Biological activity evaluation and structure-activity relationships analysis of ferulic acid and caffeic acid derivatives for anticancer

Article abstract of DOI:10.1016/j.bmcl.2012.08.038

The anticancer activities of alkyl esters and NO-donors of ferulic acid (FA) and caffeic acid (CA) were assessed by a high-throughout screening (HTS) method, and the structure-activity relationships were described. CA alkyl esters had better anticancer activities than FA alkyl esters with the same alkyl substituent. Mono-nitrates and phenylfuroxan nitrates were more potent than the dual nitrates. Phenylsulfonylfuroxan nitrates of FA, especially compounds 8b-8d, exhibited more potent activities in anticancer.

Full text of DOI:10.1016/j.bmcl.2012.08.038

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6085–6088
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biological activity evaluation and structure–activity relationships analysis
of ferulic acid and caffeic acid derivatives for anticancer
Weixia Li a, Nianguang Li a, Yuping Tang a, , Baoquan Li a, Li Liu a, Xu Zhang a,b, , Haian Fu c, Jin-ao Duan a
⇑
⇑
a Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210046, China
b College of Basic Medicine, Nanjing University of Chinese Medicine, Nanjing 210046, China
c Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The anticancer activities of alkyl esters and NO-donors of ferulic acid (FA) and caffeic acid (CA) were
assessed by a high-throughout screening (HTS) method, and the structure–activity relationships were
described. CA alkyl esters had better anticancer activities than FA alkyl esters with the same alkyl sub-
stituent. Mono-nitrates and phenylfuroxan nitrates were more potent than the dual nitrates. Phenylsulfo-
nylfuroxan nitrates of FA, especially compounds 8b–8d, exhibited more potent activities in anticancer.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 24 June 2012
Revised 7 August 2012
Accepted 10 August 2012
Available online 16 August 2012
Keywords:
Structure–activity relationship
Derivative
Ferulic acid
Caffeic acid
Anticancer
Despite of global efforts to limit the incident of cancer, it has be-
come the leading cause of death in the last 50 years.1 Lung cancer
remains the number one cancer killer in both men and women.2,3
Breast cancer is the most common cancer in females worldwide,
and mortality from breast cancer is consistently due to tumor
metastasis.4 Cervical cancer is the second most common cancer
of women worldwide, accounting for an estimated 11,070 new
cases and 3870 deaths in USA for 2008.5 Head and neck cancer is
among the 10th most common cancer worldwide, there are about
780,000 new cases per year over all the world.6 Besides that, mel-
anoma is the most serious type of skin cancer as a malignant tumor
of melanocytes.7 With the incidence of these cancers rapidly rising
in the developed and developing countries, there is an urgent need
to develop more effective drugs.
aromatic acid, had a potent inhibitory effect on 12-O-tetradeca-
noylphorbol-13-acetate (TPA)-induced tumor promotion and
TPA-induced formation of 5-hydroxymethyl-2-deoxyuridine in
DNA of mouse skin as well as an inhibitory effect on the synthesis
of DNA, RNA and protein in cultured HeLa cells.12 Furthermore, die-
tary with rich CA and FA, such as fruits and vegetables, may play a
role in the body’s defense against carcinogenesis by inhibiting the
formation of N-nitroso compounds.13 Earlier studies indicated that
FA and CA could suppress benzo(a)pyrene-induced forestomach
carcinogenesis in mice,14 inhibit the tumor promotion in mouse
skin induced by TPA or 7,12-dimethylbenz[a]anthracene,15,16 sig-
nificantly reduce the incidences of tongue neoplasms (squamous
cell papilloma and carcinoma) and preneoplastic lesions (hyperpla-
sia and dysplasia).8
Aromatic acids in the plant kingdom are now recognized as
promising chemopreventive agents. They exhibit a wide spectrum
of pharmacological activities, such as anticancer.8 Ferulic acid (4-
hydroxy-3-methoxycinnamic acid, FA), a characteristic aromatic
acid, was active against the lung carcinogenesis by benzopyrene.9
Additionally, it could decrease incidences of azoxymethane-in-
duced large bowel neoplasms, suggesting it has a potential as a
chemopreventive agent for rice germ on colonic neoplasia.10,11
Caffeic aicd (3,4-dihydroxycinnamic acid, CA), another bioactive
However, they were rapidly absorbed with low bioavailabilities
after single oral administration, which limited their clinical
use.17,18 Besides that, FA and CA are insoluble in water and oil,
which also limits their applications.19 Therefore, in order to im-
prove their liposolubility and achieve more potent anticancer
agents, it is necessary to modify the structures of FA and CA.
