A major decomposition product, citrinin H2, from citrinin on heating with moisture.

Article abstract of DOI:10.1271/bbb.66.206

Citrinin is one of the mycotoxins produced by Penicillium citrinum. We examined the decomposition products after heating citrinin in water at 140 degrees C and isolated a major product, citrinin H2 (3-(3,5-dihydroxy-2-methylphenyl)-2-formyloxy-butane). Ci

Full text of DOI:10.1271/bbb.66.206

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A Major Decomposition Product, Citrinin H2, from
Citrinin on Heating with Moisture
Mitsuru HIROTAa, Alka MENTAb, Keiko YONEYAMAa & Naofumi KITABATAKEb
a Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University
8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
b Research Institute for Food Science, Kyoto University Uji, Kyoto 611-0011, Japan
Published online: 22 May 2014.
To cite this article: Mitsuru HIROTA, Alka MENTA, Keiko YONEYAMA & Naofumi KITABATAKE (2002) A Major Decomposition
Product, Citrinin H2, from Citrinin on Heating with Moisture, Bioscience, Biotechnology, and Biochemistry, 66:1, 206-210,
DOI: 10.1271/bbb.66.206
To link to this article: http://dx.doi.org/10.1271/bbb.66.206
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Biosci. Biotechnol. Biochem., 66 (1), 206–210, 2002
Note
A Major Decomposition Product, Citrinin H2, from Citrinin
on Heating with Moisture
† Alka B. MENTA,b Keiko YONEYAMA,a and Naofumi KITABATAKE
a,
b
Mitsuru HIROTA
,
aDepartment of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University,
8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
bResearch Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
Received July 27, 2001; Accepted September 7, 2001
Citrinin is one of the mycotoxins produced by Penicil-
lium citrinum We examined the decomposition
products after heating citrinin in water at 140 C and
isolated a major product, citrinin H2 (3-(3,5-dihydroxy-
2-methylphenyl)-2-formyloxy-butane). Citrinin H2 did
not show signiˆcant cytotoxicity to HeLa cells up to a
of the decomposition products from the heated citri-
nin led to the isolation of another product, citrinin
H2. In this paper, we describe the structure of citri-
nin H2, its lower toxicity to HeLa cells, and the
course of citrinin H2 formation from heated citrinin.
The structure of citrinin H2 was analyzed from the
spectral data and identiˆed as 3-(3,5-dihydroxy-2-
.
9
concentration of 200
63 h of incubation, but citrinin showed severe toxicity at
a concentration of 25 g ml ( cytotoxicity: 73 ).
mg ml (% cytotoxicity: 39%) in
W
methylphenyl)-2-formyloxybutane by comparison to
11)
m
%
%
the reference data of Curtis et al
.
They isolated it
W
HPLC analysis of citrinin after heating under various
conditions indicates that citrinin H2 is mainly yielded
from citrinin.
from a cultivation medium of Penicillium citrinum
and proposed that citrinin H2 might be formed
biosynthetically from citrinin. Barber et al. reported
that citrinin is present in a hydrate form at the C-1 of
citrinin in aqueous solution at pH 7.4.12) Citrinin H2
is probably produced through the addition of water
at the C-1 position of citrinin followed by decarboxy-
lation and ring opening (Fig. 1).
Key words: citrinin; detoxiˆed product; HeLa cells;
cytotoxicity
Citrinin is one of the mycotoxins produced by
fungi belonging to Penicillium or Aspergillus spe-
cies.1) It is toxic, especially to the kidneys, and is sus-
pected to be a carcinogen causing renal tumors.2)
Contamination by citrinin in several foods and feed
has been reported.3–6) To avoid the direct intake of
citrinin or indirect intake through livestock, it is im-
portant to develop detoxiˆcation methods for citrinin
during food processing. Vail et al. reported a sensi-
tive detection method for citrinin using HPLC.7)
However, there are few reports on the detoxiˆcation
of citrinin. Recently, Kitabatake et al. found that
The cytotoxic eŠect of citrinin H2 on HeLa cells
was evaluated by the MTT method.13) HeLa cells
were incubated in the presence of test compounds for
63 h at 37
icity at a concentration of 200
concentration of 100 g ml, whereas citrinin showed
90 cytotoxicity at a concentration of 100
and 73 at a concentration of 25 g ml (Table 1).
9
C. Citrinin H2 showed only 39
z
cytotox-
m
g ml and 23
z
at a
W
m
W
z
mg ml
W
z
m
W
The cytotoxicity of citrinin H2 is much weaker than
that of citrinin.
Citrinin H2 has a phenolic hydroxyl group and a
formyl group. To examine the role of the functional
groups for cytotoxicity, we prepared derivatives of
citrinin H2 and examined their cytotoxicity (Fig. 2).
Compound 1 is a diacetate of citrinin H2. Compound
2 is the hydrolyzed form of citrinin H2. Compound 3
is a dimethyl ether of compound 2. As shown in
Table 1, these compounds showed only weak cyto-
heating of citrinin at 1309C in the presence of a small
amount of water caused a signiˆcant decrease in the
toxicity of citrinin to HeLa cells.8) However, the citri-
nin heated at 140
icity. This indicates that heating of citrinin in water
at 140 C or 150 C caused formation of a highly toxic
compound(s).9)
9C or 1509C showed strong cytotox-
9
9
We were interested in clarifying the degradation
compounds of citrinin and tried to purify the degra-
dation products and obtained a product, citrinin H1,
with potent cytotoxic activity.10) Further examination
toxic activity at a concentration of 200 mg ml, like
W
that of citrinin H2. The potency of the cytotoxic ac-
tivity of these compounds is weak, similar to that of
citrinin H2. The weak cytotoxic activity of these
†
To whom correspondence should be addressed. Fax: +81-265-77-1629; E-mail: hirotam
＠
gipmc.shinshu-u.ac.jp
Abbreviations: MTT, 3(4,5-dimethyl-thiazoyl-2-yl)2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; MEM, minimum essential
medium eagle; HPLC, high-pressure liquid chromatography

