Synthesis, anticancer and antioxidant activities of 7-methoxyisoflavanone and 2,3-diarylchromanones

Article abstract of DOI:10.1016/j.ejmech.2010.02.028

A convenient, fast and high yielding method for the preparation of 7-methoxyisoflavanone and 2,3-diarylchromanones has been developed by the condensation of benzyl-2-hydroxy-4-alkoxyphenylketone with arylaldehyde/paraformaldehyde in presence of diethylamine, assisted by microwave activation. All the synthesized compounds were screened for anticancer as well as antioxidant activities. Among the nine compounds, 7-methoxyisoflavanone 7 and diarylchromanone 6c shows potential anticancer activity and diarylchromanone 6b has potential antioxidant activity. Compound 6h possesses anticancer and antioxidant activity at the same concentration.

Full text of DOI:10.1016/j.ejmech.2010.02.028

European Journal of Medicinal Chemistry 45 (2010) 2447e2452
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anticancer and antioxidant activities of 7-methoxyisoflavanone
and 2,3-diarylchromanones
Kanagasabai Kanagalakshmi a, Mariappan Premanathan b, Ragunathan Priyanka b,
,
Balasubramanian Hemalatha a, Arumugasamy Vanangamudi a
*
a Centre for Research and Post-graduate studies in Chemistry, Ayya Nadar Janaki Ammal College, Sivakasi 626 124, Tamil Nadu, India
b Department of Biotechnology, Mepco Schlenk Engineering College, Sivakasi 626 005, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient, fast and high yielding method for the preparation of 7-methoxyisoflavanone and 2,3-
diarylchromanones has been developed by the condensation of benzyl-2-hydroxy-4-alkoxyphenylketone
with arylaldehyde/paraformaldehyde in presence of diethylamine, assisted by microwave activation. All
the synthesized compounds were screened for anticancer as well as antioxidant activities. Among the
nine compounds, 7-methoxyisoflavanone 7 and diarylchromanone 6c shows potential anticancer activity
and diarylchromanone 6b has potential antioxidant activity. Compound 6h possesses anticancer and
antioxidant activity at the same concentration.
Received 18 September 2009
Received in revised form
4 February 2010
Accepted 10 February 2010
Available online 14 February 2010
Keywords:
Anticancer
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antioxidant
7-Methoxyisoflavanone
7-Methoxy-2,3-diphenylchromanones
1. Introduction
2. Chemistry
Microwave assisted reactions offer a considerable advantages
over conventional thermal reactions because it results in
substantial rate enhancement in a wide range of organic reac-
tions. Moreover, majority of the microwave assisted reactions are
solvent-free reactions, hence they are considered as clean, effi-
cient and economical technology. This methodology has been
widely used in a variety of organic reactions [1]. However, the
solvent-free synthesis of 2,3-diarylchromanones has not been
reported so far.
Chromanones and flavones are integral part of human diet and
have been reported to exhibit a wide range of biological effects [2].
They also demonstrate antioxidant [3], anti-inflammatory [4],
antibacterial [5] and antitumor [6] properties. Several synthetic
analogs of the compounds such as cromokalim, demiflin and
flavaxate have been developed into useful drugs [7]. Therefore,
many methods of synthesis of 7-methoxyisoflavanone and 2,3-
diarylchromanones have been explored. Generally, these reactions
were carried out in solution and have drawbacks of using large
amount of volatile and poisonous solvents with high reaction time.
In order to find an environmentally benign procedure, a new
technique of solvent-free, microwave activated synthesis of 2,3-
diarylchromanones has been developed (Scheme 1). Using this
technique, seven new compounds of 2,3-diarylchromanones
(Table 1) and one reported 2,3-diphenylchromanone 6a and 7-
methoxyisoflavanone 7 have been synthesized in short reaction
period. Equimolar amounts of benzyl-2-hydroxy-4-alkox-
yphenylketone and arylaldehyde/paraformaldehyde were allowed
to react in the presence of diethylamine under microwaves with
good yield (Table 1). The time taken for the formation of 2,3-
diphenylchromanone 6a was found to be 16 min against 3 h in the
conventional method [8]. The time taken for the 7-methoxyiso-
flavanone 7 was found to be 30 min against 3 h in the conven-
tional method [9].