Esterification is one way of modifying the physical properties of
FA and CA.19,20 Previously, we have shown that higher yields of al-
kyl esters of FA (1a–1h) could be obtained under microwave irra-
diation, which is not only faster than using conventional heating
methods, but also potentially more efficient, clean, and safe.21 CA
alkyl esters (2a–2h) could also be obtained under the same reac-
tion condition (Scheme 1).22
⇑
Corresponding authors. Tel./fax: +86 025 85811916.
E-mail addresses: yupingtang@njutcm.edu.cn (Y. Tang), zhangxutcm@gmail.com
(X. Zhang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.038

6086
W. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6085–6088
COOH
COOH
COOR
microwave irradiation
microwave irradiation
ROH
ROH
+
R=a: CH3
HO
HO
HO
HO
b
: CH2CH3
O
FA
O
c: (CH2)2CH3
d: CH(CH3)2
1(a-h)
e
: (CH2)3CH3
COOR
f: CH2CH(CH3)2
g: (CH2)4CH3
+
h
: (CH2)2CH(CH3)2
OH
CA
OH
2(a-h)
Scheme 1. Synthesis of alkyl esters of FA (1a–1h) and CA (2a–2h).
Nitric oxide (NO) is a key mediator involved in many physiolog-
ical and pathological processes.23 High levels of NO and its
metabolic derivatives can modify functional proteins by S-nitrosy-
lation, nitration, and disulfide formation, leading to bioregulation,
inactivation, and cytotoxicity, particularly in tumor cells.24,25 In-
deed, some synthesized NO-releasing compounds have shown
cytotoxic activity against human colon carcinoma cells and human
hepatocellular carcinoma cells in vitro, inhibiting the growth and
metastasis of cancers in vivo.26,27 Furoxan (1,2,5-oxadiazole-2-oxi-
des) derivatives are biologically active compounds that are capable
of releasing high levels of NO in the presence of thiols and a lack of
tolerance.28,29 Hybrid NO-donor furoxan-based drugs are a novel
type of drug that retains the pharmacological activity of the parent
compound but also has the biological actions of NO.30 In this study,
we synthesized novel NO-donor–FA hybrids and NO-donor–CA hy-
brids according to our previous research.31,32 FA was first treated
with dibromoalkanes bearing three to six carbons in the presence
of Et3N and acetone at 50 °C, and then was further converted to
the nitrates 3a–3d or 4a–4d, respectively, with AgNO3 in THF/
CH3CN (Scheme 2). However, when FA was treated with (E)-1,4-
dibromobut-2-ene, and then was further converted to the nitrate
3e with AgNO3 in THF/CH3CN. Nitric ester–CA hybrids 5a–5c and
6a–6d were obtained through the same reaction condition but
CA was the lead compound (Scheme 2). Furthermore, five 4-hydro-
xyl-3-phenylfuroxan–FA hybrids (7a–7e) and four 4-hydroxy-
methyl-3-phenylsulfonylfuroxan–FA hybrids (8a–8d) were
synthesized through modifying the carboxyl group of FA with phe-
nylfuroxan or phenylsulfonylfuroxan. 4-hydroxyl-3-phenylfuro-
xan–CA hybrid (9) was synthesized through modifying the
carboxyl group of CA with phenylfuroxan (Scheme 3). The yield
of every synthetic compound was shown in Table 1.
In the present study, in order to explore the potential anticancer
activity of 16 alkyl esters and 26 NO-donors of FA and CA, their bio-
logical activities were determined in an in vitro human disease-ori-
ented cancer cell line screening panel using high-throughout
screening (HTS) method,33 including human lung cancers, mela-
noma, cervical, neck and head, and human breast cancer cells.
HTS, driven by the great progress in automation technology and
combinatorial chemistry, has been widely implemented in drug
discovery since the early 1990s and rapidly became one of the ma-
jor sources of drug leads.34 The results of anticancer activities of 42
FA and CA derivatives against the growth of 14 human cancer cell
lines were shown in Table 1.