Citrinin H2 from Citrinin on Heating
207
Fig. 1. Possible Mechanisms of Formation of Citrinin H2 from Citrinin by Heating.
Table 1. Cytotoxicity of Citrinin, Citrinin H2, and Citrinin H2
derivatives to HeLa Cells
Concentration (
50
m
g ml)
W
Tested
compound
25
100
200
*
Citrinin
0.150 (73
z)
0.086(84
0.490(12
0.453(18
0.486(12
0.402(27
z)
z)
z)
z)
z)
0.055(90
0.425(23
0.427(23
0.426(23
0.380(31
z)
z)
z)
z)
z)
NT
Citrinin H2
Compound 1
Compound 2
Compound 3
NT
NT
NT
NT
0.337(39
0.301(46
0.324(42
0.362(35
z)
z)
z)
z)
*
Absorbance at 540 nm: control without test compound: 0.554.
toxicity in parentheses. Cytotoxicity (control-test sample) control
100. Data are means of triplicate examinations.
z Cyto-
W
z
＝
×
compounds at high concentrations is non-signiˆcant.
To conˆrm the time course and temperature de-
pendency of citrinin H2 formation, we attempted
HPLC analysis of citrinin after heating at various
temperatures and heating times. Two hundred mg of
citrinin was heated in water, and the residue was then
dissolved in acetonitrile and analyzed by HPLC. The
HPLC analysis of citrinin H2 showed a peak at tR
7.2 min by the conditions as indicated in the Ex-
perimental section.
Fig. 2. Structures of Citrinin H1 and Citrinin H2-derivatives.
As shown in Fig. 3, heating at 140
mediately converted 200 g of citrinin to 30–45
citrinin H2. About 20 of citrinin was changed to
9
C–160
9
C im-
m
z
m
g of
under these conditions is not the non-toxic end
product and further changes to diŠerent compounds.
Other reactions might occur to decrease the amounts
of citrinin H2. These results correlate with previous
reports relating to cytotoxity tests of citrinin, heated
under various conditions, toward HeLa cells.9)
Citrinin is unstable and thermolabile in water. It
citrinin H2 by heating under these conditions.
Prolonged heating times up to 60 min did not have
any eŠect on the conversion ratio (ca. 20
of citrinin at 120 C or 130 C required 20 min to
reach a similar conversion ratio and heating of
citrinin at 100 C required 40 min–60 min. Heating at
80 C for 60 min yielded only 12 g of citrinin H2
from 200 g of citrinin.
Every conditions that we tested, except for heating
at 80 C, ˆnally produced a similar amounts of citri-
z
). Heating
9
9
9
9
degrades on heating above 80 C under aqueous con-
9
m
ditions. A major degradation product, citrinin H2,
which we isolated, is considered to be non-cytotoxic
to HeLa cells under physiological conditions. The
experiments on heating citrinin under various condi-
tions also indicated that citrinin H2 is a major
decomposition product. Under selective heating con-
ditions, citrinin decomposed to citrinin H2, and the
m
9
nin H2. Heating at higher temperature or for a
prolonged time did not increase the amount of citri-
nin H2. These results suggest that citrinin H2 formed

