Quantification of the bioactive compound eritadenine in selected strains of shiitake mushroom (Lentinus edodes)

Article abstract of DOI:10.1021/jf062559+

Cardiovascular disease is one of the most common causes of death in the Western world, and a high level of blood cholesterol is considered a risk factor. The edible fungus, shiitake mushroom (Lentinus edodes), contains the hypocholesterolemic agent eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid. This study was conducted to quantify the amount of the cholesterol reducing agent eritadenine in shiitake mushrooms, in search of a potential natural medicine against blood cholesterol. The amounts of eritadenine in the fruit bodies of four different shiitake mushrooms, Le-1, Le-2, Le-A, and Le-B, were investigated in this study. To achieve this goal, methanol extraction was used to recover as much as possible of the hypocholesterolemic agent from the fungal cells. In addition, enzymes that degrade the fungal cell walls were also used to elucidate if the extraction could be further enhanced. To analyze the target compound, a reliable and reproducible HPLC method for separation, identification, and quantification of eritadenine was developed. The shiitake strains under investigation exhibit up to 10 times higher levels of eritadenine than previously reported for other shiitake strains. Further, pretreating the mushrooms with hydrolytic enzymes before methanol extraction resulted in an insignificant increase in the amount of eritadenine released. These results indicate the potential for delivery of therapeutic amounts of eritadenine from the ingestion of extracts or dried concentrates of shiitake mushroom strains.

Full text of DOI:10.1021/jf062559+

J. Agric. Food Chem. 2007, 55, 1177−1180 1177
Quantification of the Bioactive Compound Eritadenine in
Selected Strains of Shiitake Mushroom (Lentinus edodes)
JOSEFINE ENMAN, ULRIKA ROVA,* AND KRIS A. BERGLUND
Division of Biochemical and Chemical Process Engineering,
Luleå University of Technology, SE-97187 Luleå, Sweden
Cardiovascular disease is one of the most common causes of death in the Western world, and a
high level of blood cholesterol is considered a risk factor. The edible fungus, shiitake mushroom
(Lentinus edodes), contains the hypocholesterolemic agent eritadenine, 2(R),3(R)-dihydroxy-4-(9-
adenyl)-butyric acid. This study was conducted to quantify the amount of the cholesterol reducing
agent eritadenine in shiitake mushrooms, in search of a potential natural medicine against blood
cholesterol. The amounts of eritadenine in the fruit bodies of four different shiitake mushrooms, Le-
1, Le-2, Le-A, and Le-B, were investigated in this study. To achieve this goal, methanol extraction
was used to recover as much as possible of the hypocholesterolemic agent from the fungal cells. In
addition, enzymes that degrade the fungal cell walls were also used to elucidate if the extraction
could be further enhanced. To analyze the target compound, a reliable and reproducible HPLC method
for separation, identification, and quantification of eritadenine was developed. The shiitake strains
under investigation exhibit up to 10 times higher levels of eritadenine than previously reported for
other shiitake strains. Further, pretreating the mushrooms with hydrolytic enzymes before methanol
extraction resulted in an insignificant increase in the amount of eritadenine released. These results
indicate the potential for delivery of therapeutic amounts of eritadenine from the ingestion of extracts
or dried concentrates of shiitake mushroom strains.
KEYWORDS: Eritadenine; Lentinus edodes; bioactive compounds; HPLC
INTRODUCTION
The shiitake mushroom is widely cultivated and consumed
not only as food but also as a natural medicine because of its
medical properties. This mushroom has a high nutritional value
and contains several substances with additional positive effects
on health, such as the anti-tumor agent lentinan (1). One of the
health benefits, which this mushroom possesses, is the ability
to reduce blood cholesterol as shown in both animal and human
studies (2, 3). The cholesterol reducing agent in shiitake
mushrooms is a purine alkaloid (Figure 1) designated as
eritadenine (lentinacin), 2(R),3(R)-dihydroxy-4-(9-adenyl)-bu-
tyric acid (4).
