Metabolism of helenalin by Nocardia speciesNRRL5646 and Mortierella isabellina ATCC 38063

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Metabolism of Helenalin by Nocardia species NRRL 5646 and
Mortierella isabellina ATCC 38063
Galal T. Maatooq,a* Amani M. Marzouk,a Hany N. Baraka a and John
P.N. Rosazzab
a Pharmacognosy Department, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt, E-mail: galaltm@yahoo.com,
Telefax: +2 050 224 7496.
b Division of Medicinal and Natural Products Chemistry, College of
Pharmacy, and Center for Biocatalysis and Bioprocessing, University
of Iowa, Iowa City IA 52242.
*Author for correspondence and reprint requests.
Key words : Helenalin, Asteraceae, Biotransformation
Abstract
The antitumor sesquiterpene lactone, helenalin occurs in Helenium
species and some other Asteraceae herbs. Helenalin was metabolized
by Nocardia species NRRL 5646 and Mortierella isabellina ATCC
38063. Both microorganisms catalyzed the reduction of the exocyclic
double bond to afford 11α,13-dihydrohelenalin (plenolin, 2) (4.61,
13.33 %, respectively) followed by opening of the lactone ring to give
a new product (3) (6.92, 16.66%, respectively). Only Nocardia species
NRRL 5646 was able to reduce the endocyclic double bond to give
2,3-dihydrohelenalin (1) (6.15 %), which also is a new product.
1

Introduction
Helenalin is a sesquiterpene lactone of the 10α-
methylpseudoguaianolide-type (helenanolides).
Numerous helenanolides were isolated from
many genera of Asteraceae. 1-7 They possess a
variety of biological and pharmacological
activities such as anti-microbial,
antitrypansomal, antitumor, antiinflammatory,
central nervous system and cardiovascular
effects. 8-12 Helenalin esters and 11α,13-
dihydrohelenalin (plenolin) are responsible for
the pharmacological activity of flowerheads of
Arnica montana and other species of the genus
Arnica. The α,β-unsaturated carbonyl structures
are being held responsible for their activities
since they undergo Michael additions with
biological neucleophiles such as sulfhydryl
groups of enzymes causing their deactivation.
Helenalin possesses two potential alkylating
centers and unique molecular geometry.7
Nocardia species NRRL 5646 possesses an
astonishing array of enzymes that catalyzes
numerous valuable biotransformation reactions
with many natural product substrates.13 This
article describes the metabolic study of helenalin
using Nocardia species NRRL 5646 and
Mortierella isabellina ATCC 38063 for
production of new metabolites which could be
potentially more active and or/less toxic.
Results and discussion
The microbial metabolism of helenalin using
Nocardia species NRRL 5646 and Mortierella
isabellina ATCC 38063 resulted in the isolation
and identification of three different metabolites,
two of which are new (1 and 3), while metabolite
2 is a new natural product isolated from some
members of the Asteraceae .
Metabolite 1 was identified as 2,3-
dihydrohelanalin, which is a new metabolite for
helenalin, based on spectral evidence. The ESI-
MS gave m/z 264 as M+. This indicated the
presence of a metabolite with two mass units
more than the substrate which could be a
dihydroderivative. Carbon-13 spectrum of 1, as
compared with that of the substrate, indicated the
disappearance of the carbon resonances at δ
163.9 and 129.9 assigned for 2 and 3-positions,
respectively, indicating their likely reduction.
This was further confirmed by 1H-NMR, where
the two double doublets at δ 7.69 and 6.9,
assigned for 2 and 3-positions, respectively, were
absent. The new carbon signals at δ 21.9 and 35
were correlated with the proton signals at δ
(2.02, 1.54) and (2.13, 2.47), respectively, by
HMQC. The 1H and 13C-NMR data of 1 are listed
in table 1.