208
M. HIROTA et al.
Isolation of citrinin H2. The brown residue was
put on a silica gel column (Wako gel C300, 15 g) with
benzene, and eluted with toluene (50 ml) followed by
a mixture of ethyl acetate: toluene (1:2). The eluate
(each 5 ml) was collected to give 50 fractions. TLC
analysis showed that the fractions No. 7–12 con-
tained less polar compounds than citrinin. The frac-
tions were combined and concentrated. The resultant
residue thus obtained was rechromatographed on
silica gel (Wako gel C300, 15 g) with diethyl ether-
hexane mixture (0 to 80
z diethyl ether). Each frac-
tion (7 ml) was collected, and fractions No. 40–60
containing the less polar compounds, were combined
and concentrated to obtain an oily residue (159 mg).
The residue was dissolved in acetonitrile and put on
preparative HPLC (column, Nacalai Tesque; eluant,
Fig. 3. Course of Citrinin H2 Formation in Citrinin Heated with
Water at Various Temperatures.
Heating at 80
9
C (

), 100
9
C ( ), 120

9C ( ), 1309C ( ),
 
acetonitrile: water (1:1); ‰ow rate, 7 ml min; detec-
W
$
#
140
9
C ( ), 150
9
C ( ), and 1609C ( ).
×
tion, UV 254 nm). The peaks corresponding to tR
9–12 min were corrected and concentrated to yield a
brown residue (38 mg). The residue (19 mg) was fur-
ther puriˆed on silica gel (Wako gel C300, 2 g) with
citrinin H2 content reached at a plateau level. At that
level, about 20 of the citrinin was changed to citri-
z
nin H2. Probably, citrinin H2 decomposed further to
form other complex compounds. Kitabatake demon-
strated that citrinin decomposed to a nontoxic com-
pound on heating in water only under selective condi-
tions.14) Citrinin H2, as well as citrinin H1, might be
markers of citrinin decomposition by heating in
water.
toluene-ethyl acetate mixtures with the 10
elution method (0, 10, 20, 30, 40, and 50
acetate in toluene; each fraction, 10 ml). The 20
z
stepwise-
z
ethyl
z
EtOAc eluate was evaporated to give citrinin H2
(13 mg), which was crystallized from chloroform to
give pale yellow prisms, mp 139.5–140.0
131–1339C11)). IR max (KBr) cm„1: 3380 (OH), 3200,
2900, 1700 (C O), 1620, 1600, 1510, 1230, 1140,
1130, 1105, 1100, 1010, 830; UV max (EtOH) nm ( ):
9
C (mp
n
＝
n
e
1
Experimental
4
3
3
×
×
×
J
＝
203 (2.2 10 ), 223sh. (2.1 10 ), 282 (1.2 10 ); H-
Instruments. Melting points were measured on a