It is reported that the methylation of various compounds
under microwave irradiation results in good yield in a short
period of reaction [10]. In the present study, it was noted
that the methylation/ethylation of benzyl-2,4-dihydrox-
yphenylketone
to
benzyl-2-hydroxy-4-methoxy/ethox-
yphenylketone is found to be completed within 3 min against 4 h
in the presence of large volume of solvent in the conventional
method [11,12].
* Corresponding author. Tel.: þ91 4562 224489.
E-mail address: sudhakanivanangamudi@gmail.com (A. Vanangamudi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.028

2448
K. Kanagalakshmi et al. / European Journal of Medicinal Chemistry 45 (2010) 2447e2452
OH
OH
HO
HO
ZnCl2
CH2COOH
+
O
1
3
2
i) R2SO4
ii) K2CO3
MW
R2
'
3
R3
R1
'
4
(C2H5)2NH
MW
OH
O
1
'
8
2
7
MeO
O
2
1
O
8
7
R1
'
5
2
(HCHO)n
"
2
3
'
6
6
"
"
"
2
3
3
(C2H5)2NH
5
6
"
"
R3
3
CHO
O
"
4
5
6
MW
EtOH
O
"
"
4
5
6
"
R2
5
7
4
5
6
R1
4a
4b
OMe
OEt
Scheme 1. Synthesis of 7-methoxyisoflavanone and 2,3-diarylchromanones.
3. Pharmacology
Synthesized compounds were evaluated for antioxidant activity
and cytotoxicity using HL60 cells and PBMC (Peripheral blood
mononuclear cells). Antioxidant activities of the compounds were
determined using lipid peroxidation assay (Table 3). The cytotox-
icity was assessed by the MTT assay (Table 4). In addition, treatment
with HL60 also resulted in nuclear DNA fragmentation, as seen in
agarose gel electrophoresis (Fig. 2). This is a hallmark of cells
undergoing apoptosis. Confirmation of apoptosis was performed by
staining the cells with Annexin V. Annexin V-positive cells were
defined as apoptotic cells (Fig. 3).
Table 1
Physical characterization data and elemental analysis of compounds.
Compound Reaction period Melting Yield Molecular Elemental analyses
MWI (min.)
point
(%)
formula
found % (Calcd)
without solvent (ꢁC)
C
H
N
6a
6b
6c
6d
6e
6f
16 (3 h)a
179e180 70
124e126 80
135e137 60
168e171 75
129e131 85
148e151 85
139e142 70
134e137 80
72e73 80
C22H18O3
79.91 5.28
(79.98 5.49)
30
C22H17NO5 70.33 4.28 3.73
(70.39 4.56 3.73)
C22H17NO5 70.33 4.28 3.73
(70.39 4.56 3.73)
30
30
C
22H17ClO3 72.33 4.35
(72.43 4.70)
4. Results and discussion
30
C23H20O3
80.18 5.42
(80.21 5.85)
All the compounds were characterized by elemental analysis
(Table 1), UV, FT-IR and 1H NMR spectra (Table 2). The UV-spectra
of all compounds showed two maxima one around 274e278 nm
and the other at 298e317 nm. The infrared spectra of the chro-
manones 6aeh showed carbonyl absorption in the region
1670e1677 cmꢀꢀ11, aromatic CeH stretching band in the region
3031e3076 cm and aliphatic CeH stretching band in the region
2839e2983 cmꢀ1. In the 1H NMR spectra of the chromanones,
apart from the expected aromatic protons in the region
26
C23H19NO5 70.74 5.49 3.56
(70.94 4.92 3.60)
C23H19NO5 70.74 5.49 3.56
(70.94 4.92 3.60)
6g
6h
7
30
26
C
23H19ClO3 72.87 5.01
(72.92 5.06)
30 (3 h)a
C16H14O3
75.48 5.45
(75.58 5.55)
a
The value inside the parenthesis indicates the time taken for the completion of
the reaction in the conventional method.
