Vol. 64, No. 6 (2016)
Chem. Pharm. Bull.
607
4-(3-Chloro-2,5-dimethoxyphenoxy)-3,5-dichlorophenol (ODS ϕ6×250mm, eluted with H2O for 10min and then linear
(Russuphelin D, 3D) gradient from H2O to 20% CH3CN for 50min at a flow rate
Above fr. 1 (12.8mg) was separated by preparative TLC of 1.5mL/min with monitoring at 210nm) to give 4 (3.4mg,
(silica gel, 19:1 CHCl3–MeOH) followed by further puri- tR=20.9min) as colorless syrup. Rf=0.31 (ODS, 1:4 MeOH–
fication by preparative TLC (silica gel, 4:1 CHCl3–EtOAc) H2O); IR νmax (KBr): 3735, 3468, 1732, 1594, 1398, 1188,
to give russuphelin D (3D, 2.3mg) as colorless solids. mp 1054 cm−1; LR-FAB-MS m/z 244 [M+H]+, 162 [M−C5H6O]+,
127–128°C (not recrystallized) (lit.7) 136–138°C); HR-EI-MS HR-FAB-MS m/z 244.1572 [M+H]+; Calcd for C12H22NO4,
1
m/z 347.9720 [M]+; Calcd for C14H11O4Cl3, 347.9723. H-NMR, 244.1549; [α]D31 −14.5 (H2O, c=0.96).
13C-NMR, and IR spectra of 3D were identical to those of the
reported data.6,7)
Detection of Cyclopropylacetyl-(R)-carnitine (4) in Three
Russula spp. Collected in Kyoto, Miyagi, and Saitama
2-Chloro-6-(2,6-dichloro-4-methoxyphenoxy)-1,4-
hydroquinone (Russuphelin G, 3G)
The water extract of R. subnigricans collected in Kyoto
(fruiting bodies; 7.42g/H2O; 22.3mL) was concentrated to
Above fr. 2 (6.0mg) was separated by preparative TLC dryness to give the crude extract (369mg). A partial crude
(silica gel, 19:1 CHCl3–MeOH) to give 2-chloro-6-(2,6-di- extract (10mg) was dissolved into D2O (0.75mL) contain-
chloro-4-methoxyphenoxy)-1,4-hydroquinone (3G, 2.9mg) as ing TSP (0.77mM) as an internal standard. Carnitine ester 4
1
colorless solids. mp 184–185°C (not recrystallized); Rf=0.39 was detected in H-NMR spectrum of the crude extract and
(1:19 MeOH–CHCl3); IR νmax (KBr): 3419, 1610, 1504, 1470, its content was estimated to be ca. 133mg/kg fruiting bod-
1219 cm−1; 1H-NMR (CDCl3, TMS=0.00) δ: 3.82 (3H, s, ies. Similarly, Saitama species (fruiting bodies; 6.96g/H2O;
4′-OMe), 5.92 (1H, d, J=3.0Hz, H-5), 6.56 (1H, d, J=3.0Hz, 20.9mL) and Miyagi species (fruiting bodies; 11.4g/H2O;
H-3), 6.95 (2H, s, H-3′, 5′); 1H-NMR (CD3OD, solvent 34.2mL) were extracted with H2O to give the crude extracts
peak=3.31) δ: 3.84 (3H, s, 4′-OMe), 5.77 (1H, d, J=3.0Hz, (83.0, 257mg, respectively). A part of each water extract was
1
H-5), 6.44 (1H, d, J=3.0Hz, H-3), 7.10 (2H, s, H-3′, 5′); NOE dissolved in D2O and this was checked by H-NMR. No cyclo-
(increased by 5.1%) was observed at H-3′ and H-5′ overlapped propylacetyl-(R)-carnitine (4) was detected in either extracts
signals by irradiation of 4′-OMe; 13C-NMR (CD3OD, solvent (10mg in D2O (0.75mL)).
peak=49.00) δ: 56.8 (4′-OMe), 101.5 (C5), 110.8 (C3), 116.0
(C3′, 5′), 122.8 (C2), 131.0 (C2′, 6′), 137.0 (C1), 141.9 (C1′),
Synthesis of Cyclopropylacetyl-(R)-carnitine (4)
To a stirred cyclopropylacetic acid (0.098mL, 1.05mmol)
147.9 (C6), 151.2 (C4), 158.9 (C4′); HR-EI-MS m/z 333.9547 was added thionyl chloride (0.080mL, 1.10mmol) and the
[M]+; Calcd for C13H9O4Cl3, 333.9567.
