Dimers in Soil
J ournal of Natural Products, 1998, Vol. 61, No. 2 191
under vegetation (primarily Sasa palmata and Pinus
densiflora.). For the 2D TLC test for anthraquinones,
25 of the surface soil samples (top 10-20 cm horizon)
belonging to Umbric Andosols (17 soils), Distric Cam-
bisols (7 soils), and Haplic Acrisol (1 soil) were collected
from locations ranging from the subtropical to the
subarctic region in J apan, and two of the surface soil
samples (top 10-cm horizon) belonging to Distric Cam-
bisols were collected in Nepal. These sampling sites are
indicated in Figure 1. The sampling sites in J apan were
areas under various vegetation (e.g., deciduous broad-
leaf forests, evergreen broad-leaf forests, needle-leaf
forests, Sasa glasslands, or Miscanthus glassland).8 One
of the sampling sites in Nepal was an area under
pasture of grazing grass and another was pine forests
(Pinus wallichiana). Each soil was air-dried and passed
through a 2-mm screen.
TLC on Si gel (CHCl3-EtOAc (9:1), Rf ) 0.43) to obtain
chrysotalunin tetraacetate (light yellow powder, 9 mg);
1
UV, νmax (IR), EIMS, and H-NMR (250 MHz, CDCl3)
data agreed with the literature.3
(-)-7,7′-Bip h yscion (2): orange microcrystals; HRE-
IMS 566.1142 (calcd for C32H22O10, 566.1211); UV, ORD,
1
νmax (IR), EIMS, and H-NMR (250 MHz, CCl4) data in
agreement with literature data.10
Acetyla tion of 2. A mixture of compound 2 (5 mg),
Ac2O (0.4 mL) and pyridine (0.8 mL) was heated in a
boiling H2O bath for 30 min. After evaporation in
vacuo, the reaction residue was chromatographed by
preparative TLC on Si gel (CHCl3-EtOAc 9:1, Rf ) 0.43)
to obtain chrysotalunin tetraacetate (light yellow pow-
der, 3 mg): UV, νmax (IR), EIMS, and 1H-NMR (250
MHz, CDCl3) data agreed with the literature.11
Hin a k u r in (3): orange-red microcrystals; UV (CHCl3)
λ (log ꢀ) 259 (4.59), 300 (4.40), 455 (4.28) nm; IR (KBr)
νmax 1672 (nonchelated CdO), 1626 cm-1 (chelated
CdO); [R]D 0° (CHCl3); HREIMS 566.1211 (calcd for
C32H22O10, 566.1211); EIMS m/z (rel int): 566 [M+]
(100), 535 (28), 504 (9), 297 (17), 283 (11); 1H NMR (250
MHz, CDCl3) δ 13.04 (2H, s), 12.12 (2H, s), 7.33 (2H, br
s, H-4, H-4′), 7.01 (2H, br s, H-2, H-2′), 6.76 (2H, s, H-7,
H-7′), 3.73 (6H, s, OCH3-6, OCH3-6′), 2.32 (6H, s, CH3-
3, CH3-3′).
Extr a ction a n d Isola tion . Air-dried soil samples
(15 kg) were extracted with hot CHCl3 for 1 day. The
CHCl3 extract (45 L), including precipitate, was filtered
through a membrane filter (1.0 µm). The precipitate
(27 g) on the filter was successively washed with MeOH,
H2O, Me2CO, EtOAc, and n-hexane and repeatedly
recrystallized from CHCl3, and 70 mg of 1 was obtained.
