Biotransformation of hydrocortisone by a natural isolate of Nostoc muscorum

Article abstract of DOI:10.1016/j.phytochem.2004.05.015

Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The microorganism was isolated during a screening program from soil samples collected from paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11β-hydroxytestosterone, 11β-hydroxyandrost-4-en-3,17-dione and 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one on the basis of their spectroscopic features.

Full text of DOI:10.1016/j.phytochem.2004.05.015

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2205–2209
www.elsevier.com/locate/phytochem
Biotransformation of hydrocortisone by a natural isolate of
Nostoc muscorum
a,
a
a
Mojtaba Tabatabaei Yazdi *, Homeira Arabi , Mohammad Ali Faramarzi ,
a
Younes Ghasemi , Mohsen Amini , Shadman Shokravi , Farzaneh Aziz Mohseni
b
c
d
a
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451,
Tehran 14174, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 14174, Iran
b
c
Department of Biology, Iranian Center of Culture and Education, Institute of Applied Sciences, Shahid Beheshti University, P.O. Box 19835-371,
Tehran, Iran
d
Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
Received 15 December 2003; received in revised form 1 May 2004
Available online 4 August 2004
Abstract
Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some
androstane and pregnane derivatives. The microorganism was isolated during a screening program from soil samples collected from
paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11b-hy-
droxytestosterone, 11b-hydroxyandrost-4-en-3,17-dione and 11b,17a,20b,21-tetrahydroxypregn-4-en-3-one on the basis of their
spectroscopic features.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Bioconversion; Hydrocortisone; Microalgae; Cyanobacteria; Nostoc muscorum
1. Introduction
The use of microalgae in biotransformation of ste-
roids has been mentioned in some limited articles during
the past 20 years. Abul-Hajj and Qian (1986) indicated
the conversion of androstendione to testosterone with
11 different strains of microalgae. Previtera’s group
showed the transformations of progesterone (Greca
et al., 1996a; Pollio et al., 1994, 1996) and prednisolone
(Greca et al., 1997) in several microalgal cultures within
four studies. In other investigations, steroid substrates
were 5a-androstane-3,17-dione (Fiorentino et al., 1991),
adrenosterone (Greca et al., 1996c), androsta-1,4-di-
ene-3,17-dione (Greca and Previtera, 1996) and 17-hy-
doxy-17a-methylandrosta-1,4-dien-3-one (Greca et al.,
1996b). Bioreactions observed in the mentioned re-
searches were reduction of carbonyl group (Abul-Hajj
and Qian, 1986; Fiorentino et al., 1991; Pollio et al.,
1994), oxidation (Greca et al., 1996b), hydration of C4/
C5 double bond (Greca and Previtera, 1996; Greca
et al., 1997; Pollio et al., 1996), C9/C10 bond cleavage
(Pollio et al., 1994), side chain degradation (Pollio et al.,
The algal transformation of exogenous substrates has
been investigated less than the higher plants (Giri et al.,
2001; Stohs and Rosenberg, 1975; Suga and Hirata,
1990), fungi and bacteria (Mahato and Majumdar, 1993;
Mahato and Garai, 1997). The potential of microalgae
in compound alteration has been highly focused on or-
ganic pollutants degradation (Hook et al., 1999; Pavlo-
stathis and Jackson, 2002; Semple et al., 1999) and
steroid modification (Abul-Hajj and Qian, 1986; Fio-
rentino et al., 1991; Greca et al., 1996a; Greca et al.,
1996b; Greca et al., 1996c; Greca and Previtera, 1996;
Greca et al., 1997; Pollio et al., 1994; Pollio et al., 1996)
because of their importance in environmental safety and
therapeutic roles, respectively.
* Corresponding author. Tel.: +98-216-959095/494993; Fax: +98-
216-461178.
E-mail address: mtabataba@sina.tums.ac.ir (M.T. Yazdi).
0031-9422/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.05.015

