Studies on interaction of norbixin with DNA: Multispectroscopic and in silico analysis

Article abstract of DOI:10.1016/j.saa.2015.02.049

The interaction of food colorant norbixin with calf thymus DNA (CTDNA) was investigated through UV-Visible spectroscopy, Fourier Transform Infrared (FTIR), Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), DNA melting studies, electrophoretic analysis, histological staining technique and molecular docking studies. The results indicated that norbixin interacted with CTDNA by partial intercalation mode. The binding constant (K) of norbixin with CTDNA was calculated to be 5.08 × 10⁵ Mol^-1 L. FTIR and CD studies were coupled with ¹H NMR spectra revealed that norbixin intercalates partially and binds to the groove's, phosphate group, deoxyribose sugar of DNA and also induces conformational transition of B-form to A-form DNA. Agarose gel electrophoretic and histological staining technique results further prove that, norbixin specifically binds to the DNA in the cell. Moreover, molecular docking studies on the specific binding of norbixin with CTDNA have exhibited lowest conformation energy score of -3.2. Therefore, this food colorant has the ability to interact with DNA and it could emerge as a promising class of natural DNA targeted therapeutic.

Full text of DOI:10.1016/j.saa.2015.02.049

Accepted Manuscript
Studies on Interaction of Norbixin with Dna: Multispectroscopic and In Silico
Analysis
Amrita Anantharaman, Rajendra Rao Priya, Hridya Hemachandran, Akella
Sivaramakrishna, Subramanian Babu, Ramamoorthy Siva
PII:
S1386-1425(15)00213-9
http://dx.doi.org/10.1016/j.saa.2015.02.049
SAA 13351
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
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Accepted Date:
21 September 2014
11 February 2015
12 February 2015
Please cite this article as: A. Anantharaman, R.R. Priya, H. Hemachandran, A. Sivaramakrishna, S. Babu, R. Siva,
Studies on Interaction of Norbixin with Dna: Multispectroscopic and In Silico Analysis, Spectrochimica Acta Part
A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.02.049
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STUDIES ON INTERACTION OF NORBIXIN WITH DNA: MULTISPECTROSCOPIC
AND in silico ANALYSIS
Amrita Anantharaman1, Rajendra Rao Priya1, Hridya Hemachandran1, Akella
Sivaramakrishna2, Subramanian Babu1 and Ramamoorthy Siva1*
1School of Bio Sciences and Technology, VIT University, Vellore – 632014,
Tamil Nadu, India
2School of Advanced Sciences, VIT University, Vellore – 632014,
Tamil Nadu, India
*Corresponding author :
Tel: (+91)9443448905
Fax: (+91) 416 2243092
Email: rsiva77in@rediffmail.com
1

Abstract:
The interaction of food colorant norbixin with Calf Thymus DNA (CTDNA) was investigated through
UV-Visible spectroscopy, Fourier Transform Infra Red (FTIR), Circular Dichroism (CD), Nuclear
Magnetic Resonance (NMR), DNA melting studies, electrophoretic analysis, histological staining
technique and molecular docking studies. The results indicated that norbixin interacted with CTDNA by
partial intercalation mode. The binding constant (K) of norbixin with CTDNA was calculated to be 5.08 x
105 Mol-1 L. FTIR and CD studies were coupled with H1NMR spectra revealed that norbixin intercalates
partially and binds to the groove’s, phosphate group, deoxyribose sugar of DNA and also induces
conformational transition of B-form to A-form DNA. Agarose gel electrophoretic and histological
staining technique results further prove that, norbixin specifically binds to the DNA in the cell. Moreover,
molecular docking studies on the specific binding of norbixin with CTDNA have exhibited lowest
conformation energy score of -3.2. Therefore, this food colorant has the ability to interact with DNA and
it could emerge as a promising class of natural DNA targeted therapeutic.
Key words: Norbixin, apocarotenoid, DNA, interaction, Spectroscopic, in silico analysis
2

