Z. Cao et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116074
+
4
.2.7. Inhibition experiments of self- and Cu2 -induced Aβ1-42 aggregation
After incubation, the absorbance of mixture at 200–600 nm was
and experiments of disaggregation of self-induced aggregated Aβ1-42 fibrils
We used the Thioflavin T-based fluorometric method to evaluate the
measured on the multifunctional enzyme marker (Thermo Scientific).
2
+
inhibitory activity of target compound against self- and Cu -induced
Aβ1-42 aggregation.41,42 For the inhibition experiment of self-induced
4.2.10. In vitro blood–brain barrier permeation assay
The in vitro blood–brain barrier permeation of target compound was
Aβ1-42 aggregation, 20
test compound solution (50
plate. Then the mixture was placed in oven to incubate for 24 h at 37 C.
After incubation, 160 L Thioflavin T solution (5 M in pH 8.5 glycine-
μ
L Aβ1-42 solution (50
μM in PBS) and 20
μ
L
measured by the method described by Di et al.47
4 L porcine brain lipid
μ
μ
M in PBS) were added to the black 96-well
solution (20 mg/mL in n-dodecane) was added to the filter membrane
◦
dropwise. The coated filter membrane was placed at room temperature
μ
μ
for 5 min. 200
μ
L PBS/EtOH (70:30, v/v) was added to the acceptor
NaOH buffer) was added. Then, the fluorescence was measured with
excitation and emission wavelengths at 446 nm and 490 nm. The
wells. Then, 350
μ
L test compound solution (100 g/mL in PBS) was
μ
added to the donor wells. The donor plate was placed over the receptor
◦
percent inhibition of the inhibitor was calculated by the formula: (1-IF
i
/
plate carefully, and incubated at 25 C for 18 h. After the incubation,
IF and IF are obtained in the presence and absence
c
) × 100, in which IF
i
c
150 L test compound solution in the donor and acceptor wells were
μ
of inhibitors after subtracting the background. For the inhibition
added to the 96-well quartz cuvette, respectively. Then, the absorbance
experiment of Cu2 -induced Aβ1-42 aggregation, 20
+
μ
L Aβ1-42 solution
M in pH 8.5 glycine-NaOH
L test compound solution (50 M in PBS) were added
of mixture at 200–600 nm was measured on the multifunctional enzyme
marker (Thermo Scientific). The P value was calculated by the method
e
(
50
μ
M in PBS), 20
μ
L CuCl
2
solution (75
μ
buffer) and 20
μ
μ
which was described previously.
to the black 96-well plate. Then the mixture was placed in oven to
◦
incubate for 24 h at 37 C. After incubation, 160
μL Thioflavin T solution
4.2.11. In vitro neuroprotective effect on H
The neuroprotective effect of representative compound (Z)-13c on
PC-12 cell apoptosis induced by H was measured by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid (MTT)
2 2
O -mediated PC12 cells injury
(
5
μ
M in pH 8.5 glycine-NaOH buffer) was added. Then, the fluorescence
was measured with excitation and emission wavelengths at 446 nm and
90 nm.
As for the experiments of disaggregation of self-induced aggregated
Aβ1-42 fibrils, 20 L Aβ1-42 solution (50 M in PBS) and 40 L PBS (pH
.4) was added to the black 96-well plate, and the mixture was placed in
oven to incubate for 24 h at 37 ℃. After incubation, 20 L test compound
2 2
O
4
method.48 For the cytotoxicity assay, PC-12 cells were firstly seeded into
5
μ
μ
μ
96-well cell culture plates (1 × 10 cells per mL) in DMEM (100
μ
L), and
◦
7
incubated at 37 C for 24 h. After incubation, tested samples (10
μL, at
μ
different concentrations in DMSO) were added to the plates. Then, the
treated wells were incubated for another 2 h. After incubation, MTT
solution (50
μ
M in PBS) was added. Then, the mixture was placed in
◦
oven to incubate for another 24 h at 37 C. After incubation, 160
μL
(100
μ
L, 5 mg/ml in PBS) was added to each well. The plates were
◦
Thioflavin T solution (5
μM in pH 8.5 glycine-NaOH buffer) was added.
incubated for 4 h at 37 C, and the crystal formazan was formed. The
Then, the fluorescence was measured with excitation and emission
wavelengths at 446 nm and 490 nm. The data were analysized by using
the method which was described previously.
supernatants were removed, and the formazan was dissolved in 150 L
μ
of DMSO. Then, the absorbance at 490 nm was determined by using a
Varioskan Flash Multimode Reader (Thermo Scientific). Results are
expressed as percent cell viability compared to untreated cells. As for the
assay to determine the neuroprotective effect of (Z)-13c, the procedures
were similar to the mentioned above, but after the incubation of PC-12
4
.2.8. Antiplatelet aggregation in vitro
The antiplatelet aggregation activity of 3-benzylidene/benzylphtha-
lide Mannich base derivatives was measured by the method which was
cells with tested samples, H
further incubated for 24 h.