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M.T. Yazdi et al. / Phytochemistry 65 (2004) 2205–2209
1996), hydroxylation in different positions (Abul-Hajj
and Qian, 1986; Fiorentino et al., 1991; Greca et al.,
1996a,1997; Greca and Previtera, 1996; Pollio et al.,
1994, 1996), Bayer-Villigar (Greca et al., 1996a), and
some other kinds of rearrangement (Greca et al., 1996b,
1997; Greca and Previtera, 1996). However, no literature
report has been found in hydrocortisone biotransfor-
mation by the strains belonging to Nostoc sp.
Nostoc muscorum, a freshwater blue-green alga, is
well known in producing a numerous important en-
zymes, including peroxidase (Tupyk and Los, 1978),
hydrogenase (Tamagnini et al., 2002), nitrate reductase
(Kumar et al., 1985), nitrogenase (Verma et al., 1990),
glutamine synthetase and aminoligase (Srivastava and
Alma, 1997), malate dehydrogenase (Sallal and Nimer,
1990), ascorbate peroxidase (Tel-OR and Huflejt, 1986)
and alkalin phosphatase (Subramanian et al., 1994).
This study is focused on the ability of an isolated
strain of the cyanobacterium Nostoc muscorum PTCC
1636 to convert hydrocortisone as an exogenous
substrate.
third metabolite (III) had the same Rf , melting point and
spectral data with hydrocortisone, so it was concluded
that it is unconverted substrate and the last metabolite,
11b,17a,20b,21-tetrahydroxypregn-4-en-3-one (IV), had
a Rf value less than hydrocortisone. The mass spectra of
compound I and II showed the molecular ions at m=z 302
and 304, respectively, which suggested the reduction of 60
and 58 units of m=z as compared to hydrocortisone (m=z
362). The IR spectra indicated the existence of at least one
hydroxyl group in compounds I and II. Furthermore, in
compound I, IR spectra showed two absorptions at1661
and 1735 cmꢀ1, which confirmed the existence of two
carbonyl groups in C-3 and C-17, respectively. These IR
data have been also supported by the related 13C NMR
spectra (Table 1). Two signals at d 199.3 and 218.9 in 13
C
NMR spectra have been imputed to C3 and C17, re-
spectively. In compound II, the IR spectra showed only
one carbonyl group at 1662 cmꢀ1, which was conjugated
to C4/C5 double bond. The resonances at d 3.8 and 4.38
1
in H NMR spectra clearly showed the existence of two
hydroxyl groups. The chemical shift of H-11 was re-
ported for hydrocortisone and other 11-hydroxylated
steroids in d 4.3–4.4 (Kirk et al., 1990), so the resonance
in d 3.8 has been attributed to CH–OH in C-17. These
data were supported by 13C NMR, which showed a
downfield resonance at d 84.0 for CH–OH in C-17. The
mass spectra of compound IV showed the molecular ion
at m=z 364 which indicated the addition of two units of
m=z as compared to that of hydrocortisone (m=z 362). It
2. Results and discussion
Hydrocortisone (III) was undergone the transforma-
tion reactions in the cultures of the blue-green alga
Nostoc moscurum PTCC 1636. At the end of incubation
procedure, the cultures were analyzed for metabolites by
TLC. Thus, four products with the following character-
ization were identified (see Fig. 1). 11b-Hydroxyandrost-
4-en-3,17-dione (I) and 11b,17b-dihydroxyandrost-4-en-
3-one (II), both were less polar than the substrate and the
Table 1
13C NMR signals of the substrate and metabolites (d in parts per
million (ppm) downfield from TMS, in CDCl3)
Carbon atom Compounds
I
II
37.9
III
34.1
IV
33.8
V
O
OH
1
2
35.3
33.8
34.1
H3C
H3C
HO
33.7
198.2
122.2
171.1
29.3
32.1
31.9
55.1
35.0
66.7
45.6
42.7
48.9
22.6
30.6
84.0
14.1
20.8
–
33.8
199.1
121.5
172.7
32.8
31.5
31.6
55.9
38.5
66.1
39.3
46.0
51.5
23.2
32.5
88.5
161
33.1
199.8
122.2
172.7
32.1
29.7
29.3
55.1
41.5
68.5
39.2
46.7
50.6
23.6
32.8
85.2
17.8
20.9
73.2
64.6
33.8
199.1
121.5
172.7
32.8
31.5
31.6
55.9
38.5
66.1
39.3
46.0
51.5
23.2
32.5
88.5
161.1
20.4
212.3
65.8
HO
3
199.3
122.6
171.5
31.8
31.5
36.9
56.7
41.0
67.9
39.3
46.7
52.4
21.4
35.0
218.9
15.8
21.1
–
H3C
H3C
4
5
6
O
O
7
8
(II)
(I)
9
CH OH
2
10
11
12
13
14
15
16
17
18
19
20
21
CH OH
2
CHOH
CO
OH
OH
H3C
H3C
HO
HO
H3C
H3C
O
O
20.4
212.3
65.8
(III)
(IV)
–
–
Fig. 1. The structures of hydrocortisone and the biotransformed
products. 11b-Hydroxyandrost-4-en-3,17-dione (I), 11b,17b-dihy-
droxyandrost-4-en-3-one (II), hydrocortisone (III), 11b,17a,20b,21-
tetrahydroxypregn-4-en-3-one (IV).
I––11b-hydroxyandrost-4-en-3,17-dione; II––11b,17b-dihydroxyan-
drost-4-en-3-one; III––unconverted substrate; IV––11b,17a,20b,21-
tetrahydroxypregn-4-en-3-one; V––hydrocortisone standard.