Competitive inhibitors of HMG-CoA reductase, the statins,
are produced in a large scale as cholesterol reducing pharma-
ceuticals. Unlike the statins, eritadenine does not inhibit the
biosynthesis of cholesterol in the liver but enhances removal
of blood cholesterol (5). The exact mechanism by which
eritadenine elicits its hypocholesterolemic action is not yet fully
understood. However, the hypocholesterolemic action of erita-
denine has been investigated in several studies on rats. It has
been shown that total plasma cholesterol levels are decreased
in rats fed eritadenine in their diets and that the hypocholes-
Figure 1. Chemical structure of eritadenine (Mv
) 253 g/mol).
terolemic action is caused by a decrease of the phosphatidyl-
choline (PC)/phophatidylethanolamine (PE) ratio (6-10). Erita-
denine is a very potent inhibitor of the enzyme S-adenosyl-L-
homocysteine hydrolase in rat liver cells (11), thereby causing
an increase in the S-adenosylhomocysteine concentration (12).
The increase in the S-adenosylhomocysteine concentration in
turn inhibits the PE N-methylation, thus increasing the PE
content in liver microsomes (9). Further studies on rats suggest
that eritadenine may increase the uptake of plasma lipoprotein
cholesterol by the liver and thus reduce the plasma cholesterol
* To whom correspondence should be addressed. Tel: +46(0)920-
491315. Fax: +46(0)920-491199. E-mail: ulrika.rova@ltu.se.
10.1021/jf062559+ CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/27/2007

1178 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Enman et al.
and its purity, NMR analysis was conducted for each step of the
synthesis and compared with the literature. An LC/MS run further
confirmed the final product. A stock solution (1.98 mg/mL) of the
standard was prepared by dissolving synthesized eritadenine in distilled
water.
(10). There is a possibility that the change in composition of
the membrane phospholipids may activate lipoprotein receptors
in liver cell membranes, thus regulating the uptake of plasma
lipoprotein lipids (6).
The amounts of eritadenine in shiitake mushrooms quantified
so far are in the range of 50-70 mg/100 g dry weight (dw) in
the caps, 30-40 mg/100 g in the stems (13, 14), and 73.7 mg/
100 g in shiitake mycelium (15). In rats, a diet containing
0.005% eritadenine was shown to lower the serum total
cholesterol level with 25% (4). Similar studies on humans have
not been found in the literature. Previous work indicates the
efficacy of shiitake mushrooms in cholesterol reduction; how-
ever, the concentrations reported indicate that large quantities
may have to be consumed to achieve therapeutic effects (3).
To establish the dose-response effect of shiitake mushrooms
as a cholesterol reducing product, strains producing considerable
amounts of eritadenine are favorable, as is a reliable analytical
procedure for this substance. To achieve an accurate quantifica-
tion of eritadenine, losses in the extraction procedure should
be minimized and the amount released from the mushrooms
maximized.
The goal of the present work was to evaluate the eritadenine
content of four commercially cultivated shiitake mushrooms,
Le-1, Le-2, Le-A, and Le-B, to determine if they could be used
as a viable source of eritadenine, either as a dried product or as
an extract. To achieve this objective, a reliable analytical HPLC
procedure quantifying eritadenine in shiitake mushrooms was
developed.
Methanol Extraction and Isolation of Eritadenine. The extraction
was a modified version of the method developed by Tokita et al. (17).
Powder from dried fruit bodies was weighed and extracted with 80%
(v/v) methanol for about 3 h under reflux, with a solid-liquid ratio of
1:20. The extract was filtered through Whatman No. 5 filter paper,
evaporated to dryness in vacuo at 50-60 °C, and diluted in 50 mL
distilled water. The sample was further extracted with 50 mL of diethyl
ether 3 times. To the aqueous layer, 4 volumes of 99.5% ethanol was
added and incubated at -20 °C overnight. The precipitate was removed
by filtration through Whatman OOH filter paper, and the filtrate was
evaporated to dryness in vacuo at 50-60 °C. The extract was diluted
in 50 mL of distilled water and applied to a column of Amberlite IR-
120 (H+) ion-exchange resin. The substance was eluted with 2%
ammonia, showing a high absorbance at 260 nm. The volume collected
was evaporated to dryness in vacuo at 50-60 °C, diluted in 50 mL of
distilled water, and applied to an Amberlite IRA-67 (OH-) ion-exchange
resin. The substance was eluted with 0.1 M acetic acid, and fractions
showing high absorbance at 260 nm were collected. After evaporation
to dryness in vacuo at 50-60 °C, the mushroom sample was dissolved
in 100 mL of distilled water. The completely isolated eritadenine was
confirmed with LC/MS.
