Microbial transformation of parthenolide

Article abstract of DOI:10.1016/S0031-9422(99)00066-7

Microbial transformation of the germacranolide parthenolide using Rhizopus nigricans, Streptomyces fulvissimus and Rhodotorula rubra yielded three new compounds: 11αH-dihydroparthenolide, 9β-hydroxy-11βH- dihydroparthenolide and 14-hydroxy-11βH-dihydroparthenolide. 11βH- dihydroparthenolide was the only common metabolite produced by all microorganisms.

Full text of DOI:10.1016/S0031-9422(99)00066-7

Phytochemistry 51 (1999) 761±765
Microbial transformation of parthenolide
Ahmed M. Galal*, Abdel-Rahim S. Ibrahim, Jaber S. Mossa, Farouk S. El-Feraly
Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Received 4 January 1999; accepted 20 January 1999
Abstract
Microbial transformation of the germacranolide parthenolide using Rhizopus nigricans, Streptomyces fulvissimus and
Rhodotorula rubra yielded three new compounds: 11aH-dihydroparthenolide, 9b-hydroxy-11bH-dihydroparthenolide and 14-
hydroxy-11bH-dihydroparthenolide. 11bH-dihydroparthenolide was the only common metabolite produced by all
microorganisms. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Parthenolide; Hydroxylation; Feverfew; Tanacetum parthenium; Asteraceae; Microbial; Transformation; NMR
1. Introduction
potential of using parthenolide (1) for di€erent indi-
cations, there are no studies concerning the mamma-
lian or microbial metabolism of this compound. This
investigation utilized microorganisms as models to pro-
spectively mimic and predict mammalian biotransform-
ation of parthenolide (1). Furthermore, the microbial
metabolites obtained can be used as reference stan-
dards for monitoring mammalian metabolic studies
(Lin & Rosazza, 1998). The use of microorganisms as
models for xenobiotic metabolism (Rosazza & Smith,
1979) has been successfully utilized in mammalian
metabolism studies of many biologically active natural
and synthetic compounds (Davis, 1988).
Parthenolide (1), a germacranolide sesquiterpene lac-
tone, is the major constituent of the European feverfew
(Tanacetum parthenium ) and several other members of
the Asteraceae and Magnoliaceae (Fischer, Vargas, &
Menelorou, 1991). In recent years, parthenolide (1) has
attracted considerable attention (Castaneda-Acosta,
Fischer, & Vargas, 1993), because of its wide spectrum
of biological e€ects, which include cytotoxic (Ogura,
Cordell, & Farnsworth, 1978; Picman, 1986; Abdel
Sattar, Galal, & Mossa, 1996), antibacterial and anti-
fungal activities (Picman, 1986; Fischer, 1991).
Parthenolide (1) was also found to be the most active
germacranolide against Mycobacterium tuberculosis
and M. avium (Fischer et al., 1998). Reports on its
anti-in¯ammatory (Heptinstall, Williamson, White, &
Mitchell, 1985), antirheumatic action (Patrick,
Heptinstall, & Doherty, 1989) and its action as the
active principle in European feverfew, used in treat-
ment of migraine, have renewed interest in this com-
pound. Thus, many studies on the mechanism of its
action in the treatment of migraine have been docu-
mented (Bejar, 1996; Weber et al., 1997).
2. Results and discussion
Screening-scale experiments have shown that most
microorganisms, used in this study, were capable of
converting parthenolide (1) into compound 2. Thus,
preparative-scale fermentation of 1 with Streptomyces
fulvissimus NRRL 1453B or Rhizopus nigricans NRRL
1477 gave 2 in 20±30% yield. The EIMS of compound
2 showed a parent ion peak at m/z 250. Examination
1
of the H NMR spectrum (Table 1) did not show the
Despite the diverse pharmacological actions and the
two down®eld doublets at d 6.34 and 5.62 due to the
exocyclic methylene in 1. However, a new methyl sig-
nal at d 1.25 (d, J=6.8 Hz), which was attributed to
* Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00066-7

762
A.M. Galal et al. / Phytochemistry 51 (1999) 761±765
Table 1
1H NMR assignments of compounds 1±6
Proton
H-1
1a
2
3
4
5
6
5.21 (dd, br,
4.0, 12.2)
2.09±2.24 (m)
5.15 (dd, 2.3, 11.9)
5.18 (br, d, 10.1)
5.37 (br, dd,12.3, 1.2)
2.14 (m)
5.29 (dd, 12.4, 3.9)
2.18 (br, s)
5.61 (dd, 7.7,7.7)
1.89 (m)
H-2a
H-2b
H-3a
H-3b
2.11 (dddd, 2.3,
6.0, 13.0, 13.0)
2.37 (dddd, 5.0,
11.9, 13.3, 13.0)
1.21 (ddd, 5.5,
5.6, 13.0)
1.87 (br, dd,
15, 6.7)
2.36 (m)
2.46 (ddd, 13.8,
12.2, 12.5)
1.25 (m)
2.46 (dddd, 4.5,
5.4, 12.2, 13.4)
1.12 (ddd, 5.5,
5.6, 13.0)
2.44 (dddd, 13.2,
12.8, 5.7, 5.4)
1.18 (br,d, 6.1)
2.27 (m)
1.21 (m)
2.10 (m)
1.06 masked (dd)
2.13 (m)
2.09±2.24 (m)
2.16 (m)
2.14 (m)
2.11 (dddd, 1.5.