1.Introduction:
In recent years, many studies were carried out on the binding interaction of small molecules or drug with
DNA and has created a great significance in cancer research. DNA (Deoxyribonucleic acid) is a prime
target for many chemotherapeutic agents, which has affected the replication, transcription, topoisomerase
activity and also induced DNA cleavage, noncovalent cross-linking [1,2]. Understanding, the structural
and functional interaction of drugs with DNA provides a further information on mutant genes, origin of
diseases and also structural properties of the DNA [3]. Consequently, the interaction mechanism between
the drugs and DNA occurs mainly through three noncovalent mode (i.e.,) by intercalation, groove binding
and electrostatic interaction [4-7]. The specific binding of drug with DNA could induce a change in
conformation and also thermodynamic stability in DNA [3]. Therefore, it is necessary to investigate the
in-depth interaction mechanism between the drug molecules and DNA, which could lead to the design of
novel DNA targeted drugs [8].
Norbixin is one of the major orange-red food colorant obtained from the seed of Bixa orellana
(commonly known as annatto or lipstick tree) [9]. It is used as a natural coloring agent in dairy products,
smoked fish, sausage, snack food, ice cream, cosmetics and body care product. The Acceptable Daily
Intake (ADI) of norbixin is 0-0.065mg/kg body weight/day [10,11]. Kovary et al., (2001) reported that
pro and antioxidant properties of norbixin prevented the plasmid DNA from oxidative damage induced by
H2O2 [12]. Hagiwara et al., (2003) also reported that thirteen week feeding of annatto extract containing
91.6% norbixin at 0.1% dietary level has not exerted adverse effects in Sprague-Dawley rats [13]. Though
there are various pharmacological reports available on norbixin, but the therapeutic ability of it still
remains an elusive. Furthermore, these food colorants were consumed in our day to day life, without
understanding the insight mechanism occurring in our human system. Hence, for the first time the
interaction of food colorant norbixin with DNA was systematically investigated using various
spectroscopic techniques such as UV-Visible spectrophotometer, FTIR, CD spectrum, NMR and further
evidenced through electrophoretic analysis, histological staining technique, molecular docking studies. As
3

a result, the binding mode of norbixin with DNA implies that this food colorant can be used as a model
for the development of DNA targeted therapeutics.
2.Materials and methods:
2.1.Materials
The sodium-calf thymus DNA was purchased from Sigma Chemical Co. (St. Louis, MO). Ethidium
bromide and Agarose was purchased from Himedia, India. The gel loading dye was purchased from
Merck Bioscience, India. The analytical grade solvent was used.
2.2.Preparation of CTDNA stock solution
The CTDNA stock solution was prepared by dissolving CTDNA in Tris-EDTA buffer at 4°C for 24hour
to obtain a homogeneous solution. The concentration of the CT-DNA solution was determined by UV
absorbance at 260nm using a molar extinction coefficient ε260 =6600 M−1 cm−1 [8]. The absorbance at
260nm and 280nm was recorded in order to determine the purity of CTDNA solution. The A260/A280 ratio
was found to be 1.85 depicting that the DNA was sufficiently free from protein.
2.3.Preparation of norbixin
The bixin was isolated from the seed of Bixa orellana as per Kovery et al., (2001) protocol [12]. The
purity of bixin was checked through UV-Vis spectrophotometer (Supplementary Fig.1) and compared
with the published data [12]. The final yield of bixin is 0.5g, which was obtained from 50g of Bixa
orellana seed. The norbixin was obtained through the saponification of bixin (i.e.,) by agitating with 5%
NaOH solution (bixin: NaOH, 1:3 (mol/mol)) for 24hour at 37˚C. Water was evaporated to dryness at
45˚C and then crystallized norbixin was kept at -20˚C, until further use. The formation of norbixin was
confirmed through UV-Visible spectrophotometer, Fourier Transform Infrared, High performance liquid
chromatography (Supplementary Fig. 2-4).
2.4.UV-Visible Analysis
The various absorbance and wavelength of the different peaks during each experimental setup were
determined through a wave scan range from 200 to 800nm as per the protocol of Rehman et al., 2014;
4

