Food Chemistry 113 (2009) 351355

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Food Chemistry
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Isolation and quantitative determination of ergosterol peroxide

in various edible mushroom species
Wojciech Krzyczkowski a,*, Eliza Malinowska a, Piotr Suchocki b,c, Jerzy Kleps a, Marian Olejnik d,
Franciszek Herold a
a
Chair and Department of Drug Technology, Faculty of Pharmacy, The Medical University of Warsaw, ul. Banacha 1, 02-097 Warsaw, Poland
b
Department of Drug Analysis, Faculty of Pharmacy, The Medical University of Warsaw, ul. Banacha 1, 02-097 Warsaw, Poland
c
National Medicine Institute, ul. Chemska 30/34, 00-725 Warsaw, Poland
d
Institute of Organic Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Ergosterol peroxide, the steroidal derivative with cytotoxic activity, has been isolated for the rst time
Received 16 March 2008 from the mycelium of edible and medicinal mushroom Hericium erinaceum (lions mane mushroom)
Received in revised form 24 June 2008 together with erinacine A. The new densitometric method was applied for the quantitative determination
Accepted 28 June 2008
of ergosterol peroxide in n-hexane extracts of H. erinaceum, Laetiporus sulfureus (chicken mushroom), and
Morchella esculenta (common morel) mycelia, as well as in Boletus edulis (king bolete), Suillus bovinus (Jer-
sey cow mushroom), and B. badius (bay bolete) fruiting bodies. The ergosterol peroxide content reached
Keywords:
15.98 0.78, 10.07 0.75, 13.37 0.56, 29.32 1.43, 17.27 0.84, and 12.60 0.59 mg per 100 g, respec-
Mushrooms
Hericium erinaceum
tively. What is signicant was that ergosterol peroxide was identied for the rst time, to the best of our
Boletus badius knowledge, in edible mushrooms mentioned above.
Boletus edulis 2008 Elsevier Ltd. All rights reserved.
Laetiporus sulfureus
Morchella esculenta
Suillus bovinus
Densitometry
HPTLC
2D-TLC

1. Introduction against Walker carcinosarcoma and human mammary adenocarci-

Up to now, ergosterol peroxide (EP) has been isolated from var-
ious species of edible and medicinal mushrooms e.g., Ganoderma
lucidum (El-Mekkawy et al., 1998; Ziegenbein, Hanssen, & Knig,
2006), Cordyceps sinensis (Bok, Lermer, Chilton, Klingeman, &
Towers, 1999; Nam, Jo, Kim, Hyun, & Kim, 2001), Volvariella volva-
cea (Mallavadhani et al., 2006), Armillaria mellea (Kim, Park, Min, &
Yu, 1999), microscopic fungi such as Gibberella fujikuroi and Asper-
gillus spp. (Nemec, Jernejc, & Cimerman, 1997), yeasts such as Sac-
charomyces cerevisiae (Nes, Xu, & Haddon, 1989), plants such as
Melilotus messanensis (Macas, Simonet, & Galindo, 1997), sponge
Ascidia nigra (Gunatilaka, Gopichand, Schmitz, & Djerassi, 1981),
soft coral Sinularia exibilis (Yu, Deng, van Ofwegen, Proksch, &
Lin, 2006), and halotolerant microalga Dunaliella salina (Sheffer,
Fried, Gottlieb, Tietz, & Avron, 1986).
It has been proven that EP poses a wide spectrum of biological
activity. This compound was shown to have antitumor activity
* Corresponding author. Tel.: +48 22 5720 647; fax: +48 22 5720 631.
E-mail address: wkrzyczkowski@wum.edu.pl (W. Krzyczkowski).
noma cell lines in vitro (Jong & Donovick, 1989), as well as against
human gastric tumor cell line (SNU-1), human hepatoma cell line
(SNU-354), human colorectal tumor cell line (SNU-C4), and murine
sarcoma-180. The cytotoxicity of EP is assumed to be relevant to its
conversion to ergosterol, accompanied by the formation of highly
toxic peroxide products (Nam et al., 2001). Recent studies showed
that the cytotoxicity of EP on HL60 cells results from its ability to
induce apoptosis (Takei, Yoshida, Ohnishi-Kameyama, & Kobori,
2005).