M.T. Yazdi et al. / Phytochemistry 65 (2004) 2205–2209
2207
can be imagined that one carbonyl group or double bond
in the structure of hydrocortisone has been reduced. The
IR spectra showed only one carbonyl group at 1649 cmꢀ1
that indicated that the conjugated ketone in C-3 position
has not been altered. The elimination of 20-carbonyl
group absorption in IR spectra showed that the reduction
has been done in C-20. Additional multiplet resonance at
relative to TMS. Coupling constants (J) are given in
hertz (Hz). The IR spectra were determined on a Mag-
na-IR 550 Nicolet FTIR spectrometer. Optical rotations
were measured on solutions of methanol in 1-dm cells on
a Perkin–Elmer 142 automatic spectropolarimeter.
Melting points (m.p.) were determined on a Reichart-
Jung hot stage melting point apparatus and were
uncorrected. Thin layer chromatography (TLC) and
preparative TLC were performed, respectively, on 0.25
and 0.5 mm thick layers of silica gel G (Kieselgel 60
HF254 þ 366, Merck). Layers were prepared on glass
plates and activated at 105 °C, 1 h before use. Chro-
matography was performed with chloroform/acetone
(6:4) and visualized by spraying the plates with a mix-
ture of methanol–sulfuric acid (6:1) and heating in an
oven at 100 °C for 3 min until the colors developed.
HPLC analyses were also done using HPLC (JASCO,
JAPAN) equipment on a C18 column (4.6 ꢁ 250 mm).
An isocratic column elution was monitored by UV de-
tector (JASCO 1570 UV/Visible detector). The wave-
length was set on 254 nm and the mobile phase used was
methanol–water (45:55 v/v) at a flow rate 1 ml/min.
1
3.78 in H NMR spectra as compared to the substrate
confirmed the structure of IV. The stereochemistry of C-
20 in the product IV was determined by comparison of its
melting point with the compounds having a a-hydroxyl
and b-hydroxyl groups at C-20 (for a-hydroxyl m.p. 253–
257 °C literature (Gardi et al., 1965) and for b-hydroxyl
m.p. 133–135 °C literature (Kirk et al., 1990)). Melting
point value of the product IV was similar to the com-
pound with b-hydroxyl group at C-20 position (Kirk
et al., 1990).
Winter et al. (1984) reported the isolation of com-
pound IV in 20a-hydroxyl form in the culture of Clos-
tridium scindens containing hydrocortisone. In contrast,
20b-hydroxyl form of this product was obtained using
Arthrobacter globiformis 193 (Arinbasarova et al., 1985),
Eubacterium desmolans and Clostridium cadavaris
(Bokkenheuser et al., 1986) with the same substrate.
As these results indicate, the isolated alga is able to
effect on the cyclopentane ring and the side chain at-
tached to the D-ring of the substrate. 20-Ketone re-
duction was mainly occurred to accumulate the
compound IV. Side chain degradation to prepare two
androstane derivatives (I, II) was also seen. However, no
alteration was found in the rest of the molecule. Thus,
the isolated strain of N. muscorum may be considered
useful biocatalyst for some kinds of biotransformations.
It has a potential for site- and regioselective biocon-
version of hydrocortisone and probably other pregnane
like steroids.
3.3. Collection, preservation and identification of the alga
The blue-green alga was isolated during a screening
program from soil samples collected from paddy fields
of north of Iran (Mazandaran and Golestan provinces)
from May to November 2001. Primary culturing was
done in BG-11 and modified Allen and Arnon medium
(Allen, 1968; Borowitzka, 1988). After colonization,
pure cultures of living specimens were prepared using
subculturing with agar plate method in BG-11 medium
(Allen, 1968). Preserved specimens were prepared and
the living specimens were incubated in 50 ml-tubes
bubbling by one percent carbon dioxide. Constant illu-
mination was used at 40 lE mꢀ2 Sꢀ1 intensity with white
fluorescent lamps. Temperature was 25 ꢂ 2 °C. The
identification was done using semi-permanent slides
(glycerin mount) and living specimen according to cya-
nobacter genera (Anagnostidis and Komarek, 1988).
3. Experimental
3.1. Chemicals
Hydrocortisone (pharmaceutical grade) was kindly
donated by Aburaihan Pharmaceutical Co. (Tehran,
Iran), which had been purchased from Pharmacia &
Upjohn S.A. (Guyancourt, USA). All other reagents
and solvents were of analytical grade and purchased
from Merck (Germany).
3.4. Identification of the algae
The strain was recognized by colony with indefinite
and irregular shape, black to dark brown; filaments
compact and spirally arranged; cells 3–4 in diameter,
oblong cylindric, 5–8 lm in length; heterocysts 5–7 lm
in diameter, oblong; akinestes 5 lm broad, 7–10 lm
long, several attached to each other. According to these
characters and comparing with the keys of cyanobacte-
ria genera (Anagnostidis and Komarek, 1988), the se-
lected strain was identified as a genus of Nostoc. The
classification of the isolate alga was performed by Per-
sian Type Culture Collection (PTCC), Tehran, Iran, as a
strain of N. muscorum, PTCC 1636. The strain was
3.2. General experimental procedures
The EI-MS spectra were obtained with a Finnigan
MAT TSQ-70 instrument. The 1H and 13C NMR
spectra were recorded at 400 and 100 MHz with a
FTNMR Varian Unity Plus spectrometer in CDCl3.
Chemical shifts (d) are given in parts per million (ppm)

