Professional Documents
Culture Documents
1 s2.0 S1878535214001816 Main PDF
1 s2.0 S1878535214001816 Main PDF
1 s2.0 S1878535214001816 Main PDF
ORIGINAL ARTICLE
Department of Bioscience and Biotechnology, Banasthali University, 304022 Banasthali, Rajasthan, India
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.
ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction for human health (Raskin et al., 2002; Reddy et al., 2003).
Compounds belonging to the terpenoids, alkaloids and flavo-
Phytochemistry reports thousands of newer organic molecules noids are currently used as drugs or to prevent various diseases
or compounds every year. Pharmacological testing, modifying, (Raskin et al., 2002) and in particular some of these com-
derivatizing and research on these natural products represent a pounds seem to be efficient in preventing and inhibiting vari-
major strategy for discovering and developing new drugs ous types of cancer (Reddy et al., 2003).
(Kayser et al., 2000). Moreover, plant secondary metabolites Flavonoids are a group of about 4000 naturally polypheno-
present chemical and pharmaceutical properties interesting 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
* Corresponding author at: Lab-209, Department of Bioscience and
into several subgroups including the following: flavone, flava-
Biotechnology, Banasthali University, P.O. Banasthali, Distt.: Tonk none, flavonol, isoflavonoid, anthocyanidin, and chalcones
304022, India. Tel.: +91 1438228386, +91 9352381260.
(Harborne, 1998).
E-mail addresses: drvshs@gmail.com (V. Sharma), pracheta25@
gmail.com (P. Janmeda).
The plant Euphorbia neriifolia, especially the leaves, has
Peer review under responsibility of King Saud University.
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
Production and hosting by Elsevier compounds have already been isolated (Chatterjee et al., 1978;
http://dx.doi.org/10.1016/j.arabjc.2014.08.019
1878-5352 ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
510 V. Sharma, P. Janmeda
Ng, 1990; Ilyas et al., 1998; Yadav et al., 2011) but this is the brown bands that were produced after keeping the plates in
first report to isolate flavonoid from E. neriifolia leaves. The iodine chamber were scratched and suspended in the mobile
purpose of this study was to isolate the most important group phase separately for 3–4 days. After that mobile phase was
of phytochemical class i.e. flavonoids using TLC and HPTLC sucked out, vacuum evaporated and the residue left was col-
as technique and was characterized through their spectral (IR, lected till sufficient amount for dosing and spectrophotometric
1
H NMR and MS), as well as comparison with a reference analysis was obtained. The selected purified material was sub-
materials. jected to its HPTLC and IR spectral.
2.1. Chemicals and reagents Chemical tests as described by Harborne (1998) were carried
out on the isolated compound using standard procedures.
All chemicals and reagents used in the study were of analytical
2.6. HPTLC method for the estimation of flavonoids
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 The complete CAMAG TLC equipment consists of a fully
aluminum sheets (Merck KGaA, Germany). Silica gel 60 automatic sample Linomat V sample applicator, a developing
F254, HPTLC aluminium sheets 20 · 20 cm, Merck KGaA, chamber. Finally, a Camag TLC scanner is available allowing
Germany Ord. No. 1.05554. 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
2.2. Procurement, authentication of plant material
volume was made up to 10 ml in a standard flask (1000 lg/ml).
Standard (10 mg) was taken and dissolved in methanol. This
E. neriifolia (EN) leaves were collected from medicinal garden was transferred into a standard flask and the volume was made
of the Banasthali University, Banasthali and nearby areas of up to 100 ml to prepare 100 lg/ml solution. Silica gel 60 F254
Banasthali, Rajasthan, India (Latitude N–26240 14.841400 ; and HPTLC aluminium sheets were used as adsorbent (sta-
Longitude E–73520 9.719400 ), in the month of October, 2009 tionary phase). The extracts were applied point-wise from
and were taxonomically identified by Botanist of Krishi Vig- 1000 lg/ml sample solution, 10 ll of the sample was applied
yan Kendra, Banasthali University, Banasthali, Rajasthan, In- on HPTLC aluminium sheets as different tracks in the form
dia. A voucher specimen was deposited in the herbarium of of 6 mm wide bands by using a Camag semi-automatic Linom-
Department of Bioscience and Biotechnology, Banasthali Uni- at 5 spotter at a distance of 12 mm. Nitrogen gas was also sup-
versity (No. BVH–780141A). plied for simultaneous drying of bands and then using drier for
completely drying of bands. HPTLC of different extracts was
2.3. Extraction performed by using the mobile phase n-butanol: acetic acid:
water (2:2:6). To saturate the chamber, 10 ml mobile phase
Shade dried leaves were powdered (250 g), soxhlet extracted was placed in each flat-bottomed Camag twin trough TLC
with 70% (v/v) ethanol and vacuum concentrated to dryness chamber, 30 min before the development of the PTLC plate.
