Antioxidant and antitumor activities of 4-arylcoumarins and 4-aryl-3,4-dihydrocoumarins

Article abstract of DOI:10.1016/j.biochi.2014.03.014

Five 4-arylcoumarins (1c-g) and twelve 3,4-dihydro-4-arylcoumarins (2a-l) were synthesized and tested for antioxidant activity, antitumor activity, toxicity and structure-activity relationships analysis. 4-Arylcoumarins and 3,4-dihydro-4-arylcoumarins tha

Full text of DOI:10.1016/j.biochi.2014.03.014

Biochimie xxx (2014) 1e8
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Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Antioxidant and antitumor activities of 4-arylcoumarins and
4-aryl-3,4-dihydrocoumarins
,
a
,
b
,
,d
Keyun Zhang a , Weixian Ding c, Jie Sun b, Bin Zhang a, Fujiao Lu a, Ren Lai a
,
*
Yong Zou b e, Gabriel Yedid a
,
a College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
b Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou 510650, China
c National Key Laboratory of Pharmaceutical New Technology for Chinese Medicine, Kanion Pharmaceutical Corporation, Lianyungang, China
d Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223 Yunnan, China
e School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Five 4-arylcoumarins (1ceg) and twelve 3,4-dihydro-4-arylcoumarins (2ael) were synthesized and
tested for antioxidant activity, antitumor activity, toxicity and structureeactivity relationships analysis.
4-Arylcoumarins and 3,4-dihydro-4-arylcoumarins that possess two hydroxyl groups in ortho position,
such as 1d, 1f, 2a, 2f, 2g and 2h had stronger radical scavenging properties than that of vitamin C (Vit C)
in ABTSꢀþ assay. Kinetic traces of scavenging ABTSꢀþ and DPPH radicals showed that all the reaction could
reached endpoint in 1 min, which was similar with Vit C. 4-Arylcoumarins with 30-hydroxyl-40-meth-
ylphenyl structural show more efficient NOꢀ radical scavenging activity. Three compounds 2e, 1f and 2a,
in particular had superior EC50 for NOꢀ scavenging than did Vit C. MTT assay indicated that one compound
in particular had a potential antitumor effect, inhibiting proliferation of BGC-823 cells and almost
Received 15 December 2013
Accepted 28 March 2014
Available online xxx
Keywords:
Arylcoumarin
Antioxidant
Radical scavenging
Structureeactivity relationships
Antitumor
completely killing them at a concentration 62.5 mg/L. With same concentration 100 mg/mL, hemolytic
analysis in rabbit red blood cells showed that only two compounds had hemolytic activity with a little
more than 5% hemolysis. Injection and oral toxicity tests on Galleria mellonella larvae showed that none
of the tested 4-arylcoumarins significantly affected their appetite, viability and mortality.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
molecular orbitals. Low concentrations of free radicals play very
important roles in various physiological functions, such as signal
Coumarins are found naturally in many green plants and natural
food products, and have been widely used as an aroma enhancer in
foods and pastries [1]. 4-Arylcoumarins and 3,4-dihydro-4-
arylcoumarins represent a minor class of natural coumarin de-
rivatives commonly found in fruit and vegetables, with a C6eC3e
C6 skeleton and a characteristic 4-aryl group. More than 130 nat-
ural 4-arylcoumarins and 3,4-dihydro-4-arylcoumarins have been
isolated from plants of the Clusiaceae, Fabaceae, Rubiaceae,
Asteaceae, Thelypteridaceae, Passifloraceae and Rutaceae families
[2e4]. Both natural and synthetic 4-arylcoumarins have exhibited
antioxidant [5e7], anticancer [8,9], antimicrobial [10,11], and anti-
inflammatory properties [12].
transduction, vessel pressure control, immune response among
others [13]. Over production of free radicals results in oxidative
stress that can be an important source of damage to cell structures,
such as cell membranes, proteins, and DNA [14]. As reviewed, heart
disease [15], central nervous system disorders [16], aging process
and cancer development [17] may be caused by free radical over-
produced. We have reported previously that several synthetic 4-
arylcoumarins and 3,4-dihydro-4-arylcoumarins with two hy-
droxyl groups placed ortho to each other in the benzene ring
exhibited significantly stronger DPPH free radical scavenging
properties than traditional antioxidant reagents, such as Vit C and
butylated hydroxytoluene, which indicated that these 4-
arylcoumarin derivatives may have potential in as biomedicines
[6]. However, the scavenging ability to other free radicals, the
biosecurity to animals and the relationship between structural and
antioxidant activities of those coumarins remains unclear. Mean-
while, a number of researchers have reported that some coumarins
are toxic to insects, such as furanocoumarins which have appetite
Free radicals can be defined as molecules or molecular frag-
ments containing one or more unpaired electrons in atomic or
* Corresponding author. Tel.: þ86 25 84396849; fax: þ86 25 84396542.