Metabolite 2 was identified through its spectral
data as the naturally occurring 11α,13-
dihydrohelenalin (plenolin) by comparing to
literature data.14-16 The ESI-MS gave m/z 264 for
M+, indicating the probable presence of a
dihydro-derivative. The carbon resonances at δ
140.1 and 122.1, assigned for 11 and 13-
positions, and the proton resonances at δ 6.30
and 5.79, assigned for the two exomethylene
protons at 13-position, were absent. A new
methyl carbon signal at δ 13.9 and a methine
signal at δ 37.2 which were correlated by HMQC
with the proton doublet at δ 1.37and a multiplet
at δ 2.58, were assigned for 13 and 11-positions,
respectively.
The orientation of H-11 was proved to be α
based on ROESY experiment, where cross peaks
with H-7 and H-8 were observed. The 1H and
13C-NMR data of 2 are listed in table 1.
Metabolite 3 spectral data were very close to
those of 2. The ESI-MS gave m/z 305 for (M+
+Na) suggesting the likely hydration of
metabolite 2 to give the new metabolite 3 by
opening of the lactone ring. This was further
confirmed by the significant difference in proton
and carbon resonances of positions 6, 7, 8, 11, 12
and 13 for 3 from 2 (table 1). Further structure
confirmations came through HMQC, HMBC and
ROESY spectra. The 1H and 13C-NMR data of 3
are listed in table 1. In conclusion, three
metabolites for helenalin were obtained
following microbial biotransformation. Two of
them are new, while the other is a known natural
product. Whether they are biologically active or
not is still needing experimental evidence and
will be carried out in our laboratory. However,
previous chemical modifications of the
structurally related pseudoguaianolide parthenin,
also resulted in several analogues. Those which
sowed reduction of the endo- as well as the
exocyclic double bonds had significant
biological activity compared to the parent
compound.17
Experimental
General
NMR spectra were obtained in CDCl3 using
TMS as the internal standard with chemical
shifts expressed in δ and coupling constants (J)
in Hz. Routine 1H, 13C NMR and DEPT spectra
were obtained with a Bruker NMR 300 (Bruker
Instruments, Billerica, US), operating at 300
2

MHz (1H) and 75 MHz (13C). HMQC, HMBC
and ROESY NMR experiments were carried out
using a Bruker AMX-600 high-field
spectrometer equipped with an IBM Aspect-2000
processor and with VNMR version 4.1 software.
Mass spectrometry was obtained as electrospray
ionization spectra (ESI-MS) taken on a VG-
ZAB-HF reversed-geometry (BE configuration,
where B is a magnet sector and E is an
electrostatic analyzer) mass spectrometer (MS)
(VG Analytical Inc., US).
Flash column liquid chromatography was
performed using J.T. Baker glassware with 40µ
Si gel (Baker) as the stationary phase. TLC was
carried out on precoated Si gel 60 F254 (Merck)
plates. Developed chromatograms were
visualized under UV light and by spraying plates
with 0.01% vanillin/H2SO4, followed by heating
at 100 o C for 5-10 Sec.
Microorganisms and substrate
Nocardia species NRRL 5646 and Mortierella
isabellina ATCC 38063 were maintained in the
University of Iowa, College of Pharmacy culture
collection on slants of sabouraud-dextrose agar
or sporulation agar (ATCC medium #5).18
Helenalin was obtained from Dr. Rosazza`s
inventory. The purity of the substrate was
determined by TLC, 1H- and 13C-NMR
spectrometry. 19-21, 7
Analytical scale fermentation
Nocardia species NRRL 5646 and Mortierella
isabellina ATCC 38063 cultures were grown
according to the standard two-stage fermentation
protocol.22 The experiments were carried out in
125 ml DeLong culture flasks. The culture flasks
held one fifth of their volume of the following
medium: 2% glucose, 0.5% soybean meal, 0.5%
yeast extract, 0.5% NaCl and 0.5% K2HPO4. The
pH of the medium was adjusted to 7 using 6 N
HCl before autoclaving for 20 min at 121o C and
15 psi. After inoculation with the
microorganism, stage I cultures were incubated
by shaking at 200 rpm at 30o C on New
Brunswick Scientific Innova 5000 Gyratory Tier
shaker (Edison, US) for 72 h before being used
to inoculate stage II culture flasks. Usually, 10%
inoculum volumes are recommended. Two mg
of helenalin, dissolved in 20 µl
dimethylformamide (DMF) were added to each
flask of 24-hour- old stage II cultures, which
were incubated again and sampled periodically
for analysis. Samples of 1 ml each were taken
after 12, 24, 36 and 48 h and every other day for
two weeks following substrate addition. Each
sample was extracted by shaking with 0.5 ml of
10% n-butanol/EtOAc and spun at 3.000xg for 1
m in a desk-top centrifuge. EtOAc extracts from
all samples were spotted on Si gel GF254 TLC
plates, developed in 10% MeOH/CH2Cl2 solvent
system, and spots made visible with
vanillin/H2SO4, followed by heating for 5-10 s.
