Accepted Manuscript

Isolation of quercetin from the methanolic extract of

Lagerstroemia speciosa by HPLC technique, its cytotoxicity
against MCF-7 cells and photocatalytic activity

V. Sai Saraswathi, D. Saravanan, K. Santhakumar

PII: S1011-1344(17)30454-2
DOI: doi: 10.1016/j.jphotobiol.2017.04.031
Reference: JPB 10810
To appear in: Journal of Photochemistry & Photobiology, B: Biology
Received date: 4 April 2017
Accepted date: 25 April 2017

Please cite this article as: V. Sai Saraswathi, D. Saravanan, K. Santhakumar , Isolation
of quercetin from the methanolic extract of Lagerstroemia speciosa by HPLC technique,
its cytotoxicity against MCF-7 cells and photocatalytic activity. The address for the
corresponding author was captured as affiliation for all authors. Please check if
appropriate. Jpb(2017), doi: 10.1016/j.jphotobiol.2017.04.031

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 ACCEPTED MANUSCRIPT

Isolation of Quercetin from the methanolic extract of Lagerstroemia speciosa by HPLC

technique, its cytotoxicity against MCF-7 cells and photocatalytic activity

V. Sai Saraswathia, D. Saravananb and K. Santhakumar*, c,

a
Department of Chemistry, SAS, VIT University, Vellore-632014, India.
b
Department of Pharmaceutical chemistry, Jaya College of Pharmacy, Chennai, India.
c
CO2 Research and Green Technologies Centre, VIT University, Vellore-632014, India.

Abstract

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The flavonoids present in the leaves of Lagerstroemia speciosa were extracted,
characterized by spectral methods and studied for its cytotoxicity activity against MCF- cell

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lines and photocatalytic activity against azo dye. Direct and sequential soxhlet extraction was
performed and its concentrated crude extract was subjected to high performance liquid

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chromatography. The yield obtained by the isolated compound (MEI- quercetin) from leaves
of L. speciosa was found to be 1.8 g from the methanolic extract. The phytochemical analysis
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and the Rf value of the isolated flavonoid was found to be 3.59. The isolated compound was
characterized by Infrared Spectroscopy, NMR and Mass. Based on the characterization, the
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structure was elucidated as Quercetin a flavonoid. The isolated compound showed the
significant in vitro cytotoxicity activity against MCF- 7 cell lines at 500 g/ml when
compared to the crude extract. Among the various concentrations (25, 50, 100, 250, and 500
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g/ml), at higher concentration the cell viability was pronounced and also compared with that
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of the control. It was first time to report that the isolated flavonoid showed photocatalytic
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against azo dye- methyl orange. The dye degradation was monitored by UV- Vis
spectrophotometry. The isolated compound showed dye degradation of 91.66 % with the
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crude extract 82.47 % at 160 min. Hence in the present findings, the photocatalytic
degradation of MO dye under UV irradiation was investigated over isolated compound of L.
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speciosa. Hence we expect that this can be used to treat the waste water in near future based
on the photocatalytic technique.

Key words: Quercetin, L. speciosa, HPLC, MCF- 7 cell lines, photocatalytic activity.
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1. Introduction

In general, plants possess varied secondary metabolites, which have pronounced

pharmacological activity. Several bioactive compounds like morphine, digoxin, digitoxin,
reserpine, taxol, vincristine, vinblastine etc have been successfully isolated from plant source
[1,2]. Hence discovery of lead molecules from plant source, led to the drug development
which have profound therapeutic applications. There are number of bioactive compounds
from plant sources like Alkaloids, tannins, terpenoids, glycosides, phenolic compounds, poly

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phenols, flavonoids, saponins, Steroids, Steroidal saponins, proteins, amino acids etc., which
has broad array of biological applications in combating the disease. [3]. Flavonoids are

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considered to be a poly phenolic compounds which is broadly classified into anthocyandins,
flavanols, flavanones, flavanols, flavones, Isoflavones. To understand flavonoidss functional

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hydroxyl groups are mediating the anti-oxidant activity by the free radical scavenging activity
or by chelating the metal ions [4, 5] anticancer breast, colon cancer [6,7] antibacterial and
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other aging diseases [8], Hepatoprotective activity [9], anti-inflammatory [10].

