Arabian Journal of Chemistry (2017) 10, 509–514

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Extraction, isolation and identification of flavonoid

from Euphorbia neriifolia leaves
Veena Sharma *, Pracheta Janmeda

Department of Bioscience and Biotechnology, Banasthali University, 304022 Banasthali, Rajasthan, India

Received 13 December 2012; accepted 20 August 2014

Available online 21 September 2014

KEYWORDS Abstract The flavonoids contained in Euphorbia neriifolia leaves were extracted, identified and
Euphorbia neriifolia; characterized. Direct and sequential soxhlet extraction and its concentrated fractions were subjected
Flavonoids; to thin layer chromatography and high performance thin layer chromatography. The results
Chromatography showed that maximum yield of the flavonoid (6.53 g) was obtained from ethanolic extract. The
Rf value of isolated flavonoid and phytochemical screening has been compared with standard
Quercetin. Characterization of isolated flavonoid was done by IR, 1H NMR, and MS. On the basis
of chemical and spectral analysis structure was elucidated as 2-(3,4-dihydroxy-5-methoxy-phenyl)-
3,5-dihydroxy-6,7-dimethoxychromen-4-one, a flavonoid. This compound was isolated for the first
time from this plant.
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1. Introduction
Phytochemistry reports thousands of newer organic molecules
or compounds every year. Pharmacological testing, modifying,
derivatizing and research on these natural products represent a
major strategy for discovering and developing new drugs
(Kayser et al., 2000). Moreover, plant secondary metabolites
present chemical and pharmaceutical properties interesting
* Corresponding author at: Lab-209, Department of Bioscience and
Biotechnology, Banasthali University, P.O. Banasthali, Distt.: Tonk
304022, India. Tel.: +91 1438228386, +91 9352381260.
E-mail addresses: drvshs@gmail.com (V. Sharma), pracheta25@
gmail.com (P. Janmeda).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
for human health (Raskin et al., 2002; Reddy et al., 2003).
Compounds belonging to the terpenoids, alkaloids and flavo-
noids are currently used as drugs or to prevent various diseases
(Raskin et al., 2002) and in particular some of these com-
pounds seem to be efficient in preventing and inhibiting vari-
ous types of cancer (Reddy et al., 2003).
Flavonoids are a group of about 4000 naturally polypheno-
lic compounds, found universally in plant origin. According to
the differences in functional groups and their relative positions
of the 15-carbon skeleton (aglycons), flavonoids are classified
into several subgroups including the following: flavone, flava-
none, flavonol, isoflavonoid, anthocyanidin, and chalcones
(Harborne, 1998).
The plant Euphorbia neriifolia, especially the leaves, has
been reported to possess a wide range of medicinal properties
(Ilyas et al., 1998; Janmeda et al., 2011; Pracheta et al., 2011;
Sharma et al., 2011a,b, 2012a,b), which has prompted a com-
prehensive investigation of leaves of E. neriifolia. A number of
compounds have already been isolated (Chatterjee et al., 1978;