Bhakta and Siva, 2012 with certain modification [14, 8]. The UV–visible spectra were recorded on a
Shimadzu UV-2401 PC UV–Vis spectrophotometer. The absorbance spectrum of norbixin was examined.
The interaction of norbixin with CTDNA was studied at the constant concentration of norbixin
(0.15µg/3ml) to be monitored for the spectral shift with the addition of various concentrations of CTDNA
(10, 20, 30, 40 µg).
2.5.FTIR studies
Infrared spectra were recorded on a Magna 750 FT-IR spectrophotometer (DTGS detector, Ni- Chrome
source and KBr beam splitter) with a total of 100 scans and a resolution of 4 cm-1. Solution spectra were
recorded after 1 h of incubation, using AgBr windows. Obtained spectra were analyzed using OMNIC
software specific to the spectrophotometer. In the present investigation, the binding property of norbixin
(5mg/ml) was examined with CTDNA (3mg/ml) through FTIR spectrum.
2.6.CD studies
Conformational changes were recorded on a Jasco J-715 spectropolarimeter, in a 3-cm rectangular cuvette
associated with an optical path length at 25 0.2 °C. Temperature was kept in check using a Peltier type
temperature control system. The interaction of norbixin with different concentrations of CTDNA (10, 20,
30, 40, 50 µg) were recorded for five times with a bandwidth 1.0n. and resolution of 0.5nm with a scan
speed of 100nm/min.
2.7.NMR spectroscopy
1
The interaction of norbixin with CTDNA was determined through H NMR spectral data obtained at
400MHz using Burker Ascend Model. The 10mg norbixin and 10mg CTDNA mixed thoroughly in D2O
(Deuterium oxide) before adding to the NMR tube. The chemical shift was reported in part per million
1
(ppm) based on the interaction of norbixin with CTDNA and then it was compared with H NMR
spectrum of norbixin and DNA alone.
2.8.DNA melting studies
5

The Thermal denaturation studies were determined using Shimadzu UV-2401 PC UV–Vis
spectrophotometer with a thermoelectric temperature controller. The absorbance at 260nm was observed
for the CTDNA (20µg/ml) alone and CTDNA-norbixin (1:1) ratio at different temperature. The melting
temperature region (Tm) was determined through the almost linear melting region (i.e.,) the start and end
temperature of the melting process.
2.9.Histological Staining Procedures
Histological staining of onion tissue was performed as per Bhakta et al., (2013) method [15]. Fine
sections of Allium cepa inner peel were dipped in isotonic solution (0.9% NaCl) for 10 min. The sections
were stained with norbixin (10 mg/ml) and allowed to stand for 5 min. The excess stain was washed away
with double distilled water and the stained section was observed under microscope adjusted at 10x
magnifications.
2.10.Agarose Gel electrophoresis analysis
The reaction mixture containing a constant concentration of CTDNA (1µg) with the different
concentrations of norbixin (10, 20, 30, 40, 50µg) was incubated at 37˚C for 30min. The 10µl of reaction
mixture was added to 5µl of gel loading dye (bromophenol blue). The samples were subjected to 0.8%
agarose gel prepared in TBE buffer (45mM Tris, 45mM boric acid and 1mM EDTA, pH 7.3) at a constant
voltage of 100V for 1hr (until the bromophenol blue had passed through 50% of the gel) in TBE buffer.
After electrophoresis, the gel was stained for 1hr by soaking it in a 0.5µg/ml ethidium bromide solution at
room temperature. The stained gel was photographed using Image Quant 300 Gel Imaging System.
2.11.Docking Simulation of ligand with DNA
The target 3D structure of octamer (PDBID: 1DSC) was retrieved from the protein data bank
(www.rcsb.org). The receptor DNA (1DSC) and the ligand norbixin was taken for in silico docking
studies using Auto Dock4.0. For docking calculations, the co-crystallized ligands were identified and
removed from the structure of 1DSC and the energy minimization was done for both DNA and ligand by
using UCSF Chimera. The program UCSF (http://www.cgl.ucsf.edu/chimera) was added Gasteiger
6