It is interesting that flavan- 3-ol was more effective than flavones and flavonones in
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selective inhibition of HIV-1, and HIV- 2. [11]. Epigallocatechin -3- gallate inhibits fatty acid
synthase, and lipogenesis inn prostate cancer cells [12]. Licochalcones A and C were isolated
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from roots of Glycyrrhiza inflate was studied by [13] Glabridin was isolated from
Glycyyrhiza glabra, which inhibits the LDL oxidation via free radical mechanism [14].
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Lagerstroemia speciosa, belong to Lythraceae called as pride of India, Banaba. The tea
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decoction of leaves, which contains corosolic acid as major component, was used to in the
treatment of diabetes . Hence an attempt was made to isolate flavonoids, from the leaves of
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L. speciosa, characterized by spectral methods and studied for its cytoxicity- MCF- 7 cell
lines and catalytic activity against azo dye.
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2. Materials & Methods

2.1 Materials, Collection and processing of leaf samples

Methanol were purchased from SRL, India, which were used for the extraction of the
leaves. The leaves of Lagerstroemia speciosa (Fig. 1) were collected from VIT University
garden, during the month of March June .The leaves were authenticated by Dr. P.
Jayaraman, Director, PARC- Plant Anatomy Research Centre, Chennai, Tamil Nadu, India.
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The fresh leaves were washed with double distilled water. It was shade dried and after drying
it was blended into fine powder in a mechanical blender.

2.2 Instruments

For the chromatographic separations, Shimadzu LC- 20 AT Liquid chromatograph

consist of LC- 20 AT VP pumps, Shimadzu SPD 20A UV- Vis detector, and Welchrom
C18 Column, (4.6 mm internal diameter x 250 mm, 5 micron particle size). 20 L of sample
was introduced into HPLC system. The HPLC instrument procures the data using

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Spinchrome software. To record UV- Vis spectra Double beam UV- Vis spectrophotometer
(JASCO- V- 730) was used to perform the photocatalytic activity.

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2.3 Extraction of leaves of L. speciosa

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Approximately 50 gms of shade dried leaves were subjected to soxhlet continuous
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extraction using various solvents like hexane, petroleum ether, chloroform, acetone, ethyl
acetate, methanol and ethanol. The exhaustive extraction was carried out for 48 hours for
each solvents, hence results in complete extraction. Extracts of various organic solvents were
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collected in separate clean dry beakers, and the excessive of solvent was removed by rotary
evaporator, and then dried in desiccators [15]. The crude extract was filtered using Whatman
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s filter paper. Then the filtrate was centrifuged at 1000 rpm for 10 min, the supernant was
carefully separated and extract was concentrated, dried and stored in vacuum desiccator. This
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extract was utilized for further studies. The preliminary qualitative phytochemical test was
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performed as per protocol prescribed [16, 17] to identify the various secondary metabolites
such as flavonoids, Alkaloids, Sterols, Terpenoids, Tannin etc.
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2.4 Isolation of fraction by HPLC method

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The methanolic extract was subjected to HPLC DAD- MS model 2010 A in a

shimadzu system consisting of an auto sampler LC SIL 10 AD with an electrospray
ionization. The interfacing operating in positive mode with an ion spray voltage of 3000 V
and the capillary temperature is maintained upto 350 C, Nitrogen (N2) gas was used as
nebulizing gas of 40 mL/min. The instrument was auto tuned for optimum ionization
procedure (Fabio de S. Dias 2016). Quadrupole mass spectrometer was collected in full scan
mode within a range from 100 to 600 m/z with DAD detector. A column C18, 2.1 x 7.5 mm.
and a guard column (4.6 mm internal diameter) agilent (USA) were performed. The mobile
phase was composed of water acidified with formic acid (pH = 3.0- solvent A); and methanol
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(solvent B) 30 min with a flow rate of 0.5 mL/min. UV absorbance was observed from 200 to
400 nm.

