Synthesis of phenylisothiourea derivatives as inhibitors of NO production in LPS activated macrophages

Article abstract of DOI:10.1016/j.bmcl.2009.04.001

A series of phenylisothioureas were synthesized as inhibitors of NO production in lipopolysaccharide-activated macrophages. We investigated the effect of lipophilic moiety and N- or S-substituents of the phenylisothioureas on the activity. Inhibitory acti

Full text of DOI:10.1016/j.bmcl.2009.04.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3088–3092
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of phenylisothiourea derivatives as inhibitors of NO production in LPS
activated macrophages
*
Guo Hua Jin, Da Yeon Lee, Ye-Jin Cheon, Hyo Jin Gim, Do Hee Kim, Hee-Doo Kim, Jae-Ha Ryu, Raok Jeon
College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenylisothioureas were synthesized as inhibitors of NO production in lipopolysaccharide-
activated macrophages. We investigated the effect of lipophilic moiety and N- or S-substituents of the
phenylisothioureas on the activity. Inhibitory activities of carbazole-linked phenylisothioureas were
superior to the corresponding simple phenylisothiourea derivatives. Among these compounds, 12b hav-
ing N-ethyl and S-isopropyl groups on phenylisothiourea moiety was the most potent in the inhibition of
NO production. They inhibited NO production through the suppression of the LPS-induced translocation
Received 24 January 2009
Revised 31 March 2009
Accepted 2 April 2009
Available online 7 April 2009
Keywords:
Isothiourea
Nitric oxide
iNOS
of p65 subunit of NF-jB and the followed suppression of the iNOS protein and mRNA expression.
Ó 2009 Elsevier Ltd. All rights reserved.
Nitric oxide (NO) is a small signaling molecule involved in the
regulation of many physiological and pathophysiological pro-
cesses. NO is synthesized by three types of nitric oxide synthase
Main efforts for controlling NO levels were devoted to develop
selective inhibitors of iNOS that can be applied for the treatment
of diseases accompanying high levels of NO. Regulation of iNOS
(NOS) that catalyze the oxidation of
L
-arginine to
L
-citrulline as a
can be achieved by the control of expression and/or enzymatic
activity of iNOS. iNOS inhibitors can be classified into arginine
and non-arginine types by their structural features and mechanism
co-product. The diverse biological role of NO depends on which
subtype of enzyme is involved, and how much amounts of NO is
synthesized.1
Two isoforms found in neuronal tissues (nNOS, type I) and vas-
cular endothelium (eNOS, type III) are constitutive NOS (cNOS).
The cNOS is Ca2+-dependent and releases small amounts of NO re-
quired for homeostatic function.2 The third isoform is inducible
NOS (iNOS, type II) that can be induced by lipopolysaccharide
of the action. NG-Methyl-
nine (
L
-arginine (
L L-argi-
-NMA)9 and NG-nitro-
L
-NNA)10 are the representative arginine type iNOS inhibi-
tors. Recently, various non-arginine type inhibitors have been
reported such as amidines,11 isoquinolinamines,12 and isothioure-
as.13,14 But most of the inhibitors are neither potent nor NOS iso-
forms selective enough to be applied in vivo.
(LPS) and various cytokines such as IFN-
a
, IL-1b, and TNF-
a
. The
Urea,15 thiourea,16 and isothiourea13,14,17 have been reported to
inhibit iNOS expression and/or NO production. S-ethylisothiourea
was well known iNOS selective inhibitor18,19 and the co-crystal
structure of this compound with iNOS has been reported.20 Previ-
ously, we also reported thiourea derivatives as inhibitors of NO
production in LPS activated macrophages.21 Based on these reports,
we tried to investigate thiourea and isothiourea derivatives as no-
vel inhibitors having mechanism for enzymatic inhibition and/or
down regulation of iNOS expression.
