Journal of Agricultural and Food Chemistry
Article
mixture, the organic phase was separated, and the aqueous phase was
extracted by ethyl acetate. The combined organic phases were washed
with saturated brine and dried with anhydrous sodium sulfate, and the
solvent was removed in vacuo. Target compounds f1−f2 were gained
by column chromatography using petroleum ether and ethyl acetate
as eluents.
CH3I (2.0 mmol) was added to a solution of f1−f2 (1.0 mmol) in
1,4-dioxane (15 mL). After stirring at 55 °C for 8 h, the solid was
precipitated and target compounds g1−g2 could be obtained by
washing the solid with a small amount of ethyl acetate without further
purification.
General Synthetic Procedure for Target Compounds h1−h2 and
i1−i2 (Figure 4, Route 3).44 To a solution of 3-bromoquinoline (47.3
mmol) in 1,4-dioxane was added CuI (2.4 mmol), NaI (94.5 mmol),
and N,N′-dimethyl-1,2-ethanediamine (0.5 mL). The mixture was
heated at 110 °C for 48 h. When the reaction was completed, 30 mL
of 30% ammonium hydroxide was added and stirred at room
temperature for 10 min. Then, water was added to the resulting
mixture, and the whole mixture was extracted with ethyl acetate. The
combined organic phases were washed with saturated brine and dried
with anhydrous sodium sulfate, and the solvent was removed in vacuo.
Target intermediate 3-iodoquinoline was gained by column
chromatography using petroleum ether and ethyl acetate as eluents.
To a round-bottomed flask was added 3-iodoquinoline (1.96
mmol), CuI (0.1 mmol), K2CO3 (3.92 mmol), substituted thiophenol
(1.96 mmol), and 2- isopropoxyethanol (3.92 mmol). Then, the
mixture was heated at 80 °C for 16 h. When the reaction was
completed, a solution of 10% hydrogen peroxide was added and the
whole mixture was stirred at room temperature for 10 min. Then,
water and ethyl acetate were added to the resulting mixture, the
organic phase was separated, and the aqueous phase was extracted by
ethyl acetate. The combined organic phases were washed with
saturated brine and dried with anhydrous sodium sulfate, and the
solvent was removed in vacuo. Target compounds h1−h2 were
gained by column chromatography using petroleum ether and ethyl
acetate as eluents.
where dc and dt were average diameters of the fungal colony of black
control and treatment, respectively.
Spore Germination Inhibition Assay. According to the
described method,46 the phytopathogenic fungi included in the
assay was grown on PDA plates in darkness at 25 °C for 2 weeks. The
spores were harvested from sporulating colonies and suspended in
sterile distilled water containing 0.1% (vol/vol) Tween 80. The
concentrations of spores in the suspension were determined using a
hemocytometer and adjusted to 1 × 106 spores per mL.
The stock solutions of the tested compounds dissolved in DMSO
were diluted 100-fold with sterile distilled water, which were further
prepared as 2-fold dilutions for the assay, in which the final
concentration of DMSO was 1% (vol/vol). Sterile distilled water
with 1% DMSO was used as a negative control. A 30 μL spore
suspension was placed on separate glass slide containing 30 μL of
prepared test solutions for a final volume of 60 μL. The inoculated
glass slides were incubated in a moisture chamber at 25 °C for 12 h. A
spore was considered to be germinated if the germ tube length was
longer than the short radius of the spore, and the number of spores
germinated at each concentration was counted under the microscope.
The percentage of spore germination was calculated, and the half
maximal effective concentration (EC50) values were derived from the
data analysis of the concentration-inhibition rate. The experiment was
independently performed three times under the same conditions. The
experiment was repeated three times, and the inhibition rates were
calculated according to the following formula
inhibition germination of spores (%) = [(N0 − N )/N0] × 100
1
where N0 and N1 were average values of the spore germination rates
of the black control and treatment, respectively.
