M. Ota et al.
Journal of Ethnopharmacology 278 (2021) 114262
2.6. Preparation of brain homogenate
Milford, MA, USA). The analytical column used was a Mightysil RP-18
GP II (4.6 i.d. × 250 mm, 5 μm, Kanto Chemical). For condition 1, the
The preparation of the brain homogenate was performed with
modification according to methods described in previous studies (Risa
et al., 2004; Choi et al., 2017). Rats were sacrificed by CO2 inhalation,
the heads were separated from the body, and the cerebral cortex was
collected. Cerebral cortex samples from eight rats were homogenized in
40 ml of Tris-citrate buffer (50 mM, pH 7.1), and the suspension was
centrifuged at 4 ◦C for 15 min at 2.7 × 104 × g. The sediments were
washed three times with Tris-citrate buffer. The washed sediment was
resuspended in 40 ml Tris-citrate buffer. The suspension was incubated
at 37 ◦C for 30 min and then centrifuged for 10 min at 2.7 × 104 × g. The
final sediment was resuspended in 60 ml of Tris-citrate buffer and stored
in aliquots at ꢀ 80 ◦C until use. The protein concentration of this sus-
pension was 7.94 mg/ml, as measured using a BCA™ Protein Assay Kit
(Thermo Scientific, Rockford, IL, USA).
mobile phase was passaged using a linear gradient elution system of
0.1% formic acid (A) and methanol (B), at a flow rate of 1.0 ml/min,
with the following gradient profile: increasing from 10% B to 20% (0–7
min), increasing from 20% to 25% (7–13 min), 25% (13–28 min),
increasing from 25% to 35% (28–29 min), increasing from 35% to 45%
(29–43 min), increasing from 45% to 85% (43–48 min), increasing from
85% to 100% (48–53 min), and 100% (53–63 min). For condition 2, the
mobile phase used an isocratic system with 70% B. The column tem-
perature was 40 ◦C for both conditions, and the detection wavelengths
for conditions 1 and 2 were 210–340 nm and 198 nm, respectively. This
method was based on previous studies assessing VO (Navarrete et al.,
´
2006; Lucio-Gutierrez et al., 2012). The peaks of valerenic acid and KGD
were identified by comparison with standard compounds (valerenic
acid, Extrasynthese, Lyon, France).
2.9. Quantitative analysis using LC-MS/MS for VF extracts
2.7. Binding assay for flunitrazepam sites of GABAA receptor
LC/MS/MS system (Quattro Premier XE, Waters) was used. Herein,
the mass spectrometer used an electrospray ionization source in positive
ion mode with multiple reaction monitoring. The analytical column used
The GABAA receptor binding assay was performed as described
previously (Benke et al., 2009; Choi et al., 2017; Risa et al., 2004), with
the modification using a 96-well plate instead of a glass filter. The wells
was an Inertsil ODS-4, 2.1 i.d. × 100 mm, 3 μm (GL Sciences Inc., Tokyo,
of a 96-well plate were coated with poly L-lysine solution (20 μg/ml,
Japan). The mobile phase was delivered using a linear gradient elution
system with 0.5% acetic acid (A) and acetonitrile containing 0.5% acetic
acid (B), at a flow rate of 0.25 ml/min, with the following gradient
profile: increasing from 25% B to 30% (0–0.8 min), increasing from 30%
to 60% (0.8–3.2 min), 60% (3.2–7.6 min), increasing from 60% to 80%
(7.6–7.7 min), and 80% (7.7–9.0 min). The column temperature was
maintained at room temperature. The transitions (precursor to
daughter) were monitored, with retention times detected as 339.4 to
201.2 m/z for KGD (6.2 min), 297.4 to 291.2 m/z for KG2 and KG8 (4.4
and 5.1 min, respectively), 255.4 to 219.2 m/z for KG (3.0 min), 281.4 to
Sigma Aldrich, St. Louis, MO, USA) for 1 h at room temperature. After
washing the wells with H2O, 50 l of rat brain homogenate (4 mg pro-
tein/ml) was added to each well and dried by standing on a hot plate
(40 ◦C) overnight. After washing the wells with 200
l of 0.05% Tween
20 in phosphate-buffered saline (T-PBS; 0.15 M, pH 7.2) three times,
200 l of Block Ace® solution (blocking buffer; 40 mg/ml in Tris-citrate
μ
μ
μ
buffer, pH 7.1, Snow Brand Milk Products, Tokyo, Japan) was added to
wells, and the plate was incubated at 37 ◦C for 1 h. The samples dis-
solved in ethanol was collected into the test tubes, and dried up under N2
gas flow, and then, dissolved in blocking buffer directly by sonication at
room temperature for more than 30 min. After discarding the blocking
203.2 m/z for α-KA (7.4 min), 163.1 to 130.9 m/z for CI (6.2 min), and
195.2 to 139.0 m/z for p-hydroxybenzoic acid butyl ester (PHB, Nacalai
buffer in 96-well plate, 50 μl of each sample or blank solution in blocking
Tesque, 5.7 min; used as an internal standard). Linear regressions over
buffer and 50
μ
l of [3H]-flunitrazepam (1 nM in blocking buffer,
the concentration range 7.81 ng/ml– 2.00 μg/ml for KGD, KG2, KG8,
American Radiolabeled Chemicals, St. Louis, MO, USA) were added to
the wells; the plate was incubated at 4 ◦C overnight. Diazepam (Fujifilm
Wako Pure Chemicals, Osaka, Japan) was used as the positive control.
