Synthesis, characterization, DNA interaction, and antitumor activities of mixed-ligand metal complexes of kaempferol and 1,10-phenanthroline/2,2′- bipyridine

Article abstract of DOI:10.1007/s00044-013-0863-2

Four novel mixed-ligand metal complexes with the composition [Cu(Kae)(phen)]Cl·2H₂O (1), [Cu(Kae)(bpy)]Cl·H ₂O ( 2), [Zn(Kae)(phen)]Cl·H₂O (3), and [Zn(Kae)(bpy)]Cl·H₂O (4), where Kae = kaempferol, phen = 1,10-phenanthroline, bpy = 2,2′-bipyridine were synthesized and characterized by elemental analysis, IR, UV, ¹H NMR, ¹³C NMR, HRMS ,and molar conductivity. The binding and cleavage abilities of complexes 1-4 with DNA have been studied by fluorescence spectroscopy, viscosity measurements, and gel electrophoresis under physiological conditions. The experimental results indicated that four complexes could bind to CT-DNA via an intercalative mode, and the complex 1 showed the strongest activity to cleavage DNA (pUC 19). The in vitro cytotoxicities of kaempferol and complexes 1-4 were also evaluated against human breast carcinoma cells by MTT assays. The results show that the cytotoxicities of complexes 1-4 are much higher than that of kaempferol alone, and the complex 1 shows the strongest ability to inhibit the growth of human breast carcinoma cells. The results suggest that the complex 1 may be a valuable tool in cancer chemotherapy agents. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0863-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2659–2666
DOI 10.1007/s00044-013-0863-2
ORIGINAL RESEARCH
Synthesis, characterization, DNA interaction, and antitumor
activities of mixed-ligand metal complexes of kaempferol
and 1,10-phenanthroline/2,20-bipyridine
•
Qin Wang Min Huang Yu Huang Jin-Sheng Zhang
•
•
•
•
Gui-Feng Zhou Ren-Quan Zeng Xin-Bin Yang
•
Received: 23 August 2013 / Accepted: 18 October 2013 / Published online: 30 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Four novel mixed-ligand metal complexes
with the composition [Cu(Kae)(phen)]Clꢀ2H2O (1),
[Cu(Kae)(bpy)]ClꢀH2O (2), [Zn(Kae)(phen)]ClꢀH2O (3),
and [Zn(Kae)(bpy)]ClꢀH2O (4), where Kae = kaempferol,
phen = 1,10-phenanthroline, bpy = 2,20-bipyridine were
synthesized and characterized by elemental analysis, IR,
Keywords Kaempferol ꢀ Mixed-ligand metal complexes ꢀ
DNA interaction ꢀ Antitumor activity
Introduction
1
UV, H NMR, 13C NMR, HRMS ,and molar conductivity.
Flavonoids are a large group of polyphenolic natural
compounds that are found in many high plants. Such
compounds have been receiving widespread attention of
many researchers because of their biological and pharma-
cological activities such as antioxidant, anticancer, and
antimicrobial (Fraga et al., 1987; Dugas et al., 2000; Rusak
et al., 2005; Dehghan et al., 2007). Moreover, most of
flavonoids are strong metal chelators because hydroxy and
oxo groups in flavonoid structure have the ability to form
complexes with various metal ions, and their biological
activities are influenced by the presence of metal ions.
People have showed great interest in synthesis, DNAin-
teraction, and biological activity of quercetin metal com-
plexes in recent years (Yamashita et al., 1999; Zhou et al.,
2001; Mira et al., 2002; Kang et al., 2004; Tan et al., 2009;
Dehghan and Khoshkam, 2012; Tay et al., 2013). Ka-
empferol (3,30,5,7-tetrahydroxyflavone), one of the most
abundant natural flavonoids, is found in berries, tea,
Brassica, Allium species, etc.(Scheme 1). It is also an
attractive reagent due to extensive pharmacological activ-
ities (Nguyen et al., 2003; Bestwick et al., 2005; Wang
et al., 2005; Bestwick et al., 2007). However, to the best of
our knowledge, less attention was paid to biological
activity of kaempferol metal complexes (Zhang et al.,
2010). We have previously reported on the DNA interac-
tion and antimicrobial activities of copper (II) complexes
with Schiff base ligands derived from kaempferol and
polyamines. The results indicated that the complexes could
bind to DNA by anintercalative mode and exhibit an
The binding and cleavage abilities of complexes 1–4 with
DNA have been studied by fluorescence spectroscopy,
viscosity measurements, and gel electrophoresis under
physiological conditions. The experimental results indi-
cated that four complexes could bind to CT-DNA via an
intercalative mode, and the complex 1 showed the strongest
activity to cleavage DNA (pUC 19). The in vitro cyto-
toxicities of kaempferol and complexes 1–4 were also
evaluated against human breast carcinoma cells by MTT
assays. The results show that the cytotoxicities of com-
plexes 1–4 are much higher than that of kaempferol alone,
and the complex 1 shows the strongest ability to inhibit the
growth of human breast carcinoma cells. The results sug-
gest that the complex 1 may be a valuable tool in cancer
chemotherapy agents.
