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A simple, facile method for isolation of embelin from fruits of Embelia ribes
Burm.f (Vidang).

Article  in  Indian Drugs · February 2016

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 A SIMPLE, FACILE METHOD FOR ISOLATION OF EMBELIN FROM FRUITS
oF EMBELTA RIBES BURM.F (V|DANG).
Pundarikakshudu K.., Joshi H., Shah p. and panchal S.

(Received 22 April2015) (Accepted 04 Jan 2016)

ABSTRACT
The purpose of the present investigation was to isolate embelin from the fruits of Embetia ribes Burm.f.
Embelin was isolated from E ribes truil powder by cold and hot extraction with polar and non-polar
solvents. The concentrated and dried extracts were washed with petroleum ether (60-g0"C) to get
crystalline embelin. The isolated embelin molecules were characterized by melting points, TLC, various
spectral techniques such as Ultraviolet (UV), lnfrared (lR), Proton Nuclear Magnetic Resonance (1HNMR),
and Mass Spectrometry (MS). Extraction with either chloroform or diethyl ether yielded bright orange
coloured crystals in good yields. Spectral and chromatographic studies confirmed the isolated embelin
to be pure. A simple maceration with chloroform was found to be most ideal for isolation of embelin. our
method completely eliminates the need for any column chromatography, as reported in literature. Hence,
it is economical and has the potential for scale up.

Keywords: Embelia ribes, Embelin, proton Nuclear

Magnetic Resonance (1HNMR), lnfrared Spectroscopy
(lR), Mass Spectrometry (MS), Ultraviolet Spectroscopy
(UV)
cH2(cH2)gCH:
INTRODUCTION
Fruit of vidang (Embelia rlbes Burm.f.; Family:
Myrsinaceae), is reputed in the lndian Systems of
Medicinel, used mainly in helminth parasite infestation.
The plant is an lndo-Malaysian species found in lndia,
Sri Lanka, Singapore and Malaysia. ln lndia, it grows
wildly in Central lndia, Arunachal pradesh, Assam,
Bengal, Orissa, Andhra Pradesh and Madhya pradesh2.
Anti-fertility3, anti-implantation4, analgesic5, anti-tumor5,
anti-inflammatory6and anti-oxidant activitiesT of the fruits
Fig. 1: Structure of Embellin
have been reported in the literature. The fruits were also
reported to have anti-bacterial activity against Gram-
of interest evinced in this molecule and efforts are being
positive and Gram-negative bacterial.
made to make synthetic derivatives of embelin to improve
its various therapeutic applicationss.
The fruits contain 2.5-9"/o w/w of an orange red
benzoquinone compound embelin (Fig.1). Chemically,
A number of methods have been reported for the
it is 2, 5-dihydroxy-3-undecyl-'1, 4-benzoquinone. This isolation of embelin. lndian Herbal pharmacopoeia
compound is reported to be mainly responsible for the
recommended hot extraction of embelin by refluxing the
medicinal uses of the fruit. Of late, there has been a lot
fruit powder in n-hexane for 6 hours, evaporation of ihe
extract, and recrystallization in a mixture of chloroform
*For Correspondence: and ethanole. Daniel et a/. isolated embelin from E.
Department of Pharmacognosy,
schimperiby first extracting the fruits in ethyl acetate at
L. J. lnstitute of Pharmacy, room temperalureforT2 hours, concentrating the extract,
Between Sarkhej Circle and Kataria Motors, followed by column chromatography on silica gel columns.
S.G. Road, Ahmedabad - 38221O, Gujarat, lndia Sekar efal. initiallyextracted thefruit powderof E ribeswith
E-Mail: P_kilambi@yahoo.com n-hexane by cold maceration and the concentrated extract