2.5 Characterization of compound- MEI

The isolated compound was characterized by IR, NMR, and Mass spectral studies.
The IR was performed for the isolated compound; about 2 mg of the sample was crushed in
KBr (3-4 mg) pellets using a FTIR (Fourier Transform Infrared spectroscopy). The sample
was recorded within the wavenumber range from 4000- 400 cm-1 in FTIR instrument (model

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varian 3600). Then the samples was subjected to 1H- NMR spectra DRX Bruker 500 MHz
(Mega Hz), Swizterland by employing TMS as an internal standard and DMSO was used as

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solvent. ESI mass spectra were recorded for the isolated compound and characterized [18].

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2.6 Photo degradation activity against dyes by isolated compound and extract
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The phytoremediation studies were carried for methyl orange (C14H14N3NaO3S) using
sunlight as the source during the month of Aug- Sep 2016 between 11 am to 3 pm. The light
flux was measured during the reaction, at the begin of the analysis it was 1250 W/m2 and
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increased to 1300 W/m2 following 160 minutes. [19, 20] The intensity of the light was found
to be same throughout the study. About 0.2 mg of MEI and ME was weighed and mixed to 10
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ml dye solution and the mixture was sonicated for 20 min in dark room. Similarly control was
prepared and maintained in same condition, therefore gives an idea of the colour change of
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the dye solution for comparison with the extract and isolated compound [21]. Dye
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degradation was finalized visually by the colour change. For methyl orange (MO) the
solution changes from deep orange to yellow colour. The UV absorption peak for MO was
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noticed at 460 nm. The phytoremediation process was monitored by decrease of the peak
intensity during 1.5 hrs of exposure to solar light. Optical absorption spectra were resolved
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after varying light exposure durations utilizing an UV/Vis spectrophotometer (JASCO- V-

730) to screen the rate of degradation methyl orange at the maximum wavelength (max = 460
MO). The degradation efficiency (PE) was ascertained as in condition1:

where, I0 is the initial absorption intensity of methyl orange solution at max= 460 nm (MO),
and I is the absorption intensity after photo-degradation. C0 is the first concentration of
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methyl orange solution and methylene blue respectively and C is the concentration after
photo-degradation [22-24]

2.7 In vitro cytotoxicity activity of MEI and methanolic extract against MCF-7 cell lines

2.7.1 Human cell lines

MCF- 7 cancer cell lines, was obtained from National Centre for Cell Science, Pune,
India. The cell lines were cultured in Dulbeccos phosphate buffer saline (DPBS) medium

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supplemented with 10% FBS, glutamine (2raM), and the pH was adjusted to 4.7. The cells
were cultured to 37C in a humidified 5% CO2 incubator.

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2.7.2 In vitro cytotoxicity activity

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5 mg/ml of Stock solution was prepared by dissolving the MEI and ME in dimethyl
sulfoxide (DMSO, Hi-media). The cytotoxicity activity of the MEI and ME extract and
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control was determined by MTT assay. [25] The MCF-7 cells were seeded in to 96 wells at a
density of 5 x 103 cells/well in 200 l culture medium. After the 24 h of incubation, the cells
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were treated with both MEI and ME of various concentrations (25, 50, 100 250 and 500
g/ml). Similarly the control was maintained in same condition. After treating, the media was
treated with MTT solution (10l of 5 mg/ml per well) prepared in PBS and incubated for 4
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hours at 37C in a humidified incubator with 5% CO2. The yellow MTT dye was reduced to
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purple formazan crystals by succinate dehydrogenase in the mitochondria [26]. Various