Q. Wang ꢀ G.-F. Zhou ꢀ R.-Q. Zeng ꢀ X.-B. Yang (&)
Rongchang Campus, Southwest University, Chongqing 402460,
People’s Republic of China
e-mail: yangxbqq@126.com
M. Huang ꢀ Y. Huang
Pharmacy College, Ningxia Medical University,
Yinchuan 750004, People’s Republic of China
J.-S. Zhang
School of Chemistry and Material Science, Guizhou Normal
University, Guiyang 550001, People’s Republic of China
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Med Chem Res (2014) 23:2659–2666
complex this band appears at about 1,640 cm-1. It can be
suggested that the metal coordination occurs through the
carbonyl oxygen atom and the 3-OH or 5-OH group of the
kaempferol (Zhou et al., 2001). The UV–Vis spectra of the
free kaempferol and the complexes 1–4 in ethanol were
also described in Fig. 1. Kaempferol absorbed with max-
ima at 367 nm (band I) and 267 nm (band II). Band I is
related to ring B (cinnamoyl system), and band II to ring A
(benzoyl system) (Zhang et al., 2010). After formation of
the complexes, differences of the band II were not easily
distinguished due to phen or bpy, while the band I was
shifted to the long-wavelength regions, and new absor-
bance peaks appeared at about 430 nm. Band I batho-
chromic shift could be explained by the interaction of
metal ion with the 3-hydroxyl group of ring B resulting in
electronic redistribution between kaempferol and metal to
become an extended p-bonding system. As the 3-hydroxyl
has a more acidic proton, therefore the 3-OH and 4-oxo
groups are the first sites to be involved in the complexation
process. The 5-OH group is not involved due to lesser
proton acidity and the steric hindrance caused by the first
complexation (Bukhari et al., 2009; Zhang et al., 2010).
Since no single crystals suitable for X-ray determination
could be isolated, the proposed structure of complexes was
given in Fig. 2 according to above spectra data.
Scheme 1 Kaempferol (i), 1,10-phenanthroline (ii) and 2,20-bipyri-
dine (iii)
increased antimicrobial activity in comparison to kaempf-
erol alone (Yang et al., 2012).
Metal complexes containing phen and related ligands
have been intensively investigated because of their
numerous biological activities such as antitumor, antibac-
terial, and antimicrobial since Dwyer and coworkers first
reported that a Cu(II) complex of composition of [Cu(tm-
phen)2]Cl2, where tmphen stands for 3,4,7,8 tetramethyl-
1,10-phenanthroline, inhibits the growth of Landschutz
ascites tumor in 1965 (Dwyer et al., 1965; Zoroddu et al.,
1996; Hindo et al., 2009; Liguori et al., 2010). Their
intercalating properties along with their function as artifi-
cial nucleases have also been reported (Thederahn et al.,
1989; Zelenco et al., 1997; Lu et al., 2003; Li et al., 2008).
Moreover, there are many reports about the mixed-ligand
metal complex containing phen, bpy, or its substituted
derivatives with amino acid, salicylic acid, etc. And such
complexes exhibit remarkable in vitro cytotoxicity toward
the human cancer cell lines (Ranford et al., 1993; Katsarou
et al., 2008; Buchtik et al., 2011; O’Connor et al., 2012).