E-mail address: Keyunzhang@njau.edu.cn (K. Zhang).
http://dx.doi.org/10.1016/j.biochi.2014.03.014
0300-9084/Ó 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: K. Zhang, et al., Antioxidant and antitumor activities of 4-arylcoumarins and 4-aryl-3,4-dihydrocoumarins,
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2
K. Zhang et al. / Biochimie xxx (2014) 1e8
suppress in ant and growth inhibition activities in Depressaria
pastinacella [18] and Papilio polyxenes [19]. The safety of synthetic
4-arylcoumarin derivatives still not been tested.
In this study, we examined five synthesized 4-arylcoumarins and
twelve 3,4-dihydro-4-arylcoumarins for free radical scavenging
activities, antitumor, insect toxicity, hemolysis, and investigated the
relationship between their structures and bioactivities, in order to
assess their further research potential as drug candidates.
2.4. Antitumor activity
BGC-823 cells (a human stomach cancer cell line) were cultured
in RPMI 1640 supplemented with 10% fetal calf serum and 1% an-
tibiotics at 37 ꢂC and 5% CO2. Cells were trypsinized and resus-
pended in RPMI 1640 at a concentration of 1 ꢄ 105 cells/mL. Cell
suspension was seeded in each well of a 96 well plate (50 mL per
well) and compounds were dissolved in DMSO solution at a set of
predetermined concentrations and diluted 100 times in RPMI 1640.
2. Material and methods
After cells were cultured 2 h, 50
into wells. Mixtures were cultured for 48 h at 37 ꢂC and 5% CO2.
10 L MTT dye (5 g/L in PBS) was added into each well and incu-
bated 4 h, then 100 L SDS (100 g/L in PBS) was added. Absorbance
mL compound solutions were added
2.1. Sources of reagents
m
m
The tested 4-arylcoumarins and 3,4-dihydro-4-arylcoumains in
our study were synthesized by our research team with the purity
substantially ꢁ98% [6,10]. ABTS and MTT were obtained from
Sigma, USA. DPPH was obtained from Woko, Japan. DMSO and
butylated hydroxytoluene (BHT) were obtained from Amresco,
USA. Sodium nitroprusside (SNP), sulfanilamide, 77 N-(<alpha>-
naphthyl)ethylenediamine dihydrochloride (NEDD), Vit C, sodium
dodecyl sulfate (SDS), potassium persulfate, and all other reagents
were AR purchased from China National Medicines Corporation
Ltd., Shanghai, China.
of each well was measured by micro-plate reader at 570 nm [25]
after 8 h of culturing. The tumor cell line growth inhibition was
calculated as follows: 100 ꢄ (1ꢅAc/An), where Ac was the absor-
bance of test well and An was the absorbance of the negative control
well. 50 mL cisplatin (10 mg/L) was used as a positive control; 50 mL
RPMI1640 (containing 1% DMSO) was used as a negative control.
The IC50 value was determined as the concentration of compound
that caused 50% inhibition of cell proliferation.
2.5. Hemolysis assay
2.2. ABTSꢀþ free radical scavenging activity
A hemolysis assay [26] was conducted to investigate the cyto-
toxicity of those coumarins derivatives. Whole rabbit blood was
collected by syringe, mixed with EDTA (2 mg/mL) and centrifuged
Free radical scavenging of compounds was also measured using
an improved ABTS radical cation (ABTSꢀþ) decolorization assay with
modification [20,21]. ABTSꢀþ was produced by reacting 7 mM ABTS
stock solution with 2.45 mM potassium persulfate (final concen-
tration, both solutions were prepared with distilled water), and
incubating the mixture in the dark at 25 ꢂC for 12e16 h before used.
For study of the tested compounds, the ABTSꢀþ solution was diluted
with absolute ethanol, loaded in 1 cm quartz vessel (BioCell, p/n
7272051, BioTek Instruments Inc., USA), and measured in a micro-
plate reader to adjust the absorbance at 734 nm of 0.70 ꢃ 0.02.
