J.-c. Li, et al.
Bioorganic Chemistry 92 (2019) 103266
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sterile slide to incubate. Then 0.5 mL of 2,7-dichlorodihydrofluorescein
diacetate (DCFH-DA) solution (Beyotime, China) was added and in-
cubated for half an hour at 37 °C in coverture. After incubation, the
mycelia were washed by pre-cooled PBS for three times in 15 min.
Then, the samples were observed and photographed immediately (Carl
Zeiss LSM 800, Germany).
5
.2.6. Nuclear staining assay [41]
B. cinerea mycelia tips were treated with A-0 (5 μg/mL) for three
days and then fixed with stain fixative for half an hour under re-
frigerated temperature. Following, the hyphae were washed with PBS
for two times and incubated at 25 °C for 20 min after treated with
Hoechst 33,258 (Beyotime, China). Then a coverslip was placed on, and
the samples were observed and photographed under LSM 800 laser
confocal microscope immediately (Carl Zeiss LSM 800, German).
Fig. 6. Effects of Compound A-0 on the nuclear morphology of B. cinerea my-
celia. (A) Control, 0.5% DMSO; (B) Treated with 0.5% DMSO plus Compound A-
0
at 5 µg/mL.
The fruit of tomato (Lycopersicum esculentum Mill), which was wa-
shed and treated with water and 75% aqueous ethyl alcohol in advance,
rinsed with water, and evaporation under room temperature. The fruits
were wounded (d = 5 mm) using an inoculating needle and then each
pathogen was inoculated. The test sample dissolved in 200 μL of DMSO,
then 50 μL of Tween-80 was added and distilled water at concentrations
of 25, 50 and 100 μg/mL respectively was added to form 10 mL sus-
pension. The fruits treated by the aqueous of DMSO (2%) containing
Tween-80 (0.5%) were used as the control. Commercial fungicide
boscalid was used as the positive control. All of the treated fruits were
then placed into an illumination incubator (23 ± 2 °C and 85% relative
humidity) for 4 days. This experiments were repeated 3 times. The
statistical analyses were performed by SPSS Statistic 19.0.
5.2.7. Cytotoxicity assay
The cytotoxicity against HL7702 and PC12 cell lines were per-
formed using CCK-8 method according to previously described methods
[43]. Cells were treated with different concentrations of tested samples
in the growth medium for 24 h, and the absorbance was measured at
450 nm (Thermo Scientific Multiskan MK3 microplate reader, USA).
Five replicates were performed.
5.3. Statistical analysis
Data were analyzed using SPSS software version 18.0 and expressed
as the means ± SD. The concentration for 50% of maximal effect
(EC50) were obtained from the parameters in the regression curves for
the different concentrations. The half maximal inhibitory concentration
(IC50) was calculated using the complementary log-log (CLL) model.
5
.2.3. Scanning electron microscopy (SEM) observations
To examine the effects of A-0 on B. cinerea microstructure, scanning
electron microscopy (SEM) observations were performed according to
described methods [40]. The mycelia blocks with the specification of
5
.0 mm × 4.0 mm square was cut from the growth boundary of the
Acknowledgements
fungi on PDA mediums treating with compound A-0 at 5 μg/mL. After
fixed with glutaraldehyde for 1 day and washed with PBS for three
times, the blocks were then fixed with osmium tetraoxide for 2 h. And
then the samples were washed with PBS again and dehydrated by
aqueous ethanol from 20% to 100%. After drying at critical point and
gold coating, SEM observations were carried out with a scanning
electron microscope (Hitachi, S-3400 N, Japan).
This work was supported financially by the National Natural Science
Foundation of China (21672092, 21877056) and the National Key
Research and Development Program of China (2017YFD0201404);
Support was also supplied by the Key Program for international S&T
cooperation projects of China Gansu Province (18YF1WA115).
HYPERLINK "SPS:role::contributing" Author contributions
statement
5
.2.4. Effect on the mitochondrial membrane potential (MMP)
Effect on the mitochondrial membrane potential (MMP) of B. cinerea
mycelia was performed according to the previously described method
Jun-cai Li and Ren-xuan Wang performed the chemical synthesis,
biological tests; Jun-cai Li wrote the paper; Ying-qian Liu conceived this
work; Yu Sun, Jia-kai Zhu, Zhong-min Zhao, Guan-fang Hu, Yu-ling
Wang, Rui Zhou and Yin-fang Yan carried out the bioassay experiments
and prepared the data; Jing-wen Peng conducted structure detection.
Xiao-fei Shang assisted in writing the paper and checked the manu-
script.
[
41]. The hyphae treated with A-0 at 5 μg/mL were stained with 0.5 mL
of Rhodamine 123 (Beyotime, China) and incubated in darkness
(
30 min at 37 °C). Then the samples were washed using pre-cooled PBS
for two times in 10 min. Finally, the samples were observed and pho-
tographed under LSM 800 laser confocal microscope.
5
.2.5. Production of reactive oxygen species (ROS)
The accumulation of reactive oxygen species (ROS) was measured
Declaration of competing interest
by the method previously described [42]. The mycelia tips of B. cinerea
(
d = 5 mm) treated with A-0 at 5 μg/mL for three days were placed on a
All authors declare that they have no competing interests.
Table 7
Cytotoxic activities of isolated compounds against HL7702 and PC12 cell lines (IC50 in μg/mL)*
Isocryptolepine
Demethylated Isocryptolepine
A-0
A-1
B-1
B-2
Azoxystrobin
HL7702
PC12
6.89 ± 0.43
1 <
13.93 ± 0.91
13.28 ± 1.21
59.75 ± 3.04
34.45 ± 2.60
69.54 ± 4.37
42.21 ± 3.14
> 100
> 100
> 100
> 100
> 100
> 100
*
The results are means ± SD deviation from five independent experiments.
10