mixture was stirred at room temperature (rt) for 1h. To the
Detection of Russuphelins (3) in Other Two Russula spp. crude acid chloride was directly added (R)-carnitine (86.0mg,
Collected in Saitama and Kyoto 0.533mmol) and the mixture was stirred at rt for 1.5h. After
Each EtOAc extract (see Experimental of “Detection of evaporation, the residue was dissolved into H2O and the aque-
3-hydroxybaikiain (2) in other two Russula spp. collected in ous layer was washed with EtOAc. The aqueous layer was ap-
Saitama and Kyoto”) was dissolved in CDCl3 and checked by plied to ODS column chromatography (H2O). The eluate was
1H-NMR. No russuphelins (3) were detected in either extracts neutralized by saturated aqueous NaHCO3 solution and then
(5–10mg in CDCl3 (0.75 m L)).
concentrated to dryness. The resulting residue was extracted
Cyclopropylacetyl-(R)-carnitine (4)
with MeOH and it was filtered through Celite. The filtrate
Isolation of Cyclopropylacetyl-(R)-carnitine (4) from the and washings were concentrated in vacuo to afford cyclopro-
Russula subnigricans (Collected in Kyoto) pylacetyl-(R)-carnitine (4) (60.4mg, 47% yield) as colorless
The fruiting bodies (500g) of Russula subnigricans (col- foam. mp 180°C (decomp.); Rf=0.31 (ODS, 1:4 MeOH–H2O);
lected in Kyoto) were cut into pieces and soaked in aqueous IR νmax (KBr): 3735, 3433, 1734, 1592, 1392, 1179, 1034cm−1;
0.3% AcOH (1.5L) at 4°C overnight. The extract was filtered 1H-NMR (D2O, HOD=4.79) δ: 0.15 (2H, m, H-4′, 5′), 0.52
through filter paper under suction and then the filtrate was (2H, m, H-4′, 5′), 0.99 (1H, m, H-3′), 2.28, (1H, dd, J=7.0,
concentrated to about 100mL under reduced pressure. The 16.0Hz, H-2′), 2.36 (1H, dd, J=7.4, 16.0Hz, H-2′), 2.49 (1H,
concentrated solution was dialyzed (relative molecular mass dd, J=8.0, 16.0Hz, H-2), 2.63 (1H, dd, J=5.6, 16.0Hz, H-2),
(Mr) 14000) against aqueous 0.3% AcOH (2.0L×2) overnight. 3.18 (9H, s, 4-N+Me3), 3.61 (1H, d, J=14.0Hz, H-4), 3.86 (1H,
The dialyzate was concentrated to dryness and lyophilized to dd, J=9.0, 14.0Hz, H-4), 5.63 (1H, m, H-3); 13C-NMR (D2O,
give a crude extract (27.1g). A part (4.8g) of the crude extract DSS=–2.04) δ: 4.2 (C4′, 5′), 4.4 (C4′, 5′), 6.7 (C3′), 39.7 (C2′),
was dissolved in 1% AcOH in MeOH (48mL), and then the 40.9 (C2), 54.5 (4-N+Me3), 67.5 (C3), 68.9 (C4), 175.7 (C1′),
soluble part was applied to an alumina column (aluminium 177.2 (C1); LR-FAB-MS m/z 244 [M+H]+, 162 [M−C5H6O]+,
oxide 90 standardized, Merck, 32g), which was eluted with HR-FAB-MS m/z 244.1555 [M+H]+; Calcd for C12H22NO4,
1% AcOH in MeOH (300mL). The eluate was concentrated 244.1549; [α]D34 −16.6 (H2O, c=0.67).
to 5mL, and this was diluted with aqueous 0.3% AcOH
(10mL) and chromatographed on ODS (Cosmosil 140C18
OPN, 16g) which was eluted with H2O (300mL) and 50%
Toxicity on Mice
Oral Injection
The water extract (200mg) of the mushroom was dissolved
aqueous MeOH (100mL). After removal of MeOH from the in H2O (0.2mL) and the solution was orally afforded to a
50% MeOH fraction, the aqueous solution was washed with mouse (ddy, female, 19–21g body weight) using a catheter.
EtOAc (100mL×3). The aqueous layer was concentrated in
vacuo and then chromatographed on ODS by elution with
Intraperitoneal Injection
Synthetic carnitine ester (4, 10mg) was dissolved in saline
20% MeOH. The obtained fractions which contained a cyclo- (0.2mL) and the solution was intraperitoneally injected to a
propane derivative were concentrated (16.2mg) and purified mouse (ddy, female, 9.5–10.5g body weight).
by preparative TLC (ODS, 20% CH3CN) followed by HPLC
Within 24h, when the mouse died, the extract was regarded