2.0, 7.5, 5.4)
2.68 (d, 9.1)
3.79 (t, 9.1)
H-5
H-6
2.79 (d, 8.9)
3.86 (dd, 8.9, 8.3)
2.69 (d, 9, 10)
3.80 (dd, 8.4,
9, 10)
2.73 (d, 9.0)
3.96 (t, 9.3)
2.60 (d, 8.9)
3.8 (t, 8.6)
2.75 (d, 9.4)
3.82 (t, 9.6)
H-7
2.78 (m)
2.09±2.24 (m)
1.73 (m)
2.09±2.24 (m)
2.38 (m)
±
2.28 (m)
1.80 (m)
2.28 (m)
1.80 (m)
2.25 (m)
±
2.36 (m)
1.55 (m)
2.10 (m)
2.10 (m)
2.36 (m)
2.71 (m)
±
1.96 (m)
1.96 (m)
1.86 (m)
4.16 (m)
±
1.87 (m)
1.87 (m)
1.89 (m)
H-8a
H-8b
H-9a
H-9b
H-11a
H-11b
H-13a
H-13b
H-14
2.4 (dd, 13.7, 5.4)
2.27 (m)
1.56 (m)
1.73 (m)
1.87 (m)
2.72 (dd, 6.2, 7.2)
±
2.27 (m)
±
2.27 (m)
±
±
2.27 (dq, 6.8, 10.3)
1.25 (d, 6.8)
±
2.29 (m)
1.30 (d, 7.0)
±
2.27 (dq, 7.0, 5.2)
1, 24 (d, 7.0)
±
6.34 (d, 3.6)
5.62 (d, 3.1)
1.72 (s)
±
1.24 (d, 7.0)
±
1.18 (d, 10.1)
1.65 (s)
1.68 (s)
1.73 (s)
4.04 (d, 11.8)
and 4.38 (d, 11.8)
1.20 (s)
4.10 (d, 12.7)
and 4.03 (d, 12.7)
1.53 (s)
H-15
1.31 (s)
1.27 (s)
1.29 (s)
1.31 (s)
a Taken from the reference by Ruangrungsi et al. (1987) and included for the purpose of comparison.
H-13 was observed. The physical and spectral proper-
ties (see Section 3) of compound 2 were indistinguish-
an oxygenated methine at d 80.0. The presence in the
IR spectrum of a broad band at nmax 3500 cm
1
able
from
those
reported
for
11bH-
suggested the presence of a hydroxylated methine. The
13C NMR spectra were most informative in determin-
ing the position of the hydroxyl group in 4. A com-
parison of 13C NMR spectra of 4 and 2 indicated
major di€erence in the region around C-9 (Table 2). In
the 13C NMR of 4, C-8 is shifted down®eld by 8.5
ppm, while C-7 is shifted up®eld by about 3 ppm.
These shifts established that the hydroxyl must be
placed at C-9 (Breitmair & Voelter, 1974). The physi-
cal and spectral data of 4 were found to be indistin-
dihydroparthenolide (Ruangrungsi et al., 1987;
Ruangrungsi, Rivepiboon, Lange, & Decicco, 1988;
Fischer et al., 1991; Castaneda-Acosta et al., 1993).
Metabolite 3 could only be formed by two organ-
isms of those tried, namely, Rhizopus nigricans
NRRL1477 and Rhodotorula rubra NRRL y1592.