6.47e8.16 d, two doublets each integrating to one proton were

K. Kanagalakshmi et al. / European Journal of Medicinal Chemistry 45 (2010) 2447e2452
2449
Table 2
Spectral data of compounds 4b, 6aeh and 7.
Compound
UV (lmax
)
IR (cmꢀ1) (KBr)
1H NMR (CDCl3) (
d ppm)
4b
321, 280
3446, 3060, 2983, 1637,
1587, 1290, 1197, 1035,
858, 813
1.35(t, CH3, 3H); 4.0(q, OCH2, 2H); 4.14(s, CH2, 2H); 12.64(s, OH, 1H);
7.66(d, H-3, 1H); 6.33e6.37(m, H-4 & H-6, 2H); 7.19e7.27(m, phenyl ring, 5H)
6a
6b
6c
6d
6e
6f
312, 278
307, 274
308, 277
312, 276
298, 276
312, 277
3031, 2839, 1672, 1616,
1446, 1251, 1163, 1022, 831
3.8(s, OCH3, 3H); 4.1(d, H-3, 1H); 5.6(d, H-2, 1H); 6.96(d, H-6, 1H);
6.52(s, H-8, 1H); 6.98e7.06 (m, C-3 phenyl-H, 5H); 7.93(d, H-5, 1H);
7.18e7.23(m, C-2 phenyl-H, 5H)
3.88(s, OCH3, 3H); 4.0(d, H-3, 1H); 5.61(d, H-2, 1H); 6.71(d, H-6, 1H);
6.54(s, H-8, 1H); 6.95(m, H-300, 500, 2H); 7.22e7.35(m, 50, 60, 200, 400, 600, 5H);
8.07(d,H-20,40,2H); 7.93(d, H-5, 1H)
3.88(s, OCH3, 3H); 4.03(d, H-3, 1H); 5.61(d, H-2, 1H); 6.71(d, H-6, 1H);
6.54(s, H-8, 1H); 6.96(m, H-300, 500, 2H); 7.21(m, H-200, 400, 600, 3H);
7.93 (d, H-5, 1H); 8.16(s, H-30, 50, 2H); 7.36(s, 20, 60, 2H)
3.85(s, OCH3, 3H); 4.02(d, H-3, 1H); 5.49(d, H-2, 1H); 6.64(d, H-6, 1H);
6.51(s, H-8, 1H); 7.14(s, H-20, 60, 2H); 7.21(m, H-30, 50, 200, 400, 600, 5H);
7.93(d, H-5, 1H); 6.96(m, H-300, 500, 2H)
1.43 (3H, t, CH3); 4.00 (2H, q, OCH2); 4.05 (1H, d, H-3); 5.53 (1H, d, H-2);
6.49 (1H, d, H-8); 6.65 (1H, dd, H-6); 7.15e7.25(10H, m, C-2 & C-3 phenyl-H);
7.88 (1H, d, H-5)
3064, 2940, 1677, 1616,
1537, 1440, 1352, 1251,
1161, 831
3046, 2930, 1670, 1616,
1521, 1446, 1348, 1249,
1163, 1108, 833
3040, 2840, 1672, 1616,
1581, 1575, 1446, 1361,
1249, 1163, 1110, 831
3050, 2983, 1683, 1608,
1442, 1359, 1251, 1178,
1105, 840, 813
3076, 2926, 1680, 1608,
1529, 1442, 1352, 1253,
1170, 1107, 1037, 846.