Table 1
Structures of five 4-arylcoumarins and twelve 4-aryl-3,4-dihydrocoumarins studied
in this work.
The diluted ABTSꢀþ solution (180
mL) was mixed with 20 mL of tested
compound in 96 well plate. Finally, the plate was quickly loaded
into the micro-plate reader at 30 ꢂC, moderately shaken for 5 s, and
absorbance at 734 nm measured after 6 min. Ethanol was used as a
negative control; Vit C and BHT were used as positive controls.
Inhibition
percentage
was
calculated
as
follows:
100 ꢄ [(AꢅAblank)ꢅ(AcꢅAc-blank)]/(AꢅAblank), where A was the
absorbance of only ABTSꢀþ solution, Ablank was the absorbance of
ethanol, Ac was the absorbance of the examined compound and Ac-
blank was the absorbance of ethanol in the presence of compound.
2.3. NOꢀ scavenging capacity
Compound
R6
R7
R8
R3
R4
R5
To estimate possible NOꢀ scavenging capacities of the tested
compounds, SNP was used as a NOꢀ donor in our experiments.
100 mL SNP (10 mM) was mixed with 100 mL of each compound at
4-Arylcoumarins
1c
1d
1e
1f
H
H
H
H
H
OH
OH
OCH3
OH
OCH3
H
OH
H
OH
H
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
set of predetermined concentrations in a 96 well plate. All solutions
were prepared with 0.1 M phosphate buffer (PB, pH ¼ 7.4). As SNP is
an inorganic complex that can release NOꢀ when exposed to visible
light [22,23], the plate was incubated on light (a 5 W 38 lm/W lamp
and lighting at the distance of 50 cm in a dark room). After incu-
bation for 120 min at 25 ꢃ 2 ꢂC, Griess’ reagent was added to the
1g
H
4-Aryl-3,4-dihydrocoumarins
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
OH
H
H
H
H
OH
H
OH
OH
OH
H
OH
OH
OH
OH
H
OH
H
H
H
H
OH
OH
OH
H
H
H
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
H
OCH3
H
H
OCH3
H
H
H
OCH3
H
OCH3
OCH3
OCH3
OH
OCH3
OCH3
H
H
H
OCH3
OCH3
mixture solution [24]. Briefly, sulfanilamide solution (50
5% phosphoric acid) was added, incubated 5 min at room temper-
ature, and then 50 L NEDD solution (0.1% in distilled water) was
mL, 2% in
m
added. Finally, the plate was loaded in the micro-plate reader,
incubated for 30 min at 25 ꢂC, and the absorbance of wells at
2j
2k
2l
OCH3
OCH3
H
H
OH
540 nm were measured. For this assay, 100
mL PB was used as a
H
negative control; Vit C and BHT were used as positive controls.
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K. Zhang et al. / Biochimie xxx (2014) 1e8
3
Table 2
100 ꢄ (AcꢅAc-blank)/(AꢅAblank), where Ac was the absorbance of the
examined compound, Ac-blank was the absorbance of PBS in the
presence of the compound, A was the absorbance of positive control
and Ablank was the absorbance of PBS.
The SC50 values of tested compounds.
Compound
SC50
ABTSꢀþ
(m
M)a
DPPHb
NOꢀ
1c
1d
1e
8.66 ꢃ 0.7
ND
ND
2.6. Injection and oral toxicity to insect
3.91 ꢃ 0.01
3.24 ꢃ 0.08
133.1 ꢃ 16.5
ND
ND
ND
1f
1g
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
Vit C
BHT
3.25 ꢃ 0.01
2.63 ꢃ 0.12
ND
63.8 ꢃ 3.2
ND
Injection toxicity of each compound was assayed by the
method of Hamamoto et al. [27]. 1 mL compound solutions, dis-
solved in DMSO and diluted by 20% DMSO, were injected into the
hemocoel of Galleria mellonella fifth instar larvae (body mass with
ND
2.35 ꢃ 0.01
912.9 ꢃ 64.2
162.6 ꢃ 10.6
196.3 ꢃ 33.2
111.8 ꢃ 11.2
5.03 ꢃ 0.16
5.48 ꢃ 0.17
5.48 ꢃ 0.10
73.7 ꢃ 14.4
ND
2.69 ꢃ 0.09
105.6 ꢃ 6.4
235.7 ꢃ 23.0
256.6 ꢃ 7.3
245.8 ꢃ 49.7
26.4 ꢃ 0.6
288.1 ꢃ 27.8
155.6 ꢃ 27.7
ND
ND
ND
ND
ND
ND
ND
250 ꢃ 25 mg) using a micro-injector. An injection of 1
mL 20%
925.6 ꢃ 83.3
247.6 ꢃ 49.3
3.29 ꢃ 0.12
3.17 ꢃ 0.12
2.32 ꢃ 0.10
365.2 ꢃ 23.3
ND
DMSO was used as a negative control. Each compound used 20
larvae, and replicated three times for each treatment. Mortality
(M%), cocooning (C%), nymphosis (N%) and eclosion (E%) were
recorded every 12 h. And the additive effect of insect growth (AG)
was calculated as AG(%) ¼ C% þ N% þ E% ꢅ M%. The inhibition
effect of compounds to the growth of insects (GIint) was calcu-
lated as GIint ¼ (AG of compound ꢅ AG of control)/AG of
control ꢄ 100.