iNOS is Ca2+-independent and produces micromolar levels of
NO.3 The physiological concentrations of NO produced by cNOS
have roles in the regulation of blood pressure and neurotransmis-
sion. Low concentrations of NO produced by iNOS possess benefi-
cial roles in host defence mechanism against pathogens,4 while
the excessive amounts of NO can cause various inflammatory dis-
eases. Large amounts of NO can rapidly react with superoxide an-
ion radical (ÅO2ꢀ) to produce peroxynitrite anion (ONOOꢀ) which is
thought to be a key mediator of NO-derived toxicity. The implica-
tion of NO and peroxynitrite in pathology of a wide range of dis-
eases such as rheumatoid arthritis, cancer, and atherosclerosis,
has led to the discovery of novel iNOS inhibitors as potential ther-
apeutic agents.5–8
Herein, we report the design and synthesis of phenylisothiourea
derivatives in which R can be simple alkyl or aryl, or carbazolyleth-
oxy as depicted in Figure 1. Their effects on the NO production and
H
N
N
R1 = alkyl, benzyl
2 = H, Et
R2
R
S
R
R1
* Corresponding author. Tel.: +82 2 710 9571; fax: +82 2 715 9571.
E-mail address: rjeon@sookmyung.ac.kr (R. Jeon).
Figure 1. Representative structure of phenylisothiourea.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.001

G. H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3088–3092
3089
H
N
H
H
NH2
N
N
N
a
b
S
S
R
R
R
1a : R = Cl
2a-e
3a-e
1b : R = Me
1c : R = Pr
1d : R = OEt
1e : R = Ph
Scheme 1. Preparation of phenylisothiourea derivatives. Reaction conditions : (a) SCNEt, K2CO3, THF; (b) 2-iodopropane, NaH, DMF.
expression of iNOS were evaluated in LPS-activated macrophage
cell culture system.
the LPS-treated control. Meanwhile, phenylisothiourea 3b, 3d,
and 3e showed 62%, 71%, and 100% activities at 50 lM,
Simple phenylthioureas and phenylisothioureas were prepared
starting from p-substituted anilines as shown in Scheme 1. Con-
densation of anilines 1 with ethylisothiocyanate offered thioureas
2. The following S-alkylation of thioureas with 2-iodopropane in
the presence of NaH gave desired S-isopropylisothioureas 3.
Carbazole-linked phenylisothioureas were also prepared to
investigate the effect of the lipophilic segment at the para-position
of phenyl ring. The preparation of the carbazole-linked phen-
ylisothiourea derivatives is outlined in Scheme 2. Hydroxyethyl
group was introduced at nitrogen of carbazole and the resulting
alcohol 5 was mesylated to give compound 6. Alkylation of com-
pound 6 by treatment of 4-nitrophenol in the presence of NaH gave
compound 7. Following reduction of nitro compound 7 over 10%
Pd/C under atmospheric pressure of hydrogen gas provided amine
8. Condensation of amine 8 with the appropriate isothiocyanates
was followed by S-alkylation of the resulted thioureas 9 and 11
to offer the desired respective isothioureas 10 and 12.
respectively.
Among these compounds, bisphenylisothiourea 3e was the
most potent showing 11.2 M of IC50 value. This result suggested
l
that appropriate substitution at para-position of phenyl ring might
be beneficial for their activities. Our previous study with thiourea
derivatives showed that the existence of suitable lipophilic moiety
such as the carbazole group had beneficial effect on inhibitory
activity of NO production.21,24 Therefore, we investigated biological
activities of the carbazole-linked phenylisothioureas.
The activities of carbazole-linked phenylisothioures for the
inhibition of NO production are given in Table 2. Most of the carba-
zole-linked phenylisothioureas showed higher activity than simple
phenylisothioureas. This result was consistent with the previous
observation found in thiourea derivatives.
Effect of the alkyl substitution at N3 position of isothiourea was
found to be significant. Compound 12a with ethyl substituent at
N3 position revealed four fold higher activity than 10a. On the base
of IC50 values of N3-ethylisothiourea derivatives, S-isopro-
pylisothiourea 12b is the most potent. But the inhibition (%) of
The inhibitory activities of the prepared compounds for the NO
production in LPS-activated macrophages were evaluated. Murine
macrophage cell line, RAW 264.7 cells were stimulated with
NO production at 20 lV is lower than those of the derivatives
1
l
g/mL of LPS in the presence or absence of samples for 20 h.