1.43 (3H, t, CH3); 4.05(2H, q, OCH2); 4.07 (1H, d, H-3); 5.6 (1H, d, H-2);
6.49 (1H, d, H-8); 6.64 (1H, dd, H-6); 8.08e8.14(2H, m, H-20, 40);
7.34e7.38 (2H, m, H-50, 60); 6.93e6.96 (2H, m, H-300, 500);
7.19e7.26(3H, m, H-200, 400, 600); 7.92 (1H, d, H-5)
6g
6h
7
317, 275
312, 277
275, 234
3050, 2929, 1670, 1616,
1521, 1446, 1348, 1249,
1108, 1004, 833
3034, 2982, 1670, 1612,
1492, 1446, 1359, 1251,
1176, 1107, 1045, 823, 748
3050, 2964, 1681, 1614,
1577, 1242, 698
1.45 (3H, t, CH3); 3.59 (2H, q, OCH2); 4.1(1H, d, H-3); 5.6 (1H, d, H-2);
6.52 (1H, s, H-8); 6.9 (1H, dd, H-6); 7.2e7.4(5H, m, C-3 phenyl-H);
7.68 (2H, d, H-20, 60); 7.95 (2H, d, H-30, 50); 8.1 (1H, d, H-5)
1.43 (3H, t, CH3); 4.05 (2H, q, OCH2); 4.10 (1H, d, H-3); 5.49 (1H, d, H-2);
6.47 (1H, s, H-8); 6.62 (1H, d, H-6); 6.94e7.26 (9H, m, C-2 & C-3 phenyl-H);
7.8 (1H, d, H-5)
3.8 (s, OCH3, 3H); 3.9 (t, H-3, 1H); 4.65 (d, H-2, 1H); 6.44 (s, H-8, 1H);
6.62 (d, H-6, 1H); 7.26e7.35 (m, C-3, phenyl-H, 5H); 7.88 (d, H-5, 1H)
observed in the regions 5.49e5.61
d
and 4.0e4.1
d
were assigned to
4.2. Cytotoxicity assay
C-2 and C-3 protons, respectively. These two doublets indicated
the formation of the products 6aeh. Further, the coupling constant
value (11e12 Hz) suggested that the protons at C-2 and C-3 are
trans oriented [13]. The formation of 7-methoxyisoflavanone 7 was
confirmed by UV, IR and 1H NMR spectra. In 1H NMR spectra of the
7-methoxyisoflavanone 7, apart from the expected aromatic
MTT assay was used to assess the cell viability based on its
reduction by mitochondrial dehydrogenase enzyme of the viable
cells to purple formazan product [17]. The cytotoxicity of the
chromanone derivatives was studied in the acute myeloblastic
leukemia HL60 cells and PBMC. The cytotoxicity of the compounds
was determined after five days of exposure and its CC50 values were
calculated. Among the tested compounds, 7-methoxyisoflavanone
7 and diarylchromanone 6c exhibited highest cytotoxicity with
protons, C-2 proton appeared as a doublet at 4.35
appeared as a triplet at 3.9
d and C-3 proton
d
.
CC50 of 82.58 mM and 73.16 mM respectively, while the two diary-
4.1. Free radical scavenging activity
lchromanones 6g and 6h exhibited significant cytotoxicity
(Table 4). Their cytotoxicity seems to be specific for tumor cells
since normal human lymphocytes were not susceptible. It is real-
ized, however, that normal and neoplastic cells have different rates
of proliferation and it is not surprising that an active drug is inef-
fective on slow-growing normal cells.
Increase in lipid peroxidation denotes cytotoxicity and hepato-
cellular dysfunction on mice [18]. The EC50 and CC50 values were
compared using one-way analysis of variance ANOVA (Fig. 1). This
study showed that the compounds which possess high antioxidant
Testing the antioxidant activity of the 7-methoxyisoflavanone 7
and eight diarylchromanones 6aeh were measured by the inhibi-
tion of lipid peroxidation. Table 3 shows the radical modulation
activity of the compounds. It was found that the chromanone 6b
possesses highest activity with EC50 value of 129.86
6d, 6f, 6a and 6 h with EC50 value of 164.86, 193.29, 499.56 and
578.39 M, respectively. The higher activity of the chromone is
mM followed by
m
attributed to the conjugation of double bonds and the methoxy
group. It can possibly contribute to the activity of this compound as
a scavenger of free radicals. This is confirmed by the isolation of the
3-[2-(3,5-dimethoxyphenyl)ethenyl]-2methylchromone from Eru-
caria microcarpa and it was proved to be active as a scavenger [14].