ND
ND
95.9 ꢃ 21.2
9.39 ꢃ 0.20
22.6 ꢃ 1.1
0.59 ꢃ 0.04
15.35 ꢃ 1.29
451.53 ꢃ 64.2
4.50 ꢃ 0.14
143.58 ꢃ 4.62
e
121.9 ꢃ 2.1
ND
For oral toxicity test, compounds were diluted with absolute
ethanol and added into insect diet at maximum concentration of
Resveratrol
Pterostilbene
54.6 ꢃ 0.2
ND
e
100 mg/g. The mixed well diets were flattened and dried 30 min to
ND: not detectable in the tested concentrations.
a
Confidence limits were 95% and n ¼ 4.
evaporate ethanol, then loaded into 24 well plates (1 g per well).
Third-instar larvae were carefully moved into plates (three larvae
each well), and were raised in the dark at 30 ꢂC and 65% RH. Larval
mortality, weights of surviving larvae, remaining feed, and larvae
producing stool were observed and recorded after 10 days. Abso-
lute ethanol was used as negative control. Four replicates of each
treatment were made. Relative food consumption rate (RCR), rela-
tive growth rate of insects (RGR), approximate digestibility (AD),
food digestion and utilization efficiency of conversion of ingested
food (ECIF) and other indicators were investigated to assay the
compounds toxicity to G. mellonella. Nutritional indices were
calculated as described previously [28e30] and modified in this
study. The following parameters were calculated: RGR ¼ (AꢅB)/
(B ꢄ days), where A is the weight of live insects on the tenth day
(mg)/number of live insects on the tenth day, B is the original
weight of insects (mg)/original number of insects; RCR ¼ D/
(B ꢄ days), where D is the biomass ingested (mg)/number of live
b
The data as reference [6,32].
for 10 min at 700 ꢄ g. The plasma was removed and blood cells
(RBCs) were washed three times with 0.9% saline solution, after
which they were suspended in PBS at
a concentration of
1 ꢄ 108 RBCs/mL. Assayed compounds were dissolved in DMSO
solution with set of predetermined concentrations and diluted 50
times with PBS. 100 mL RBCs suspension and 400 mL compounds
solution were mixed in 1.5 mL Eppendorf tubes, incubated for
60 min at 37 ꢂC, then centrifuged for 10 min at 12,000 ꢄ g. The
supernatant was collected, and its absorbance was measured at
541 nm. Distilled water was used as the positive control, and PBS
(containing 2% DMSO) was used as the negative control. Percent
hemolysis, measured at different concentrations of the assayed
compounds, was calculated by the following equation:
Fig. 1. Kinetic traces of scavenging DPPH (A) and ABTSꢀþ (B) radicals.
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4
K. Zhang et al. / Biochimie xxx (2014) 1e8
Table 3
insects on the tenth day; AD ¼ dry weight of digested food/dry
weight of ingested food ꢄ 100; ECIF ¼ (RGR)/(RCR) ꢄ 100.
IC50 of compounds in BGC-823 cell line.