Enzymatic Pretreatment. The enzyme preparation, NS 33075, was
kindly supplied by Novozymes, Bagsvaerd, Denmark. NS 33075 is a
multicomponent carbohydrase preparation originating from Trichoderma
harzianum. The main components of this enzyme mixture are various
R- and â-glucanases, but it also contains some side activities like
chitinases and proteases. According to the supplier, this enzyme mixture
shows a fairly high activity at pH 4.8 and 50 °C. The enzyme powder
was dissolved in water to a stock solution of 2% (w/v). A volume of
the stock solution corresponding to 0.2 mg of enzyme per gram of
mushrooms was added to either distilled water (pH 6.0) or 0.1 M acetate
buffer (pH 4.8). The volume of the water and the buffer used was 10
mL per gram of mushrooms. The enzyme-water or enzyme-buffer
mixture was added to the weighed mushroom powder, and the reaction
was incubated at 50 °C for 3 h with gentle stirring. Following the
enzymatic treatment, a methanol extraction was preformed as previously
described.
In an attempt to further increase the amount of eritadenine
released, enzymes involved in the breaking of bonds between
the polymers in fungal cell walls were used in this study. The
cell walls of shiitake mushrooms are mainly composed of the
polysaccharides chitin (â-1,4-N-acetylglucosamine) and glucans
(â-1,3 and â-1,6), which can be degraded by hydrolytic enzymes
possessing chitinase or glucanase activity (16). Hence, enzymes
with these properties were used to enhance the extraction
procedure by macerating the fungal cells.
HPLC Analysis. The eritadenine concentration in shiitake fruit
bodies was analyzed by HPLC (Series 200 Quaternary LC pump and
UV-vis detector, TotalChrom software, Perkin-Elmer, Wellesley, MA)
and separated over a C18 column (RESTEK Ultra Aqueous, 5 µm,
150 mm × 4.6 mm). Prior to analysis, the samples from the extractions
were diluted twice with the initial mobile phase and filtered through a
0.2 µm syringe filter. The HPLC analysis was conducted at 23 °C,
with a flow rate of 1 mL/min and UV detection at 260 nm. The initial
mobile phase was 0.05% TFA in aqueous solution/0.05% TFA in
MeCN, in the proportions of 98:2 followed by a linear change to 40:
60 over 10 min, and then returned to the initial condition for 15 min.
All data were collected and processed using Perkin-Elmer’s TotalChrom
analytical software. Peak areas from the chromatograms were evaluated
on the basis of a reference curve prepared from standard samples of
eritadenine diluted in the initial mobile phase to concentrations in the
range of 0.0124-0.198 mg/mL.
MATERIALS AND METHODS
Fungal Material. Four different commercial shiitake mushrooms
were used in this study. The fruit bodies of the Lentinus edodes-1 (Le-
1) and Lentinus edodes-2 (Le-2) strains were kindly supplied by Dr.
Gary L. Mills, Diversified Natural Products, Inc., Scottville, MI. The
fruit bodies of the two other shiitake mushrooms were bought at local
stores and denoted here as Lentinus edodes-A (Le-A) and Lentinus
edodes-B (Le-B). The supplier of Le-A was Limax, Horst, The
Netherlands, and the supplier of Le-B was Mykora Oy, Kiukainen,
Finland. The fruit bodies were dried in a mushroom dryer. To eliminate
individual differences among the mushrooms, dried fruit bodies were
subsequently homogenized in a blender, making one batch of 50 g of
homogeneous mushroom powder for each strain. The extraction
procedures were all repeated 3 times, using 3 g of the homogeneous
batches for each extraction.