http://dx.doi.org/10.1016/j.arabjc.2014.08.019
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510
Ng, 1990; Ilyas et al., 1998; Yadav et al., 2011) but this is the
first report to isolate flavonoid from E. neriifolia leaves. The
purpose of this study was to isolate the most important group
of phytochemical class i.e. flavonoids using TLC and HPTLC
as technique and was characterized through their spectral (IR,
1
H NMR and MS), as well as comparison with a reference
materials.
2. Experimental
2.1. Chemicals and reagents
All chemicals and reagents used in the study were of analytical
grade and were purchased from reliable firms and institutes
[Sd-fine chemicals, SIGMA, SRL (India), MERCK, RANB-
AXY, and SUYOG]. Silica gel (G) 60F and 0.25 readymade
aluminum sheets (Merck KGaA, Germany). Silica gel 60
F254, HPTLC aluminium sheets 20 · 20 cm, Merck KGaA,
Germany Ord. No. 1.05554.
2.2. Procurement, authentication of plant material
E. neriifolia (EN) leaves were collected from medicinal garden
of the Banasthali University, Banasthali and nearby areas of
Banasthali, Rajasthan, India (Latitude N–26240 14.841400 ;
Longitude E–73520 9.719400 ), in the month of October, 2009
and were taxonomically identified by Botanist of Krishi Vig-
yan Kendra, Banasthali University, Banasthali, Rajasthan, In-
dia. A voucher specimen was deposited in the herbarium of
Department of Bioscience and Biotechnology, Banasthali Uni-
versity (No. BVH–780141A).
2.3. Extraction
Shade dried leaves were powdered (250 g), soxhlet extracted
with 70% (v/v) ethanol and vacuum concentrated to dryness
under reduced pressure at 60 ± 1 C. After drying in hot air
oven (40–45 C), it was stored in an air tight container in
refrigerator at 5 C. The residue was designated as hydro-
ethanolic extract of E. neriifolia (HEEN).
Dried leaves of E. neriifolia (250 g) were extracted succes-
sively with pet-ether, benzene, chloroform, ethyl acetate, and
ethanol and finally macerated with distilled water (non-polar
to polar) to get respective extracts. Flavonoid contents and
their presence were determined by the method of Harborne
(1998), using quercetin as a standard. The extracts were ana-
lysed by means of TLC.
2.4. Thin layer chromatography (TLC)
Thin layer chromatography was performed using standard
methods (Harborne, 1998). Small quantities of samples
(2 mg/ml) were dissolved in their respective solvents. Quercetin
(1 mg) standard was dissolved in methanol. Different mobile
phases with varying concentrations were employed in the
screening programme and selected the one in which separation
of flavonoid was clear: n-butanol: acetic acid: water (2:2:6). All
plates were visualized directly after drying and with the help of
UV at 254 nm and 366 nm in UV TLC viewer. The Rf value of
the different spots that were observed was calculated. The dark
V. Sharma, P. Janmeda
brown bands that were produced after keeping the plates in
iodine chamber were scratched and suspended in the mobile
phase separately for 3–4 days. After that mobile phase was
sucked out, vacuum evaporated and the residue left was col-
lected till sufficient amount for dosing and spectrophotometric
analysis was obtained. The selected purified material was sub-
jected to its HPTLC and IR spectral.
2.5. Phytochemical screening of the isolated compound
Chemical tests as described by Harborne (1998) were carried
out on the isolated compound using standard procedures.
2.6. HPTLC method for the estimation of flavonoids
The complete CAMAG TLC equipment consists of a fully
automatic sample Linomat V sample applicator, a developing
chamber. Finally, a Camag TLC scanner is available allowing
densitometric evaluation of chromatograms and CATS 4 soft-
ware for interpretation of data. About 10 mg of the extracts of
the EN was taken and dissolved in respective solvents and the
volume was made up to 10 ml in a standard flask (1000 lg/ml).
Standard (10 mg) was taken and dissolved in methanol. This
was transferred into a standard flask and the volume was made
up to 100 ml to prepare 100 lg/ml solution. Silica gel 60 F254
and HPTLC aluminium sheets were used as adsorbent (sta-
tionary phase). The extracts were applied point-wise from
1000 lg/ml sample solution, 10 ll of the sample was applied
on HPTLC aluminium sheets as different tracks in the form
of 6 mm wide bands by using a Camag semi-automatic Linom-
at 5 spotter at a distance of 12 mm. Nitrogen gas was also sup-
plied for simultaneous drying of bands and then using drier for
completely drying of bands. HPTLC of different extracts was
performed by using the mobile phase n-butanol: acetic acid:
water (2:2:6). To saturate the chamber, 10 ml mobile phase
was placed in each flat-bottomed Camag twin trough TLC
chamber, 30 min before the development of the PTLC plate.
The chamber was sealed with parafilm and covered with a steel
lid. The developed plates were then dried and scanned using a
TLC scanner 3 with Wincats software under 364 nm (Wagner
and Bladt, 1996). All plates were visualized directly after dry-
ing and a fingerprint profile was photo documented using a
Camag Reproster – 3 under 254 nm and 366 nm in UV and
visible light. The digital images of the chromatograms were
evaluated with the programme CAMAG Video Scan. The cap-
tured image was subjected to a visual inspection on the com-
puter screen. The differences found, are specified by the
HPTLC system in which the difference is detected and by
the Rf value (and colour) of a compound in the system.
2.7. Compound characterization
For characterization of compound IR, NMR and Mass
spectra were performed. IR was obtained for the isolated
flavonoid. The sample (1–2 mg) was crushed in KBr
(3–4 mg) pellets using a FTIR (Fourier transform Infrared
spectroscopy; Model - Varian 3600) with the help of mechan-
ical pressure. They were recorded within the wavenumber
range 4000–400 cm1 in an FTIR instrument. 1H NMR
spectra was recorded on DRX-Bruker 300 MHz (Mega Hz),
Switzerland. Nuclear magnetic field was obtained for the
Extraction, isolation and identification of flavonoid from Euphorbia neriifolia leaves 511