TNDTAN DRUGS s3 (02) FEBRUARY 2016 23


was chromatographed on a column of silica gel (100-200
mesh). Elution with benzene afforded an orange colored
embeiin powder which, on recrystallization with diethyl
ether, yielded orange plates of embelin (yield: 0'325%
w/w)r'0.- Latha isolated embelin by microwave-assisted
extraction process employing acetone apd reported that
non-polar solvents such as n-hexane and di chloromethane
1' We
were not eff ective for selective extraction of embel in1
had earlier reported gravimetric estimation of embelin
f rom
E. rlbesf ruits by using simple extraction with diethyl ether
for 20 minutes, followed by washing of the residue with
petroleum ether12. Keeping in vtew these varying isolation
methods reported in the literature, we undertook a study
wherein embelin was isolated by different processes
to find out a viable and less cumbersome method' The
isolated embelin was characterized by recording melting
points, TLC chromatography, and study of U'V'' FTIR'
Mass and 1H NMR spectra. The merits and demerits of
the different methods are discussed and the most facile
and convenient method is recommended'
MATERIAL AND METHODS
EXPERIMENTAL
Chemicals
All the chemicals used in the isolation were of
laboratory grade and obtained f rom S'D' Fine Chemicals'
Mumbai, lndia or {rom Qualigens, Mumbai, lndia'
Chemicals for TLC, spectral analyses like UV, NMR
were of analytical grade. Reference standard of embelin
(98% pure) was purchased from Yucca Enterprises
(Mumbai, lndia).
Plant Material
Fruits of E. ribeswere purchased f rom an established
local dealer and identified with the help of available
literaturel'13. A specimen of the seeds is preserved in
the pharmacognosy museum of the author's institute
(specimen No. LJIP/cog l2O1 41006)'
lnstrumentation
Extracts were concentrated to dryness on a water
bath (Labtronics, Ahmedabad, lndia)' Melting points
were uncorrected and recorded in an open capillaryon a
Toshniwal melting point apparatus. TLC was performed
on aluminum plates pre-coated with silica gel 60Fruo
(cat. No. 1.05548, E. Merck, Darmstadt, Germany)'
Samples were applied on plates and developed
in glass (2OX2O cm) TLC chamber' UV absorption
spectra were recorded in methanol on a Shimadzu
double-beam UV-Visible spectrophotometer, model
1800 with UV probe software Version 2'3' lR spectra
were recorded on a Shimadzu FTIR-8400S with
ATR-MIRACLE 1 0 spectrophotometer' Mass spectra
were taken on an Advion Expression CMS (Compact
Mass Spectrometer) using ESI as ionization source'
NMR spectra was taken on Bruker 400 MHz Bruker
Proronon NMR sPectrometer.

from E ribes fruits

Table l: Different solvents and methods on isolation ol Embelin
No of Drying of filtrate Washing of the
Method Solvent for Ref lux/Maceration residue with
extraction extractions
Petroleum Ether
One To a dry residue on a water bath Yes
1 Diethyl ether Maceration(10 min)
One To a dry residue on a waterbath Yes
2 Diethyl ether Reflux(30 min)
One To a dry residue on a waterbath Yes
3 Chloroform Maceration(24 h)
One To a dry residue on a waterbath Yes
4 Chloroform Reflux (2 h)
One To a dry residue on a waterbath Yes
5 Methanol Maceration(24 h)
One To a dry residue on a waterbath Yes
6 Methanol Reflux (2 h)
One To a dry residue on a waterbath Yes
7 n-Hexane Maceration(24 h)
One To a dry residue on a waierbath Yes
8 n-Hexane Reflux (6 h)
One To a dry residue on a waterbath Yes
9 Ethyl Maceration(24 h)
acetate
One To a dry residue on a waterbath Yes
10 Ethyl Feflux (2 h)
acetate
One To a dry residue on a waterbath Yes
11 Acetone Maceration(24 h)
To a dry residue on a waterbath Yes
12 Acetone Reflux (2 h) One