In this study, we focus our attention on the synthesis and
structural characterization of four novel mixed-ligand metal
complexes of [Cu(Kae)(phen)]Clꢀ2H2O (1), [Cu(Kae)(b-
py)]ClꢀH2O (2), [Zn(Kae)(phen)]ClꢀH2O (3), and [Zn(Kae)(b-
py)]ClꢀH2O (4), where Kae = kaempferol, phen = 1,10-
phenanthroline, bpy = 2,20-bipyridine (Scheme 1). DNA
binding and cleaving abilities of four complexes were studied,
respectively. Furthermore, the antitumor activities were
investigated in vitro.
DNA binding and cleavage studies
In order to understand the binding mode of complexes and
DNA, the fluorometric competitive binding experiment
was carried out using ethidium bromide (EB) as a probe.
Ethidium bromide has weak fluorescence, but its emission
Results and discussion
Characterization
The likely composition of complexes 1–4: [Cu(Kae)
(phen)]Clꢀ2H2O (1), [Cu(Kae)(bpy)]ClꢀH2O (2), [Zn(Kae)
(phen)]ClꢀH2O (3), and [Zn(Kae)(bpy)]ClꢀH2O (4) were
obtained through elemental analysis, IR, 1H NMR, 13C
NMR, HRMS, and molar conductivity. On the compari-
sonof the IR spectra of the kaempferol with that of com-
plexes 1–4, an important information was revealed: the
characteristic stretching (C=O) mode of the kaempferol
occurs at 1,670 cm-1, while due to the formation of
Fig. 1 Absorption spectra of free kaempferol (a), complex 1 (c),
complex 2 (b), complex 3 (e), and complex 4 (d) in ethanol,
respectively
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Med Chem Res (2014) 23:2659–2666
2661
Fig. 2 The proposed structure
of the complexes 1–4. The pink
for copper atom, the dark gray
for Zinc atom, the red for
oxygen atom, the blue balls for
nitrogens, the gray balls for
carbons, and the white balls for
hydrogens (Color figure online)
respectively, which suggested that the interactions of
complexes with DNA were moderate intercalative mode.
Spectroscopic data are necessary, but not enough to
explain binding mode between DNA and the complex. The
viscosity measurement of CT-DNA is an effective tool to
study the binding mode of compound to DNA in the
absence of crystallographic structural data and NMR. A
classical intercalative mode would cause elongation of
DNA polymer as base pairs were separated to accommo-
date the bound compound, resulting in an increase in its
viscosity. In contrast, a partial and/or nonclassical inter-
calation of compound may bend (or kink) the DNA helix
resulting in a decrease in its effective length with a con-
comitant decrease in its viscosity (Lerman, 1961; Satyan-
arayana et al., 1993). The effects of complexes on the
viscosity of CT-DNA were given in Fig. 4. It could be seen
that the relative viscosity of DNA increased with the
addition of increasing amounts of complexes. The results
further revealed that the complexes could bind to DNA by
intercalation mode, which were consistent with the above
fluorescence spectral results.
intensity in the presence of DNA could be greatly enhanced
because of its strong intercalation between the adjacent
DNA base pairs. It was previously reported that this
enhanced fluorescence could be quenched, at least partly by
the addition of a competing agent (Kuwabara et al., 1986;
Thederahn et al., 1990). The quenching curves of EB
bound to CT-DNA in the absence and presence of com-
plexes 1–4 with different concentrations were given in
Fig. 3. The addition of these complexes to DNA pretreated
with EB caused appreciable decrease in the emission
intensity, which indicated that these complexes completed
with EB in binding to DNA through intercalative binging
mode. The above data were analyzed according to the
classical Stern–Volmer Eq. (1) (Eftink and Ghiron, 1981):
F0=F ¼ 1 þ KSV½Qꢁ
ð1Þ
where F0 and F are the fluorescence intensities in the
absence and presence of complexes, KSV is a linear Stern–
Volmer quenching constant, and [Q] is the concentration of
the complexes. The quenching plots (insets of respective
figures) illustrated that the fluorescence quenching of EB
bound to DNA by these complexes were in good agreement