The diarylchromanone derivative 6b showed the highest radical
modulation activity compared to other tested compounds due to
the presence of nitro group in the meta position. Free radicals are
known to induce cellular damage and may play a role in heart
disease, rheumatoid arthritis, cancer, inflammatory disorders as
well as aging processes [15]. The flavonoids are most common
natural antioxidants [16]. These are not only the defense molecules
in prevention of different pathological disorders but also commonly
used in industry for the prevention of oxidative degradation of
polymers, synthetic and natural pigments. In the present study, five
diarylchromanones were found to have antioxidant activity.
Table 3
Radical modulation assay of compounds 6aeh and 7.
Test compound
EC50 (mM)
6a
6b
6c
499.56 ꢂ 123.07
129.86 ꢂ 025.58
>2500
6d
6e
6f
6g
164.86 ꢂ 009.45
876.81 ꢂ 597.17
193.29 ꢂ 034.41
>2500
6h
7
578.38 ꢂ 037.64
964.71 ꢂ 239.93
754.14 ꢂ 7.03
Ascorbic acid

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K. Kanagalakshmi et al. / European Journal of Medicinal Chemistry 45 (2010) 2447e2452
Table 4
cell form within. Disassembly creates changes in the phospholipid
MTT assay of compounds 6aeh and 7.
content of the cytoplasmic membrane outer leaflet. Phosphati-
dylserine (PS) is translocated from the inner to the outer surface of
the cell for phagocytic cell recognition. The human anticoagulant,
Annexin V, is a Ca2þ-dependent phospholipid protein with a high
affinity for PS. Annexin V labeled with fluorescein can identify
apoptotic cells in the population (Fig. 2). It is a confirmatory test for
apoptosis. It was also reported that the flavonoid Berberine exhibits
the ability to induce apoptosis in HL60 cells [20].
Test compound
CC50
(mM)
HL60
PBMC
6a
6b
6c
6d
6e
6f
2331.25 ꢂ 499.36
>2500
73.16 ꢂ 007.84
2414.02 ꢂ 176.49
>2500
>2500
>2500
594.81 ꢂ 43.31
>2500
>2500
>2500
>2500
6g
6h
7
202.92 ꢂ 015.36
514.21 ꢂ 040.58
82.58 ꢂ 007.84
0.07 ꢂ 0.01
1359.74 ꢂ 123.81
>2500
5. Conclusion
520.51 ꢂ 59.66
NT
5-Fluorouracil
The present study, is a quick and simple method for the synthesis
of 2,3-diarylchromanones in a solvent-free environment by micro-
wave irradiation with good yield. Further, it is demonstrated that
the compounds are potential anticancer and antioxidant agents.
Among the nine compounds prepared, diarylchromanone 6b
possesses highest antioxidant activity and 7-methoxyisoflavanone
7 has highest anticancer activity. The diarylchromanone 6h shows
Values represent the mean ꢂ standard deviation of three separate experiments
(p < 0.05).
activity have less cytotoxicity and the compounds which showed
high cytotoxicity have less antioxidant activity.
The 7-methoxyisoflavanone 7 showed highest cytotoxicity than
2,3-diarylchromanones except diarylchromanone 6c due to the
absence of phenyl ring in C-2 position. Among the three diary-
lchromanones 6c posses highest cytotoxicity than 6g and 6h due to
the presence of methoxy group in C-7 position and the nitro group
in the C-30 position. Generally, in diarylchromanones presence of
methoxy group and nitro group will enhance the activity and the
presence of ethoxy group will decrease the activity. Thus, the
7-methoxyisoflavanone 7 and the three diarylchromanones 6c, 6g
and 6h have been selected for further studies.
antioxidant activity with EC50 value of 578.38
mM and cytotoxicity
with CC50 value of 514.21 M. Since it shows both antioxidant
m
activity and cytotoxicity at almost the same concentration, it can
prevent the free radical induced cancer as prophylactic agent and
kill the cancer cells by apoptotic process as a chemotherapeutic
agent. It will be the candidate compound for further studies to be
developed as a viable anticancer drug.
