ISOLATION OF ILLUDIN S FROM THE MATURE BASIDIOCARP OF
OMPHALOTUS ILLUDENS, AND
ISOLATION OF ILLUDIN S AND M FROM THE FERMENTATION BROTH
OF OMPHALOTUS OLEARIS
Thomas G. McCloud,* Paul A. Klueh,* Kay C. Pearl,*
Laura K. Cartner,* Gary M. Muschik,* and Karyol K. Poole$
*Chemical Synthesis and Analysis Laboratory and
$Fermentation Production Facility,
SAIC Frederick, NCI-Frederick Cancer Research and
Development Center, Frederick, MD
Presented at the American Society of
Pharmacognosy - 37th Annual Meeting - July 27-31, 1996,
Santa Cruz, CA
In response to a request from the
Developmental Therapeutics Program (DTP) of NCI for
illudin M for research purposes, the Natural Products
Support Group, Chemical Synthesis and Analysis Laboratory,
FCRDC, undertook the investigation of several routes by
which illudin M could be obtained in quantity. An
examination was made of the mature fungal basidiocarp of Omphalotus
illudens, which was found to contain only illudin S,
and subsequently, two species of Omphalotus growing
in liquid shake culture, both of which produced illuden M
and S. Normal and reverse phase chromatographic methods
where developed that allowed for rapid quantitation of the
desired compounds from fermentation broth. Fermentation
conditions where optimized for the production of illudin M
by O. olearis and 4.4 gm of the desired compound
PART 1: Isolation of illudin M from mature
The fruiting bodies of Omphalotus
illudens are found throughout eastern North America,
often in the fall of the year, occurring in large clumps
at the base of decaying trees or stumps. The common name
for O. illudens is 'Jack-O'-Lantern' mushroom both
because the basidiocarp has the orange color of a ripe
pumpkin from which a 'Jack-O'-Lantern is carved, and
because they sometimes exhibit bioluminescence. Although
the toxicity of Omphalotus species is well known,
occasional human poisonings occur when collectors confuse
them with edible Chanterelles or with Laetiporus
sulphureus, the 'Chicken-of-the-Woods' mushroom.5,
Omphalotus illudens was
collected (TGM) on Catoctin Mountain, Frederick County,
MD, on October 4, 1992. These where quickly frozen,
lyophilized, finely ground in a Wiley mill (4mm mesh)
yielding 2.63 kg, which was extracted exhaustively with
dichloromethane (DCM): methanol (MeOH)(1:1). A combined
dry weight of 242 gm of crude extract was obtained.
Extraction of the marc was continued with 100% MeOH and
afforded an additional 86.3 gm of extract. Thin layer
chromatographic analysis (tlc)(on glass backed silica 60 (EM
5729-6) developed onece with chloroform: MeOH (9:1, M:Rf
0.56, S:Rf 0.24) and visualized with vanillin-sulfuric
acid) demonstrated that both of these extracts contained
illudin S. They where combined, resolubilized in
MeOH:water (1:1), and partitioned against n-hexane. Thge
hexane layer was withdrawn, and the aqueous layer waas
re-partitioned against DCM. The DCM solubles where dried in
vacuo to give 12.7 gm of illudin S enriched crude
extract. This was adsorbed onto 25 gm silica (Davison
Chemical, grade 22, 60-200 mesh, lot #12/89) and flash
chromatographed9 on 1.27 kg of the same silica
using hexane: ethyl acetate (1:1) isocratic. Ten pools
where formed based on similar appearance on tlc. No spot
corresponding in Rf to illudin M was observed.
Crystallization of pool 7 (245 mg) from DCM/hexane gave
139.0mg of colorless needles. This crystalline product
exhibited identical NMR, MS, & UV spectra to a
standard of illudin S obtained form the DTP Repository and
to data reported in the literature.3, 12 Purity
of these crystals was shown to be greater than 99% by both
reverse and normal phase HPLC.
A single, mature, freeze-dried
basidiocarp was separated into cap, gill, and stem
portions, each separately solvent extracted and analyzed
for illudin content by reverse-phase HPLC. The three
chromatograms were similar, and indicated that illudin S
was present in all parts, though in lesser concentration
in the stem. No illudin M was detected in the mature
fungal basidiocarp. The ammount of illudin S in an avarage
sized basidiocarp has been calculated to be ~0.5 mg.
*Most mycologist consider O. illudens and O.
olearis to be synonymous, however, both names are
still used. Separation into two species was
formerly based on the larger spore size of O. olearis.
PART II. Isolation of illudin S and M from
Cultures of Omphalotus illudens
(UT 02212) and Omphalotus olearis (American Type
Culture Collection (ATCC) 11719) were grown on
soy-peptone-glucose-starch (SGSM) and PNSM media, shaking,
and on Potato-Dextrose broth (PDB), stationary. Ethyl
acetate extracts from each were assayed by tlc and HPLC
(figure 2) for the production of the desired compound. In
order to quantify illudin during fermentation both normal
and reversed-phase HPLC analytical methods were developed.