Preparative scale fermentation
Thirteen 125 ml DeLong culture flasks stage II
cultures of Nocardia species NRRL 5646 each
received 10 mg helenalin in DMF. After
incubation for 10 days under the usual condition,
the cultures of Nocardia species NRRL 5646
were combined, and exhaustively extracted with
3x2 liter of 10% n-butanol/EtOAc. The extract
was dried over anhydrous Na2SO4 and
evaporated under reduced pressure to yield a
crude residue of 134 mg.
Four 125 ml DeLong culture flasks containing
stage II cultures of Mortierella isabellina ATCC
38063 received 5 mg, each, of helenalin in
DMF. After incubation for 12 days under the
same conditions the cultures of Mortierella
isabellina ATCC 38063 were combined and
exhaustively extracted with 3x0.5 liter of 10% n-
butanol/EtOAc. The extract was dried over
anhydrous Na2SO4 and evaporated under reduced
pressure to yield a crude residue of 34 mg.
Isolation and purification of the metabolites
Nocardia species extract (130 mg) was column
chromatographed by the flash method, 20 g Si
gel 0.5x30 cm. The elution was achieved using
300 ml each of Hexane-CH2Cl2 (1:1) containing
increasing proportions of MeOH (1-5%). Ten ml
fractions were collected. Fractions eluted with 2-
3% MeOH showed one spot which gave red,
colour changing into purple and then grey on
spraying with vanillin/H2SO4 spray reagent and
heating. These afforded one product (52 mg, Rf=
0.71, 10% MeOH/CH2Cl2). Purification on
RpC18 silica gel column using 50% MeOH/H2O
afforded two fractions. Final purification of the
first fraction was achieved on preparative TLC
using 20% isopropyl alcohol /Hexane to afford 9
mg of metabolite 3 as a white powder (6.92 %)
(Rf= 0.37, 20% isopropyl alcohol/Hexane). Final
purification of the second fraction was achieved
on preparative TLC using 12.5% isopropyl
alcohol /Hexane to afford 6 mg of metabolite 2
and 8 mg of metabolite 1 as white powders
(4.61 and 6.15 %, respectively) (Rf = 0.42
3

and 0.5, respectively, 20% isopropyl alcohol
/Hexane).
Mortierella isabellina extract (30mg) was
repeatedly purified on preparative TLC using
20% isopropyl alcohol /Hexane (double
development) to give metabolites 2 (4mg) and 3
(5mg) as white powders (13.33 and 16.66%,
respectively).
ESI-MS of 1 and 2 gave ion peaks at m/z = 264
(M+), while that for 3 gave ion peaks at m/z 305
(M+ + Na), 287 (M+ +Na-H2O), 261 (M+ +Na-
CO2) in addition to cluster ions at m/z 588 (2M+
+1+Na) and 870 (3M+ +1+Na). 1H and 13C-NMR
data of helenalin and its metabolites 1-3 are
listed in table 1.
References
1. Romo, J., De Vivar, A.R., Herz, W.
"Constituents of Helenium species XIV.
The structure of mexicanin E"
Tetrahedron (1963) 19, 2317-2322.
2. Romo, J., Joseph-Nathan, P., Diaz, F.
"The constituents of Helenium
aromaticum (Hook) Bailey. The structure
of aromatin and aromaticin" Tetrahedron
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3. Herz, W. "Constituents of Helenium
species. XII. Sesquiterpene lactones of
some southwestern species" Journal of
Organic Chemistry (1962) 27, 4043-4045
(and references therein).
