Carnitine-esters from the mushroom Suillus laricinus

Article abstract of DOI:10.1016/j.phytochem.2006.09.014

Carnitine-esters (1-8) including, a compound (R)-3-hydroxybutanoyl-(R)-carnitine (5), were isolated from the mushroom Suillus laricinus. Their structures were determined by spectroscopic analyses and by total synthesis. One of these, (R)-3-hydroxy-2-methylpropanoyl-(R)-carnitine (4), promoted hyaluronan-degradation by human skin fibroblasts.

Full text of DOI:10.1016/j.phytochem.2006.09.014

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 2676–2680
www.elsevier.com/locate/phytochem
Carnitine-esters from the mushroom Suillus laricinus
a,b,
b
c
c
Hirokazu Kawagishi
*, Hiroaki Murakami , Shingo Sakai , Shintaro Inoue
a
Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
b
c
Skin Science Research Group, Basic Research Laboratory, Kanebo COSMETICS INC., 3-28, 5-Chome,
Kotobuki-cho, Odawara-City, Kanagawa 2500002, Japan
Received 13 April 2006; received in revised form 3 August 2006
Available online 19 October 2006
Abstract
Carnitine-esters (1-8) including, (R)-3-hydroxybutanoyl-(R)-carnitine (5), were isolated from the mushroom Suillus laricinus. Their
structures were determined by spectroscopic analyses and by total synthesis. One of these, (R)-3-hydroxy-2-methylpropanoyl-(R)-carni-
tine (4), promoted hyaluronan-degradation by human skin fibroblasts.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
the structure determination and the activity against HA-
degradation of the carnitine-esters.
Hyaluronan (HA) is a non-sulfated glycosaminoglycan
composed of repeating disaccharide units of N-acetylglu-
cosamine and glucuronic acid. There are many reports
showing that hyaluronan is elevated in disorders such as
fibrosis, diseases and tumors (Satoh et al., 2000). For exam-
ple, during the progression of hepatitis, accumulated HA
causes fibrosis and eventually cirrhosis of the liver (Satoh
et al., 2000). An increase of HA in the endocervical canal
at the appropriate stage of pregnancy can result in miscar-
riage (El Maradny et al., 1997). Overproduction of HA
accelerates tumor growth and is associated with cancer
metastasis (Itano et al., 1999; Jacobson et al., 2002). These
results show that the balance of production and degrada-
tion of HA in tissues is strictly regulated. Regulators tar-
geting HA degradation may be expected as useful drugs.
We have already reported hyaluronan-degradation inhibi-
tors, orirubenones, from the mushroom Tricholoma oriru-
bens. (Kawagishi et al., 2004; Sakai et al., 2005).
2. Result and discussion
Fresh fruiting bodies of S. laricinus were extracted with
EtOH and then acetone. The extracts were partitioned
between CHCl3 and H2O and then between EtOAc and
H2O. Among the three fractions, the water-soluble fraction
only showed significant activity. Therefore, this fraction
was further separated using bioassay-guided fractionation.
As a result, eight compounds (1–8) were purified.
Compounds 1–4 and 6–8 were identified as (R)-carni-
tine and its esters based on analysis of their NMR spectro-
scopic data (Tables 1 and 2) as well as from additional
spectroscopic data; most of their structures were also con-
firmed by synthesis (Duncombe and Rising, 1972; Fukao,
2003; Guilbert and Chung, 1974; Holland and Sherratt,
1973; Koeberl et al., 2003; Paul, 2000; Quistad et al.,
1978a,b; Quistad et al., 1978c; Quistad et al., 1986; Quis-
tad et al., 1979; Rashed et al., 1995; Roeschinger et al.,
1885; Shigematsu et al., 1994; Silva et al., 2001; Zytkovicz
et al., 2001). The absolute configuration of 4 was deter-
mined by synthesis of both diastereomers of the (R)-carni-
tine-ester.
In the present study, we found promoting activity in the
extract from the mushroom Suillus laricinus against HA-
depolymerization. We wish to report here the isolation,
*
Corresponding author. Tel./fax: +81 54 238 4885.
E-mail address: achkawa@agr.shizuoka.ac.jp (H. Kawagishi).
0031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.09.014

