1
2
T. Morikawa et al. / Phytochemistry xxx (2014) xxx–xxx
by filtration. Evaporation of the solvent from the filtrate under re-
duced pressure gave a residue, which was purified by HPLC [Cos-
mosil 5C18-MS-II, MeOH–1% aqueous AcOH (60:40, v/v)] to give
Effects on cytotoxicity induced by
hepatocytes
The hepatoprotective effects of the constituents were deter-
mined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) colorimetric assay using primary cultured
mouse hepatocytes (Matsuda et al., 2009; Morikawa et al.,
D-GalN in primary cultured mouse
1
0 (2.1 mg, 97.2%).
Alkaline hydrolysis of potentillanoside D (4)
2
010a). Hepatocytes were isolated from male ddY mice (30–35 g)
4
by a collagenase perfusion method. A cell suspension at 4 ꢁ 10
cells in 100 L William’s E medium containing FBS (10%), penicillin
G (100 units/mL), and streptomycin (100 g/mL) was inoculated in
a 96-well microplate and pre-incubated for 4 h at 37 °C under 5%
CO atmosphere. After pre-incubation, 100 L of fresh medium con-
taining -GalN (2 mM) with or without the test sample was added
to the medium. After 44 h incubation, the medium was exchanged
with 100 L of fresh medium, and 10 L of MTT [5 mg/mL in phos-
phate buffered saline (PBS(–))] solution was added to the medium.
After 4 h incubation, the medium was removed, and 100 L of iso-
A mixture of 4 (6.8 mg), 1,4-dioxane (1.0 mL) and 5% aqueous
KOH (1.0 mL) was stirred at 80 °C for 1 h. After being cooled, the
l
+
l
reaction mixture was neutralized with Dowex HCR W2 (H form),
and the resins were removed by filtration. Removal of the solvent
under reduced pressure gave a residue, which was partitioned in
an EtOAc–H O (1:1, v/v) mixture to give an EtOAc-soluble fraction
2
and an aqueous phase. The EtOAc-soluble fraction was subjected to
HPLC [Cosmosil 5C18-MS-II, MeOH–1% aqueous AcOH (70:30, v/v)]
to give cecropiacic acid (17, 5.1 mg, 98.5%).
2
l
D
l
l
l
propanol containing 0.04 M HCl was added to dissolve formazan
produced in the cells. The optical density (O.D.) of the formazan
solution was measured by microplate reader at 570 nm (reference:
Methylation of potentillanosides D (4) and E (5) with TMSCHN
2
A
solution of
4
(3.3 mg) and trimethylsilyldiazomethane
6
55 nm). Inhibition (%) was obtained by following formula.
(
2
TMSCHN , 10% in hexane, ca. 0.2 mL) in MeOH (0.5 mL) was stir-
red at room temperature for 1 h. Removal of the solvent under re-
duced pressure gave 4a (3.1 mg, 90.2%). In a similar manner, 5
Inhibitionð%Þ ¼ ½ðO:D:ðsampleÞ ꢂ O:D:ðcontrolÞÞ=ðO:D:ðnormalÞ
ꢂ O:D:ðcontrolÞÞꢀ ꢁ 100
(
3.3 mg) wad derived to 4a (2.8 mg, 81.5%).
Compound 4a
2
D
1
+ 7.2 (c 0.32, MeOH); for H and 13C
1
Effects on production of NO in LPS-activated mouse peritoneal
macrophages
Screening tests for NO production using TGC-induced mouse
peritoneal macrophages were performed as described previously
An amorphous powder, ½
a
ꢀ
NMR spectroscopic data, see Tables 2 and 3; Positive-ion FABMS m/
+
+
z: 731 [M+Na] ; HRFABMS m/z: 731.3979 [M+Na] (calcd for C38-
12Na, 731.3982).