As illustrated in Table 1, almost all FA and CA derivatives exhib-
ited different potent anticancer activities against the growth of dif-
ferent human cancer cell lines. About the nine kinds of lung cancer
cells, most compounds except 1a, 1d, 1g, 4a–4d, 6b–6d, 7b, 7d, 7e
and 8a–8d had better activities on A549, H157 and 1299 cells; had
weaker activities on H460 and Calu 1 cells; had the weakest activ-
ities on 1792, H266, Hop62 and 292G cells. Most compounds had
better activities on LOX-IMVI cell than on M14 cell. Almost all com-
pounds had significant cytotoxic against Hela cells with small IC50
values, indicating that they were probably good agents for the
treatment of human cervical cancer.
The anticancer activity of most NO-donors including mono-ni-
trates 3 and 5, phenylfuroxan nitrates 7 and 9, and phenylsulfonylf-
uroxan nitrates 8, in general but not always, were superior to the
alkyl esters 1 and 2. On the other hand, the NO-donors had a slightly
higher anticancer activity in this test system. In the series of alkyl
esters, CA derivatives (2a–2h) had better anticancer activities than
FA (1a–1h) with the same alkyl substituent, such as compounds 2a
versus 1a and 2g versus 1g. This result was consistent with the
COORONO2
COORBr
AgNO3, CH3CN
3(a-e)
HO
HO
50 °C
COOH
O
O
Et3N, acetone
60 °C
BrRBr
+
HO
HO
COORBr
COORONO2
O
FA
AgNO3, CH3CN
BrRO
HO
4(a-d)
50 °C
O2NORO
O
O
COORONO2
COORBr
AgNO3, CH3CN
50 °C
5(a,b,e)
HO
COOH
BrRBr
OH
OH
OH
Et3N, acetone
60 °C
+
COORBr
AgNO3, CH3CN
COORONO2
OH
CA
BrRO
6(a-d)
50 °C O2NORO
OH
R=a: -(CH2)3-, b: -(CH2)4-, c: -(CH2)5-, d: -(CH2)6-, e: -CH2CH=CHCH2-
Scheme 2. Synthesis of nitric ester–FA hybrids (3a–3e, 4a–4d) and nitric ester–CA hybrids (5a, 5b, 5e, 6a–6d).

W. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6085–6088
6087
Ph
Ph
N
COR1OCH2
COR1OCH2
(iii)
(iii)
N
N
7(a-e)
N
EtO2CO
O
HO
O
O
COOH
(i)
O
COOH
(ii)
(Ι)
O
O
SO2Ph
N
SO2Ph
N
COR2O
HO
HO
EtO2CO
COR2O
(ΙΙ)
O
FA
O
8(a-d)
N
N
EtO2CO
O
O
HO
O
O
O
O
Ph
N
Ph
COOH
(i)
COOH
COOCH2
COOCH2
(iii)
(ii)
(Ι)
N
N
N
EtO2CO
EtO2CO
HO
O
O
O
O
OH
CA
OH
OH
OH
9
R1
R2
a
b
c
d
e
= : /, : m-O-Ph-, : p-O-Ph- : m-OCH2-Ph-, : p-OCH2-Ph-
a
b
c
d
= : (CH2)2O, : (CH2)2CH(CH3)O, : (CH2)4O, : (CH2)2O(CH2)2O
Scheme 3. Synthesis of 4-hydroxyl-3-phenylfuroxa–FA hybrids (7a–7e), 4-hydroxymethyl-3-phenylsulfonylfuroxa–FA hybrids (8a–8d) and 4-hydroxyl-3-phenylfuroxa–CA
hybrid (9). Reagents and conditions: (i) ClCO2Et, 1 N NaOH, 50 °C; (ii) DCC, DMAP, CH2Cl2; (iii) NH2(CH2)2OH, 95%EtOH; (I) 4-hydroxyl-3-phenylfuroxan; (II) 4-
hydroxymethyl-3-phenylsul-fonylfuroxan.
Table 1
The yield (%) and in vitro anticancer activities in human cancer cell lines (IC50 in
hybrid
lM) of alkyl esters of FA and CA, NO-donors–FA hybrids and 4-hydroxyl-3-phenylfuroxa-CA
No.