Preparative-scale fermentation, of parthenolide (1)
with the ®rst organism gave 3 in 13% yield. The MS
and H and 13C NMR spectral data for 3 were gener-
1
ally similar to those of 2, except for some minor di€er-
ences. Most notably, the C-13 13C NMR signal in 3
resonated at d 11.2, versus 13.2 in 2. Furthermore, H-
7, which occupies a pseudoaxial position in 2, was
more deshielded in 3, while, H-13 was more shielded
in 2 than 3 (see Table 1). This observation strongly
suggested that 3 was epimeric with 2 at C-11. Thus
compound 3 is characterized as 11aH-dihydroparthe-
nolide and is hitherto unreported from any other
source.
guishable
from
those
obtained
for
9b-
hydroxydihydroparthenolide. The latter was prepared
by reduction of 9b-hydroxyparthenolide (7), which was
obtained by isolation from Anvillea garcinii (Abdel
Sattar et al., 1996). Compound 4 was also obtained by
incubation of 2 with S. fulvissimus NRRL 1453B.
Microbial hydroxylation of C-9 of germacranolides
might prove to be particularly useful, because naturally
occurring germacranolides with oxygenation at this
position are quite rare.
Metabolism of parthenolide (1) by Streptomyces ful-
vissimus NRRL 1453B gave, in addition to compound
2, a highly polar minor metabolite, compound 4, in a
3% yield. This new metabolite possessed gross NMR
spectral data that were generally similar to those of 2,
except for the presence in the 13C NMR spectrum of
Compound 5 was obtained as a minor metabolite by
Rhizopus nigricans NRRL 1477 (4% yield). It exhibited
1H NMR and 13C NMR signals that were similar, but
not identical, to those of 2, with the C-13 methyl
group resonating at d 13.6 and thus, maintaining its a
disposition. This was further con®rmed by the pro-

A.M. Galal et al. / Phytochemistry 51 (1999) 761±765
763
Table 2
13C NMR assignments of compounds 1±6
Carbon 1a
2
3
4
5
6
1
125.2 d 125.1 d 125.1 d 126.6 d 128.8 d 127.3 d
2
24.1 t
36.3 t
61.5 s
66.3 d
82.4 d
47.6 d
30.6 t
41.1 t
24.0 t
36.3 t
61.4 s
66.3 d
82.1 d
51.9 d
29.7 t
41.1 t
24.0 t
36.8 t
61.4 s
66.6 d
82.2 d
46.8 d
26.2 t
41.0 t
24.2 t
36.8 t
61.8 s
66.5 d
81.7 d
48.9 d
38.2 t
80.0 d
23.9 t
37.0 t
61.7 s
66.7 d
82.6 d
52.0 d
30.6 t
36.5 t
24.0 t
37.0 t
60.4 s
63.9 d
81.7 d
46.8 d
24.6 t
27.0 t
3
4
5
6
7
8
9
10
11
12
13
14
15
134.6 s 134.4 s 134.3 s 136.9 s 138.3 s 140.0 s
139.2 s 42.4 d 41.2 d 42.5 d 43.0 d 41.9 d
169.2 s 179.6 s 178.6 s 177.4 s 178.1 s 178.4 s
121.1 t
16.5 q
17.2 q
13.2 q
16.8 q
17.1 q
11.2 q
16.8 q
17.1 q
13.6 q
11.3 q
17.7 q
13.6 q
59.8 t
17.2 q
13.4 q
65.9 t
18.3 q
a Taken from the reference by Castaneda-Acosta et al. (1993) and
included for the purpose of comparison.
the aerial parts of the local plant Tarchonanthus cam-
phoratus (Al-Sheddi, private communication). M.p.'s
were determined in open capillary tubes using an
Electrothermal 9100 capillary melting-point apparatus
and are uncorr. IR spectra were recorded in KBr using
a
PYE Unicam infrared spectrophotometer and
speci®c rotations were obtained at amb. temperature
on a Perkin-Elmer digital polarimeter model 241 MC.
The H and 13C NMR spectra were obtained in CDCl3
1
on a Bruker DRX-500 NMR spectrometer operating
at 500 and 125 MHz, respectively. The chemical shift
values are reported as ppm using tetramethylsilane
(TMS) as internal standard and coupling constants are
expressed in Hz. Electron ionization (EI) mass spectra
were taken on a Shimadzu QP500 OGC/mass spec-
trometer. Thin layer chromatographic analyses were
carried out on pre-coated silica gel 60 F254 (Merck)
using n-hexane±EtOAc mixtures as solvent systems
and spots were visualized under short wavelength UV
light or by spraying with p-anisaldehyde spray reagent.
The adsorbent used for column chromatography was
silica gel 60/230±400 mesh (EM Science).
duction of 5 by fermentation of 2, using the same
organism. The 13C NMR spectrum of 5, however,
lacked the C-14 methyl signal and instead, it exhibited
a hydroxymethyl group at d 59.8. The broad band at
1
nmax 3500 cm in the IR spectrum con®rmed the pre-
sence of this group. The presence of the less deshielded
methyl signal at d 1.20, due to H-15, established that
hydroxylation, indeed, involved C-14, rather than C-
15, of 1.