4. Lee, K-H., Imakura, Y., Sims, D.,
McPhail, A.T., Onan, K.D. "Antitumor
sesquiterpene lactones from Helenium
microcephalium: Isolation of mexicanin-E
and structural characterization of
microhelenin-B and –C" Phytochemistry
(1977) 16, 393-395.
5. Bohlmann, F., Tsankova, E., Jakupovic, J.
"Pseudoguaianolides and guaianolides
from Helenium puberulum"
Phytochemistry (1983) 22, 1822-1824.
6. Maldonado, E., Flores, E., Ortega, A.
"Constituents of Helenium
scorzoneraefolium" Journal of Natural
Products (1986) 49, 1152.
7. Schmidt, T.J. "Helenanolide type
sesquiterpene lactone. Part 1.
Conformations and molecular dynamics
of helenalin, its esters and 11,13-dihydro
derivatives" Journal of Molecular
structure (1996) 385, 99-112 (and
references therein).
8. Lyb, G., Schmidt, T.J., Pahl, H. L.,
Merfort, I. "Anti-inflammatory activity of
Arnica tincture (DAB 1998) using the
transcription factor NF-kappaB as
molecular target" Pharmaceutical
Pharmacology Letters (1999) 9, 5-8.
9. Schmidt, T.J., Burn, R., Willuhn, G.,
Khalid, S.A. "Anti-trypansomal activity of
helenalin and some structurally related
sesquiterpene lactones" Planta Medica
(2002) 68, 750-751.
10. Hall, I.H., Williams, W.L., chaney, S.G.,
Gilbert, C.J., Lee, K.H. "Antitumor
agents. Part 68. Effects of a series of
helenalin derivatives on P-388
lymphocytic leukemia nucleic acid and
protein synthesis" Journal of
Pharmaceutical Sciences (1985) 74, 250-
254.
11. Willuhn, G., Rottger, P.M., Quack, W.
"Investigation of the sesquiterpene
lactones of Arnica flowers" Pharm. Zeit.
(1982) 127, 2183-2185.
12. Lee, K.H., Haruna, M., Huang, H.C., Wu,
B.S., Hall, I.H. "Antitumor agents, 23.
Helenalin, an antitumor principle from
Anaphalis morrisonicola Hay" Journal of
Pharmaceutical Sciences (1977) 66, 1194-
1195.
13. Maatooq, G.T., Rosazza, J.P.N.
"Metabolism of daidzein by Nocardia
species NRRL 5646 and Mortierella
isabellina ATCC 38063" Phytochemistry
(2005) 66, 1007-1011.
14. Itoigawa, M., Kumagai, N., Sekiya, H.,
Ito, K., Furukawa, H. "Isolation and
Structures of Sesquiterpene Lactones from
North-Carolina Helenium-autumnale L.
2" Yakugaku Zasshi-Journal of the
Pharmaceutical Society of Japan (1981)
101, 605-613.
15. Ebert, M. "Inhaltsstoffe von Arnica
viscosa und Arnica nevadensis"
Dissertation. Mathematisch-
Naturwissenschaftlichen, Fakultät der
Universität Düsseldorf (1985).
16. Kos, O. "Phytochemische und
pharmakologisch-biologische Unter-
suchungen von Arnica montana und
Vernonia triflosculosa. Der Fakultät für
Chemie" Dissertation, Pharmazie und
Geowissenschaften der Albert-Ludwigs-
Universität Freiburg Im Breisgau (2005).
17. Ramesh, C., Harakishore, K., Murty,
U.S.N and Das B. "Analogues of
parthenin and their antibacterial activity"
ARKIVOK (online journal) (2003), IX,
126-133.
4

18. Gherna, R., Pienta, P.and Cote, R. "ATCC
Catalogue of Bacteria and
Bacteriophages" 18th ed., American Type
Culture Collection: Rockville, MD, (1982)
P 415.
19. Delgado, G., Alvarez, L., Huerta, E., De
Vivar, A.R. "Carbon-13 NMR spectra of
some pseudoguaianolides" Magnetic
Resonance Chemistry (1987) 25, 201-202.