isolated flavonoid. Isolated compound was dissolved in respec-

tive solvent CDCl3 and about 600 ll was poured in NMR tube
and observed on the applied magnetic field. Mass was obtained
by TOF MS ES + 5.18e4, TOF MS ES + 477 and TOF MS
ES + 174.80ES + 3 (ABHAY_BIOTECH 28).
3. Results and discussion
Chromatographic methods (TLC & HPTLC) for detection of
flavonoids from E. neriifolia, have not been reported in the
literature.
3.1. Chromatographic separation
Among all, the yield of ethanol (48.9 g) and aqueous (89.5 g)
extract was found in excess compared to all (Fig. 1). Available
all seven extracts, ethanol extract contained bulk of flavonoids
and dark brown sticky semi solid ethanol extract (48.9 g) was
used for chromatographic separation using quercetin as stan-
dard. Initially, various solvent phases with varying ratios were
tried (Sharma and Janmeda, 2013a), for all fractions of E.
neriifolia. Out of all the n-butanol: acetic acid: water (BAW,
2: 2: 6) was found to be the most appropriate solvent system
for separation of flavonoids from ethanol extract of EN. After
TLC three spots resolved that were nomenclatured as F1, F2
and F3 have Rf values of 0.60, 0.79 and 0.90 respectively
(Fig 1). Percentage yield of compounds isolated from ethanolic
extract of EN is depicted in Table 1. Maximum yield was ob-
tained in F2 fraction (0.65 g). The standard Rf value of
kaempferol was greater than quercetin and rutin, thus sus-
pected that the F3 (0.90) compound was related to kaempferol
and F1 coincides with rutin. When the developed plates were
sprayed with ammonia and iodine vapours, it showed deep yel-
low colour of quercetin. Rf value (0.79) of F2 isolated from the
Table 1 Percentage yield of compounds isolated from 500 g
ethanolic extract of E. neriifolia.
IC Rf Value Yield of IC (g) % yield of IC
IF1 0.60 2.121 0.424
IF2 0.79 6.532 1.306
IF3 0.90 5.263 1.052
IC: Isolated compounds.
ethanol extract which coincides with the Rf value of standard
quercetin. F2 carefully crystallized with ethanol, gives solid
pale yellow crystal, soluble in water and in organic solvents
and was designed as E. neriifolia isolated flavonoid (ENF).
ENF compound was selected for further study which includes
repeated TLC, identifying the nature of compound by per-
forming several qualitative tests, confirmation by HPTLC
and IR of this compound. Meena and Patni (2008) adopted
same procedure for the isolation & identification of flavonoid
‘‘quercetin’’ from Citrullus colocynthis. (See Fig. 2)
3.2. Phytochemical screening of the isolated compound (ENF)
The results of various qualitative tests performed in the labo-
ratory are outlined in Table 2. The presence of flavonoids
(ENF) was confirmed by screening tests, which illustrated a
positive test for flavonoids and negative for steroids, terpe-
noids and alkaloids.
3.3. HPTLC fingerprinting profile
Authentic marker of flavonol (quercetin) obtained commer-
cially was co-chromatographed. HPTLC chromatogram
showed that a maximum number of components were