Compound 5 was found to be di€erent from 6,
obtained by chemical hydroxylation of the allylic C-14
hydroxyl group of 2, with inversion of con®guration
from trans to cis (Haruna & Ito, 1981). It was then
possible to conclude that the microbial reaction has
proceeded with the retention of the trans con®guration
of the double bond. It is interesting to add that both
metabolites 4 and 5 are products of allylic oxidation
reactions, which are considered common microbial
bio-conversions of many unsaturated steroids and ter-
penoids (Fonken & Johnson, 1972).
3.1. Microorganisms
Microorganisms were obtained from either
American Type Culture Collection (ATCC) or
Northern Regional Research Laboratories (NRRL);
organisms were maintained on Sabourad dextrose agar
(Oxoid) slants at 48C and were used for the prelimi-
nary screening. The twenty microorganisms, used in
this study were: Aspergillus alliaceous NRRL 315,
Aspergillus ¯avipes ATCC 11013, Aspergillus niger
NRRL 599, Aspergillus niger NRRL 2295, Aspergillus
ochraceous NRRL 398, Aspergillus ochraceous NRRL
3. Experimental
( )-Parthenolide (1) was isolated and puri®ed from

764
A.M. Galal et al. / Phytochemistry 51 (1999) 761±765
+
405,
Candida
albicans,
laboratory
isolate,
m/z 266 (>1) [M] ; H and 13C NMR (CDCl3) d: see
Tables 1 and 2, respectively.
1
Cunninghamella blackesleeana MR 198, C. echinulata
NRRL 1382 (ATCC 42616), C. elegans NRRL 1392
(ATCC 10028a), Gymnascella citrina NRRL 6050,
Lindera pinnespora NRRL 2237, Penicillium chryso-
genum ATCC 10002, P. chrysogenum ATCC 10002 k,
P. purpureus UI 193, P. vermiculatum NRRL 10009,
Rhizopus nigricans NRRL 1477, Rhodotorula rubra
NRRL y1592, Saccharomyces cerevisae (Baker's yeast)
and Streptomyces fulvissimus NRRL 1453 B.
3.5. Fermentation of parthenolide (1) with Rhizopus
nigricans NRRL 1477
Parthenolide (1, 350 mg), dissolved in 8.75 ml of
Me2CO, was evenly distributed among 35 ¯asks each
containing 50 ml of stage II cultures. Fermentation
was stopped after 6 days and broth extracted three
times with equal vols of CHCl3. Evapn of the com-
bined extracts gave 490 mg of a semisolid residue.
Fractionation of the crude extract on silica gel column
using 25% EtOAc in hexane gave compounds 2 (30
mg) and 3 (22 mg), respectively.
3.2. Media (Ibrahim, Galal, Mossa, & El-Feraly, 1997)
All fermentation experiments were carried out in a
medium of the following composition: 10 g dextrose,
10 ml glycerol, 5 g yeast extract, 5 g peptone, 5 g
K2HPO4, 5 g NaCl and 1000 ml distilled water. The
pH was adjusted to 6.0 before autoclaving at 1218C
for 15 min.
Compound 2, Rf 0.44 (n-hexane±EtOAc, 7:3); m.p.
156±1578C; [a ]D
(Ruangrungsi et al., 1987); EIMS (m/z) (% relative
intensity): 250 (M+) (>1).
74.08 (c 0.021; CHCl3) ( 628)
Compound 3, Rf 0.39 (30% EtOAc in n-hexane);
m.p. 151±1528C; [a ]D +27.78 (c 0.084; MeOH); IR
(nmax) (KBr) cm 1: 1765 (lactone CO); EIMS (m/z)
(% relative intensity): 250 (M ) (>1); for the H and
13C NMR for 2 and 3 see Tables 1 and 2, respectively.
Compound 5 (8 mg) was obtained by further elution
with 60% EtOAc in n-hexane; colorless gum; Rf 0.36
3.3. Fermentation procedures (Ibrahim et al., 1997)
+
1
Cells of microorganisms were transferred from two-
week old slants into sterile culture media and kept on
gyratory shaker for 72 h to give stage I culture. Stage
I cultures (5 ml) were used as inocula for stage II cul-
tures (50 ml/250 ml ¯ask). After 24 h incubation of
stage II cultures, parthenolide (1) was added as a soln
in Me2CO (10 mg 0.25 ml 1). Both substrate and
organism controls were made. Each fermentation was
sampled by extracting 5 ml of the culture medium with
CHCl3 (5 ml). After evapn of the solvent, the residue
was chromatographed on silica gel G plates using
EtOAc±hexane (1:1) as the mobile phase.