Scanning Electron Microscopy Observations. (After treating
with a3 at a concentration of 0.05 μg/mL for 72 h, mycelia blocks
(5.0 mm × 4.0 mm) were cut from the fungi.). All the samples were
treated by 4% glutaraldehyde for 4 h, washed three times with 0.01 M
PBS (pH = 7.2), and then fixed with 1% osmium tetraoxide solution
(wt/vol) for 2 h. After that, each sample was dehydrated with graded
ethanol series (20, 50, 80, and 90%) for 10 min. Subsequently, the
samples were dried at a critical point and gold-sprayed and observed
by using a scanning electron microscope (Hitachi, S-3400N, Japan).
Determination of Cell Membrane Permeability. Effects of
compound a3 on the cell membrane permeability were determined by
the conductivity method.47 The mycelial disk of B. cinerea (5 mm)
was placed in 60 mL of PD broth medium and shook at 140 rpm for 4
days at 27 °C. After that, the mycelia were filtered and added into the
solution of a3 with different concentrations (50, 25, 10, and 5 μg/
mL). Eventually, the conductivity values were determined with a
conductivity detector (0 h was marked as L0, and 0.5, 1, 2, 4, 6, 8, 10,
12, and 24 h were marked as L1). The conductivities of samples
treated by boiling water for 30 min were remarked as L2. The relative
permeability rate of the cell membrane was calculated by the following
formula
CH3I (2.0 mmol) was added to a solution of h1−h2 (1.0 mmol) in
1,4-dioxane (15 mL). After stirring at 55 °C for 8 h, the solid was
precipitated and target compounds i1−i2 could be obtained by
washing the solid with a small amount of ethyl acetate without further
purification.
General Synthetic Procedure for Target Compound j1 (Figure 5).
To a solution of compound b1 in acetone was added potassium
hydroxide (1.5 mmol) and CH3I (1.2 mmol). The mixture was
reacted at room temperature for 15 min. When the reaction was over,
the solvent was removed in vacuo, and the residue was purified by
silica gel column chromatography to give target compound j1.
In Vitro Antifungal Assay. Cryptolepine and its derivatives were
tested by the mycelium growth rate method45 for their antifungal
activities against four fungi, including R. solani, B. cinerea, F.
graminearum, and S. sclerotiorum. All the synthetic compounds were
dissolved in DMSO and then were added to PDA medium that was
prepared and sterilized to obtain a series of concentrations (50, 25,
10, 5, 2.5, 1, 0.5, 0.1, 0.05, and 0.01 μg/mL). The antifungal activities
of target compounds a1−a24, b1−c3, c1−c3, d1−d4, e1−e4, f1−f1,
g1−g2, h1−h2, i1−i2, and j1 in detail were included in the
DMSO (vol/vol) mixed with PDA (The same amount of DMSO was
added to the sterile medium as a blank control.), and azoxystrobin
was used as a positive control. The mycelial disks (5 mm) of
phytopathogenic fungi were inoculated on PDA plates and then were
incubated at 25 °C in the dark. Each sample was measured in
triplicates, and its diameters (mm) of inhibition zones were measured
by the cross-bracketing method. The growth inhibition rates were
calculated when the blank control hyphae grew to the edge of the
Petri dish according to the following formula
relative electric conductivity (%)
= [(L1 − L0)/(L2 − L0)] × 100
ROS Production of B. Cinerea. The accumulation of ROS was
measured by the method previously described.48 Mycelia tips (0.5
cm) were treated with compound a3 (0.05 and 0.1 μg/mL) for 48 h
and then placed on a sterile slide in a culture dish. After the samples
were cultured at 25 °C for 24 h, the PDA medium was removed
carefully and the hyphae were stained with 10 μM DCFH-DA
solution (Beyotime, Shanghai, China). The hyphae were incubated at
37 °C for 30 min in the darkness and washed twice with phosphate-
buffered saline (PBS). A coverslip was placed on the hyphae, and the
samples were observed and photographed using the microscopic
morphology of fungal hyphae that was observed by a scanning
electron microscope (Hitachi, S-3400 N, Japan).
MMP of B. Cinerea. Effects of compound a3 on the MMP of B.
cinerea mycelia were analyzed according to the previously described
method.49 After staining with rhodamine 123 solution (Beyotime,
mycelial growth inhibition (%)
= [(dc − dt)/(dc − 5 mm)] × 100
1263
J. Agric. Food Chem. 2021, 69, 1259−1271