2208
M.T. Yazdi et al. / Phytochemistry 65 (2004) 2205–2209
maintained on BG-11 agar slope and freshly subcultured
before using in transformation experiment.
3.7.3. (III) Hydrocortisone
This compound was crystallized from methanol; (305
mg), m.p. 217 °C, [a]D +163–164° (MeOH), lit. (Hill
et al., 1991) IR mmax 3431, 2931, 1710, 1639 cmꢀ1; MS
(EI) m=z (%) 362 (38) (Mþ, C21H30O5), 344 (20), 329
(16), 303 (25), 285 (65), 242 (62), 227 (100), 173 (30),
3.5. Fermentation conditions
The experiment was conducted in twenty 500-ml
conical flasks, each containing 100 ml of BG-11 liquid
medium, illuminated continuously with fluorescent
lamps at 40 lE mꢀ2 Sꢀ1, and incubated at a temperature
of 25 ꢂ 2 °C without shaking for five days. Hydrocorti-
sone (1 g) was dissolved in 40 ml of absolute ethanol.
Two milliliters of the ethanol solution was added to each
500-ml conical flask (final concentration of the substrate
was 0.05% in each flask). Incubation was continued for
another seven days at the same conditions and the con-
trol was similarly processed without the microorganism.
1
161.1 (29), 123 (52), 91 (45), 74 (35), 55 (45); H NMR
(CDCl3) d 0.74 (3H, s, H-18),1.36 (3H, s, H-19), 4.62
(1H, m, H-11), 5.55 (1H, s, H-4); Rf in chloroform/
acetone (6:4) 0.49.
3.7.4. (IV) 11b,17a,20b,21-Tetrahydroxypregn-4-en-3-
one
This compound was crystallized from methanol; (215
mg), m.p. 128–130°C, [a]D +91° (MeOH), lit. (Carvajal
et al., 1959) m.p. 133–135°C, [a]D +85°; IR mmax 3462,
1649 cmꢀ1; MS (EI) m=z (%) 364 (18) (Mþ, C21H32O5),
346 (18), 331 (7), 315 (56), 303 (46), 285 (100), 267 (31),
3.6. Analytical procedure
1
227 (64), 148 (38), 124 (40), 91 (82), 79 (55); H NMR
At the end of incubation, the contents of the flasks
were extracted with three volumes of chloroform. The
extract was dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The residue was
loaded on preparative TLC and fractionated with
chloroform/acetone (6:4) solvent system and then puri-
fied metabolites were crystallized in methanol. Purified
metabolites were identified by melting points and spec-
(CDCl3) d 1.08 (3H, s, H-18), 1.46 (3H, s, H-19), 3.66
(2H, m, H-21), 3.78 (1H, m, H-20), 4.36 (1H, m, H-11),
5.62 (1H, s, H-4); Rf in chloroform/acetone (6:4): 0.1.
Acknowledgements
The authors appreciate Mr. Khosro M. Abdi, Majid
Darabi and Afshin Dalvandi for their kind collabora-
tion in spectral studies.
1
tral data (13C NMR, H NMR, FTIR and MS). The
purity as well as the amount of individual metabolites
was checked with HPLC analysis.
3.7. Bioconversion of hydrocortisone
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