charges (computed using ANTECHAMBER) and running 10,000 steps for energy minimization. Docking
was performed to obtain a possible conformations and orientations for the ligand at the binding site.
Using the software, polar hydrogen atoms were added to the DNA and its nonpolar hydrogen atoms were
merged. All bonds of ligand were set to be rotatable. The docking program was done using the
Lamarckian Genetic Algorithm (LGA) method [16]. The best conformation was chosen with the lowest
docked energy, after the docking search was completed. The interaction of complex DNA with norbixin
was analyzed using python molecular viewer software.
3.Results and discussion:
3.1.Absorption spectrum
The binding mechanism of the molecules with DNA was studied through the most convenient absorption
spectroscopy technique [17]. Based on the interaction of drugs or molecules with DNA, the absorption
spectra shows hyperchromism and hypochromism, that confirms the change in the duplex structure of the
DNA. The hyperchromism with blue shift arises from the disruption of the secondary structure of DNA
duplex and the hypochromism with red shift indicates the stabilization of DNA duplex through the
intercalation or electrostatic binding of the molecule with the DNA [18, 19]. The absorption spectrum of
norbixin displayed two maximum absorption at 480nm and 450nm wavelength (Supplementary Fig. 1).
With the addition of CTDNA, the absorption peak at 480nm and 450nm decreased suddenly and then
gradually increases at higher concentration of CTDNA without any wavelength shift (Fig 1). This kind of
interaction with DNA indicates, the partial insertion of molecules with the DNA and also it can be a
minor or major groove binding of a molecule with DNA as reported by Darabi et al., (2014) [19]. The
overall affinity of the molecule towards the DNA mainly depends upon the structure of the molecule,
especially on the geometric size, hydrogen bonding and hydrophobicity [20]. The absorption relationship
between the norbixin and CTDNA is expressed by double reciprocal equation [21]
1/(A0-A)=1/A0+1/(K*A0*CDNA
)
Equation (1)
7

where A0 and A is the absorbance of norbixin without and in the presence of CTDNA, respectively. K is
the binding constant between the norbixin and CTDNA. CDNA is the concentration of CTDNA. The
dependence of 1/( A0 - A) on the reciprocal value of the CTDNA concentration
1/CDNA is linear, with a slope equal to the value of 1/K A0. The value of 1/ A0 is fixed on the ordinate. The
constant K is the quotient of an ordinate 1/ A0 and slope 1/K A0.
From Fig. 2, the double reciprocal plots of 1/ (A0 -A) versus 1/CDNA were linear. The binding constant of
norbixin with CTDNA is K=5.08x105 Mol-1 L was calculated from Equation (1). Similarly, the binding
constant value of ethidium bromide-CTDNA (K=4.3x105 Mol-1 L) is equal to the binding constant value
of Norbixin-CTDNA [22]. The result from UV-Visible spectrum suggested that the partial intercalation
and also groove binding was exhibited by norbixin towards the CTDNA.
3.2.FTIR spectroscopy
Infrared band at 1614 cm-1, 1606 cm-1, 1664 cm-1 and 1493 cm-1 shows the spectrum of CTDNA, which
was assigned to the vibration stretches of guanine, adenine, thymine and cytosine nitrogenous base
respectively [23]. The stretching vibration at 1664 cm-1 due to the thymine (C2=O2) band, which was
shifted to 1658 cm-1 in norbixin-CTDNA adduct (Fig. 3). Moreover, the thymine oxygen group present in
the minor groove of the DNA. The change in the band of the thymine oxygen group indicates the minor
groove binding of norbixin to CTDNA [23]. The cytosine band at 1493 cm-1 exhibited a shift to 1479 cm-1
that shows the norbixin does not intercalate with guanine –cytosine base pair and also never binds to the
major groove of CTDNA.
The spectrum observed for the CTDNA phosphate asymmetric and symmetric stretching of bands at 1224
cm-1and 1086 cm-1 [23] was shifted to 1236 cm-1 and 1093 cm-1, after the addition of CTDNA to norbixin
(Fig. 3). The vibrational band of CTDNA backbone and deoxyribose sugar at 969 cm-1 and 896 cm-1
respectively [23] gets shifted to 964 cm-1 and 864 cm-1 respectively in norbixin-CTDNA complex. Ghosh
et al., (2013) stated that the disappearance of certain characteristic peak indicates the probable interaction
of dye-CTDNA adduct formation [24]. The disappearance of a characteristic vibration stretch of norbixin
8