(40% Me2CO in CH2Cl2); [a]D
280.08 (c 0.04;
CHCl3); IR (nmax) (KBr) cm 1: 3610 (OH) and 1760
(lactone CO); EIMS (m/z) (% relative intensity): 266
+
(M ) (>1); for the H- and 13C NMR assignments see
1
Tables 1 and 2, respectively.
3.6. Preparation of 9b-hydroxydihydroparthenolide (4)
from 7
9b-Hydroxy- parthenolide (7) was isolated and puri-
®ed from Anvillea garcinii as reported in the literature
(Abdel Sattar et al., 1996). To compound 7 (55 mg),
dissolved in 4 ml absolute EtOH, sodium borohydride
(35 mg) was added and the mixture was stirred for 1 h
at room temperature. The reaction was quenched by
addition of 0.3 ml of 25% acetic acid, the solvent was
distilled o€, residue dissolved in EtOAc, washed with
10% NaHCO3 and dried over anhydrous Na2SO4.
After evapn of the solvent, the residue was chromato-
graphed on a silica gel column. Elution of the column
using n-hexane±EtOAc (1:1) gave 20 mg of colorless
cubes that were indistinguishable from those of com-
pound 4.
3.4. Fermentation of parthenolide (1) with
Streptomyces fulvissimus NRRL 1453B
Parthenolide (1, 650 mg), dissolved in 16 ml of
Me2CO, was evenly distributed among 65 ¯asks con-
taining stage II cultures. Fermentation was stopped
after 6 days. The mixture was ®ltered and fermentation
broth was extracted three times with equal vols of
CHCl3. The combined extracts were dried over anhy-
drous Na2SO4 and solvent evaporated to give 1.3 g of
a brownish residue. The crude residue was subjected to
preliminary puri®cation by passing its EtOAc solution
through a short column to give 0.78 g of residue,
which was then loaded on a silica gel column. Elution
with n-hexane±EtOAc (2:1) gave 200 mg of compound
2. Further elution of the column with hexane±EtOAc
(1:1) yielded 4, which crystallized from ether to give 20
mg of cubic crystals; Rf 0.26 (n-hexane±EtOAc, 1:2);
3.7. Preparation of 6 from 2
Compound 2 (67 mg) was dissolved in CH2Cl2 (5
ml) and treated with selenium dioxide (16 mg) and
tert-butylhydroperoxide (0.14 ml). The mixture was
stirred at room temperature for 19 h, then CH2Cl2 (50
m.p. 1648C; [a]D 268 (c 0.03; CHCl3); IR (nmax
)
(KBr) cm 1: 3460 (OH) and 1750 (lactone CO); EIMS

A.M. Galal et al. / Phytochemistry 51 (1999) 761±765
765
Castaneda-Acosta, J., Fisher, N. H., & Vargas, D. (1993). J. Nat.
Prod., 56, 90±98.
ml) was added. The reaction mixture was washed with
a 2% soln of sodium bisul®te and the solvent was dis-
tilled o€ to leave an oily residue that was crystallized
from a mixture of n-hexane and EtOAc to give 27 mg
of 6 as cubic crystals, m.p. 195±1968C; Rf 0.28 (20%
MeCN in CHCl3); [a ]D 99.78 (c 0.06; CHCl3); IR
(nmax) (KBr) cm 1: 3450 (OH), 1780 (CO); EIMS (m/
z) (% relative intensity): 266 (M+) (>1); for the 1H
and 13C NMR assignments see Tables 1 and 2, respect-
ively.
Davis, P. J. (1988). Devel. Indust. Microbiol., 29, 197.
Fischer, N. H. (1991). In B. V. Charlwood, & D. V. Banthorpe,
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Fischer, N. H., Vargas, D., & Menelorou, M. (1991). In N. H.
Fischer, M. B. Isman, & H. A. Sta€ord, Modern phytochemical
methods. New York and London: Plenum press.
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Acknowledgements
Ibrahim, A.-R. S., Galal, A. M., Mossa, J. S., & El-Feraly, F. S.
(1997). Phytochemistry, 46, 1193.
Lin, S. T., & Rosazza, J. P. (1998). J. Nat. Prod., 61, 922.
The authors are grateful to Dr. Charles D. Hu€ord,
School of Pharmacy, University of Mississippi, USA,
for the NMR spectra.
Ogura, M., Cordell, G. A.,
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& Farnsworth, N. R. (1978).
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