under reduced pressure at 60 ± 1 C. After drying in hot air The chamber was sealed with parafilm and covered with a steel
oven (40–45 C), it was stored in an air tight container in lid. The developed plates were then dried and scanned using a
refrigerator at 5 C. The residue was designated as hydro- TLC scanner 3 with Wincats software under 364 nm (Wagner
ethanolic extract of E. neriifolia (HEEN). and Bladt, 1996). All plates were visualized directly after dry-
Dried leaves of E. neriifolia (250 g) were extracted succes- ing and a fingerprint profile was photo documented using a
sively with pet-ether, benzene, chloroform, ethyl acetate, and Camag Reproster – 3 under 254 nm and 366 nm in UV and
ethanol and finally macerated with distilled water (non-polar visible light. The digital images of the chromatograms were
to polar) to get respective extracts. Flavonoid contents and evaluated with the programme CAMAG Video Scan. The cap-
their presence were determined by the method of Harborne tured image was subjected to a visual inspection on the com-
(1998), using quercetin as a standard. The extracts were ana- puter screen. The differences found, are specified by the
lysed by means of TLC. HPTLC system in which the difference is detected and by
the Rf value (and colour) of a compound in the system.
2.4. Thin layer chromatography (TLC)
2.7. Compound characterization
Thin layer chromatography was performed using standard
methods (Harborne, 1998). Small quantities of samples For characterization of compound IR, NMR and Mass
(2 mg/ml) were dissolved in their respective solvents. Quercetin spectra were performed. IR was obtained for the isolated
(1 mg) standard was dissolved in methanol. Different mobile flavonoid. The sample (1–2 mg) was crushed in KBr
phases with varying concentrations were employed in the (3–4 mg) pellets using a FTIR (Fourier transform Infrared
screening programme and selected the one in which separation spectroscopy; Model - Varian 3600) with the help of mechan-
of flavonoid was clear: n-butanol: acetic acid: water (2:2:6). All ical pressure. They were recorded within the wavenumber
plates were visualized directly after drying and with the help of range 4000–400 cm1 in an FTIR instrument. 1H NMR
UV at 254 nm and 366 nm in UV TLC viewer. The Rf value of spectra was recorded on DRX-Bruker 300 MHz (Mega Hz),
the different spots that were observed was calculated. The dark Switzerland. Nuclear magnetic field was obtained for the
Extraction, isolation and identification of flavonoid from Euphorbia neriifolia leaves 511
Figure 2 HPTLC baseline display of ethyl acetate, ethanol, aqueous extract and isolated flavonoid (ENF) compared with quercetin.
References Raskin, I., Ribnicky, D.M., Komarnytsky, S., Ilic, N., Poulev, A.,
Borinker, N., Moreno, D.A., Ripoll, C., Yakoby, N., 2002. Plants
Agrawal, P.K., Bansal, M.C., 1989. Flavonoid glycosides. In: Agrawal, and human health in the twenty-first century. Trends Biotechnol.
P.K. (Ed.), Carbon-13 NMR of Flavonoids. Elsevier, Amesterdam, 20, 522–531.
pp. 283–364. Reddy, L., Odhav, B., Bhoola, K.D., 2003. Natural products for
Alves, C.N., Pinheiro, J.C., Camargo, A.J., Ferreria, M.M.C., cancer prevention: a global perspective. Pharmacol. Ther. 99, 1–13.
Romero, R.A.F., da Silva, A.B.F., 2001. A multiple linear Schultz, S.C., Bahraminejad, S., Asenstorfer, R.E., Riley, I.T., 2008.
regression and partial least squares study of flavonoid compounds Analysis of the antimicrobial activity of flavonoids and saponins
with anti-HIV activity. J. Mol. Struct. 541, 81–88. isolated from the shoots of oats (Avena sativa L.). J. Phytopathol.
Chatterjee, A., Saha, S.K., Mukhopadhyay, S., 1978. Lewis acid- 156, 1–7.
catalysed rearrangement of glut-5-en-3$-y1 acetate and glut-5(10)- Sharma, V., Janmeda, P., 2013a. Chromatography fingerprinting
en-3$-y1 acetate. Indian J. Chem. 16, 1038–1039. profile studies on the flavonoids of Euphorbia neriifolia (Linn.)
de Melo, E.B., Martins, J.P.A., Jorge, T.C.M., Ferreira, M.M.C., leaves. Int. J. Drug Dev. Res. 5 (1), 286–296.