The system consisted of a Waters 600E Multisolvent
delivery system with Millenium 2010 Chromatography Manager
v. 2.00 with eluate monitored by a Waters 996 photo diode
array detector. HPLC columns were Rainin Dynamax 8µ
silica, 4.6 x 300 mm with 4.6 x 15 mm guard column,
solvent system: 95% DCM: 5% MeOH isocratic @ 1 ml/min,
retention time; M:4.4 min, S: 7.8 min for normal phase and
Waters Bondpack 10 µ C18, 3.9 x 300, MeOH/water 8:2,
isocratic, @ 1.0 ml/min, retention time; M: 4.4 min, S:
3.2 min for reverse phase. Solvents were HPLC grade and
sparged with helium at 5 cc/min. The detector response was
found to be linear between ~0.1 and 1.0 g/ml @ 254 nm for
pure standards of illudin S and M, which were obtained
from the DTP Repository.
Both cultures were found to produce
illudin S and M, but the quantityand ratio between the two
was variable and greatly affected by culture conditions
FIGURE 2. C-18 reverse phase HPLC
quantitation of illudin S and M in ethyl acetate
extract of O. olearis fermented on SGSM (top)
and PNSM (bottom)
Large Scale Fermentation
When grown shaking on PNSM media,
[dextrose (25 gm/l), soluble starch (10 gm/l), lexein (10
gm/l), NZ Amine (4 gm/l), blackstrap molasses (5 gm/l),
MgSO4*7H2O (5 gm/l), and CaCO3
(2 gm/l)], O. olearis, ATCC 11719, produced ~10
mg/L of the illudins in a 9/1 ratio of M to S, (figure 3).
Extract of the whole broth of this organism grown on PNSM
waas relatively free of contaminating materials with an
HPLC analysis that chloroform preferentially extracted
illudin M, leaving illudin S in the broth.1
O. olearis seed cultures for
scaled up fermentation were grown on PDB, shaking, in 4 L
Erlenmeyer flasks, then used to inoculate two fermentors,
one of 50 L (@ 8.6% v/v), the other 80 L (@ 10.9% v/v)
volume. The production medium was PNSM, pH 5.7, with 0.5%
v/v Dow Corning type A antifoam added. A downward trend
over time to pH 2.5 was observed in the 50 L fermentor,
which was not pH adjusted during fermentation. The pH of
the 80 L fermentor was adjusted upward during fermentation
to maintain a pH of 5.5. The agitation rate was 250 rpm
giving a tip speed of 237 ft/min. Temperature was
controlled at 25% ± o.5%C. Dissolved O was maintained at
20% by adjusting air flow. Aliquots were taken each day
for HPLC quantitation. Illudin M was detected after 5
days, and reached an apparent plateau at 7 days. No
significant difference in illudin production was noted
between the two fermentors. Harvest of both fermentors
occurred at 164 hours. The combined broths were filtered
to remove cellular debris. HPLC analysis of an ethyl
acetate extract of cell debris showed an insignificant
amount of illudin S or M.
Purification of illudin M and S
The clarified broth was first
extracted with chloroform. Solvent was removed by rotary
evaporation and the extract was high vacuum dried, giving
40 gm. The crude extract was resolubilized in chloroform,
adsorbed to 170 gm of Davison grade 22 silica
chromatographic media, and flash chromatographed through
2,000 gm of the same silica in a 10 cm diameter x100 cm
glass column (Kontes 584800-4000) using nitrogen pressure
<5 psi. Elution was started with hexane and progressed
in a step-gradient fashion through 10, 25, 35, and 50%
ethyl acetate in hexane, followed by a strip with MeOH.
Eighteen fractions (1 to 4 liters) were collected, rotary
evaporated and examined by tlc. Based on similarity of
appearance, six pools were made. The second of these, a
yellowish solid weighing 6.8 gm, was resolubilized in
hexane/ethyl acetate and, after sitting overnight at room
temperature, 4.4 gm of colorless needles were obtained.
Analysis by normal and reverse phase HPLC showed this
material to be >99% pure illudin M. Identity was
confirmed by NMR and MS comparison to an authentic sample.
The fermentation broth was
re-extracted exhaustively with ethyl acetate to yield 25
gm dry weight of crude material. Both tlc and HPLC
analysis showed this extract contained illudin S and M.
This extract was flash chromatographed on silica in a
manner analogous to before. Illudin M was detcted by tlc
in pool 2 (0.372 gm) and illudin S was present in pools 5
and 6 (combined weight 2.26 gm). Each pool was
resolubilized in hexane/ethyl acetate and, after sitting
at room temperature overnight, 80 mg of illudin M and 46
mg of illudin S were obtained, respectively.
Illudin S has been isolated from the
mature basidiocarp of Lampteromyces japonicus in 4
x 10-4% yield without illudin M being found.3
Likewise, only illudin S was reported from the mature
basidiocarp of Omphalotus olivascens, in similar
quantity.4 In this work only illudin S has been
in the mature basidiocarp of Omphalotus illudens at
5x 10-3%. This suggests that illudin S itself
must be the toxic compound responsible for instances of
human poisoning. The uncertain availability of the
basidiocarp and low yield makes the fruiting body an
unreliable source from which to obtain illudin S in
quantity, and illudin M appears not to be present in the
basidiocarp of any species so far examined.
Both cultures of Omphalotus were
producers of illudin S and M, but the ratio of these
products was found to be variable and influenced by the
composition of the media. In the work reported here,
culture conditions and medium were optimized for the
production of illudin M, and an isolated yield of ~88 mg/L
was obtained. It is probable that further improvements in
yield of either product could be achieved by manipulation
of fermentation conditions. The prospects for producing
either illudin M or S in quantity by fermentation appears
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