Ergosterol peroxide inhibits 12-O-tetradecanoylphorbol-13-
acetate (TPA)-induced inammatory ear oedema (Yasukawa
et al., 1994) and proves to be selectively active against PLA2 en-
zymes secreted from Crotalus adamenteus venom (Gao et al.,
2007). This suggests its possible medicinal use as an antivenom
and anti-inammatory agent (Keyzers & Davies-Coleman, 2005).
EP also shows antimicrobial activity against Bacillus subtilis,
Staphylococcus aureus, Sarcina lutea, Pseudomonas sp., Candida albi-
cans, and A. niger (Lu, Zou, Meng, Hu, & Tan, 2000). On the other
hand, EP was found to be inactive against S. aureus ATTC 25923,
Escherichia coli ATTC 25922, P. aeruginosa ATTC 27753, and 11

0308-8146/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.06.075
352 W. Krzyczkowski et al. / Food Chemistry 113 (2009) 351355
strains of Candida spp. Moreover, it showed poor activity against
Mycobacterium tuberculosis H37Rv (Cateni et al., 2007).
The mechanism of EP biosynthesis and its authenticity as a
metabolite has been controversial since the sixties. Adam, Camp-
bell, and McCorkindale (1967) found this compound to be an arti-
fact that arises from oxidation or photooxidation of ergosterol
induced by air. However, the study of either Nes et al. (1989) or
Nemec et al. (1997) refuted it.
Ergosterol peroxide is believed to be either an intermediate in
the H2O2-dependent enzymatic oxidation in the biosynthetic path-
way of steroidal dienes or a product of detoxication of reactive
oxygen species. Its content in cells depends on many factors on
the level of reactive oxygen species or individual ratio between
EP formation and its conversion back into ergosterol (Batrakov
et al., 2004; da Graa Sgarbi et al., 1997).
Erinacine A is a cyathane-xyloside diterpene isolated from H.
erinaceum mycelium and fruiting body and is known to have po-
tent stimulating activity on nerve growth factor (NGF) synthesis
as well as activity against Methicillin-resistant S. aureus (Kawagishi
et al., 1994; Kawagishi, Masui, Tukuyama, & Nakamura, 2006).
Our paper describes the isolation and identication of EP and
erinacine A from H. erinaceum mycelium. A sensitive and precise
method for the determination of EP has been developed. We exam-
ined the possibility of EP formation during an UVvis radiation. A
quantitative determination of EP in medicinal and edible mush-
rooms H. erinaceum (lions mane mushroom), Boletus edulis (king
bolete), Morchella esculenta (common morel), Laetiporus sulfureus
(chicken mushroom), Suillus bovinus (Jersey cow mushroom), and
B. badius (bay bolete) was performed.
2. Materials and methods
2.1. General methods
High-resolution electrospray ionization mass spectra were re-
corded on a PE Biosystems Mariner mass spectrometer. 13C and
1
H NMR (125.88 and 500.60 MHz, respectively) spectra were
established in CDCl3 as an internal standard on a Varian UNITY-
plus-500 instrument. A FTIR spectrometer 8300 (Shimadzu, Japan)
was used to establish IR bands. Melting points were determined on
a Melt-Temp apparatus with open capillary tubes and values were
uncorrected. Dry matter was measured by a moisture analyzer
Sartorius Goettingen, Germany. HPTLC analyses were carried out
with a CAMAG TLC-Scanner III supplied with WINcats software.