12a and 12f. That might come from the poor solubility of 12b in
cell culture media as we observed the formation of crystal at high
dose of 12b. In order to find optimal substituent on sulfur, S-ben-
zylisothioureas 12c–f having para-fluoro, methoxycarbonyl, or car-
boxyl group were investigated. Among these benzylisothioureas, S-
(p-carboxylbenzyl)isothiourea 12f was the most potent implying
the importance of the appropriate polarity for its activity. IC50 val-
ues of compounds 3e, 12a, 12b, and 12f with higher than 50%
The amounts of NO released into culture media were determined
by the Griess method22 in the form of nitrite.23
The inhibitory activities of simple phenylthioureas and phenyl-
isothioures are given in Table 1. It was found that activities of
phenylisothioureas were higher than those of the corresponding
phenylthioureas. The inhibitory activities of NO production of
phenylthioureas 2 were lower than 50% at 50
lM compared with
a
d
c
b
NO2
N
H
N
N
N
OH
OMs
O
4
5
6
7
H
N
e
g
f
NH2
N
NH2
R
NH2
N
N
N
S
S
O
O
O
8
9
10
H
N
H
N
H
h
N
N
N
S
N
S
O
R
O
11
12
Scheme 2. Preparation of carbazole-containing derivatives of phenylisothiourea. Reaction conditions: (a) ICH2CH2OH, NaH, DMF; (b) MsCl, TEA, DMF; (c) 4-nitrophenol, NaH,
DMF; (d) H2, 10% Pd/C, MeOH/THF; (e) NaSCN, K2CO3, THF; (f) RI, NaH, DMF; (g) EtNCS, K2CO3, THF; (h) RBr, NaH, DMF (or THF).

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G. H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3088–3092
Table 1
Inhibitory activities of phenylthioureas and phenylisothioureas on the NO production in LPS-activated macrophages
H
N
H
N
H
N
N
S
S
R
R
2
3
Inha (%) 5
l
M
Inh (%) 20
lM
Inh (%) 50
lM
IC50 (lM)
b
Compounds
R
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
OEt
Cl
Me
n-Pr
Ph
OH
OEt
Cl
Me
n-Pr
Ph
NDc
ND
ND
ND
ND
ND
ND
ND
ND
ND
13
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
85
12
11
14
23
23
20
22
62
14
71
96
11.2 2.2
a
Values mean the inhibition (%) of NO production of compounds relative to the LPS control (n = 3).
Values are means SD of three experiments.
ND means not determined.
b
c
activity at 20
respectively.
l
M were determined as 14.8, 6.7, 1.4, and 5.6
l
M,
To understand the further mechanism of action of isothioure-
as, we determined the effects of 3e, 12a, 12b and 12f on iNOS
expression in LPS-activated RAW 264.7 cells. The expression lev-
els of iNOS protein was analyzed by Western blot analysis after
20 h incubation with compounds during LPS activation of macro-
phages.25 All the compounds tested showed the significant inhibi-
The inhibitory activity (%) means the suppressing potency of NO
accumulation compared with LPS control by the treatment of sam-
ples during LPS activation of cells. The activity of phenylisothiourea
derivatives might come from the inhibition of iNOS enzymatic
activity and/or the suppression of signal pathway of iNOS expres-
sion. When we treated the compounds after the completion of
iNOS induction by LPS (post-treatment), they showed weak activ-
ity compared with the results of the co-treatment of the com-
pounds with LPS. Even the most potent compound 12b showed
tion of iNOS protein levels at 10
most potent showing 79% inhibition of iNOS protein expression at
V (Fig. 2). At RT-PCR analysis,26 the amounts of iNOS mRNA
lV. The compound 12b was the
5
l
was increased markedly by LPS-activation for 6 h. Compound
12b significantly suppressed the induction of iNOS mRNA at
28% inhibition at 5
l
V by post-treatment. These results suggested
5
l
V (Fig. 3). Curcumin was used as a positive control for the
expressional experiments of iNOS. The gene expression of iNOS
can be modulated by nuclear factor- B (NF- B) which can be
activated by the degradation of I- B- and the followed translo-
that phenylisothiourea derivatives exhibited their activities mainly
through the inhibition of iNOS expression with marginal inhibition
against enzymatic activity. This result was controversial since sev-
eral isothioureas have been reported to inhibit the enzymatic
activity of iNOS acting through posttranscriptional mecha-
nism.13,14,17–19
j
a
j
j
cation into nucleus. To elucidate the further mechanism of isot-
hiourea derivatives for the inhibition of iNOS expression, we
investigated their effects on the nuclear translocation of NF-jB.