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solubilising agent like acidified isopropanol, DMSO, DMF, SDS etc is being used to
solubilize the dye. After adding the solubilizing agent the plates were gently incubated for
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few minutes and absorbance was recorded at 570 nm by microtiter plate reader (MIOS
Junior, Merck) and the percentage cell viability (CV) was calculated using the following
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formula:

= 100

2.7.3 Morphological assessment of cancerous cells

After the treatment of the cancerous cells with MEI and ME extract of various
concentrations, in 35 mm polyvinyl coated cell culture plates, the cells were seeded and kept
for incubation at 37C for 24 h in CO2 incubator. DMF was serving as control and similar
procedure was maintained. The morphological changes of cancerous cell MCF-7 were
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compared to the control and with treated samples by viewing the cells with phase contrast
inverted microscope [27]. The images were photographed.

2.7. 4 Statistical Analysis

The results were analysed by MeanSD for triplicates from the same samples. Data were
analysed and compared by one way ANOVA by using the software SPSS 15.0 for windows.

3. Results and Discussion

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3.1 Continuous extraction and phytochemical screening

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Continuous extraction was carried out sequentially from non-polar to polar solvents, hence
the methanol was finally extracted and subjected to qualitative analysis showed the presence

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of major phytoconstituents except saponins, gums and mucilage as reported in Santhakumar
et al., 2017 [28].
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3.2 HPLC isolation of the methanolic extract
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The compound was identified based on the retention times, UV- Vis spectra and mass
spectra. The absorption spectrum was obtained at 254 nm wavelength (Fig. 2), showing the
Rf value of 3.59 with 91.39% purity. Also no maximum peaks were noticed during the
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separation of fractions by HPLC technique.

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3.3 IR Characterization
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The isolated sample from methanolic extract was given as MEI. The IR absorption
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spectrum of MEI showed absorption peaks (Fig. 3) at 3334 cm-1 (OH stretching), 2943 cm-1
(CH2 stretching), 2831 cm-1 (CH stretching), 1724 cm-1 (C=O), 1375 cm-1 (C-OH vibration),
1022 cm-1 (CH deformation). The FTIR spectroscopic analysis of MEI compound exhibited
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characteristic broad peak centered at 3334 cm-1 which are characteristic of the OH stretching
in the ring that suggesting the presence of hydroxyl group. The absorption bands at 2943 cm -1
and 2831 cm-1 correspond to CH2 and CH stretching. A carbonyl group stretching was
observed at 1724 cm-1. The absorption bands at 1375 cm-1 which is characteristic of C-OH
vibration) and the absorption bands at 1022 cm-1 correspond to CH deformation.
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3.4 NMR spectral analysis

3.4. 1 1H-NMR spectra

The MEI -Compound showed the signals of 1H-NMR (DMSO D6, 500MHz- Fig. 4a)
spectrum, at 6.19 (1H, d, H-6), 6.40 (1H, d, H-8), 6.88-6.90 (1H, d, H-5), 7.53-7.55 (1H,
dd, H-6), 7.68 (1H, d, H-2), 9.35 (1H, broad, C14 and C15-OH), 9.59 (1H, s, C11-OH), 10.79
(1H, s, C12-OH) and 12.49 (1H, s, C13-OH).
Two characteristic peaks was observed by 1H-NMR spectrum at 6.18-6.19 and 6.40-

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6.41 which are corresponds to meta protons H-6 and H-8 on A ring and an ABX system at
6.88-6.90, 7.53-7.55 and 7.68 corresponding to the catechol protons on B ring. A hydroxyl

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group at 5th and 7th position of the A ring showed a broad singlet at 10.79 and 12.49

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respectively. A hydroxyl group at 3rd and 4th position of the B ring showed a broad merged
singlet peak at 9.35. A hydroxyl group at 3rd position of the X ring showed a singlet peak at
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12.49.