The concentrated syrupy residue (84 g), obtained by
removal of the solvent from the filtrates and washings
in vacuo, was partitioned between CHCl3 and H2O. The
former layer was concentrated under reduced pressure,
and the resulting syrupy residue was chromatographed
on Si gel (600 g) using CHCl3 as eluent. The eluate (23
g) was concentrated and rechromatographed on Si gel
(200 g) by eluting with a stepwise gradient of hexane-
C6H6 (1:1) to C6H6. Individual fractions were combined
(TLC monitoring) to provide three positive hydroxyan-
thraquinone fractions. The first fraction (1.4 g) was
subjected to preparative TLC (Si gel, 2 mm, petroleum
benzine-EtOAc-formic acid-H2O 89:10:1:saturated)
and yielded an upper yellow band (5, Rf ) 0.55, 15 mg)
and a lower yellow band (6, Rf ) 0.33, 18 mg). The
second fraction (2.6 g) was dissolved in CCl4, and the
CCl4 solution was filtered through a membrane filter
(0.2 µm). The concentrated filtrate was rechromato-
graphed on preparative TLC (Si gel, 2 mm, hexane-
Me2CO-H2O 10:10:7), and the eluate from the major
yellow band was successively recrystallized from EtOH,
hexane, and MeCN to produce 4 (11 mg). The third
fraction (2.7 g) was subjected to column chromatography
(Cosmosil 75 C18-open) with 75% EtOH. The first yellow
band on a C18 column was then collected, concentrated,
and rechromatographed on preparative TLC (Si gel, 2
mm, hexane-EtOAc-H2O 9:1:saturated). The major
yellow band (Rf ) 0.45) on preparative TLC was eluted
with EtOH, and the eluate was recrystallized from
EtOH (3, 8 mg). The second yellow band on the C18
column was collected, and the eluate was recrystallized
from EtOAc (2, 15 mg).
Clea va ge of 3. A mixture of compound 3 (2 mg), 1N
NaOH (2 mL), and Na2S2O4 (0.1 g) was heated at 70 °C
for 20 min. After extraction with CHCl3 followed by
acidification with HCl, the CHCl3 layer was evaporated
in vacuo. The residue was chromatographed on a TLC
plate by developing with petroleum ether-EtOAc-H2O
(75:25:3.5). The major compound was identified as 5
by co-TLC and EIMS data by comparison with an
authentic sample.
Micr oca r p in (4): orange microcrystals; HREIMS
1
506.0996 (calcd for C30H18O8, 506.1000); H NMR (250
MHz, CDCl3) δ12.44 (1H, s), 12.36 (1H, s), 12.00 (1H,
s), 11.92 (1H, s), 7.97 (1H, d, J ) 8.0 Hz, H-5′), 7.86
(1H, dd, J ) 8.0, 1.1 Hz, H-5), 7.83 (1H, br s, H-4), 7.70
(1H, t, J ) 8.0 Hz, H-6), 7.69 (1H, s, H-4′), 7.62 (1H, d,
J ) 8.0 Hz, H-6′), 7.31 (1H, dd, J ) 8.0, 1.1 Hz, H-7),
7.13 (1H, br s, H-2′), 2.49 (3H, s, CH3-3′), 2.30 (3H, s,
CH3-3); assignments are based on interpretation of
COSY NMR data, and UV, ORD, νmax (IR), and EIMS
data in close agreement with literature data.14 1H-NMR
data are reported for the first time.
Acetyla tion of 4. A mixture of compound 4 (1.5 mg),
Ac2O (0.05 mL), and pyridine (0.1 mL) was heated at
60 °C for 30 min. After evaporation in vacuo, the
reaction residue was subjected to preparative TLC on
Si gel (CHCl3, Rf ) 0.55) to obtain microcarpin tetraac-
etate (orange yellow powder, < 1 mg): UV, νmax (IR),
EIMS, and 1H-NMR (250 MHz, CDCl3) data in close
agreement with the literature.14
Ch r ysop h a n ol (5): obtained as an orange-yellow
Ch r ysota lu n in (1): orange amorphous solid; HRE-
IMS 506.1011 (calcd for C30H18O8, 506.1000); [R]D 0°
(CHCl3); other spectroscopic data (EIMS, UV, IR) were
in agreement with literature data.3
powder and identified by comparison of its TLC, IR, UV,
1
EIMS, and H-NMR data with an authentic sample.
P h yscion (6): obtained as orange needles and identi-
1
fied by comparison of its TLC, IR, UV, EIMS, and H-
Acetyla tion of 1. A mixture of compound 1 (15 mg),
Ac2O (1 mL), and pyridine (2 mL) was heated in a
boiling H2O bath for 30 min. After evaporation in
vacuo, the reaction residue was subjected to preparative
NMR data with an authentic sample.
Detection of Soil Hyd r oxya n th r a qu in on es. Soil
samples (20 g) were extracted with CHCl3 using Soxhlet.