H. Kawagishi et al. / Phytochemistry 67 (2006) 2676–2680
2677
Table 1
1H NMR spectroscopic data for 1–8 (in D2O)
Position
2
1
2
3
4
5
6
7
8
2.30
2.39
2.35
2.40
2.39
2.37
2.38
2.36
(dd, 15.6, 6.6) (dd, 15.4, 7.6) (dd, 15.4, 7.7) (dd, 15.5, 7.5) (d, 7.0)
2.35 2.53 2.48 2.50 2.51
(dd, 15.6, 7.2) (dd, 15.4, 5.5) (dd, 15.4, 5.5) (dd, 15.5, 5.8) (m)
(dd, 15.6, 7.7) (dd, 15.7, 7.8) (dd, 15.6, 7.6)
2.51
2.52
2.48
(m)
(dd, 15.7, 5.3) (dd, 15.6, 5.8)
3
4
4.47 (m)
3.32
5.49 (m)
3.50
5.47 (m)
3.47
5.54 (m)
3.50
5.51 (m)
3.49
5.50 (m)
3.49
5.49 (m)
3.48
5.49 (m)
3.48
(m)
(d, 14.4)
3.74
(d, 14.3)
3.71
(d, 14.4)
3.77
(d, 14.6)
3.74
(d, 14.5)
3.75
(d,14.7)
3.73
(d, 14.4)
3.73
(dd, 14.4, 8.6) (dd, 14.3, 8.9) (dd, 14.4, 8.7) (dd, 14.6, 8.7) (dd, 14.5, 8.5) (dd, 14.7, 8.6) (dd, 14.4, 8.7)
N+(CH3)3 3.12 (s)
20
3.07 (s)
2.02 (s)
3.04 (s)
2.35
3.06 (s)
2.67
3.06 (s)
2.39
3.05 (s)
2.51
3.05 (s)
2.48
3.05 (s)
1.61
(q, 7.7)
(m)
(m)
(m)
(d, 6.4)
(dddd, 6.5, 6.5, 6.4, 6.7)
2.51 (m)
4.12
(m)
30
0.95
(t, 7.7)
3.59
(d, 6.1)
4.00
(m)
1.05
1.18
(s)
0.88
(m)
40
1.10
(d, 6.4)
(d, 6.4)
1.00
(t, 7.1)
20-CH3
30-CH3
1.00
(d, 7.0)
1.18 (s)
Table 2
13C NMR spectroscopic data for 1–8 (in D2O)
Position
1
2
3
4
5
6
7
8
1
2
3
4
178.9
43.8
64.9
71.0
54.9
177.2
40.7
67.4
68.7
54.3
173.3
21.2
177.2
40.9
67.4
69.0
54.5
176.7
28.2
8.7
177.9
40.8
67.4
68.7
54.3
176.8
42.6
63.9
177.1
40.9
67.6
69.0
54.5
173.4
44.0
65.2
22.7
176.9
45.5
66.3
67.5
53.1
175.0
40.0
67.4
18.7
9.6
176.2
40.6
67.4
68.6
54.2
172.4
47.6
70.3
28.4a
177.2
41.0
67.4
68.9
54.5
177.0
13.5
9.4
N+(CH3)3
10
20
30
40
9.5
20-CH3
30-CH3
a
12.9
29.0a
Interchangeable.
1
degrees of unsaturation in the molecule. The H and 13C
NMR spectra of 5, along with DEPT and HMQC analyses,
suggested that this compound was also an ester of (R)-car-
nitine (Tables 1 and 2). The COSY and HMBC spectra
indicated a contiguous sequence of coupled signals of the
acid part of the ester, CH3–CH–CH2–COO– [dH 1.10
(3H, d, J = 6.4), dC 22.7; 4.12 (1H, m), 65.2; 2.39 (1H,
m), 2.51 (1H,m), 44.0; 173.4]. This sequence and the molec-
ular formula suggested that this compound was a 3-
hydroxybutanoate ester of (R)-carnitine. Its absolute con-
figuration was determined by preparation of both diaste-
1
reomers. The H and 13C NMR spectroscopic data, and
[a]D of (R)-3-hydroxybutanoyl-(R)-carnitine were com-
pletely identical with those of 5.
Compounds 1–8 were evaluated in a hyaluronan-degra-
dation promoting assay. Among them, (R)-3-hydroxy-2-
methylpropanoyl-(R)-carnitine (4) only promoted the
degradation; 24% and 28% of HA was degraded at 0.4
and 0.8 mM, respectively.
Compound 5 was isolated as a clear amorphous solid
and its FABMS showed a molecular ion peak at m/z 248
([M+H]+). Its molecular formula, C11H21NO5, as deter-
mined by HRFABMS, indicated the presence of two