with the linear Stern–Volmer equation. The quenching
constant (KSV) values were 1.32 9 104, 1.12 9 104,
1.49 9 104, 1.10 9 104 M-1 for complexes 1, 2, 3, and 4,
Besides the above methods, interactions between the
complexes 1–4 and DNA were also investigated by the
cleavage assay of plasmid DNA (pUC 19). The DNA
cleavage was analyzed by monitoring the conversion of
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Med Chem Res (2014) 23:2659–2666
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Med Chem Res (2014) 23:2659–2666
2663
b Fig. 3 Fluorescence spectra of EB bound to CT-DNA in the presence
of complex (i) 1, (ii) 2, (iii) 3, and (iv) 4. [EB] = 50 lM,
[DNA] = 200 lM, [complexes] = 0, 16, 32, 48, 64, 80, 96 lM,
respectively. kex = 340 nm. The arrow shows the intensity changes
on increasing the complex concentration. The inset is Stern–Volmer
quenching plots of DNA-EB system by the complex
supercoiled circular DNA (form I) to nicked circular DNA
(Form II) under physiologic conditions. The amounts of
strand scission were assessed by agarose gel electropho-
resis. Firstly, we compared the cleavage abilities of free
kaempferol and the complexes at a concentration of
200 lM and an incubation time of 4 h. Figure 5 showed
the relative cleavage efficiency of kaempferol and com-
plexes 1–4. Kaempferol induced little DNA cleavage (lane
2). It was obvious that complex 1 showed the strongest
DNA cleavage activities among four mixed-ligand metal
complexes (Lanes 3–6). Therefore, our subsequent efforts
were focused on the DNA cleavage abilities of various
concentrations of complex 1.
Then, the concentration effect of complex 1 on DNA
cleavage was studied for incubation time of 4 h under
physiological conditions. As shown in Fig. 6, the results
showed that complex 1 catalyzed the cleavage of plasmid
DNA (pUC 19) in a dose-dependent manner. When the
concentration of complex 1 increased to 200 lM, most of
supercoiled circular DNA (form I) was converted to nicked
circular DNA (Form II).
Cytotoxic activity
Fig. 4 Effects of increasing amount of complexes 1 (filled square), 2
(filled circle), 3 (filled triangle), and 4 (filled down pointing triangle)
on the viscosity of CT-DNA, [DNA] = 100 lM, [complexes] = 0, 4,
8, 12, 16, 20 lM, respectively
The in vitro cytotoxicities of kaempferol and complexes 1–4
were evaluated against human breast carcinoma cells
(MDA-MB-231) by MTT assays. The concentration which
showed 50 % (IC50) inhibition of the cell viability was
Fig. 5 Effect of different complexes (200 lM) on the cleavage
reactions of pUC 19 DNA (12.5 lg/mL) in a Tris–HCl buffer
(100 mM, pH 7.4) at 37 °C for 4 h. a Agarose gel electrophoresis
diagram: lane 1, DNA control; lane 2, kaempferol; lane 3, complex 1;
lane 4, complex 2; lane 5, complex 3; and lane 6, complex 4.
b Quantitation of % plasmid relaxation (Form II %) relative to
plasmid DNA per lane
Fig. 6 Effect of concentration of complex 1 on the cleavage of
pUC19 DNA (12.5 lg/mL) in a Tris–HCl buffer (100 mM, pH 7.4) at
37 °C for 4 h. a Agarose gel electrophoresis diagram: lane 1, DNA
control; lanes 2–5, [complex 1] = 50, 100, 150, and 200 lM.
b Quantitation of % plasmid relaxation (Form II %) relative to
plasmid DNA per lane
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Med Chem Res (2014) 23:2659–2666
analyzed by linear fitting. All tested compounds have the
ability to inhibit the growth of human breast carcinoma cells
in a dose-dependent manner. The IC50 of kaempferol com-
plexes 1–4 were found to be 39.76 1.06, 17.39 1.42,
23.71 1.12, 29.89 1.19, and 27.49 1.42 lM,
respectively. The results indicated that the cytotoxicities of
complexes 1–4 are much higher than that of kaempferol
alone, and the complex 1 shows the strongest ability to
inhibit the growth of human breast carcinoma cells.
650. HRMS-ESI: m/z: calcd. for C25H17N2O6Cu 504.
0383[M]?, found: 504.0381[M]?. Anal. Calcd. For
C25H19N2O7ClCu: C, 53.77; H, 3.43; N, 5.02 %. Found: C,
54.07; H, 3.38; N, 4.83 %. Conductance: 21 S cm2 mol-1
in DMSO solutions.