Yield (%)
Lung cancer
H266
Melanoma
LOX-IMVI
Cervical
Hela
Neck & head
M4E
Breast
SKBR
A549
H157
H460
1792
Hop62
1299
292G
Calu1
M14
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
4a
4b
4c
4d
5a
5b
5e
6a
6b
6c
6d
7a
7b
7c
7d
7e
8a
8b
8c
8d
9
79.0
81.0
77.0
69.0
73.0
63.0
58.0
46.0
75.0
73.0
72.0
71.0
68.0
64.0
70.0
69.0
29.9
91.7
83.0
89.4
46.2
22.2
99.3
86.0
17.0
87.0
90.0
55.3
82.0
79.0
95.7
83.4
89.1
63.9
78.9
73.0
91.4
82.3
97.7
92.0
86.6
44.0
>50.0
12.5
15.6
>50.0
8.23
34.6
>50.0
43.6
7.54
7.22
4.39
>50.0
3.51
13.6
5.75
8.76
13.2
8.82
6.39
37.4
5.95
15.8
>50.0
>50.0
>50.0
10.8
22.6
0.40
0.69
0.41
0.45
>50.0
2.06
0.72
3.39
>50.0
17.2
>50.0
0.42
0.40
0.41
2.71
>50.0
32.3
15.2
>50.0
9.03
28.3
>50.0
40.2
6.25
7.62
7.16
11.0
2.62
8.55
2.28
8.25
20.5
12.9
13.4
32.1
4.29
>50.0
>50.0
>50.0
>50.0
4.34
5.19
1.36
5.71
>50.0
>50.0
>50.0
4.53
24.7
43.1
>50.0
>50.0
>50.0
0.41
0.42
0.44
>50.0
>50.0
29.1
25.2
>50.0
19.4
>50.0
>50.0
45.0
14.8
13.1
7.01
10.5
5.62
16.0
1.91
16.5
29.0
19.5
12.3
>50.0
4.98
>50.0
>50.0
>50.0
>50.0
3.31
3.52
2.90
9.58
>50.0
>50.0
>50.0
7.03
11.6
15.8
29.4
44.2
>50.0
0.42
0.45
0.43
2.54
>50.0
25.6
34.1
>50.0
15.0
>50.0
>50.0
37.7
6.09
18.4
22.1
>50.0
5.75
>50.0
6.35
31.9
28.5
20.8
13.0
48.7
4.86
>50.0
>50.0
>50.0
>50.0
18.1
15.4
0.41
6.61
>50.0
>50.0
>50.0
11.1
>50.0
8.15
>50.0
>50.0
>50.0
0.40
>50.0
28.1
20.4
>50.0
18.3
35.7
>50.0
48.1
>50.0
>50.0
34.4
>50.0
11.3
>50.0
7.24
33.8
44.0
24.8
21.2
>50.0
9.79
>50.0
>50.0
>50.0
>50.0
>50.0
23.3
>50.0
26.8
22.4
>50.0
14.9
>50.0
>50.0
31.2
3.86
12.5
7.81
>50.0
5.59
>50.0
5.11
15.7
19.4
13.1
12.5
>50.0
5.13
>50.0
>50.0
>50.0
>50.0
10.0
>50.0
36.8
40.3
>50.0
8.23
38.0
>50.0
31.5
3.53
4.93
5.85
30.8
5.81
37.7
4.69
17.9
28.3
4.51
20.8
>50.0
32.0
35.9
>50.0
22.9
>50.0
>50.0
48.4
3.11
5.25
7.58
>50.0
6.46
37.5
7.73
27.6
39.4
28.4
24.9
>50.0
21.9
>50.0
>50.0
>50.0
>50.0
13.5
16.7
7.95
4.98
>50.0
>50.0
>50.0
16.5
>50.0
31.0
>50.0
>50.0
>50.0
1.77
>50.0
>50.0
48.9
>50.0
17.2
>50.0
>50.0
32.2
5.50
17.2
17.7
>50.0
11.7
>50.0
8.57
20.2
>50.0
30.1
26.3
>50.0
2.48
1.42
>50.0
>50.0
27.8
>50.0
>50.0
15.2
>50.0
19.3
>50.0
>50.0
48.4
21.8
6.21
7.50
>50.0
9.54
>50.0
3.38
8.03
29.5
20.4
>50.0
>50.0
2.36
>50.0
>50.0
>50.0
>50.0
18.9
>50.0
>50.0
28.1
>50.0
18.1
>50.0
>50.0
45.8
8.86
9.82
13.5
>50.0
11.1
>50.0
7.48
30.6
42.6
32.5
24.5
>50.0
3.89
>50.0
>50.0
>50.0
>50.0
24.8
5.55
2.13
5.62
31.1
10.3
28.7
>50.0
>50.0
3.77
1.96
1.51
9.65
0.59
4.70
2.02
9.39
6.70
6.35
13.4
16.3
1.00
3.62
0.74
>50.0
>50.0
3.11
2.25
0.40
>50.0
3.93
5.73
1.35
7.82
>50.0
12.1
5.64
>50.0
>50.0
0.40
0.46
0.43
1.23
>50.0
33.4
18.5
>50.0
15.0
>50.0
>50.0
30.5
8.89
11.2
12.4
>50.0
8.66
36.8
6.80
29.1