Preparation of Eritadenine Standard. Since eritadenine is not
commercially available, it was synthesized. In the first step, methyl
2,3-O-isopropylidene-â-D-ribofuranoside was synthesized (18). This
product was further processed to give the compound methyl 2,3-O-
isopropylidene-5-O-p-toluenesulfonyl-â-D-ribofuranoside (19). The third
step was a reaction of sodium salt of adenine with methyl 2,3-O-
isopropylidene-5-O-p-toluenesulfonyl-â-D-ribofuranoside. This reaction
gave the product methyl 5-(6-aminopurin-9H-9-yl)-2,3-O-isopropyl-
idene-5-deoxy-â-D-ribofuranoside. Hydrolysis of this product resulted
in 5-(6-aminopurin-9H-9-yl)-5-deoxy-D-ribofuranose. The final step was
an air oxidation of the previous compound to obtain the product 2(R),3-
(R)-dihydroxy-4-(9-adenyl)-butyric acid (i.e., D-eritadenine (20)). All
chemicals were of analytical grade. To verify the correct product
RESULTS AND DISCUSSION
The usage of shiitake mushroom extracts as food additives
against blood cholesterol may have some benefits. Since the
active substance eritadenine is water soluble, no excessive fat
has to be ingested as is the case with phytosterols used for the
same purpose. Eritadenine consumed in combination with
cholesterol reducing statins might reduce severe side effects
since the mechanism of action for eritadenine differs from the
corresponding one for statins. Using shiitake mushroom as a
cholesterol reducing natural medicine requires a reliable method

Eritadenine in Selected Strains of Shiitake Mushroom
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1179
Figure 2. HPLC chromatograms at 260 nm of a methanol extract from shiitake mushrooms (A), completely isolated eritadenine from shiitake mushrooms
(B), synthesized eritadenine (C), methanol extract from shiitake mushrooms treated with hydrolytic enzymes in buffer (pH 4.8) (D), and methanol extract
from shiitake mushrooms treated with hydrolytic enzymes in water (pH 6.0) (E). Eritadenine is eluted at a retention time of 4.9 min and is represented
by E in the chromatograms of methanol extracts.
of quantifying eritadenine amounts and careful dose-response
studies on humans.
Table 1. Eritadenine Content Measured in the Fruit Bodies of Four
Different Shiitake Mushrooms from Various Treatments by HPLC
To make the quantification of eritadenine as accurate as
possible, the losses from the extraction procedure should be
minimized. By comparing the HPLC chromatogram from simple
methanol extraction (Figure 2A) with the HPLC chromatogram
of isolated eritadenine (Figure 2B), it is clearly shown that
methanol extraction, without further purification, is reliable
enough for quantification. Further, the chromatogram of the
synthesized standard (Figure 2C) coincides with the chromato-
gram of isolated eritadenine (Figure 2B). The chromatogram
resulting from methanol extraction preceded by enzymatic
hydrolysis in either buffer (Figure 2D) or water (Figure 2E) is
not as clean as that from pure methanol extraction but acceptable
for quantification. From recovery studies of isolated eritadenine,
the accuracy values were about 50%. Since the chromatographic
separation was acceptable, methanol extracts were used for
quantification, and further isolations were omitted. Samples from
the extraction procedures stored in a refrigerator were stable
for at least 1 week.
Analysis
eritadenine content (mg/g mushrooms (dw))a
Le-1 Le-2 Le-A Le-B
0.07 3.24 0.27 6.33 0.03
treatment
methanol extraction
enzymatic pretreatment
in acetate buffer pH 4.8
enzymatic pretreatment
in water pH 6.0
3.50
±
±
0.26 3.17
±
±
±
3.82
0.30 NAb
NA
NA
3.60
±
0.11 NA
NA
NA
a All values are mean
±
SD from triplicate analyses. b NA: not analyzed.
study show a statistically significant difference between Le-B
and the other shiitake varieties (p < 0.05). Each extraction was
repeated 3 times, and the spread of the measurements by means
of standard deviations was within a reasonable range. There
were no statistically significant differences between Le-1, Le-
2, and Le-A (p > 0.05). These results indicate the importance
of the source for high eritadenine content, which can be due to
both strain specific properties and cultivation conditions.