(j2-O,O-kaempferol)(j2-N,N-1,10-phenanthroline)] zinc
(II) chloride (complex 3) This complex was obtained as
pale yellow solid. Yield: 77 %. Mp: 220 °C (decomposi-
tion). IR (KBr, cm-1): 3420, 1644, 1600, 1424, 1368, 843,
1
725, 644. H NMR (d6-DMSO 400 MHz) d (ppm): 3.34
(H2O); 6.16 (s, 1H, H6-Kae); 6.46 (s, 1H, H8-Kae); 6.90 (d,
Materials and methods
0
J = 8.4 Hz, 2H, H3 ,5 -Kae); 8.04 (m, 2H, H3,8-phen); 8.13
0
0
0
General
(d, 2H, H2 ,6 -Kae); 8.28 (2H, H5,6-phen); 8.44 (2H,
4,7-phen); 8.91 (2H, H2,9-phen). 13C NMR (d6-DMSO
100 MHz) d (ppm): 177.5 (C4-Kae); 164.4 (C7-Kae); 159.0
H
Elemental analyses were performed using a Carlo-
Elba1106 elemental analytic instrument. The IR spectra
were recorded on a Shimadzu FTIR-4200 spectrometer as
KBr pellets in the range of 4,000–400 cm-1 region. NMR
spectral data were measured on a Varian INOVA-400
spectrometer with tetramethylsilane (TMS) as an internal
standard. HRMS spectral data were recorded on Bruker
Daltonics Bio TOF. Fluorescence spectra were recorded on
an F-7000 spectrofluorometer. UV–Vis absorption spectra
were measured on TU-1901 spectrophotometer. Conduc-
tivity measurements were performed at room temperature
using a DDS-11A conductivity meter.
0
(C9-Kae); 157.9(C5-Kae); 155.6 (C4 -Kae) 149.3
(C2,9-phen); 148.9 (C2-Kae); 139.8 (C3-Kae); 128.7 (C4,7
-
-
0
phen); 128.2 (C2 ,6 -Kae); 127.1(C5,6-phen); 125.9 (C3,8
0
0
0
0
phen); 124.5 (C1 -Kae) 115.3 (C3 ,5 -Kae); 102.3 (C10-Kae);
99.8 (C6-Kae); 94.0 (C8-Kae). HRMS-ESI: m/z: calcd. For
C27H17N2O6Zn 529.0378[M]?, found: 529.0388[M]?.
Anal. Calcd. For C27H19N2O7ClZn: C, 55.50; H, 3.28; N,
4.79 %. Found: C, 55.39; H, 3.19; N, 4.86 %. Conduc-
tance: 11 S cm2 mol-1 in DMSO solutions.
(j2-O,O-kaempferol)(j2-N,N-2,20-bipyridine) zinc (II)
chloride (complex 4) This complex was obtained as pale
yellow solid. Yield: 75 %. Mp: 217 °C (decomposition).
IR (KBr, cm-1): 3410, 1645, 1594, 1468, 1372, 841, 735,
644. 1H NMR (d6-DMSO 400 MHz) d (ppm): 3.54 (H2O);
6.16 (s, 1H, H6-Kae); 6.46 (s, 1H, H8-Kae); 6.91 (d, J = 6.
General procedure for preparation of complexes 1–4
To a solution of kaempferol (2 mmol, 0.572 g) in methanol
(10 mL) and sodium hydroxide (2 mmol, 0.08 g) was
added a solution of copper (II) chloride dihydrate or Zinc
chloride (2 mmol) in methanol (10 mL) at room tempera-
ture for 1 h, followed by the dropwise addition of phen or
bpy (2 mmol) in methanol (10 mL). The reaction mixture
was refluxed for 4 h. The obtained solid product was fil-
tered off, washed with ethanol, and dried in vacuo.