Compound
IC50
(m
M)
Compound
IC50(mM)
2.7. Lipinski’s rule of five
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
120.8 ꢃ 10.1
59.0 ꢃ 4.7
124.7 ꢃ 16.1
29.1 ꢃ 1.3
>500
2f
2g
2h
2i
2j
2k
2l
>500
>500
237.8 ꢃ 66.4
444.4 ꢃ 83.9
>500
Lipinski’s “rule of five” [31] was used to evaluate the drug
likeness of those compounds. Generally, orally bioavailable drugs
following these rules: number of hydrogen bond donator less than
5; no more than 10 hydrogen bond acceptors; molecular weightless
than 500 Da; an octanolewater partition coefficient, log P no more
than 5.
135.8 ꢃ 19.9
125.5 ꢃ 16.5
>500
>500
>500
Umbelliferone
Resveratrol
Pterostilbene
450.6 ꢃ 61.7
17.1 ꢃ 1.3
7.3 ꢃ 0.8
390.9 ꢃ 87.9
214.4 ꢃ 29.6
2.8. Statistical analysis
3. Results and discussion
Statistical analysis was performed with SPSS Statistics software
(version 17.0, SPSS Inc. Chicago, USA). One-way ANOVA and Tukey
multiple comparison tests were employed to analyze all the results.
Results were firsts analyzed with one-way ANOVA, followed by
Tukey multiple comparison tests to further examine any significant
differences between treatments, at a 0.05 level of significance. All
results were expressed as mean ꢃ SD.
3.1. Chemistry of the complexes
Five 4-aryl-3,4-dihydrocoumarin (1aee) and twelve 4-
arylcoumarins (2ael) in this study were synthesized as previ-
ously described [6,10,32]. The structures of all the synthesized
Fig. 2. NOꢀ inhibition rate change treated with different concentrations of Vit C at different illumination time (A, B). Distance from light source of 50 cm; light with 5 W 38 lm/W
lamp; reaction temperature of 25 ꢃ 2 ꢂC. The NOꢀ released were influenced by different illumination conditions (C): in the darkness, radom three time intervals in one day (avoid
direct sunlight), serveral distances from the lamp and all the illumination time weresetted120 min. Dose-dependent histograms of several compounds inhibiting the NOꢀ generation
from SNP (D). The error bars show 95% confidence interval, n ¼ 4.
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K. Zhang et al. / Biochimie xxx (2014) 1e8
5
Fig. 3. Dose-dependence histograms of several compounds inhibiting the growth of BGC-823 tumor cells.
compounds were supported by FT-IR, MS, 1H NMR, and 13C NMR
spectral data [10,32] (Scheme S1, Tables S1-8).
4-arylcoumarins with 7,8-dihydroxy groups significantly enhanced
their ABTSꢀþ scavenging ability, but the number of methoxy groups
in those 4-arylcoumarins had no significant correlation with their
ABTSꢀþ radical scavenging capacity (Table 2). The SC50 values,
2i < 2l < 2e < 2c < 2d < 2b, showed that 3,4-dihydro-4-
arylcoumarins with 6-hydroxy group had stronger ABTSꢀþ radical
scavenging capacity than those with 7-hydroxy group, and 3,4-
dihydro-4-arylcoumarins with more methoxy groups had weaker
ABTSꢀþ radical scavenging capacity. The DPPH radical scavenging
activity of these compounds were similar to their ABTSꢀþ scav-
enging activity (Table 2) [6,32]. The DPPH radical scavenging ca-
pacities of compounds 2b, 2c, and 2e were similar (P > 0.05),
which showed that the presence of a 4-(3-hydroxy)-aryl groups in
7,8-dihydroxycoumarins did not significantly enhance their DPPH
scavenging ability. The DPPH radical scavenging capacities of
2g < 2f < 2h (P < 0.05) and 2d < 2l < 2i (P < 0.05) showed that
more methoxy groups in 4-aryl group may reduce their antioxi-
dant capability. Furthermore, 2c > 2l (P < 0.01) and 2b > 2d
(P < 0.01) which indicated a hydroxyl group in the 6th position
3.2. ABTSꢀþ and DPPH free radical scavenging activity
SC50 were calculated to test scavenging ability of those com-
pounds on the two free radicals (Table 2). Results showed that 4-
arylcoumarins with 7,8-dihydroxy groups (1d and 1f) or with 7-
dihydroxy group (1c) and 3,4-dihydro-4-arylcoumarins with 7,8-
dihydroxy groups (2a, 2f, 2g and 2h) had stronger ABTSꢀþ radical
scavenging ability than BHT and Vit C. These results were similar
to those for 7,8-dihydroxycoumarins and ortho dihydrox-
ycoumarins [5,32,33]. The EC50 of compound 2a was higher than
that of 1f (P < 0.05), whereas SC50 of compound 1d was higher
than that of 2f, which suggested that the presence of 3,4-
dihydrogen influenced their ABTSꢀþ radical scavenging capacity,
which was positively correlated with 30-hydroxy group (Tables 1
and 2). The SC50 value of 2a was smaller than those of 2f, 2g
and 2h (P < 0.05), indicating that 30-hydroxy group in 3,4-dihydro-
Table 4
The percent of RBCs hemolysis after 60 min incubated with different concentrations of the tested compounds at 37 ꢂC.