at 3439 cm−1, 3143 cm-1, 2470 cm-1 shows the plausible interaction between dye and CTDNA (Fig. 3). The
result of UV-Visible spectrophotometer is slightly accordance with FTIR spectroscopy, which shows that
norbixin-CTDNA complex established by the minor groove binding and also with the phosphate group,
deoxyribose sugar of the CTDNA.
3.3.CD analysis
The change in the conformation of DNA occurs through various binding modes of a molecule, which was
detected by powerful sensitive CD technique. The CD spectrum of free DNA shows the positive band at
279nm, which was due to the base stacking and it is the main characteristics of right hand B-DNA [14].
Ivanov et al., (1973) reported that the interaction of small molecules with DNA could induce change in
the positive band [25]. Groove binding and electrostatic interaction of small molecules shows less or no
perturbation on the base stacking and helical band [26]. In our present study, the positive band at 279nm
was increased with the addition of increasing concentration of CTDNA to norbixin (Fig. 4). Hegde et al.,
(2012) reported that increased positive band at 279nm occurs, due to the conformational transition of B-
DNA to A-DNA [7]. The increase in CD band was associated with each base transition, which occurs due
to the coulombic interaction and also close contact between hydrophobic base stacking of nucleotides and
polymer [27]. The CD studies further confirm that norbixin induces conformation change upon the
interaction with CTDNA.
3.4.NMR studies
The interaction of norbixin with CTDNA was indicated through the line widths and chemical shifts of the
1H-NMR signal. Three distinct types of interaction mode were determined by 1H-NMR signal. In Type 1
binding, intercalation of the molecules between the base pair of the DNA was shown as total line
broadening in 1H-NMR signal. For Type 2 binding, the line broadening and upfield chemical shift of the
signal originates mainly for two reasons: the weak restrictions of molecular tumbling in the DNA-
complex and also slow rate exchange between the various DNA binding sites and the unbound state. This
type 2 binding mode indicates that the molecule may partially intercalate with DNA. If no line broadening
9