0
4 Hz, 2H, H3 ,5 -Kae); 7.67 (m, 2H, H4,4 -bpy); 8.15 (d,
0
0
0
0
0
J = 3.2 Hz, 2H, H2 ,6 -Kae); 8.44 (2H, H5,5 -bpy); 8.65
(2H, H6,6 -bpy); 8.79 (2H,H3,3 -bpy). 13C NMR (d6-DMSO
0
0
100 MHz) d (ppm): 177.6 (C4-Kae); 164.4 (C7-Kae); 159.1
0
(C9-Kae); 157.9(C5-Kae); 155.6 (C4 -Kae) 153.2 (C1,1
0
-
0
bpy); 148.9 (C2-Kae); 144.8(C3,3 -bpy) 140.2 (C3-Kae);
0
131.9 (C5,5 -bpy); 128.2 (C2 ,6 -Kae); 126.1 (C4,4 -bpy);
0
0
0
(j2-O,O-kaempferol) (j2-N,N-1,10-phenanthroline) copper
(II) chloride (complex 1) This compound was obtained as
dark yellow solid. Yield: 71 %. Mp: 253 °C (decomposi-
tion). IR (KBr, cm-1): 3410, 1646, 1590, 1422, 1358, 845,
721, 650. HRMS-ESI: m/z: calcd. for C27H17N2O6Cu 528.
0383[M]?, found: 528.0464[M]?. Anal.Calcd. For C27H21
N2O8ClCu: C, 54.01; H, 3.53; N, 4.67 %. Found: C, 53.26;
H, 3.54; N, 4.98 %. Conductance:27 S cm2 mol-1 in DMSO
solutions.
124.5 (C1 -Kae) 121.7 (C6,6 -bpy); 115.3 (C3 , 5 -Kae); 102.
3 (C10-Kae); 100.0 (C6-Kae); 93.7 (C8-Kae). HRMS-ESI:
m/z: calcd. for C25H16N2O6Zn 504.0300[M-H]?, found:
504.0386[M-H]?. Anal. Calcd. For C25H19N2O7ClZn: C,
53.59; H, 3.42; N, 5.00 %. Found: C, 54.01; H, 3.37; N, 5.
06 %. Conductance: 8 S cm2 mol-1 in DMSO solutions.
0
0
0
0
DNA binding and cleavage experiments
Fluorescence quenching experiments were carried out by
adding the increasing amounts of complex (0, 16, 32, 48,
64, 80, and 96 lM) to EB-DNA system (CEB = 50 lM,
CDNA = 200 lM, 0.1 M Tris–HCl buffer solution, pH
7.4). All measurements were recorded on an F-7000
(j2-O,O-kaempferol)(j2-N,N-2,20-bipyridine)] copper (II)
chloride (complex 2) This complex was obtained as dark
yellow solid. Yield: 68 %. Mp: 256 °C (decomposition).
IR (KBr, cm-1): 3430, 1648, 1608, 1426, 1370, 843, 729,
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Med Chem Res (2014) 23:2659–2666
2665
spectrofluorimeter. Emission spectra were carried out on
3 mL quartz cuvette with 340 nm excitation light, and
emission was measured at 590 nm.
expressed as % inhibition in cell growth of human breast
carcinoma cells compared with the control. The IC50 which
inhibits growth of 50 % of cells relative to nontreated
control cells was calculated as the concentration of tested
compound by linear fitting.
Viscosity experiments were carried on an Ubbelodhe
viscometer, immersed in a thermostated water bath main-
tained at 25 °C. The concentration of DNA was 100 lM in
Acknowledgments This study was financially supported by the
National Science Foundation of China (No. 21172182) and the
Fundamental Research Funds for the Central Universities
(XDJK2010C004).
buffer solution (0.1 M Tris–HCl, pH 7.4). Data were pre-
1/3
sented as (g/go)
versus the concentration of the com-
plexes, where g is the viscosity of DNA in the presence of
complex (4, 8, 12, 16, and 20 lM), and go is the viscosity
of DNA alone.
Plasmid DNA (pUC 19) cleavage activity of the com-
plexes was monitored by using agarose gel electrophoresis.
In a typical experiment, supercoiled DNA (pUC 19)
(50 lg/mL, 5 lL) in Tris–HCl (100 mM, pH 7.4) was
treated with different concentrations of complexes
(0–200 lM), followed by dilution with the Tris–HCl buffer
to a total volume of 20 lL. The samples were then incu-
bated at 37 °C, and loaded on a 0.7 % agarose gel con-
taining 1.0 lg/mL of ethidium bromide. Electrophoresis
was carried out at 80 V for 40 min in TAE buffer and run
in duplicate. Bands were visualized by UV light and pho-
tographed, then the intensity of the DNA bands were
estimated by a Gel Documentation System.
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