Compound
100
m
g/mL
200
mg/mL
400
m
g/mL
700
m
g/mL
1000 mg/mL
1c
1d
1e
1f
1g
2a
2b
2c
0.22 ꢃ 0.17
0.98 ꢃ 0.32
ND
7.78 ꢃ 0.29
0.62 ꢃ 0.37
3.58 ꢃ 0.12
3.18 ꢃ 0.22
13.37 ꢃ 0.77
6.98 ꢃ 1.44
0.54 ꢃ 0.07
1.02 ꢃ 0.06
ND
7.10 ꢃ 0.57
1.17 ꢃ 0.28
4.94 ꢃ 0.58
5.24 ꢃ 0.76
21.71 ꢃ 1.74
12.08 ꢃ 0.36
3.01 ꢃ 0.27
10.35 ꢃ 0.83
ND
12.48 ꢃ 0.92
1.98 ꢃ 0.62
5.39 ꢃ 0.62
16.05 ꢃ 1.07
43.48 ꢃ 1.36
14.44 ꢃ 0.52
7.46 ꢃ 0.55
16.90 ꢃ 1.62
ND
ND
2.10 ꢃ 0.76
2.63 ꢃ 0.44
ND
ND
5.73 ꢃ 0.62
11.56 ꢃ 0.18
3.11 ꢃ 0.27
ND
ND
ND
ND
ND
ND
ND
2d
2e
2f
2g
2h
2i
2j
2k
2l
ND
0.43 ꢃ 0.08
3.82 ꢃ 0.35
ND
1.34 ꢃ 0.17
16.23 ꢃ 0.84
12.03 ꢃ 2.17
17.26 ꢃ 0.98
8.86 ꢃ 1.75
17.66 ꢃ 0.61
2.01 ꢃ 0.04
1.98 ꢃ 0.09
ND
1.83 ꢃ 0.11
26.24 ꢃ 2.34
25.78 ꢃ 1.69
44.98 ꢃ 2.73
24.29 ꢃ 3.95
21.08 ꢃ 1.16
8.44 ꢃ 0.39
3.32 ꢃ 0.37
1.58 ꢃ 0.21
15.53 ꢃ 0.31
100
3.74 ꢃ 0.25
31.87 ꢃ 0.26
32.07 ꢃ 2.05
51.42 ꢃ 1.69
77.02 ꢃ 1.28
30.17 ꢃ 0.94
12.72 ꢃ 0.61
6.12 ꢃ 0.74
7.81 ꢃ 0.33
24.27 ꢃ 0.17
100
1.01 ꢃ 0.04
ND
2.01 ꢃ 0. 06
ND
2.56 ꢃ 0.91
ND
6.80 ꢃ 0.29
9.47 ꢃ 0.33
0.61 ꢃ 0.42
1.06 ꢃ 0.18
ND
ND
100
ND
0.38 ꢃ 0.06
ND
ND
Umbelliferone
Resveratrol
Pterostilbene
10.24 ꢃ 0.26
100
100
ND : no detectable hemolysis in the tested concentrations.
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6
K. Zhang et al. / Biochimie xxx (2014) 1e8
NOꢀ radical scavenging capacity of 4-arylcoumarins. 3,4-Dihydro-4-
arylcoumarin with 7,30-dihydroxy and 40-methoxy groups (2e) had
the strongest NOꢀ radical scavenging capacity. The SC50 values,
2e < 2a (P < 0.05), 2c < 2f (P < 0.05) and 2b < 2g (P < 0.05)
suggested that the presence of a 7-hydroxy group was essential for
the NOꢀ radical scavenging capacity of 3,4-dihydro-4-
arylcoumarins, whereas an 8-hydroxy group reduced the scav-
enging effectiveness of those compounds. The series of SC50 values,
2g < 2b < 2d < 2c < 2f (P < 0.05) indicated that 3,4-dihydro-4-
arylcoumarins with trimethoxy groups had stronger NOꢀ radical
scavenging capacity than those with dimethoxy groups.