2
O
2
(100 M) was added and the plates were
μ
described previously.4
3,44
The blood was taken from rat aorta abdomi-
nalis and mixed with 3.8% sodium citrate (9:1, v/v) to anticoagulate.
After centrifugation (800 r/min, 10 min), the supernate was collected to
obtain the platelet-rich plasma (PRP). The remaining blood was further
centrifuged at 3000 r/min for 15 min to get platelet-poor plasma (PPP).
4.2.12. In vivo study
The passive avoidance test was carried out by the method described
previously.26,49 Kun Ming mice (approval number, SCXK-Sichuan
2018–19) were provided by the Laboratory Animal Center, Sichuan
Academy of Chinese Medicine Sciences (Chengdu, China). Forty-eight
mice (males and females in half) weighting 18–22 g were randomly
divided into six groups including control group, model group, donepezil
group, low dose group, medium dose group and high dose group, each
group contained 8 mice. Before the step-down test was carried out, mice
in each group were treated with corresponding agents by intragastric
administration (i.g.) once a day for a period of seven days. Mice in
control group and model group were treated with 0.5% CMC-Na solu-
tion; mice in donepezil group were treated with donepezil hydrochloride
(5.0 mg/kg); mice in low dose group, medium dose group and high dose
group were treated with (Z)-13c (0.19, 0.95 and 4.75 mg/kg, respec-
tively). On the sixth day, mice were trained 1 h after the intragastric
administration. And except for the control group, mice in other groups
were treated with scopolamine hydrobromide (3.0 mg/kg, i.p.) 30 min
before training. The mice in control group were treated with physio-
logical saline at equal volume. The training equipment included two
parts, platform and copper grid floor. The mice were placed on the
platform initially, and if they step down they will be shocked by the
electricity on the grid floor. The latency time which the mice used to
jump off the platform for the first time and the error times which the
mice were shocked within 5 min were recorded. The step-down test was
carried out again 24 h after the training to evaluate the effects of these
agents on the improvement of cognitive impairment.
Then, the PRP was diluted with PPP to make the platelet concentration
4
of the plasma to 6–7.5 × 10 /
μ
L. The mixture of plasma (270 L) and
μ
test compound solution (20
μ
L, 0.5 mM in physiological saline) was
◦
placed in platelet aggregometer and incubated at 37 C for 30 min. Then,
the solution of ADP (10
μ
L, 10 M, final concentration) was added,
μ
followed by recording the maximal aggregation percentage within 5
min. The inhibition rate was calculated by the formula: 100 × [(1 ꢀ (the
maximal platelet aggregation in samples with the tested compound)/
(
the maximal platelet aggregation in control samples)].
4
.2.9. Metal-chelating studies
We used UV spectrum method to measure the metal-chelating ac-
4
5
tivity of representative compounds. 200
75 M in methanol) and 200 L different kinds of metal solution (75
in methanol) or methanol were added to a vial. The mixture was placed
at room temperature for 2 h. Then, 200 L reaction mixtures were taken
μ
L test compound solution
(
μ
μ
μM
μ
from the vial and added to the 96-well quartz cuvette. After addition, the
absorbance of mixture at 200–600 nm was measured on the multi-
functional enzyme marker (Thermo Scientific).
Compound (Z)-13c, which can interact with different metals, was
chosen to evaluate the stoichiometry of the compound-Cu2 complex.
+
The CuCl
Then, 100
different concentrations of CuCl
2
was dissolved in methanol to obtain 3.75 to 150
μ
M solution.
μ
L test compound solution (75 M in methanol) and 100 L
μ
μ
2
solution were added to 96-well quartz
◦
cuvette. The plate was placed in oven to incubate for 30 min at 37 C.
These mice were sacrificed after passive avoidance test immediately,
1
2
Cao, Zhongcheng