3.4.2 13C-NMR Spectra

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The MEI- compound showed the signal of 13C-NMR (DMSO D6, 500MHz- Fig. 4b)
spectrum, at ppm 147.264 (C-2), 136.179 (C-3), 176.294 (C-4), 161.175 (C-5), 98.635 (C-6),
164.343 (C-7), 93.804 (C-8), 156.594 (C-9), 103.465 (C-10), 122.409 (C-1), 115.520 (C-2),
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145.513 (C-3), 148.157 (C-4), 116.055 (C-5) and 120.430 (C-6).

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About 15 signals including carbonyl carbon signals at 176.294, were recorded in

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compound 1 in13C-NMR spectrum of compound 1. The chemical shifts was observed for the
carbon nucleus at 136.179 (C-3), 161.175 (C-5), 164.343 (C-7), 145.513 (C-3) and 148.157
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(C-4) which suggested the 3, 5, 7, 3 and 4 oxygenated flavones nucleus. The peaks at
147.264 (C-2), 176.294 (C-4), 98.635 (C-6), 93.804 (C-8), 156.594 (C-9), 103.465 (C-10),
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122.409 (C-1), 115.520 (C-2), 116.055 (C-5) and 120.430 (C-6) were observed for other
carbons.
3.4.3 Electron impact mass spectra
The EIMS spectrum (positive in mode- Fig. 5) the molecular formula of MEI was
encountered as C15H10O7 and which is showing the molecular ion at m/z 302 [M+H]+. Based
on the spectral analysis and the consistent of the data given in the literature, the structure was
elucidated and identified as quercetin (Fig. 6).
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We are the first to develop the HPLC method for the isolation of MEI- quercetin from
the leaves of L. speciosa. Quercetin and isoquercitrin was isolated by the conventional
method from the leaves of L. speciosa and studied for its Insulin like glucose uptake and
adipocyte differentiation inhibition in 3T3 L1 cells at Jersey [29]. Aqueous acetone leaf
extract of L. speciosa, kaempferol, quercetin and isoquercitrin was isolated successfully [30].
hence based on the geographical location, the active compounds were found to be
predominant and the concentration might be varying upon the climate change. Narendran et

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al., isolated and characterized the flavonoids and flavone glycoside from methanolic extract
of Cotoneaster bacillaris Wall. Ex. Lindl [31]. Maria Paula Junqueira Goncalves et al.,
isolated the phenolic and anthocyanin profile of murta (Ugni molinae) fruit, and evaluated for

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its antioxidant and antimicrobial activity of ethanolic and methanolic extracts. LC/MS of

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ethanolic extract showed the presence of caffeic acid 3-glu, quercetin-3- glu and quercetin
and in methanolic extract cyaniding -3-glcoside, pelargonidin -3-arabinose and delphinidin 3-
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glucoside. [32]

3.5 Photocatalytic activity of MEI and Methanolic extract

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The photocatalytic activity was performed for azo dye- methyl orange, using the
methanolic crude extract from the leaves of L. speciosa and isolated compound- MEI. Sun
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was taken as the source of light. The absorption spectra of the methyl orange were found at
270 nm and 460 nm. The UV- Vis spectrum of the MO solution was well explained for
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Methanolic extract and MEI in the Fig. 7a & b respectively. The azo dye was degraded
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82.47% for methanolic extract and 91.66 % MEI (Isolated compound) after irradiating with
sunlight for 160 min. It was observed that there was no shift of peak of MO when treated
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with crude extract and with the isolated compound [33-35]. The photolysis progress was
observed 3% and 5% for blank and in dark respectively as tabulated in table- 1. The complete
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protocol was carried similar to the blank test and showed no degradation. Hence the hot
continuous method of extraction was adopted to isolate the active compound from the leaves
of L. speciosa was well proved for the photocatalytic activity at 160 min compared to the
methanolic extract.