hot stage and are uncorrected. The following instru-
ments were used; a Bruker AC250 FT-NMR spec-
trometer for 1H- and 13C-NMR spectra using
tetramethylsilane as internal standard, a Hitachi
M80B and a JEOL JMS700 mass spectrometers for
mass spectra, and a JASCO IR spectrometer for IR
spectra. The HPLC analysis was done on Hitachi
HPLC equipment: Model L-6200 pump and L-4000
UV detector.
NMR (CDCl3) H: 7.92 (s, 1H), 6.34 (d, 2.4 Hz,
d
＝
2.4 Hz, 1H), 5.29 (br.s, OH), 5.20
1H), 6.21 (d,
J
(dq,
J
＝
7.2, 6.3 Hz, 1H), 3.26 (dq,
J
＝
7.2, 7.1 Hz,
＝
1H), 2.16 (s, 3H), 1.26 (d,
J
6.3 Hz, 3H), 1.20 (d,
C: 160.8, 154.5,
J
7.1 Hz, 3H); 13C-NMR (CDCl3)
d
＝
154.0, 143.8, 114.7, 105.9, 101.0, 74.0, 39.3, 17.3,
16.5, 10.5; EIMS m z: 224 (M, 36 ), 195 (1 ), 178
z
z
W
(17
(19
z
z
), 164 (15
), 123 (17
z z
), 151 (base peak, 100 ), 137
z
); HREIMS m z (M+): calcd. for
W
C12H16O4, 224.1048; found, 224.1014.
Materials. Citrinin was isolated from a cultivated
medium of Penicillium citrinum, as described in the
reference.10)
Acetylation of citrinin H2. Citrinin H2 (2 mg) was
dissolved in dry pyridine (0.5 ml) and acetic anhy-
dride (0.25 ml) and the reaction mixture was left at
room temperature overnight. After the usual wor-
kup, followed by silica gel column chromatography
(Wako gel C300, 0.3 g) with toluene-ethyl acetate
mixtures, citrinin diacetate was obtained (1. 1.9 mg).
9
Heating of citrinin. Citrinin was heated at 140 C
as described in our previous paper.10) Brie‰y, citrinin
(500 mg) and water (375 ml) were distributed in ˆfty
10-ml glass vials. After the vials were sealed, the mix-
ture was heated in a thermo-regulated pressure vessel
1H-NMR (CDCl3) dH
:
2.3 Hz, 1H), 6.76 (d, 2.3 Hz, 1H), 5.18 (m,
＝
6.6 Hz, 1H), 3.33 (m, J 7.0 Hz, 1H), 2.32 (s,
7.95 (s, 1H), 6.89 (d,
＝
＝
＝
J
J
J
from room temperature to 140
9
C at the rate of
＝
7.0 Hz,
3
9
C min. After cooling to room temperature, the
3H), 2.27 (s, 3H), 2.15 (s, 3H), 1.26 (d,
J
W
6.5 Hz, 3H); HREIMS m z (M+):
＝
heated citrinin was collected and extracted with
chloroform. The chloroform layer was washed with
water, dried over sodium sulfate, and evaporated, to
give a dark brown residue (420 mg).
3H), 1.24 (d,
J
W
calcd. for C16H20O6, 308.1257; found, 308.1240.
Alkaline degradation of citrinin. Alkaline degrada-