Table 2
Inhibitory activities of carbazole-linked phenylisothioureas on the NO production in LPS-activated macrophages
H
N
N
NH2
R
N
N
S
N
S
O
O
R
12
10
Inha (%) 5
lM
Inh (%) 20
lM
Inh (%) 50
lM
IC50 (lM)
b
Compounds
R
c
10a
10b
10c
12a
12b
12c
12d
12e
12f
Me
Et
i-Pro
Me
ND
22
32
22
99
48d
31
36
14
90
24
64
30
ND
ND
ND
ND
ND
ND
ND
39
100
2.9 0.6
1.3 0.5
d
i-Pro
CH2C6H5
CH2(p-F–C6H4)
CH2(p-CO2Me–C6H4)
CH2(p-CO2H–C6H4)
33
40
17
100
5.3 0.8
a
Values mean the inhibition (%) of NO production of compounds relative to the LPS control (n = 3).
Values are means SD of three experiments.
ND means not determined.
b
c
d
Values reflect crystal formation of the compounds in cell culture media at the given concentration.

G. H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3088–3092
3091
Figure 4. Effects of isothioureas on the nuclear translocation of p65 in LPS-
activated macrophages. Cells were pretreated with samples (3e, 12a, 12b, 12f and
curcumin) for 30 min and followed by further incubation with LPS (1 lg/mL) for
30 min. The protein levels of p65 in nuclear extracts were determined by Western
blotting. The relative p65 protein levels were normalized with the respective
amounts of PARP.
of samples did not show any specific toxicity against macro-
phages compared to control group.
Figure 2. Effects of isothioureas on the expression of iNOS protein in LPS-activated
macrophages. RAW 264.7 cells were treated for 20 h with compounds 3e, 12a, 12b,
12f and curcumin (cur: as positive control) during LPS (1 lg/mL) activation. Cell
lysates were prepared and the iNOS and actin protein levels were determined by
Western blotting. The relative iNOS protein levels were normalized with the
respective amounts of actin. Values represent mean SD of three independent
densitometric analyses of bands. *p < 0.01 indicate significant difference between
LPS alone control and sample treatment.
In summary, we prepared a series of phenylisothiourea deriva-
tives and evaluated their inhibitory activities of NO production in
LPS-activated macrophages. Several phenylisothioureas showed
promising inhibitory activities for NO production. The SAR study
demonstrated that phenylisothiourea is superior to phenylthiourea
and appropriate substituents at para-position of phenyl ring have a
beneficial role for their activities. Also, their inhibitory activities
were highly influenced by changing the substituents on sulfur of
the isothioureas. Western blot and RT-PCR analysis suggested that
the prepared isothioureas inhibited NO production through the
inhibition of translocation of NF-jB into nucleus, and the followed
suppression of iNOS expression. Further biological study of the de-
tailed mechanism for the activity of isothiourea derivatives is
underway. The result provided our phenylisothiourea derivatives
can be good leads for the development of therapeutic agents for
the management of NO-related diseases.
Acknowledgments
This work was supported by the SRC program of KOSEF (Re-
search Center for Women’s Diseases) and Seoul R&BD Program
(10541).