The HPLC analyses of eritadenine content in the fruit bodies
of the four different shiitake mushrooms (Table 1) used in this

1180 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Enman et al.
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Methanol extraction preceded by enzyme hydrolysis in acetate
buffer (pH 4.8) gave the highest amounts of eritadenine for Le-
1, followed by enzymatic hydrolysis in water (pH 6.0) with a
slightly lower yield of eritadenine, which is reasonable since
the pH for the reaction was not optimized in this case. The
results indicate that methanol extraction preceded by enzyme
hydrolysis may, to some extent, improve the extraction of
eritadenine from shiitake mushrooms. However, the difference
between methanol extraction preceded by enzymatic hydrolysis
in either buffer or water and pure methanol extraction was not
statistically significant (p > 0.05), and hence, the efficiency of
pretreating the mushrooms with cell wall degrading enzymes
can be considered unimportant. There is also a possibility that
a maximum yield was reached in this case (i.e., there is no more
eritadenine to be released from the fungal cells).
In comparison to other studies (13, 14), the amounts of
eritadenine in shiitake mushrooms are significantly higher in
this study, up to 10 times. There is no information in the
previous studies on what specific strains were used, and the
eritadenine content might be strain dependent. Another factor
that might contribute to the fairly high difference between the
amount of eritadenine found in the present study and previous
ones is the extraction procedure. In all cases, quantification was
preceded by methanol extraction, but there is either no informa-
tion on how the extraction procedure was performed (14) or
the temperature, time, and solid-liquid ratio obviously differ
from previous studies (13). Also, in this study, the mushrooms
were thoroughly crushed into fine particles to make a homo-
geneous fungal material, highly accessible for the subsequent
extraction procedure. Finally, the analytical procedures for
quantification differ between the studies. The amount of
eritadenine has been determined by column chromatographic
fractionation without any reference samples (14) or by GC (13).
No data have been found in the literature pertaining to HPLC
quantification of eritadenine. Since eritadenine is a nonvolatile
compound, it has to undergo derivatization prior to GC analysis;
no such modification has to be done to the target compound
for HPLC analysis.
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To validate the reliability of the proposed HPLC method, a
reference curve was obtained by triplicate measurements of five
different concentration levels in the range of 0.0124-0.1980
mg/mL. This method showed a linear response, r2, of >0.999
and a degree of reproducibility expressed as a relative standard
deviation (RSD%) of <2.1%. Furthermore, the retention peak
obtained for eritadenine in this study indicates a high column
efficiency, signifying sufficient resolution for quantification.
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compounds. Acta Aliment. 1997, 26, 271-277.
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sides. J. Heterocycl. Chem. 1966, 3, 485-489.
In this study, it is clearly shown that HPLC analysis of
eritadenine is highly applicable, and it offers a simple and
sensitive method for separation, identification, and quantification
of this compound.
ACKNOWLEDGMENT
(19) Levene, P. A.; Stiller, E. T. Acetone derivatives of D-ribose. J.
Biol. Chem. 1934, 106, 421-429.
We acknowledge Diversified Natural Products, Inc. (DNP),
Scottville, MI for the contributions of shiitake strains and
Novozymes, Bagsvaerd, Denmark for the enzyme preparations.
(20) Kawazu, M.; Kanno, T.; Yamamura, S.; Mizoguchi, T.; Saito,
S. Studies on the oxidation of reversed nucleosides in oxygen.
I. Synthesis of eritadenine and its derivatives. J. Org. Chem.
1973, 38, 2887-90.
LITERATURE CITED
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Received for review September 6, 2006. Revised manuscript received
January 2, 2007. Accepted January 2, 2007. This work was financially
supported by Teknikbrostiftelsen and Norrbottens Forskningsråd,
Luleå, Sweden and Diversified Natural Products, Inc. (DNP), Scottville,
MI.
JF062559+