Citrinin H2 from Citrinin on Heating
209
tion was done by the method of Barber et al
nin (42 mg) was dissolved in 10 aqueous sodium
hydroxide, and the solution was heated to 120 C for
.
15) Citri-
200
m
g of citrinin was added to 150
m
l of water. The
z
sealed vials were heated under various heating condi-
9
tions from 80
60 min the vials were cooled at room temperature. To
each vial, acetonitrile (100 l) was added and the dis-
solved sample was analyzed by HPLC (conditions:
×
column, TOSO TSK gel C18, 4 mm i.d. 20 cm; ‰ow
rate, 0.7 ml min; eluent, 45
detection at 280 nm).
9 9
C to 160 C. After heating for 0 min to
4 h. After cooling to room temperature, the reaction
mixture was treated with barium hydroxide and
m
ˆltered. The ˆltrate was neutralized with 2
N
HCl and
extracted with ethyl ether. The ethyl ether layer was
dried over magnesium sulfate and evaporated to give
a residue, which was chromatographed on silica gel
z
acetonitrile in water;
W
(Wako gel C300, 500 mg) to give decarboxylated
1
compound 2 (8 mg). H-NMR (pyridine-d5)
d
H: 7.07
＝
2.3 Hz, 1H), 4.29 (m,
Acknowledgments
＝
2.3 Hz, 1H), 6.93 (d, J
6.2 Hz, 1H), 3.45 (m,
(d,
J
＝
＝
7.0 Hz, 1H), 2.57 (s,
J
J
This work was supported in part by a grant-in-aid
for Cancer Research from the Ministry of Education,
Science, and Culture of Japan. We thank Ms. M.
Hattori of the Faculty of Agriculture, Shinshu
University, for measuring the NMR spectra and Ms.
Eiko Tuchida of the Faculty of Engineering, Shinshu
University for developing the mass spectra.
＝
＝
6.2 Hz,
3H), 1.41 (d,
J
7.0 Hz, 3H), 1.39 (d,
J
3H); 13C-NMR (pyridine-d5)
d
C: 158.0, 157.6, 146.4,
115.0, 106.6, 101.9, 71.5, 43.4, 21.0, 17.5, 12.0.
HREIMS m z (M+): calcd. for C11 H16O3, 196.1098;
W
found, 196.1062.
Methylation of compound 2. Compound 2 (4 mg)
was dissolved in dry acetone and treated with
dimethyl sulfate (2 ml) and dry potassium carbonate
(37 mg). The reaction mixture was re‰uxed for 20 h
and ˆltered. The solid was washed with dry acetone.
The ˆltrate and the washing were combined and
evaporated to give a residue, which was dissolved in
ethyl acetate. The ethyl acetate solution was washed
with water and sodium bicarbonate, then dried over
magnesium sulfate and ˆltered. The ˆltrate was
evaporated to give a dark brown residue. The residue
was chromatographed on silica gel (Wako gel C300,
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0.5 g) with benzene-ethyl acetate mixture to give
1
compound 3 (3 mg). H-NMR (CDCl3)
d
H: 7.26 (s,
＝
＝
2.4 Hz,
1H), 6.43 (d,
1H), 3.90 (dq,
J
2.4 Hz, 1H), 6.36 (d,
J
J
＝
8.2, 6.2 Hz, 1H), 3.81 (s, 3H), 3.80
＝
8.2, 7.0 Hz, 1H), 2.15 (s, 3H),
(s, 3H), 3.05 (dq,
J
＝
＝
7.0 Hz, 3H);
1.27 (d,
J
6.2 Hz, 3H), 1.18 (d,
J
HREIMS m z (M+): calcd. for C13 H20O3, 224.1411;
W
found, 224.1425.
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(1.15 mg) was dissolved in FBS (22
with MEM (208 l) to obtain the original test solu-
tion (5.0 mg ml). The solution was further diluted
ml) and diluted
m
, 535, 317–323
W
with MEM containing 10 FBS to 400
z
mg ml,
W
200 g ml and 100 g ml (50 mg ml in the case of
m
m
W
W
W
×
citrinin) for the cytotoxicity test. HeLa cells (3
103 cells well) were incubated with test samples at
,
W
37
9
C under a 5
z
carbon dioxide atmosphere for
63 h. The cells were further incubated for 4 h after
the addition of MTT, and the resultant blue
formazan was dissolved in acidic isopropanol.
Cytotoxicity was evaluated with the absorption of
isopropanol solution at 540 nm.
, 58,
Quantitative analysis. To each pressure vial,

210
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