60
H O
(
Matsuda et al., 2009). Briefly, peritoneal exudate cells were col-
lected from the peritoneal cavities (100 L) of male ddY mice
and were suspended in RPMI 1640 supplemented with 10% FBS,
penicillin G (100 units/mL) and streptomycin (100 g/mL), and
pre-cultured in 96-well microplates (5 ꢁ 10 cells/well) at 37 °C
in 5% CO in air for 1 h. Nonadherent cells were removed by wash-
ing with PBS(–), and the adherent cells (10 g/mL) were cultured in
00 L of a fresh medium containing LPS and various concentra-
tions of test compounds for 20 h. NO productions in each well were
l
Bioassay
Reagents
l
5
LPS (from Salmonella enteritidis), minimum essential medium
MEM), and William’s E medium were purchased from Sigma–Al-
2
(
l
drich Chemical (St. Louis, MO, USA); fetal bovine serum (FBS)
was from Life Technologies (Rockville, MD, USA); and other chem-
icals were from Wako Pure Chemical Industries, Co., Ltd. (Osaka, Ja-
pan). 96-Well microplates were purchased from Sumitomo
Bakelite Co., Ltd. (Tokyo, Japan).
2
l
–
2
assessed by measuring the accumulation of nitrite (NO ) in the cul-
ture medium using Griess reagent. Cytotoxicity was determined by
the MTT colorimetric assay, after 20 h incubation with test com-
pounds. Each test compound was dissolved in DMSO, and the solu-
Animals
tion was added to the medium (final DMSO concentration was
Male ddY mice were purchased from Kiwa Laboratory Animal
Co., Ltd., (Wakayama, Japan). The animals were housed at a con-
stant temperature of 23 ± 2 °C and were fed a standard laboratory
chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). All the experi-
ments were performed with conscious mice unless otherwise
noted. The experimental protocol was approved by the Experimen-
tal Animal Research Committee of Kinki University.
G
0
.5%). N -Monomethyl-
L-arginine
(L
-NMMA) and caffeic acid
phenetyl ester (CAPE) were used as reference compounds. Inhibi-
tion (%) was calculated using the following formula (N = 4).
Inhibitionð%Þ ¼ ½ðA ꢂ BÞ=ðA ꢂ CÞꢀ ꢁ 100
–
A–C: NO
2
concentration (lM) [A: LPS (+), sample (–); B: LPS (+),
sample (+); C: LPS (–), sample (–)].
Effects on D-GalN/LPS-induced liver injuries in mice
The method described by Tiegs et al. was modified and used for
this study (Tiegs et al., 1989). Briefly, male ddY mice weighing about
Inhibitory effects against TNF- -induced cell death in L929 cells
a
L929 cells (RIKEN) were maintained in a Minimum Essential
2
5–30 g were fasted for 20 h before the experiment.
kg) and LPS (10 g/kg) dissolved in saline were injected intraperito-
neally to produce liver injuries. Each test sample was given orally
h before the -GalN/LPS injection. Blood samples were collected
from the infraorbital venous plexus 10 h after -GalN/LPS injection.
D-GalN (350 mg/
Medium Eagle (MEM, Sigma–Aldrich) containing 10% FBS, 1%
l
MEM Non-Essential Amino acids (Invitrogen), penicillin
(100 units/mL), and streptomycin (100 g/mL) at 37 °C under 5%
CO atmosphere. Cells were inoculated in a 96-well tissue culture
plate [5 ꢁ 10 cells/well in 100
tion, 100 mL/well of medium containing TNF-
G
l
1
D
2
3
D
lL/well in MEM]. After 20 h incuba-
sAST and sALT levels were determined using Transaminase CII Test
Wako (Wako Pure Chemical Industries, Co., Ltd.). Hydrocortisone
was used as a reference compound. Test samples were suspended
with 5% arabic gum solution, and the suspension was administered
orally at 10 mL/kgineach experiment, whilethevehiclewas given or-
ally at 10 mL/kg in the corresponding control group.
a
(2 ng/mL) and a
test sample. After 44 h incubation, viability of the cells was as-
sessed by the MTT colorimetric assay (vide ante) (Matsuda et al.,
2009; Morikawa et al., 2010a). Each test compound was dissolved
in DMSO, and the solution was added to the medium (final concen-
tration in DMSO 0.5%).
(
Morikawa, Toshio