20. Pettit, G.R., Budzinski, J.C., Cragg, G.M.,
Brown, P., Johnston, L. "Antineoplastic
agents. 34. Helenium automnale L"
Journal of Medicinal Chemistry (1974)
17, 1013-1016.
21. Herz, W., Govindan, S.V., Bierner, M.W.,
Blount, J.F. "Sesquiterpene lactone of
Hymenoxys insignis. X-ray analysis of
hymenograndin and hymenosignin"
Journal of Organic Chemistry (1980) 45,
493-497.
22. Betts, R.E., Walters, D.E. and Rosazza
J.P.N "Microbial transformation of
antitumor compounds. 1. Conversion of
acronycine to 9-hydroxyacronycine by
Cunninghamella echinulata" Journal of
Medicinal Chemistry (1974) 17, 599-602.
5
O
OH
O
O
O
OH
O
O
OH
OH
O
O
OH
O
O
OH
O
Fig. 1. Metabolites of helenalin by Nocardia species NRRL 5646 and
Mortierella isabellina ATCC 38063
Helenalin
Metabolite 2
1
2
3
4
5
6
7
8
9
11
12
13
14
15
10
1
2
3
4
5
6
7
8
9
11
12
13
14
15
10
Metabolite 1
1
2
3
4
5
6
7
8
9
11 12
13
14
15
10
1
2
3
4
5
6
7
8
9
11
12
13
14
15
10
Metabolite 3

Table 1. 1H and 13C-NMR data for helenalin and its metabolites
*In CDCl3 at 300 MHz (1H) and 100 MHz (13C). Chemical shifts (δ) are expressed in ppm.
Assignments based on DEPT, HMQC, HMBC and ROESY.
Helenalin Metabolite 1 Metabolite 2 Metabolite 3
13C1H (J = Hz) 13C1H (J = Hz) 13C1H (J = Hz) 13C1H (J = Hz)
1 52.2 3.09, ddd
(6.3, 1.5, 1.6)
46.4 2.23, ddd
(6.3, 1.5, 1.6)
50.0 2.91, br ddd 52.2 3.13 ddd
(6.0, 2.7, 2.1)
2 163.9 7.69, dd (1.5, 6) 21.9 2.02, m(α)164.5 7.69, dd(1.8, 5.9) 163.8 7.70, dd (1.8, 6)
1.54, m (β)
3 129.9 6.9, dd (3, 6) 35.4 2.13, (a) dd (8.9 )
2.47, (β) dd (8.9)
130.7 6.13, dd (2.7, 5.9) 129.7 6.10, dd (3, 6)
4 211.0 220.4 212.6 212.9
5 57.6 56.0 57.2 57.6
6 74.9 4.46, br s 77.7 4.27, d (2.4) 70.0 4.24, br s 70.0 4.37, br s
7 52.1 3.56, m48.2 3.50, m54.5 2.59, m50.9 2.98, m
8 79.1 4.98, dt (7, 2) 78.9 4.86, ddd (2.2, 2.2,
2.1)
76.5 4.95, ddd (2.2, 2.2,
2.1)
80.1 4.79, ddd (2.2,
2.2, 2.1)
9 40.7 1.38, m (α)
2.48, m (β) 38.3 1.53, m (α)
2.21, m (β)
35.7 1.80, m (α)
2.29, m (β)
40.5 1.74, m (α)
2.42, m (β)
10 26.9 2.27, m27.0 2.01, m26.6 1.98, m25.8 2.18, m
11 140.1 138.5 37.2 2.58, m40.3 3.05, m
12 170.3 170.4 177.6 179.2
13 122.1 6.30, d (2.7)
5.79, d (3.0 )
124.6 6.36, d (2.5 )
5.83, d (2.3 )
13.9 1.37, d (6.5) 11.4 1.36, d (6.6)
14 20.3 1.27, d (6.6) 20.3 1.05, d (6.6 ) 20.6 1.33, d (6.8) 19.8 1.22, d (7.2)
15 18.9 1.00, s 13.7 0.76, s 18.1 1.19, s 18.0 0.98, s

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