6. Experimental protocols
4.3. DNA fragmentation assay
6.1. Chemistry
The DNA profile of the cells treated with 7-methoxyisoflavanone
7 and diarylchromanone 6aeh along with control cells was
analyzed. In control cell the DNA was not fragmented whereas
when the cells are treated with diarylchromanones 6aeh and
7-methoxyisoflavanone 7 have shown apoptosis fragmentation of
DNA (Fig. 2). Cleavage of DNA at the internucleosomal linker sites
yielding DNA fragments is regarded as a biochemical manifestation
of apoptosis [19].
Melting points were determined in open capillaries and are
uncorrected. The IR spectra were recorded on a 8400S SHIMADZU
spectrophotometer and the UV-spectra on a SHIMADZU UV-1700
UVevis spectrophotometer. The 1H NMR spectra were obtained on
a Bruker 200 MHz spectrometer in CDCl3 (Chemical shifts in d, ppm
4.4. Apoptosis detection
For further confirmation of apoptosis, FACS analysis was carried
out. Apoptosis is an active, genetically regulated disassembly of the
Fig. 1. Comparison between EC50 and CC50 values. Each bar represents the mean ꢂ SD
for three separate experiments. Statistical analysis was performed by ANOVA
(p < 0.05).
Fig. 2. DNA fragmentation assayof 7-methoxyisoflavanone and 2,3-diarylchromanones.

K. Kanagalakshmi et al. / European Journal of Medicinal Chemistry 45 (2010) 2447e2452
2451
Fig. 3. FACS analysis for apoptosis induction. HL60 cells treated with test compound and then stained with FITC-Annexin V. Shaded histograms represent cellular fluorescence of the
control cells and open histograms represent the cellular fluorescence resulting from the specific binding of FITC-Annexin V to apoptotic cells.
relative to TMS as an internal standard). Elemental analyses were
done on Elementar Vario EL III.
treated with ice cold water and neutralized with concentrated
hydrochloric acid, when benzyl-2-hydroxy-4-methoxy/ethox-
yphenylketone separated as a solid. It was crystallized from ethanol
as white crystals. It gave deep red colour with neutral ferric chlo-
ride. [4a: m.p. 88 ꢁC (lit. [11,12] m.p. 88 ꢁC); 4b: m.p. 77 ꢁC].
6.1.1. Synthesis of benzyl-2-hydroxy-4-methoxy/ethoxyphenyl-
ketone (4a e methoxy; 4b e ethoxy)
Benzyl-2,4-dihydroxyphenylketone (4.56 g) was dissolved in
dry acetone (15 mL) in a 100 mL conical flask. To this, dimethyl/
ethylsulphate (2 mL) and anhydrous potassium carbonate (5 g)
were added. A funnel was kept in the conical flask over which
a small round bottom flask filled with water was kept which acted
as a condenser. The suspension was irradiated under microwave for
3 min with the time interval of 5 s at 300 W. After the completion of
the reaction (monitored with TLC), the reaction mixture was
filtered and potassium salt was washed with several portions of
acetone. The combined filtrates were evaporated. The residue was
6.1.2. General procedure for the synthesis of 2,3-diarylchromanones
To a mixture of benzyl-2-hydroxy-4-methoxy/ethoxyphenyl
ketone (0.005 mmol), arylaldehyde (0.005 mmol) and diethyl-
amine (0.001 mmol) were taken in a 50 mL conical flask. A funnel
was kept in the conical flask over which a small round bottom flask
filled with ice-water was kept which acted as a condenser. The
mixture was irradiated in a microwave oven [SAMSUNG M 197 DL]
at 100 W for 16 min with the time interval of 30 s. The reaction
mixture was treated with ice-water and then neutralized with

2452
K. Kanagalakshmi et al. / European Journal of Medicinal Chemistry 45 (2010) 2447e2452
concentrated hydrochloric acid. The solid obtained was filtered and
crystallized from ethanol as colorless crystals. It was negative
towards neutral ferric chloride solution.
induction was analyzed using the apoptotic, necrotic, and healthy
cell quantification kit (Biotium inc., USA) following the manu-
facturer's protocol for flow cytometry (FACS caliber, BD Biosciences,
USA) assay [23].