For the nonspecific binding (NSB) group, another excessive concentra-
α
-KA, KG, and CI were examined using the peak-area ratios of the
compounds to their internal standards and the least-squares method (r2
> 0.98).
tion of clonazepam (20 μM, Fujifilm Wako) was selected based on the
2.10. Animal experiments
previous literature (Benke et al., 2009; Risa et al., 2004). In addition,
total binding (TB) was determined using a blank blocking buffer. After
The Vf6 ethanol extract was mixed with the same amount of dextrin
(VIANDEX-BH®, Showa Sangyo, Tokyo, Japan), and the following
samples were prepared: sample A, glycerin fatty acid ester (GFE; EXCEL
S-95®, Kao, Tokyo, Japan) and sesame oil (Kuki Sangyo, Mie, Japan)
mixed in a ratio of 0.5:14 (w/w). Then, 0.79 g of Vf6 extract/dextrin
mixture was mixed with 10 ml of GFE/sesame oil mixture; sample B,
0.79 g of Vf6 extract/dextrin mixture was mixed with 10 ml of 5% gum
arabic (GA; Kanto Chemical, Tokyo, Japan). The difference between
these two emulsifiers is that GFE uses oil as the solvent, and GA uses
water. The Vf6 extract/dextrin mixture (0.79 g) corresponded to 10 g of
the Vf6 sample.
washing the wells with 200 μl of T-PBS five times, 100 μl of 10% sodium
lauryl sulfate (SDS, Nacalai Tesque, Kyoto, Japan) solution was added to
the wells, and the plate was incubated at room temperature for 1 h. The
contents of each well were transferred into liquid scintillation vials
containing 3 ml Clear-sol® (Nacalai Tesque), and the radioactivity
(disintegration per minute, DPM) was measured using a liquid scintil-
lation counter.
The following equation was employed to determine the displacement
percentage of radioligand binding:
(
)
DPMsample – DPMNSB
DPMTB – DPMNSB
Binding displacement (%) = 1 ꢀ
× 100
Mice were fasted overnight and then orally administered 10 ml/kg of
sample A or B. Thirty minutes after administration, mice were sacrificed
by CO2 inhalation, and blood samples were collected from the abdom-
inal vein into heparinized tubes; brain tissues were then separated. The
blood samples were centrifuged at 4 ◦C for 7 min at 1.4 × 104 × g, and
the plasma samples were maintained at ꢀ 20 ◦C until analysis. The
preparation of brain samples was performed in accordance with previ-
ously described methods, with slight modifications (Hashiguchi et al.,
2001; Fong et al., 2017). Individual brain tissue was washed with
ice-cold PBS, gently wiped with paper to remove excess water, and then
the weight of brain tissue was recorded. The brain tissue was dipped into
2-fold weight of ethanol, and homogenized using Pestle tissue grinder.
The half-maximal effective concentrations (EC50) were calculated
from the least square regression line plotted from 3 points that crossed
50% of the control logarithmic concentration values.
2.8. Qualitative analysis using HPLC
For each powdered sample (Vf1–5 and Vo1–4), 200 mg was soni-
cated in 10 ml of ethanol for 30 min. After centrifugation at 3500 rpm
for 10 min, the supernatants were filtered through a 0.45 μm membrane
filter and analyzed using an HPLC system (Alliance HPLC, Waters,
4
Ota, Misato