18.4
20.3
21.8
>50.0
9.38
>50.0
>50.0
>50.0
>50.0
20.0
17.5
4.31
14.1
>50.0
>50.0
>50.0
3.37
>50.0
24.9
24.5
>50.0
13.2
44.8
>50.0
41.5
5.15
4.16
3.87
31.5
2.17
16.2
2.96
13.1
17.2
8.92
8.06
15.2
6.53
>50.0
>50.0
>50.0
>50.0
5.77
4.55
0.41
4.81
3.23
2.71
39.7
4.98
14.2
11.0
4.18
11.7
>50.0
0.40
0.42
0.41
0.70
>50.0
2.34
>50.0
>50.0
>50.0
>50.0
8.03
10.7
21.7
0.42
12.5
10.0
4.65
10.0
3.14
0.41
>50.0
0.43
6.94
>50.0
7.23
>50.0
13.8
19.9
1.49
8.96
10.3
>50.0
>50.0
>50.0
>50.0
13.4
>50.0
>50.0
>50.0
>50.0
>50.0
0.43
>50.0
>50.0
>50.0
>50.0
12.9
>50.0
28.5
>50.0
>50.0
>50.0
0.41
>50.0
>50.0
>50.0
8.66
>50.0
30.4
>50.0
>50.0
>50.0
0.42
>50.0
>50.0
>50.0
22.9
>50.0
28.8
>50.0
>50.0
>50.0
0.45
>50.0
>50.0
>50.0
14.0
>50.0
>50.0
>50.0
>50.0
>50.0
0.41
49.4
44.6
>50.0
>50.0
>50.0
>50.0
0.41
0.44
0.42
13.3
>50.0
>50.0
>50.0
>50.0
1.18
0.84
1.12
12.9
0.42
1.07
3.78
0.40
0.42
14.2
0.41
0.48
6.12
0.42
0.49
>50.0
1.93
2.88
9.17
0.41
0.45
7.68
0.40
0.41
12.5
Note: >50.0 means that the data were not applicable.
literature in which it was reported that CA derivatives (2a–2h)
showed stronger activities than FA (1a–1h) with the same alkyl
substituent on colon-HCT 116, breast-MCF-7 and lung-NCI H460
cells.35 In the series of FA alkyl esters 1, 1e showed the highest
anticancer activity. However, 1a, 1d, 1f and 1g had the anticancer
activity almost at the highest concentration (IC50 50 lM). In the

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series of CA alkyl esters 2, all compounds except 2d and 2f showed
significant anticancer activities. Generally, the results also showed
that compounds with straight-chain substituent had better anti-
cancer activities than those with branched-chain substituent, such
as compounds 1b versus 1d and 2a versus 2f.
In the series of NO-donors, phenylsulfonylfuroxan nitrates 8b–
8d had the very potent anticancer activity against all the human
cancer cells. Their IC50 values were all less than 10
approximately 70 times more active than compound 1g (IC50
>700 M). The results indicated that phenylsulfonylfuroxan ni-
lM, which were
l
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trates of FA were the most potent compounds among the FA and
CA derivatives tested. It was probably because the phenylsulfonylf-
uroxan group can produce high levels of NO.28–30 Generally, mono-
nitrates 3 and 5 and phenylfuroxan nitrates 7 and 9 were more
potent than the dual nitrates 4 and 6, suggesting the FA or CA phe-
nolic hydroxyl group was required for anticancer activity. For the
series of mono-nitrates 3 and 5, the unsaturated nitrates 3e and
5e showed higher anticancer activities than the saturated ones.