Agar, casein peptone, malt extract, and yeast extract were obtained
from Biocorp Sp. z o.o., Warsaw, Poland. Corn steep liquor was ob-
tained from ScienceLab.com, Inc., Houston, TX, USA. Glucose, ethyl
alcohol 99.8%, methyl alcohol, CHCl3, ethyl acetate, n-hexane, acetic
anhydride, pyridine, phosphotungstic acid, NaCl, MgSO4  7H2O,
ZnSO4  7H2O, and KH2PO4 were of analytical grade and were pur-
chased from POCh SA, Gliwice, Poland. Silica gel 60 (230400 mesh
ASTM) for ash column chromatography, TLC pre-coated silica gel
plates 60 F254, and HPTLC pre-coated silica gel plates 60 F254 with con-
centrating zone 10  2.5 cm were obtained from Merck KGaA, Darms-
tadt, Germany. Aluminium oxide for column chromatography
Brockmann Activity I, neutral, was obtained from Fluka (Buchs, Swit-
zerland). All solvents were glass-distilled before use.
2.2. Microorganisms and culture conditions
Dried fruiting bodies of B. edulis, S. bovinus, and X. badius were ob-
tained from Fungopol Sp.j., Kinice, Poland. Voucher specimens (coded
BE012, SB002, and XB008, respectively) are deposited in Chair and
Department of Drug Technology, Medical University, Warsaw, Poland.
H. erinaceum, M. esculenta, and L. sulfureus mushrooms came
from the Medicinal Mushroom Culture Collection of Chair and
Department of Drug Technology, Medical University, Warsaw,
Poland. All nutrient media were sterilized at 121 C for 20 min.
All mushrooms were provided on slants of malt agar at 4 C and
shifted onto the new slant after 3 months. Basic nutrient medium
with the composition of glucose 50 g L1, yeast extract 10 g L1,
casein peptone 10 g L1, KH2PO4 1.0 g L1, and distilled water, pH
5.0, was inoculated with several 10-mm-diameter discs of myce-
lium taken from 21-d colony growing on malt agar. Cultivation
was carried out in 500 mL Erlenmeyer asks containing 200 mL
of nutrient medium on rotary shaker at 25 C for 14-d with
110 rpm. The obtained seed cultures were used for inoculation.
2.3. Isolation and purication of ergosterol peroxide (1) and erinacine
A (3)
The mycelium of H. erinaceum was cultivated at 25 C in a 10 L
bioreactor on liquid medium containing glucose 50 g L1, casein
peptone 5 g L1, corn steep liquor 10 g L1, NaCl 5 g L1, Mg2+
0.5 g L1 (as MgSO4  7H2O), Zn2+ 0.1 g L1 (as ZnSO4  7H2O), and
distilled water. After 28-d, harvested mycelium (193.5 g as dry
weight) was ltered through the gauze and extracted with ethyl
alcohol (80 C, 3  5 L, 18 h). The combined extracts were then con-
centrated on rotary evaporator at 50 C to remove ethyl alcohol and
partitioned between water and ethyl acetate (4  500 mL). Ethyl
acetate extract was then washed with saline, dried over anhydrous
MgSO4, and evaporated to remove solvent. Obtained viscous extract
(35.4 g) was chromatographed over silica gel. The column was
eluted with n-hexane EtOAc mixtures by increasing polarity to
give ten fractions. Fraction 4 was then rechromatographed over
Al2O3 column eluted with EtOAc. The progress in separation was
monitored by TLC. Fractions that showed a single spot on the TLC
plate were collected and evaporated to give EP (1), which was crys-
tallized from MeOH as white needles (26 mg). Fraction 9 was
rechromatographed over silica gel (CHCl3MeOH 95:5 v/v) to give
erinacine A (3) as yellow amorphous powder (420 mg) (see Fig. 1).