Table 5
The inhibition effect of compounds to the growth state of G. mellonella after
injection.
Compound
24 h
AG
96 h
AG
120 h
AG
240 h
AG
GIint
GIint
GIint
GIint
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
100
90
90
90
90
60
90
100
90
90
90
90
100
90
80
60
90
60
90
60
0
10
10
10
10
40
10
0
10
10
10
10
0
10
20
40
10
40
10
40
160
130
140
170
150
120
180
180
180
140
140
150
160
120
130
110
180
100
180
130
11
28
22
12
17
33
0
180
140
170
200
200
180
200
200
200
180
190
180
200
160
180
160
190
120
200
140
10
30
15
0
0
10
0
0
0
10
5
10
0
20
10
20
5
40
0
230
220
210
250
240
230
250
250
250
230
240
230
250
200
230
210
240
140
250
200
0.8
1.7
16
0
0.4
0.8
0
0
0
0
0
3.4. Antitumor activity
22
22
17
11
33
28
39
0
0.8
0.4
0.8
0
20
0.8
16
0.4
44
0
The MTT assay was used to assess antitumor activity in 4-
arylcoumarins and 3,4-dihydro-4-arylcoumarins (Table 3). The se-
ries of IC50values, 1f < 1d < 1c < 1e < 2k < 2a (P < 0.05) showed
that without 3,4edihydrogen groups and more hydroxyl groups in
4-arylcoumarins enhanced their antitumor activity. Of the seven-
teen 4-arylcoumarins, compound 1f had the strongest antitumor
2l
Umbelliferone
Resveratrol
Pterostilbene
44
0
28
activity with an IC50 of 29.1 ꢃ 1.3
mM inhibited the proliferation of
BGC-823 cells, while a concentration of 62.5 mg/L was almost
completely lethal to all cells (Fig. 3).
30
20
conferred stronger radical scavenging ability than in the 7th po-
sition (Table 2). The kinetic curves of DPPH and ABTSꢀþ radical
scavenging indicated that the scavenging process was very fast
and plateaued in 1 min (Fig. 1).
3.5. Hemolysis
Red blood cells (RBCs) of rabbit were used as target to investi-
gate the cytotoxicity of our 4-arylcoumarin compounds. None of
the seventeen compounds showed strong cytotoxicity toward RBCs
(Table 4). Of all the compounds, compound 2j had the highest
3.3. NOꢀ scavenging capacity
cytotoxicity and only induced 6.8% RBCs hemolysis at 100
mg/mL.
As the generation of NOꢀ from SNP is a photolytic reaction [34],
illumination time significantly influences the result of an NOꢀ
radical scavenging capacity experiment. We firstly used Vit C to
determine an optimal illumination time of 2 h (Fig. 2). The NOꢀ
radical scavenging capacity assay showed that 4-arylcoumarins
with 7,8,30-trihydroxy-40-methoxy groups (1f) and 3,4-dihydro-4-
arylcoumarin with 7,8,30-trihydroxy-40-methoxy groups (2a) had
stronger NOꢀ radical scavenging activities than Vit C. 1f had almost
double the NOꢀ radical scavenging capacity of 2a, which indicated
that the presence of a 3,4-dihydro groups significantly reduced the
Compounds 2a, 2f and 2e (200 g/mL) had very weak cytotoxicity
to RBCs and induced less than 3.82% RBCs hemolysis. Compounds
1d, 1f, 2b, 2c, 2d, 2g and 2i showed no detectable cytotoxicity to
RBCs even with concentration 200 mg/mL.
m
3.6. Injected and oral toxicity to insect
G. mellonella larvae were used as targets to estimate injection
and oral toxicity of 4-arylcoumarins and 3,4-dihydro-4-
arylcoumarins to insect. Results showed that none of the tested
Fig. 4. Influence of DMSO on growth and development of Galleria mellonella (A) and oral toxicity of two 4-arylcoumarins and three 3,4-dihydro-4-arylcoumarins against G.
mellonella (B). *P < 0.05, **P < 0.001 compared with control.
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K. Zhang et al. / Biochimie xxx (2014) 1e8
7
Fig. 5. The structureeactivity relationships in tested 4-arylcoumarins and 3,4-dihydro-4-arylcoumarins.
compounds significantly affected larval viability. No significant
mortality and no appetite-suppressant effect were detected in the
tested larvae (Table 5, Fig. 4).