Supplementary data
Figure 3. Effects of isothioureas on the expression of iNOS mRNA in LPS-activated
macrophages. RAW 264.7 cells were treated for 6 h with compounds 3e, 12a, 12b,
12f and curcumin (cur: as positive control) during LPS (1 lg/mL) activation. The
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.001.
mRNA levels of iNOS and b-actin were determined by RT-PCR from total RNA
extracts. The relative iNOS mRNA levels were normalized with the respective
amounts of b-actin. Values represent mean SD of three independent densitometric
analyses of bands. *p < 0.01 indicate significant difference between LPS alone
control and sample treatment.
References and notes
1. Forstermann, U.; Schmidt, H. H.; Pollock, J. S.; Sheng, H.; Mitchell, J. A.; Warner,
T. D.; Nakane, M.; Murad, F. Biochem. Pharmacol. 1991, 42, 1849.
2. Bredt, D. S.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 682.
3. Lowenstein, C. J.; Glatt, C. S.; Bredt, D. S.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 6711.
As shown in Figure 4, treatment of isothioureas suppressed the
4. Cook, H. T.; Cattell, V. Clin. Sci. (London) 1996, 91, 375.
5. Mocellin, S.; Bronte, V.; Nitti, D. Med. Res. Rev. 2007, 27, 317.
6. Squadrito, G. L.; Pryor, W. A. Free Radical Biol. Med. 1998, 25, 392.
7. Ricciardolo, F. L.; Nijkamp, F. P.; Folkerts, G. Curr. Drug Targets 2006, 7, 721.
8. Fitzpatrick, B.; Mehibel, M.; Cowen, R. L.; Stratford, I. J. Nitric Oxide 2008, 19,
217.
9. Olken, N. M.; Marletta, M. A. Biochemistry 1993, 32, 9677.
10. Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P. Biochemistry 1993, 32,
8512.
11. Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerome, G. M.; Kornmeier, C. M.; Tjoeng,
F. S.; Currie, M. G. Bioorg. Med. Chem. 1996, 4, 1559.
12. Beaton, H.; Hamley, P.; Nicholls, D. J.; Tinker, A. C.; Wallace, A. V. Bioorg. Med.
Chem. Lett. 2001, 11, 1023.
LPS-induced translocation of NF-jB subnit, p65 into nucleus.
PARP was used as an internal control for normalizing the loading
amounts of nuclear extract onto gel. These results indicated that
the phenylisothiourea derivatives decreased the expression of
iNOS through the inhibition of NF-jB activation via the suppres-
sion of p65 nuclear translocation. Furthermore we tested the rad-
ical scavenging activity of isothiourea derivatives by monitoring
fluorescence of the oxidation product of dihydrorhodamine 123
by authentic peroxynitrite in cell free assay system.27 Several of
them showed significant scavenging activity of peroxynitrite that
can be formed from NO and superoxide anion. Compound 12f
13. Paesano, N.; Marzocco, S.; Vicidomini, C.; Saturnino, C.; Autore, G.; De Martino,
G.; Sbardella, G. Bioorg. Med. Chem. Lett. 2005, 15, 539.
14. Raman, C. S.; Li, H.; Martasek, P.; Babu, B. R.; Griffith, O. W.; Masters, B. S.;
Poulos, T. L. J. Biol. Chem. 2001, 276, 26486.
was the most potent with SC50 of 14
lM. This activity can be an-
other mechanism of the isothiourea derivatives. All the treatment

3092
G. H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3088–3092
15. Prabhakar, S. S.; Zeballos, G. A.; Montoya-Zavala, M.; Leonard, C. Am. J. Physiol.
1997, 273, C1882.
16. Goodyer, C. L. M.; Chinje, E. C.; Jaffar, M.; Stratford, I. J.; Threadgill, M. D. Bioorg.
Med. Chem. 2003, 11, 4189.
17. Shearer, B. G.; Lee, S.; Oplinger, J. A.; Frick, L. W.; Garvey, E. P.; Furfine, E. S. J.
Med. Chem. 1997, 40, 1901.
18. Garvey, E. P.; Oplinger, J. A.; Tanoury, G. J.; Sherman, P. A.; Fowler, M.; Marshall,
S.; Harmon, M. F.; Paith, J. E.; Furfine, E. S. J. Biol. Chem. 1994, 269, 26669.