2.3.1. Ergosterol peroxide (1)
White crystalline needles, mp 179181 C (uncorr.). Molecular
formula was determined as C28H44O3 by HR-ESI-MS of the
[M+Na]+ ion peak m/z 451.31541 (required 451.31827). IR Vmax
(KBr) 3398, 2959, 2870, 1458, 1377, 1296, 1072, 1042, 968, 860
(OO), 775.3 cm1. 1H NMR (500 MHz, CDCl3); d 0.82 (3H, s,
18), 0.82 (3H, d, 26), 0.83 (3H, d, 27), 0.89 (3H, s, 19), 0.91 (3H, d,
28), 1.00 (3H, d, 21), 3.97 (1H, m, 3), 5.15 (1H, dd, 23), 5.22 (1H,
dd, 22), 6.25 (1H, d, 6), 6.50 (1H, d, 7).13C NMR (125 MHz, CDCl3);
d 12.9 (18-Me), 17.6 (28-Me), 18.2 (19-Me), 19.6 (26-Me), 19.9
(27-Me), 20.6 (11), 20.9 (21-Me), 23.4 (15), 28.6 (16), 30.1 (2),
33.1 (25), 34.7 (1), 36.9 (4), 36.9 (20), 39.3 (10), 39.7 (12), 42.8
(24), 44.6 (13), 51.1 (9), 51.7 (14), 56.2 (17), 66.5 (3), 79.4 (5),
82.1 (8), 130.7 (7), 132.3 (23), 135.2 (6), 135.4 (22).
2.3.2. Erinacine A (3)
Yellow amorphous powder, mp 7274 C Molecular formula
was determined as C25H36O5 by HR-ESI-MS of the [M+Na]+ ion peak
m/z 455.24264 (required 455.24041). IR Vmax (KBr) 3364, 2932,
1674, 1574, 1169, 1045, 892, 851 cm1.
2.4. HPTLC analysis of ergosterol peroxide
2.4.1. Cultivation of mushrooms
H. erinaceum, L. sulfureus, and M. esculenta mushrooms were cul-
tivated in 500 mL Erlenmeyer asks containing 200 mL of nutrient
medium with the following composition: glucose 50 g L1, yeast
extract 10 g L1, casein peptone 10 g L1, KH2PO4 1.0 g L1 and dis-
 W. Krzyczkowski et al. / Food Chemistry 113 (2009) 351355 353

Fig. 1. The structures of ergosterol peroxide (1), ergosterol (2), and erinacine A (3). Compounds (1) and (3) were isolated from the mycelium of Hericium erinaceum (lions
mane mushroom).

tilled water, pH 5.0, on rotary shaker at 25 C for 21-d with
110 rpm. After cultivation, biomass was ltered through the lter
paper on the Buchners funnel under reduced pressure, washed
with 200 mL of distilled water, and dried at 40 C. Mycelia were
then pulverized, and a small portion of pulverized biomass was
put on the moisture analyzer to measure dry mass content at
105 C.
2.4.2. Sample preparation
Two grams of either powdered mycelium of H. erinaceum, L. sul-
fureus, and M. esculenta or 2.00 g of powdered fruiting bodies of B.
edulis, S. bovinus, and X. badius were extracted successively with n-
hexane in a Soxhlet apparatus within 6 h. The obtained extracts
were evaporated to dryness on rotary evaporator at 40 C. The
dry residue was then dissolved in EtOH, transferred to a 10 mL vol-
umetric ask and made up to the volume with EtOH. The received
solutions were used for HPTLC analysis. Each extraction was per-
formed in triplicate.
2.4.3. Standard solution preparation
A standard solution of EP with a concentration of 1.0 mg mL1
was prepared by dissolving 10.0 mg of ergosterol peroxide in EtOH
in a 10 mL volumetric ask. Of this solution, 1.00 mL and 0.10 mL
were diluted with EtOH in a 10 mL volumetric ask to a concentra-
tion of 0.10 mg mL1 and 0.01 mg mL1, respectively.

Fig. 2. HPTLC densitograms of ergosterol peroxide standard (A) and n-hexane extracts from Boletus badius (B), Morchella esculenta (C), Boletus edulis (D), Laetiporus sulfureus
(E) and Hericium erinaceum (F); 1 peak of ergosterol peroxide.