1
and up-field shift was observed in the H-NMR signal that confirms the high binding affinity of the
molecule towards the DNA. The Type 3 binding mode observed mainly due to the binding of the
molecule with the groove’s of DNA [28]. Our result shows that several new signals were formed based
1
on the significant interaction between the CTDNA and norbixin. The comparative H-NMR spectra of
norbixin in the presence and absence of CTDNA were given in Fig. 5 (a-d). After the addition of
1
CTDNA, H-NMR signal of dicarboxylic acid of norbixin C-1 and C-25, C-15 at δ6.5 and δ5.9, gets
shifted to δ6.7 and δ6 respectively, which indicate the line broadening and upfield shift. It confirms that
the norbixin binds with CTDNA through Type 2 binding mode. The norbixin exhibited strong interaction
with the CTDNA, which was indicated by no line broadening and upfield shift at δ3.9-3.4, δ5.7-5.1, δ2.1-
1
1.1. The result from H-NMR accordance with the absorption spectrum (i.e.,) norbixin binds to CTDNA
through partial intercalation.
3.5.Melting curve studies
The melting curve of CTDNA in the presence and absence of norbixin was given in Fig. 6. At increasing
temperature, the absorption spectrum of CTDNA at 260nm was raised due to the dissociation of double
stranded CTDNA to single stranded CTDNA [29]. In DNA melting studies, the intercalation of the
compound with the CTDNA indicated through increase in melting temperature (Tm) of 13-14°C and the
non-intercalative binding mode shows no apparent change in the Tm value [30]. The melting temperature
of the CTDNA alone is 64 0.2°C. After the addition of norbixin to CTDNA (1:1) ratio, the Tm value
increases to 70 0.2°C. The small increase in Tm (6°C) indicates external binding and partial intercalation
of the norbixin with CTDNA [30].
3.6.Electrophoretic studies
The electrophoretic pattern shows the binding of norbixin with CTDNA (Fig. 7). Vujcic et al., (2013)
reported that the interaction of molecules could cause DNA damage, which can be accessed through
agarose gel electrophoresis [18]. Ouyang et al., (2008) also reported that norbixin never induced DNA
damage [31]. The binding of norbixin has quenched the fluorescence of ethidium bromide in the entire
10

sample, when compared to the CTDNA alone (control). The interference of norbixin in the intercalation
mechanism of ethidium bromide with CTDNA was demostrated more in lane 3 and followed by lane 4, 5
as shown in Fig. 6. The result has suggested that norbixin quenched the binding affinity of the ethidium
bromide towards the DNA.
3.7.Histological staining
The binding mode of the dye with bio-macromolecular DNA is studied through simple staining technique,
which also reveals the cell nucleus morphology. The visualization and quantification of the cellular
constituents can be determined only by histological staining of specific organelle in the cell [32].
Eventhough, there are many synthetic dyes were used for the nuclei staining, in which most of them are
considered to be harmful to the environment and also carcinogenic. In order to replace such synthetic
dyes, natural dyes are non toxic and environmental friendly [33]. Fig. 8 indicated the specific binding of
norbixin with the nucleus. The norbixin is a basic dye with pH=12 [34]. Furthermore, Michaelis (1947)
reported that Paul Ehrlics’s research has shown that basic dye binds to the highly acidic nature of the
DNA in the cell [35]. Finally, the histological staining of Allium cepa by basic norbixin dye implies that
the preferential interaction of norbixin dye with the cell nuclei.
3.8.Low conformational energy of norbixin
The binding pattern was analyzed to predict the small molecule how it binds to the receptors of known 3D
structure. The flexible docking was performed for the ligand norbixin with target CTDNA (Fig. 9). The
investigation of CTDNA interaction with norbixin was performed to find the conformational energy of
norbixin through Autodock server. The negative and lowest value of ∆G bind specifies strong favorable
bonds between CTDNA and the novel ligand. Hence, norbixin is subjected for its validation of binding
energy and ligand efficacy. In our study the norbixin shows negative value (-3.2) which represent valid
and reasonable potential binding modes of the inhibitors. The docking results illustrations that Van der
Waals (VDW) force in association with the hydrogen bond and dissolved energies predicted from
norbixin. If the VDW energies are positive, it is a sign that the ligand is not fitting well into the active site
11

