mg)
TLC: (R F 0.54, chloroform: methanol 8.5:1.5)
PR-04 Lobophytum sp.
[Molecular formula: C 28 H 50 O 4 ]: (24S)-24-Methylcholestane-3,5,6,25-tetrol (50 mg)
TLC: (R F 0.43, chloroform: methanol 9:1)
PR-05 Sinularia grandilobata Verseveldt and Lobophytum sp.
[Molecular formula: C 28 H 48 O 2 ]: (24S)-24-Methylcholest-5-ene-3, 25-diol (40 mg)
TLC: (R F 0.35, pet.ether: EtoAc 9:1)
PR-06 Lobophytum sp.
[Molecular formula: C 30 H 52 O 6 ]: (24S)-24-Methylcholestane-1, 3,5,6,25-pentol-25- monoacetate (65 mg)
TLC: (R F 0.46, chloroform: methanol 9:1)

PR-07 Sinularia grandilobata Verseveldt
[Molecular formula: C 21 H 32 O 2 ]: Pregn-5-ene-20-one-3-ol (40 mg)
TLC: (R F 0.35, pet.ether: EtoAc 4:1)
P. Radhika et al. / Steroids 69 (2004) 439–444 443
The structure of compounds PR-01–PR-07 is shown in
Fig. 1.
3.2. Conversion of testosterone to dihydrotestosterone in P.
crustosum broths
Since 5-reductase is present in P. crustosum broths [5],
it was of interest to determine the effect of the steroids
PR-01–PR-07 isolated from marine soft corals of Andaman
and Nicobar Islands on the conversion of [ 3 H]T to [ 3 H]DHT
in this cultures.
The extracts from P. crustosum broths were subjected to
TLC analysis. The zone corresponding to DHT standard
(R F value 0.68) of each experimental chromatogram was cut
from the plate and the fraction was placed into a counting
vial containing Ultima Gold as counting vehicle. The results
obtained from two separate experiments are shown in Fig. 2.
These data indicate an appreciable difference (P 0.05). On the other hand PR-07 stimulated
(P

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