2678
H. Kawagishi et al. / Phytochemistry 67 (2006) 2676–2680
25
3. Experimental
3.1. General
Compound 5: ½aꢀD ꢁ28 (H2O, c 0.55). IR (neat) mmax
cmꢁ1: 3347, 1735; FABMS (matrix, glycerol) m/z 248
(M+H)+; HRFABMS m/z 248.1490 [calcd for
C11H22NO5 (M+H)+, 248.1492].
1
The H NMR spectra (one- and two-dimensional) were
recorded on a JEOL lambda-500 spectrometer at 500 MHz,
while 13C NMR spectra were recorded on the same instru-
ment at 125 MHz. The FABMS and HR-FABMS spectra
were recorded on a JEOL DX-303HF. A JASCO grating
infrared spectrophotometer was used to obtain the IR spec-
tra. The [a]D spectrum was measured by using a JASCO
DIP-1000 spectropolarimeter. MPLC was carried out using
a YAMAZEN MPLC-system (Japan) and an UltraPack
ODS-S50D column (50 · 300 mm, YAMAZEN, Japan).
HPLC separations were performed with a JASCO Gulliver
system. YMC-Pack polyamine II columns (20 · 250 mm,
YMC, Japan) or Wakosil II columns (20 · 250 mm, Wako
pure chemicals, Japan) were used for HPLC. Silica gel plate
(Merck F254) and silica gel 60 N (Merck 100–200 mesh)
were used for analytical TLC and for flash CC, respec-
tively. Sepharose CL-2B was purchased from Pharmacia
Fine Chemicals (Sweden). Methyl (R or S)-3-hydroxybut-
anoate and methyl (R or S)-3-hydroxy-2-methylpropano-
ate were products of Sigma-Aldrich, USA. Hyaluronan
variants extracted from pigskin (4–6 · 104 Da) and human
umbilical cord (8–12 · 105 Da) were obtained from Sei-
kagaku Corporation (Japan).
3.4. Preparation of 4, 5 and their diastereomers
(R)-3-Hydroxybutanoic acid (20.8 mg, 0.20 mmol)
derived from its methyl ester by hydrolysis with 1 M
KOH was dissolved in DMSO (0.2 ml) and N-(3-dimeth-
ylaminopropyl)-N0-ethylcarbodiimide
(31.0 mg,
0.20
mmol) was added to the solution. The mixture was stirred
at room temperature for 1 h and (R)-carnitine (32.2 mg,
0.20 mmol) was added to the mixture. The reaction mixture
was stirred at 80 ꢁC for 24 h and lyophilized. The dried
reaction mixture was separated by reversed-phase HPLC
(1% aqueous MeOH), giving (R)-30-hydroxybutanoyl-(R)-
carnitine (7.2 mg, 0.029 mmol).
(S)-30-Hydroxybutanoyl-(R)-carnitine (5.0 mg), (R)-30-
hydroxy-20-methylpropanoyl-(R)-carnitine (2.3 mg), and
(S)-30-hydroxy-20-methylpropanoyl-(R)-carnitine (2.8 mg)
were also prepared by the same method.
25
(R)-30-Hydroxybutanoyl-(R)-carnitine; ½aꢀD þ 29 (H2O,
c 0.20).
25
(S)-30-Hydroxybutanoyl-(R)-carnitine; ½aꢀD ꢁ 5:1 (H2O,
1
c 0.20). H NMR (D2O): d 1.08(3H, d, J = 6.4), 2.37(1H,
m), 2.40(1H, m), 2.50(1H, m), 2.52(1H, m), 3.04(9H, s),
3.48(1H, d, J = 14.3), 3.73(1H, dd, J = 14.3, 8.7),
4.11(1H, m), 5.48(1H, m).
3.2. Fungus materials
25
(R)-30-Hydroxy-20-methylpropanoyl-(R)-carnitine; ½aꢀD ꢁ
Mature fruiting bodies of Suillus laricinus (Berk. in
Hook.) O. Kuntze were collected at Narusawa village,
Yamanashi Prefecture in Japan and identified by the one
of the authors (H. K.). A voucher specimen of the organ-
ism (SL-02-09) is located in Shizuoka University.
26 (H2O, c 0.10).
25