However, compounds containing 3,4-dihydro groups showed
significantly reduced antitumor activity (Fig. 5).
Compounds containing 30-hydroxy, 6-hydroxyl, 7,8-dihydroxy
and 7-methoxy groups together with 3,4-dihydro groups were
correlated with increased antimicrobial activities [10] (Fig. 5).
These compounds with only 7-hydroxy group showed no detect-
able antimicrobial activities. Compounds with 3,4-dihydro groups
had stronger antimicrobial activity against gram-negative bacteria
and weaker antimicrobial activities against gram-positive bacteria
and fungi than unmodified 4-arylcoumarin compounds [10]
(Fig. 5).
3.7. Structureeactivity relationships of seventeen novel 4-
arylcoumarin derivatives
For free radical scavenging activity, 4-arylcoumarin derivatives
containing 7,8-dihydroxy groups, 6-hydroxy group, 7-hydroxy
group and 30-hydroxy group were positively correlated with
ABTSꢀþ and DPPH free radical scavenging activities [6] (Fig. 5).
Compounds containing 7-hydroxy and 30-hydroxy groups were
positively correlated with NOꢀ free radical scavenging activity,
whereas the presence of an 8-hydroxy group was negatively
correlated (Fig. 5).
3.8. Potential of 4-arylcoumarin derivatives as novel drug
candidates
4-Arylcoumarin compounds containing 7,8-dihydroxy and 30-
hydroxy groups were positively correlated with antitumor activity.
Two 4-arylcoumarins, 1f and 1d, and four 3,4-dihydro-4-
arylcoumarins, 2a, 2f, 2g and 2h, had stronger ABTSꢀþ and DPPH
free radical scavenging activities than those of Vit C and pter-
ostilbene. Kinetic traces of radical scavenging showed that scav-
enging of DPPH radicals by compounds 2a, 2g and 2h were fast,
occurring 1 min into the platform stage. This was also the case for
scavenging of ABTSꢀþ radicals by compounds 2a, 2f, 1d and 1f
(Fig. 3). 7,30-Dihydroxy-40-methoxy-3,4-dihydro-4-arylcoumarin,
2e, had stronger NOꢀ radical scavenging activity than that of Vit C,
resveratrol and pterostilbene, but with much weaker hemolytic
activity against vertebrate blood cells than that of the latter three
compounds. 7,8,30- Trihydroxy-40-methoxy-4-arylcoumarin, 1f,
showed similar antitumor activity as resveratrol and very weaker
hemolysis activity to vertebrate blood cells than that of resvera-
trol and pterostilbene. Four 3,4-dihydro-4-arylcoumarin de-
rivatives, 2a, 2f, 2g and 2h, had significant antimicrobial activity
against gram-negative bacteria, with similar minimal inhibitory
concentration as ampicillin, and a three times weaker inhibition
effect against probiotics (such as Bacillus subtilis) than that of
ampicillin [10]. All of these compounds may have well drug-
likeness potential for none of them violated the rule of five
(Table 6).
Table 6
Some common molecular descriptors of tested compounds.
Compounda
MW
AlogP
H-bond Acceptors
H-bond donors
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
298.29
3.103
2.861
3.329
2.635
3.345
2.464
2.915
2.932
2.915
2.706
2.69
2.673
2.706
2.948
3.174
3.141
2.932
5
6
5
6
4
6
6
5
6
5
6
7
5
4
4
6
5
1
2
0
3
0
3
1
1
1
2
2
2
2
1
0
0
1
314.289
312.317
300.263
282.291
302.279
330.332
300.306
330.332
286.279
316.305
346.331
286.279
270.28
284.307
344.358
300.306
a
all the compounds absolutely obeyed the “rule of five”.
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8
K. Zhang et al. / Biochimie xxx (2014) 1e8
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Conflict of interest
The authors confirm that there are no known conflicts of in-
terest associated with this publication and there has been no sig-
nificant financial support for this work that could have influenced
its outcome.
Acknowledgments
This work was supported by Key projects of drug discovery
initiative of Ministry of Science and Technology of People’s Republic
of China (2009ZX09103091-002), Science & Technology Program of
Guangdong Province (S2013010014278, 2012B091100241) and
by National Science Foundation of China (J1210056, 21272280,
81201716).
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Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.03.014