Furthermore, the results showed that, in the series of mono-ni-
trates 3, the anticancer activity increased as the number of atoms
in the nitric esters increased, such as compounds 3a–3c; but de-
creased significantly when the number of atoms in the nitric esters
was six (compound 3d). In the series of phenylfuroxan nitrates 7
and 9, phenylfuroxan–CA 9 showed a much higher anticancer
activity than phenylfuroxan–FA 7. No substituent between FA
and phenylfuroxan 7a showed a much higher anticancer activity
than analogues with benzene ring substituent 7b–7e. Besides that,
benzene ring substituent without methylene between FA and phe-
nylfuroxan showed higher activity than the benzene ring with
methylene substituent, such as 7b, 7c and 7d, 7e, indicating that
the NO release activity decreased with the chain between FA and
phenylfuroxan became much longer. For the series of phenylsulfo-
nylfuroxan nitrates 8, the butyl ether derivatives (8b and 8c) and
dual diethyl ether derivative 8d had a much higher anticancer
activity than the mono-diethyl ether derivative 8a.
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33. Human lung cancers (A549, H157, H460, 1792, H266, Hop62, 1299, 292G and
Calu1), melanoma (LOX-IMVI and M14), cervical (Hela), neck and head (M4E)
and human breast cancer (SKBR) were from American Type Culture Collection
(ATCC, USA) grown in RPMI 1640 containing 10% foetal calf serum (FCS),
100 UI/mL penicillin G and 100 mg/mL streptomycin. Dimethyl sulphoxide
(DMSO) were purchased from Sigma Chemical Co. (St. Louis. MO), Alamar blue
were from Promega. Cells were seeded into 384-well plates (Costar# 3712)
In summary, the present study clearly described the structure–
activity relationships between the alkyl esters and NO-donors of FA
and CA in anticancer. Both the substituent group and the level of
releasing NO were essential for potent activity. Alkyl esters and
NO-donors of FA and CA possessed activities on the growth of hu-
man cancer cell lines, and most of them produced notable selective
cytotoxicity against Hela cells. The NO-donors of FA and CA had a
slightly higher anticancer activity in this test system. More impor-
tantly, the results suggest that phenylsulfonylfuroxan nitrates of
FA, especially compounds 8b–8d, may be considered to be promis-
ing anticancer agent for further studies.
(800–1000 cell/well or 20–25 cells/lL, 45 lL medium/well) using a Liquid
dispenser (Thermo Fisher Multidrop Combi) in a bio-safety cabinet. Plates were
placed in an incubator overnight to allow for attachment and recovery.
Compound plates were utilized and prepared to yield 10 mM of compound in
DMSO (neat) by Robot (Sciclone software), to generate 8 concentrations with
series dilution, wells were reserved on each plate for background and vehicle
control (0.5% DMSO). Using the liquid handling system, the following day the
cells were treated with drug for 72 h, the final concentrations used in the assay
Acknowledgments
This research was financially supported by National Key Tech-
nology R&D Program (2008BAI51B01), the Specialized Research
Fund for the Doctoral Program of Higher Education of China
(20113237110010), Key Research Project in Basic Science of Jiang-
su College and University (06KJA36022, 07KJA36024), National S. &
T. Supporting Program in Chinese Medicine for the 11th Five-Year
Plan (2006BAI11B08-01), 2009’ Program for New Century Excellent
Talents by the Ministry of Education (NCET-09-0163), 2009’ Pro-
gram for Excellent Scientific and Technological Innovation Team
of Jiangsu Higher Education, A Project Funded by the Priority Aca-
demic Program Development of Jiangsu Higher Education Institu-
tions (ysxk-2010).
are 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39
lM in triplicate. A volume of
5
lL/well Alamar blue was transferred into the assay plates for a
final
concentration of 10% Alamar blue. The plates were exposed to an excitation
wavelength of 530 nm, and the emission at 560 nm was recorded to determine
whether any of the test compounds fluoresce at the emission wavelength and
thus interfere with the assay. Plates were returned to incubator and the
fluorescence was read at 4 h. The percent viability was expressed as
fluorescence counts in the presence of test compound as a percentage of that
in the vehicle control. The mean value and standard error for each treatment
was determined and the percentage of cell viability relative to control (0.5%
DMSO) was calculated. The IC50 was defined as the concentration of drug that
killed 50% of the total cell population as compared to control cells at the end of
the incubation period.
34. Brunsch, T.; Raisch, J.; Hardouin, L. Control Eng. Pract. 2012, 20, 14.
35. Jayaprakasam, B.; Vanisree, M.; Zhang, Y. J.; Dewitt, D. L.; Nair, M. G. J. Agric.
Food Chem. 2006, 54, 5375.
References and notes
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