Figure 1 Schematic diagram for the isolation of flavonoids from E. neriifolia.

512 V. Sharma, P. Janmeda

Figure 2 HPTLC baseline display of ethyl acetate, ethanol, aqueous extract and isolated flavonoid (ENF) compared with quercetin.

observed under UV and florescence absorbance mode. Fig. 2

Table 2 Analysis of phytochemicals in compound isolated confirmed the results obtained by TLC, in which the chro-
from ethanolic extract of E. neriifolia leaves and standard. matogram spectral clearly showed that Rf values of all men-
S. no. Phytochemicals ENF Standard (quercetin) tioned extracts and isolated compound (ENF) coincided with
that of standard quercetin. The results are supported by previ-
1 Phenols ++ ++
ous reports (Lakshmi et al., 2012; Sharma and Janmeda,
2 Steroids – –
2013a; Sharma and Pracheta, 2013a). Putative therapeutic ef-
3 Alkaloids – –
4 Flavonoids +++ +++ fects of many traditional medicines might be ascribed to the
5 Terpenoids – – presence of flavonoids (Schultz et al., 2008). The presence of
anti-carcinogenic (Janmeda et al., 2011; Sharma et al.,
2011b; Sharma et al., 2012b) and antioxidative (Pracheta

Figure 3 Mass spectrum of isolated compound.