(S)-30-Hydroxy-20-methylpropanoyl-(R)-carnitine; ½aꢀD þ
0:68 (H2O, c 0.10). 1H NMR (D2O): d 0.99(3H, d, J = 7.0),
2.37(1H, dd, J = 15.6,7.8), 2.49(1H, dd, 15.6, 5.5), 2.62(1H,
m), 3.04(9H, s), 3.48(1H, d, J = 14.3), 3.56(1H, dd, J =
11.0, 7.0), 3.61(1H, dd, J = 14.3,8.9), 3.75(1H, dd,
J = 11.0, 3.9), 5.49(m).
3.3. Extraction and isolation
Fresh fruiting bodies of S. laricinus (17.1 kg) were
extracted with EtOH (10 l, 4 times) and then acetone
(5 l). The extracts were combined, concentrated under
reduced pressure, and partitioned between CHCl3 and
H2O. The H2O layer was further partitioned between
EtOAc and H2O. The residue (253.4 g) obtained after
removing H2O was fractionated by silica gel flash CC
(CHCl3/MeOH/H2O = 5.5/4.0/0.5, 4.5/4.5/1.0, 3.5/5.5/
1.0, 0/8.0/2.0, 0/5.0/5.0, each 3 L) to obtain 13 fractions.
Fraction 10 (6.5 g) was further separated by reversed-phase
MPLC (1% aqueous MeOH) and 14 fractions were
obtained. Fraction 10-1 (349.1 mg) was separated by poly-
amine HPLC (1% aqueous MeOH) to afford compounds 1
(3.9 mg), 2 (6.2 mg), 3 (2.6 mg), 4 (1.3 mg), 5 (1.5 mg), 6
(2.3 mg), 7 (1.2 mg), and 8 (1.0 mg).
3.5. Bioassay
3.5.1. Cell lines and culture procedure
Detroit 551 human skin fibroblasts purchased from the
American Type Culture Collection (USA) were grown to
confluence on 12-well plates in Eagle’s minimal medium
(ICN Biomedicals Ltd., USA) supplemented with non-
essential amino acids, 1 mM sodium pyruvate, and 10%
(v/v) FBS (JRH Pharmaceutical Co., USA). Cultures were
grown at 37 ꢁC in a humidified atmosphere of 5% CO2 and
95% air.
3.5.2. Preparation of [3H]HA
[3H]HA was prepared by the method described by
Underhill and Tool with some modification. (Underhill
and Toole, 1979) Briefly, confluent Detroit 551 fibroblasts
were incubated with 10lCi/ml of [3H]glucosamine (Amer-
ican Radiolabeled Chemicals Inc., USA) at 37 ꢁC. The
conditioned medium was pooled, digested with pronase
25
Compound 4: ½aꢀD ꢁ25 (H2O, c 0.11). IR (neat) mmax
cmꢁ1: 3342, 1740; FAB-MS (matrix, glycerol) m/z 248
(M+H)+; HR-FAB-MS m/z 248.1499 [calcd for
C11H22NO5 (M+H)+, 248.1492].

H. Kawagishi et al. / Phytochemistry 67 (2006) 2676–2680
2679
(0.3 mg/ml) (Calbiochem, USA) at 37 ꢁC for 24 h, and
References
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fuged again (12,000 · g, 20 min), precipitated with four
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Depolymerizing activityðdpm=wellÞ
¼ Cell Freeꢂ ꢁ Culturedꢂꢂ
(Cell Free* is the total of the radioactivities of fractions 1–8
after culture without cells, and Cultured** is the total of the
radioactivities of fractions 1–8 of the CL-2B column after
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Financial support of this research was partially provided
by a Grant-in-Aid for Scientific Research on Priority Areas
‘Creation of Biologically Functional Molecules’ (No.
17035037) from the Ministry of Education, Science, Sports
and Culture of Japan.

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