3.6 Cytotoxicity activity of MEI and ME extract

The In vitro cytotoxicity against breast cancer cell lines (MCF-7) was studied for
various concentrations (25, 50, 100, 250 and 500g/ml) and compared with control. The
morphological alteration of MCF-7 cells and the control was observed under phase contrast
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light microscope and it was shown in photograph (Fig. 8 (i) a- control, 8b- ME and 8c- MEI).
The number of dead cells increased with the increase in the concentration of the isolated
compound when compared to the crude extract. At 500g/ml the enlargement of cells were
noticed, nearly 30-5 % of the cells showed small protrusions of the membrane. The apoptotic
bodies were observed in the MEI was good compared to the crude extract of the treated cells.
The cells showed big vacuoles in the cell cytoplasm indicating the autophagy (similar to cell
death mechanism). The percentage of cell viability of ME extract and MEI was explained in

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Fig. 8 (ii) (a) and (b) respectively. Our results possess the IC50 of 24 % when compared to the
control [34]. It was observed that the pronounced activity might be seen in the higher
concentration stating the phytoconstituents concentration is less, which had capped the active

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compound - quercetin. Also among extracts, methanolic showed the presence of Gallic acid

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which was determined quantitatively by HPTLC [35]. Hence the photocatalytic activity might
be the synergistic effect of phytoconstituents present in the extracts.
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4. Conclusion

To conclude this research finding, HPLC- High Performance Liquid Chromatography

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was the convenient and easiest method of isolation of flavonoids, which was adopted in the
isolation of quercetin. The spectral studies further confirmed the isolation of active flavonoid
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from the leaves of L. speciosa, which was collected from the Eastern Ghats. Also the sample
was subjected to cytotoxicity against MCF-7 cell line and compared with that of the crude
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extract. It was interesting that the flavonoids exhibited the photocatalytic activity against azo
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dye- methyl orange. The finding exhibited that the isolated compound has 91. 66 % dye
degradation, which is eco-friendly and helping for the environmental remediation, when
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compared to the crude extract 82.47 %. Hence more active compounds will be targeted in
near future to explore its biological applications.
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Conflict of interest

The authors at this juncture report no conflicts of interest in this work.

Acknowledgement
This study was supported by DST, New Delhi under Young Scientist Scheme (Grant No.
YSS/2015/001104), CSIR New Delhi under Extramural Research (Grant No.
01(2865)/16/EMR-II), VIT University under RGEMS Fund (2015-2016).
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List of Table

Table 1. Photocatalytic degradation efficiency of MEI and ME extract

List of Figures

Fig. 1 Lagerstroemia speciosa

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Fig. 2 HPLC chromatogram of isolation of compound- MEI from methanolic extract

Fig.3 IR spectra of MEI compound

Fig. 4a 1H NMR spectra of MEI compound

Fig. 4b C13 NMR spectra of MEI compound

Fig. 5 ESI mass of MEI compound

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Fig. 6 Isolated compound from leaves of L. speciosa Quercetin

Fig. 7a Photocatalytic activity against Methyl orange dye Methanolic extract

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Fig. 7b Photocatalytic activity against Methyl orange dye- MEI

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Fig. 8i (a, b and c) Cytotoxicity activity against MCF- 7 cell lines
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Fig. 8ii (a and b) Percentage inhibition against MCF-7 Cell lines
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Table 1: Photocatalytic degradation efficiency of MEI and ME extract

Sample Light Dye Time Degradation efficiency (%)

Blank Dark Photocatalysis
MEI Solar Methyl 160 Min 3% 5% 91.66%
Radiation Orange
ME- Solar Methyl 160 Min 3% 5% 82.47 %

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crude radiation orange
extract

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Graphical abstract

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Highlights

Flavonoids were isolated from the leaves of Lagerstroemia speciosa

Cell viability was studied against MCF- 7 breast cancer cell lines.
Flavonoids exhibits photocatalytic property
Isolated compound exhibits 92 % Methyl Orange degradation

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