19. Nakane, M.; Klinghofer, V.; Kuk, J. E.; Donnelly, J. L.; Budzik, G. P.; Pollock, J. S.;
Basha, F.; Carter, G. W. Mol. Pharmacol. 1995, 47, 831.
25. Western blot analysis of iNOS protein expression—RAW 264.7 cells
(1.5 ꢁ 106 cells/60 mm dish) were stimulated with LPS (1
lg/mL) in the
presence or absence of test compounds. After incubation for 20 h, the cells
were washed and lyzed with lysis buffer. Twenty micrograms of protein of cell
lysates were applied on 8% SDS–polyacrylamide gels and transferred to PVDF
membrane by a standard method. The membrane was probed with antibody
for anti-mouse iNOS (Transduction Laboratories, Lexington, KY) and anti-actin
(Sigma, St. Louis, MO). The bands were visualized using an enhanced
chemiluminescence (ECL) detection kit (Amersham Bioscience, Piscataway,
NJ) according to the manufacturer’s instruction.
20. Fischmann, T. O.; Hruza, A.; Niu, X. D.; Fossetta, J. D.; Lunn, C. A.; Dolphin, E.;
Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula, S. K.; Weber, P. C. Nat. Struct.
Biol. 1999, 6, 233.
26. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of iNOS
mRNA expression—RAW 264.7 cells (1.8 ꢁ 106 cells/60 mm dish) were
stimulated for 6 h with LPS (1 lg/mL) in the presence or absence of test
21. Kim, Y. J.; Ryu, J. H.; Cheon, Y. J.; Lim, H. J.; Jeon, R. Bioorg. Med. Chem. Lett. 2007,
17, 3317.
22. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
23. Cell culture and nitrite assay in LPS-activated RAW 264.7 cells—cells in 10%
fetal bovine serum (FBS)–DMEM medium, were plated in 48-well plates
(1 ꢁ 105 cells/mL), and then incubated for 24 h. The cells were replaced with
fresh media with 1% FBS, and then incubated for 20 h in the presence or
compounds. After washing twice with phosphate buffered saline, total RNA
was isolated from cell pellet, using an RNA isolation reagent (Trizol, Invitrogen,
Carlsbad, CA). Two microgram of RNA was reverse transcribed into cDNA using
reverse transcriptase and random hexamer. The PCR samples, contained in the
reaction mixture, were comprised of mixture buffer, dNTP, Taq DNA
polymerase (Promega, Madison, WI) and primers (sense and antisense). The
sense and antisense primers for iNOS were 50-ATGTCCGAAGCAAACATCAC-30
and 50-TAATGTCCAGGAAGTAGGTG-30, respectively. The sense and antisense
primers for b-actin were 50-TGTGATGGTGGGAATGGGTCAG-30 and 50-
TTTGATGTCACGCACGATTTCC-30, respectively. The PCR amplification was
performed under following conditions; 25 cycles of denaturation at 94 °C for
30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, using thermal
cycler (Gene Amp PCR system 2400, Applied Biosystems, Foster City, CA). The
amplified PCR products were separated on a 2% agarose gel.
absence of test compounds with LPS (1
was assessed by measuring the accumulated nitrite in culture supernatant.
Samples (100 L) of media were incubated with Griess reagent (150 L) for
lg/mL). NO production in each well
l
l
10 min at room temperature in 96 well microplate. Absorbance at 570 nm was
read using an ELISA plate reader. A standard calibration curve was prepared
using sodium nitrite as a standard. A dose–response curve was prepared, and
the results were typically expressed as IC50 values.
27. Lee, M. H.; Kim, J. Y.; Yoon, J. H.; Lim, H. J.; Kim, T. H.; Jin, C.; Kwak, W. J.; Han, C.
K.; Ryu, J. H. Phytother. Res. 2006, 20, 742.
24. Tsao, L. T.; Lee, C. Y.; Huang, L. J.; Kuo, S. C.; Wang, J. P. Biochem. Pharmacol.
2002, 63, 1961.