Extraction, isolation and identification of flavonoid from Euphorbia neriifolia leaves
et al., 2011) properties of extract of Euphorbia neriifolia leaves
can be attributed due to the presence of flavonoids.
3.4. Identification and Spectral analysis of isolated flavonoid
Pale yellow needles were obtained after purification: m.p.
191–193 C, Rf: 0.79 (BAW). IR spectroscopy provides a
fingerprint of the drug through which we can identify the nat-
ure of bonding and types of functional groups in the samples.
All values of IR and NMR were in the decreasing order. IR
spectrum of ENF in KBr pellet exhibits peaks at 3624, 3473,
3286 cm1 (OH), 1707 cm1 (>C‚O), 1649 cm1,
1 1
1408 cm (CAC‚C), 1275, 1245, 1045 cm (OCH3), and
769.8 cm1 (CAH). The peaks revealed the number of func-
tional groups that have a great significant towards medicinal
prospects of E. neriifolia.
The 1H NMR spectrum of ENF was taken as 300 MHz
operating frequency in CDCl3 and spectrum of ENF displayed
signals corresponding to a tri-substituted b-ring of the
flavonoid nucleus at d ppm: 8.04 (9H, s, –OCH3), 7.27 (3H, s,
Ar–H), 6.73 (4H, s, –OH), and 2.89–2.97 (2H, d, Non-Ar–H).
The 1H NMR spectrum of the extracted compound revealed
the signals of aromatic protons, which are close to those
reported for Quercetin moiety (Harborne and Mabry, 1982;
Agrawal and Bansal, 1989). Therefore, isolated compound
ENF could be quercetin related flavonoid which is not previ-
ously reported in the titled plant.
The molecular weight of the extracted compound was ob-
served by TOFMS ES+ m/z 378.33 calculated for C18H18O9
(m/z 378) as depicted in Fig. 3. The molecular ion peak with
base peak due to the most stable molecular ion was observed
at 240.25 calculated for C15H12O3 (m/z 240).The base ion
peaks correspond to basic moiety of flavonol. The main com-
ponent of the extract was characterized as 2-(3,4-dihydroxy-5-
methoxy-phenyl)-3,5-dihydroxy-6,7-dimethoxychromen-4-one
and designed as ENF (E. neriifolia flavonoid) a flavonoid by
employing mass spectrometry as depicted in Fig. 4.
We have already seen the anticarcinogenic activity of this
novel flavonoid in mice and it was noticed that the isolated fla-
vonoid was able to protect the tissue architecture and was
capable of minimizing the abnormalities and carcinogenicity
caused by N-nitrosodiethylamine (Sharma and Janmeda,
2013b; 2014). This might be due to ROS radical inhibition &
neutralizing the free radicals which provides scientific evidence
of ethno-medicinal therapeutic use of ENF for treating cancer.
IR spectra of isolated flavonoid confirmed the presence of
the OH group in free and binding states which acts as binding
site of enzymes due to which isolated flavonoid exhibited
anti-carcinogenic activity to the best extent by retarding and
modulating the phase I enzymes (cytochrome P450 and b5)
peroxidation level, biochemical enzymes (catalase, superoxide
dismutase, aspartate transaminase, alanine transaminase, alka-
line phosphates, total protein and total cholesterol) altered by
N-nitrosodiethylamine (Sharma and Janmeda 2013b; 2014;
Sharma and Pracheta, 2013b).
Quantitative structure–activity relationship (QSAR) is an
accepted means for establishing quantitative relationship be-
tween biological activity and descriptors representing
physicochemical properties of the compounds using statistical
methods (Kiralj and Ferreira, 2009) and it helps to precisely
513
Figure 4 Structure of flavonoid 2-(3,4-dihydroxy-5-methoxy-
phenyl)-3,5-dihydroxy-6,7-dimethoxychromen-4-one (C18H18O9).
predict the biological activities of newly designed analogues
(de Melo et al., 2010). Successful prediction of anti-HIV
(Alves et al., 2001) and anti-tumour activities (Moriani et al.,
2002) of flavonoid compounds has been reported. According
to these studies, the antioxidant activity of flavonoids depends
strongly on the number and position of hydroxyl groups in the
molecule. In the present findings dihydroxylated B-ring (cate-
chol structure), presence of hydroxyl and methoxy functional
groups and in the C-ring presence of the hydroxyl group are
also presumed to increase the antioxidant capacity. Presence
of the hydroxyl group on 3, 5, 30 and 40 is depicted to enhance
the antioxidant activity of isolated flavonoid.
4. Conclusion
Concluding the aforementioned studies it was analysed that
the studied plant contained flavonoids. Isolated flavonoid 2-
(3,4-dihydroxy-5-methoxy-phenyl)-3,5-dihydroxy-6,7-dimeth-
oxychromen-4-one (C18H18O9) extracted from Euphorbia
neriifolia posses significant potential to scavenge free radicals,
ROS and also inhibits lipid peroxidation and hence have got
anticarcinogenic potential. The presence of high concentration
of flavonoids in Euphorbia neriifolia which is depicted from our
results, are responsible for imparting the antioxidative mecha-
nism that inturns are the causitive factors of various degener-
ative diseases.
Contributors
Both authors have materially participated in the research
and/or article preparation. Veena Sharma helped in designing
the experiment and in writing the article. Pracheta Janmeda
performed the experimental work.
Acknowledgements
The authors are grateful to the CDRI, Lucknow for their
help in providing the IR, 1H NMR and mass spectra. One of
the authors (P. Janmeda) is grateful to the University Grants
Commission (UGC) for providing financial assistance (Grant
/F. No. 37-68/2009; SR) and authorities of the Banasthali
University, Rajasthan for providing the rest of necessary
facilities.
514
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