2010. Multivariate QSAR study on the antimutagenic activity of Sharma, V., Janmeda, P., 2013b. N-nitrosodiethylamine-induced renal
flavonoids against 3-NFA on Salmonella typhimurium TA98. Eur. histopathological changes in mice attenuated by Euphorbia nerii-
J. Med. Chem. 45, 4562–4569. folia (Linn). Toxicol. Int. 20 (1), 101–107.
Harborne, J.B., 1998. Phytochemical Methods, A Guide to Modern Sharma, V., Janmeda, P., 2014. Protective assessment of Euphorbia
Techniques of Plant Analysis, 3rd ed. Springer Pvt. Ltd., New neriifolia and its isolated flavonoid against N-Nitrosodiethylamine-
Delhi, India. induced hepatic carcinogenesis in male mice: a histopathological
Harborne, J.B., Mabry, T.J., 1982. The Flavonoids Advances in analysis. Toxicol. Int. 21 (1), 56–62.
Research. Chapman & Hall, London, New York. Sharma, V., Janmeda, P., Singh, L., 2011a. A review on Euphorbia
Ilyas, M., Parveen, M., Amin, K.M.Y., 1998. Neriifolione, a triterpene neriifolia (sehund). Spatulla DD 1, 107–111.
from Euphorbia neriifolia. Phytochemistry 48 (3), 561–563. Sharma, V., Pracheta, 2013a. Microscopic studies and preliminary
Janmeda, P., Sharma, V., Singh, L., Paliwal, R., Sharma, S., Yadav, pharmacognostic evaluation of Euphorbia neriifolia (L.) leaves.
S., Sharma, S.H., 2011. Chemopreventive effect of hydro-ethanolic Indian J. Nat. Prod. Res. 4 (4), 348–357.
extract of Euphorbia neriifolia leaves against DENA-induced renal Sharma, V., Pracheta, Paliwal, R., Sharma, C., 2012a. Elucidation of
carcinogenesis in mice. Asian Pac. J. Cancer Prev. 12, 677–683. analgesic activity of hydro-ethanolic extract of Euphorbia neriifolia
Kayser, O., Kidderlen, A.F., Croft, S.L., 2000. Natural products as leaves in swiss albino mice. J. Plant Dev. Sci. 4, 183–189.
potential anti-parasitic drugs. Acta Trop. 77, 307–314. Sharma, V., Janmeda, P., Paliwal, R., Sharma, S.H., 2012b. Anti
Kiralj, R., Ferreira, M.M.C., 2009. Basic validation procedures for hepatotoxic activity of Euphorbia neriifolia extract against N-
regression models in QSAR and QSPR studies: theory and nitrosodiethylamine-induced hepatocarcinogenesis in mice. J. Chin.
applications. J. Braz. Chem. Soc. 20, 770–787. Integr. Med. 10, 1303–1309.
Lakshmi, T., Rajendran, R., Madhusudhanan, N., 2012. Chromato- Sharma, V., Pracheta, 2013b. Remedial effect of Euphorbia neriifolia
graphic fingerprint analysis of Acacia Catechu ethanolic leaf extract leaves and isolated flavonoid against N-nitrosodiethylamine
by HPTLC technique. Int. J. Drug Dev. Res. 4, 180–185. induced renal-carcinogenesis in mice. Indian J. Biochem. Biophys.
Meena, M.C., Patni, V., 2008. Isolation and identification of flavonoid 50, 521–528.
‘‘ouercetin’’ from Citrullus colocynthis (Linn.) schrad. Asian J. Exp. Sharma, V., Pracheta, Paliwal, R., Singh, L., Sharma, V., Sharma,
Sci. 22, 137–142. S.H., 2011b. Anticarcinogenic potential of Euphorbia neriifolia
Moriani, M.Z., Galati, G., O’Brien, P.J., 2002. Comparative quanti- leaves against N-nitrosodiethylamine-induced nephrotoxicity in
tative structure toxicity relationships for flavonoids evaluated in mice. J. Biochem. Cell. Arch. 11, 393–398.
isolated rat hepatocytes and HeLa tumor cells. Chem. Biol. Wagner, H., Bladt, 1996. Plant Drug Analysis: A thin layer chroma-
Interact. 139, 251–264. tography Atlas, 2nd Edn. Springer-Verlag, Berlin Heidelberg
Ng, A.S., 1990. Diterpenes from Euphorbia neriifolia. Phytochemistry London, New York, 349–354.
29, 662–664. Yadav, R.P., Patel, A.K., Jagannadham, M.V., 2011. Neriifolin S, a
Pracheta, Sharma, V., Paliwal, R., Sharma, S., 2011. In vitro free dimeric serine protease from Euphorbia neriifolia Linn.: purification
radical scavenging and antioxidant potential of ethanolic extract of and biochemical characterisation. Food Chem. 132, 1296–1304.
Euphorbia neriifolia Linn. Int. J. Pharm. Pharm. Sci. 3, 238–242.