[8, 24]. However, the result of the study shows that VDW force energies are considerably negative for
norbixin which fitting well into the DNA molecule. Each Autodock run shows ten conformations which
are present in rank order based on the scores generated by the docking server. Hence, the first
conformation is the best conformation and having lowest binding energy. Therefore norbixin shows
lowest conformation energy -3.2, thus we hope that the norbixin can be used for anti-therapeutic agent
with the desired biological activity.
4.Conclusion:
In our present study, the binding affinity of apocarotenoid norbixin with CTDNA was examined through
various techniques, in which each technique reveals the interaction mechanism of norbixin with CTDNA.
The partial intercalation and groove binding of norbixin with CTDNA was shown through absorption
spectrum, DNA melting and NMR studies. Further, the FTIR result demonstrated the in-depth binding
mechanism of norbixin with CTDNA (i.e.,) it interacts with the minor groove and also with phosphate
group, deoxyribose sugar of CTDNA. The norbixin has induced conformational transition of B- form to
A- form DNA, which was indicated by the increased positive band in the CD spectrum. The simple
visualization techniques such as gel electrophoresis and histological staining have recommended that
norbixin have the ability to interact with the DNA. In addition, the molecular docking studies have proven
that norbixin interacts with CTDNA through lowest conformation energy score of -3.2. Finally, our result
has shown that the norbixin binds with CTDNA through partial intercalation, groove binding, phosphate
group and deoxyribose sugar. So, the utilization of this food colorant significantly leads to the discovery
of novel DNA-targeted therapeutics.
Acknowledgements:
We express our sincere gratitude to Science and Engineering Research Board – Department of
Science and Technology, New Delhi, India for the support extended through the project [SR/FT/LS-
75/2011]. The authors are thankful to the management of VIT University for their constant support. We
12

wish to thank VIT-SIF lab, SAS, chemistry division for FTIR, NMR and GC-MS. We wish to thank
Nithya JN and Dr G. Jayaraman, School of bioscience and Technology, VIT University for CD spectrum.
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Figure legend:
Fig. 1: Interaction of norbixin with CTDNA was investigated using UV-Visible spectrum.
Fig. 2: The double reciprocal plot obtained from the equation 1 giving the binding constant value of
norbixin-CTDNA adduct, K=4.3x105 Mol-1 L.
Fig. 3: FTIR spectrum of (a) CTDNA with norbixin (b) Norbixin
Fig. 4: CD spectrum of CTDNA (10-50µg) in the absence and presence of constant a concentration of
norbixin.
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Fig. 5: H1 NMR studies (a) CTDNA alone (b) Norbixin alone (c) CTDNA-Norbixin adducts (d) chemical
structure of norbixin.
Fig. 6: DNA melting curve of CTDNA (20µg/ml) in the presence and the absence of norbixin (20µg/ml).
Fig. 7: Electrophoretic analysis of the interaction of CTDNA with norbixin (1,2,3,4,5 µg) (lane 2-6)
respectively; lane 1: Control CTDNA (without norbixin).
Fig. 8: Histological staining technique (a) Tissue section of Allium cepa peel without staining of norbixin
(blue arrow denotes nucleus) under 10x magnification; Histological staining of Allium cepa peel with
norbixin (black arrow denotes stained nucleus) under 10x magnification.
Fig. 9: Molecular docking of norbixin with DNA (a) 3D structure of norbixin; (b) 1DSC DNA; (c)The
docked structure of norbixin with 1DSC DNA . The binding energy of norbixin-CTDNA adducts is -3.2.
Supplementary Figure
Supplementary Fig.1: UV-Visible absorption spectra of bixin isolated from Bixa orellana seed with
absorption wavelength of 502nm and 470nm in chloroform solvent.
Supplementary Fig.2: UV-Visible absorption spectra of norbixin obtained through saponification of bixin.
The absorption wavelength of norbixin is 480nm and 450nm in water.
Supplementary Fig.3: FTIR spectra of norbixin.
Supplementary Fig.4: HPLC chromatogram of norbixin was detected at particular wavelength of 450nm;
moblie phase- acetonitrile: water: glacial acetic acid (70:29:1); flow rate: 1ml/min and the retention time
of norbixin is 9.89 min.
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Highlights:
• The interaction between food colorant norbixin with CTDNA
• Partial intercalation and groove binding of norbixin with CTDNA
• Lowest conformation energy of norbixin.
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