6.1.3. Synthesis of 7-methoxy-3-phenylchroman-4-one
An ethanolic solution of benzyl-2-hydroxy-4-methox-
yphenylketone (1.21 g, 0.005 mmol), paraformaldehyde (0.3 g,
0.005 mmol) and diethylamine (1.05 mL, 0.001 mmol) were taken
in a 50 mL conical flask. A funnel was kept in the conical flask over
which a small round bottom flask filled with ice-water was kept
which acted as a condenser. The mixture was irradiated in
a microwave oven [SAMSUNG M 197 DL] at 100 W for 30 min with
the time interval of 30 s. The reaction mixture was treated with
ice-water and then neutralized with concentrated hydrochloric
acid. The solid obtained was filtered and crystallized from ethanol
as colorless crystals. It was negative towards neutral ferric chloride
solution. [m.p. 72e73 ꢁC (lit. [9] m.p. 73 ꢁC)].
6.2.5. Statistical analysis
Data were analyzed using the software SigmaPlot for Windows
(Version 11.0). Values were expressed as mean ꢂ standard devia-
tion of the mean values of three independent experiments followed
by student t-test. EC50 and CC50 values were compared using
one-way analysis of variance ANOVA. Statistical significance was
acceptable to a level of p < 0.05.
Acknowledgement
The authors are thankful to the Management, Principal of the
Ayya Nadar Janaki Ammal College and Mepco Schlenk Engineering
College, Sivakasi, Tamil Nadu, India for providing the necessary
facilities to carry out this work. One of authors Dr. AV is grateful to
University Grants Commission, New Delhi for financial support in
the form of Minor Research Project. FACS analysis was done in
TIFAC-CORE in Diabetic Retinopathy, Aravind Medical Research
6.2. Bioassays
6.2.1. Free radical scavenging activity
The free radical scavenging activity of different compounds was
determined using lipid peroxidation assay [21]. Briefly, Lipid per-
oxidation was induced in liposome prepared from egg lecithin by
Foundation (AMRF), Aravind Eye Hospital
& PG Institute of
Ophthalmology, Madurai, Tamil Nadu, India with the help of Gowri
Priya of AMRF. The authors are also thankful to the Centralised
Instrumentation Centre of the ANJA College, Sivakasi for the sample
analysis.
adding 5 mL of 400 mM FeCl3 and 5 ml of 200 mM L-ascorbic acid.
To this, different concentrations of the test compound were added.
The control was prepared which contained no compound. The
samples were incubated at 37 ꢁC for 60 min. The reaction was
inhibited by adding 1 mL of stopping solution which contains
0.25 N HCl, 1.5% Trichloroacetic acid, 0.375% Thiobarbutric acid.
These reaction mixtures were kept in boiling water bath for 15 min,
cooled and centrifuged. The absorbance of the resulting solution
was measured at 532 nm. The activity was calculated by using the
formula: 50% inhibition (EC50) ¼ [(control OD ꢀ sample OD)/control
OD] ꢃ 100.
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100 mg/mL streptomycin sulphate, 40 mg/mL gentamycin, 100 U/mL
penicillin as well as 10% heat inactivated fetal calf serum.
An environment of humidified air containing 5% CO2 was main-
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obtained from healthy donors and cultured as described [22].
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microtiter plates (200 m
L/well) and incubated at 37 ꢁC in a CO2
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viability was measured by MTT assay [17], from which 50% cyto-
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HL60 cells were incubated with appropriate concentration of the
test compound with their CC50 value. After 48 h, DNA was extracted
using DNA isolation kit (Genei, Bangalore, India), evaluated on 0.8%
agarose gel using ethidium bromide and DNA pattern was docu-
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6.2.4. Apoptosis detection by Annexin V marker
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