354 W. Krzyczkowski et al. / Food Chemistry 113 (2009) 351355
2.4.4. Sample application
The limit of detection (LOD) was determined by applying the
standard at the amounts of 10, 25, 50, 100, and 200 ng. Different
amounts of standard solution (0.125, 0.250, 0.500, 1.00, and
2.00 lg) were applied on HPTLC plates to establish the range of lin-
earity. The method of standard addition was used for quantica-
tion. On a HPTLC plate, 20 lL of sample and 20 lL of sample
with 1.00 lL (1.0 mg mL1) of standard solution in different tracks
were applied. All analyses were performed in triplicate. The follow-
ing equation was used to evaluate the quantity of EP in the ana-
lyzed samples.
YB
mB mA
YA
where mB is the concentration of EP in sample; mA the concentra-
tion of EP in standard; YB the peak height of sample; YA the peak
height of sample + standard.
2.4.5. HPTLC conditions
HPTLC plates, 10  10 cm, with 2.5  10 cm concentration zone
were developed in vertical saturated chamber with mobile phase
n-hexane EtOAc 1:1 v/v; Rf 0.3010.323. Solvent front distance
was 70 mm (from the end of concentrating zone). Next, the devel-
oped plates were sprayed with 15% w/v solution of phosphotung-
stic acid in EtOH and heated with hot air (100 C) during 5 min.
Densitometric analyses were performed at 515 nm (pink) due to
the maximum absorption wavelength (kmax) of ergosterol peroxide
(see Fig. 2). In situ absorption spectra were measured from 190 to
700 nm.
2.4.6. Linearity
The linearity range was 0.1251.00 lg of EP per spot. Regres-
sion equation: y = 107.78x + 5.13. Correlation coefcient: r =
0.9989, R2 = 0.9977.
2.5. 2D-TLC
The following experiment was performed to check the possibil-
ity of transformation of ergosterol (2) into EP (1) after UVvis light
exposure. The hexane extract of mycelium was dropped on 10  10
TLC plates. The plates were developed in vertical saturated cham-
ber with mobile phase n-hexane EtOAc 9:1 v/v to a distance of
6.5 cm and exposed to daylight, UV254 or UV366, each during 24 h.
After the exposure, plates were rotated 90 clockwise and devel-
oped using n-hexane EtOAc 1:1 as eluent, and visualized as men-
tioned above. New spots of EP were not observed.
3. Results and discussion
The mycelium of H. erinaceum was cultivated on liquid medium
in a bioreactor over a period of 28-d. The harvested mycelium
(193.5 g as dry weight) was then extracted with EtOH. The alcohol
extract after concentration under reduced pressure was parti-
tioned between water and ethyl acetate. Repetitive silica gel and
Al2O3 column chromatography of ethyl acetate extract, followed
by crystallization, gave compound (1) as white crystalline needles
(26 mg) and compound (3) as yellow powder (420 mg).
The molecular formula of (1) was determined as C28H44O3 from
the [M+Na]+ ion peak in HR-ESI-MS, indicating the presence of se-
ven degrees of unsaturation. The 1H NMR spectrum exhibited four
signals due to secondary methyl groups [d 1.00 (3H, d, J = 6.5 Hz, H-
21), 0.91 (3H, d, J = 7.0 Hz, H-28), 0.82 (3H, d, J = 7.0 Hz, H-26), 0.83
(3H, d, J = 7.0 Hz, H-27)] and two signals from tertiary methyl
groups of d 0.82 (3H, s, H-18) and 0.89 (3H, s, H-19). The 13C
NMR spectrum showed the presence of two disubstituted olens
[d 130.7 (C-7), 132.3 (C-23), 135.2 (C-6) and 135.4 (C-22)], indicat-
ing that the sterol fragment of (1) is an ergosterol derivative. Two
oxygenated quaternary carbons of d 79.4 (C-5) and 82.1 (C-8) sug-
gested the presence of a peroxide structure.
The 13C and 1H NMR spectra and HR-ESI-MS ion peak m/z
[M+Na]+ 451.31541 of (1) correspond to EP (5,8-epidioxy-5a,8a-
ergosta-6,22E-dien-3b-ol) and match the published data (Bok
et al., 1999; Cateni et al., 2007; da Graa Sgarbi et al., 1997; Nam
et al., 2001; Yue, Chen, Lin, & Sun, 2001).
This is the rst report about the isolation of EP from H. erinace-
um mycelium.
The 13C and 1H NMR spectra and [M+Na]+ HR-ESI-MS ion peak
of (3) (m/z 451.31541, required 455.24041) correspond to molecu-
lar formula C25H36O5 indicating erinacine A (Kawagishi et al.,
1994).
To check the possibility of transforming ergosterol (2) into EP
(1) after UVvis light exposure, the n-hexane extract of H. erinace-
um was submitted to two-dimensional thin layer chromatography
combined with UVvis radiation between each separation. On the
basis of our results (no new spots on TLC plate were observed), we
established that EP is not an artifact. These observations were con-
sistent with those reported by Nes et al. (1989) and Nemec et al.
(1997).
A new specic and precise densitometric method was devel-
oped for the quantitative determination of EP in mushroom ex-
tracts. The method is based on the separation of n-hexane
extracts on HPTLC silica gel plates with concentrating zone using
the solvent system n-hexane EtOAc 1:1 v/v. The characteristic
pink spots of EP, after spraying with an alcoholic solution of phos-
photungstic acid and heating, reach in situ the maximum absorp-
tion at 515 nm. The Rf value lies in the range of 0.3010.323,
depending on the amount of EP. The limit of detection amounts
to 50 ng of EP per spot. Our method is suitable for serial determi-
nations, e.g., in food industry or in phytochemical investigations.
The HPTLC analyses of extracts from H. erinaceum, L. sulfureus
and M. esculenta mycelia as well as from B. edulis, S. bovinus, and
X. badius fruiting bodies reveal the presence of EP. To the best of
our knowledge, this is the rst report about quantitative determi-
nation of EP in such species of mushrooms. The results concerning
EP content are summarized in Table 1.
A thorough analysis enabled us to establish that EP is present in
every examined mushroom species. As this compound has been
found previously in a variety of mushrooms (Bok et al., 1999;
Cui, Kim, & Park, 2005; El-Mekkawy et al., 1998; Keller, Keller,
Maillard, & Hostettmann, 1997; Kim et al., 1999; Mallavadhani
et al., 2006; Nam et al., 2001; Rsecke & Knig, 2000; Ziegenbein
et al., 2006), it can be assumed that EP is a common compound
found in higher fungi. It seems that the EP content of various spe-
cies of mushrooms depends either on the level of reactive oxygen
species or on the relationship between EP formation and its con-
version back into ergosterol (Batrakov et al., 2004; da Graa Sgarbi
et al., 1997). It is possible that the presence of substances with
antioxidant properties in biomass may have an impact on the ratio
of ergosterol to EP.
Table 1
Amount (mg) of ergosterol peroxide per 100 g of dried mushroom
Mushroom Ergosterol peroxide (mg per 100 g)a
Morchella esculenta 13.4 0.56
Boletus edulis 29.3 1.43
Hericium erinaceum 16.0 0.78
Laetiporus sulfureus 10.1 0.75
Suillus bovinus 17.3 0.84
Boletus badius 12.6 0.59
a
Results are given as the average SD (standard deviation, n = 3).
 W. Krzyczkowski et al. / Food Chemistry 113 (2009) 351355
In view of the wide-ranging medicinal properties of EP, includ-
ing cytotoxic activity (Jong & Donovick, 1989; Nam et al., 2001;
Takei et al., 2005), the requirement of its determination in edible
mushrooms used extensively in many countries should be
considered.
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