INDIAN DRUGS 53 (02) FEBRUARY 2016

24
 Table ll: Physico-chemical characteristics and yields of embelin isolated from
E.ribes fruits by different methods
Method Solvent for Reflux/ % Extraction Washing with % Yield (mean Melting TLC Rf
extraction Maceration (mean t Petroleum t SEM) point Solvent
(Min/ h) SEM) Mw Ether w/w 0c Svstem
1 Diethyl Macerafion 6.6 t 0.358 Yes 2.09 t 0.035 145 Toluene: 0.8
ether (10 min) Ethyl
2 Diethyl Reflux 7.4 t 0.124 Yes 2j7 tO.36 146 acetate: 0.8
ether (30 min) Formic
3 Chloroform Maceration 8.4 t 0.'159 Yes 2.50 t 0.37 135 acid 0.8
(24 h) (5:5:0.5,
4 Chloroform Ref lux 7.6 + 0.256 Yes 1.89 t 0.21 142
vNN) 0.8
(2 h)
5 Methanol Maceration 11.1 x 0.214 Yes Dark and N.P.
(24 h) stickv residue
6 Methanol Ref lux (2 h) 13.4 t 0.234 Yes Dark and N.P.
sticky residue
7 n-Hexane Maceration 5.3x.0.412 Yes Negligible N.P.
(24 h)
B n-Hexane Reflux 4.3+ 0.12 Yes Negligible N.P.
(6 h)
I Ethyl Maceration 7.0 t 0.156 Yes 1.97 + 0.13 150 Toluene: Tailing
acetate (24 h) Ethyl
10 Ethyl Reflux 6.5 t 0.189 Yes 2.'13 t 0.16 160 acetate: Tailing
acetate (2 h) Formic
11 Acetone Maceration 6.7 x.O.578 Yes 1.93 t 0.1 'l
Above acid Tailing
(24 h) 240 (5:5:0.5,
12 Acetone Reflux 7.2 r 0.612 Yes 'l .99 t 0"13 162 v/v/v) Tailing
(2 h)

Extraction and lsolation of Embelin
The fruits were reduced to a powder of 40 # and
stored in an ambercolored glass bottle tillfurther use. The
powdered drug was extracted and embelin was isolated
by different solvents like chloroform, n-hexane, methanol,
diethyl ether, aceione and ethyl acetate as described in
Table l. Each isolation method was done in triplicate and
the yields are expressed as mean t SEM.
ldentification of lsolated Compounds
Melting poinfs: Melting points were taken in an open
capillary tube on a melting point apparatus and are
.lncorrected.
Thin layer chromatography. 10 mg. of the isolated
:ompound in each case was dissolved in 10 mL of
:nloroform. These solutions were spotted manually as
a band on pre-coated Silica gel GFrurplates. Reference
standard of embelin prepared in a similar manner was
also spotted along with test compounds. The plates were
developed in a TLC chamber saturated with the solvent
syslem tol uene: ethyl acetate: f ormic acid (5:5:0.5, V N lV),
removed f rom the chamber after development and solvent
evaporated under a ceiling fan. The spots were first
visualized in day light and later under a UV chamber.
Ultra-violet spectrum:1 0 mg of the isolated compound
in each case was dissolved in methanol and the solution
was scanned between 200-800 nm on a UV - visible
spectrophotometer in a 1 cm quarlz cell.
lnfra-red spectrum:The powdered compound was
placed on the surface of ATR of FTIR instrument and the
spectrum was recorded.
Mass Specfrurn; Mass speclra were taken on an
Advion Expression CMS (Compact Mass Spectrometer,
using ESI as ionization source.
1H NMR
spectra:Prolon NMR spectra of the rso a:e:
compoundswere recorded in CDCI.on a400 MHz 3'-.,:'
NMR spectrometer.

'.JDIAN DRUGS 53 (02) FEBRUARY 2016 z:)


Fig. 2: UV spectrum of Embelin lsolated with Chloroform
Glsgnrlozrr
RESULTS
Extraction with n-hexane or methanol
yielded sticky mass both by maceration and
hot extraction methods. lt was not possible
to obtain any crystalline material in spite
of repeated washings with various organic
solvents. This may be due to the very high
resinous materials that get readily extracted
in these solvents along with embelin. The
crystalline material from diethyl ether
and chloroform extracts is bright and
showed melting points 145'C and '146'C
respectively. This is quite in agreement
with the values given in literatures. The
crystals obtained with ethyl acetate were
dull in colour and had melting points from
150 -160"C. We could not get embelin with
n-hexane extraction as recommended in
lndian Herbal Pharmacopeiae. Similarly,
extraction with acetone as reported by
Sekar et a1.10 did not give satis{actory
results.

Extraction with diethyl ether and

l
chloroform yielded crystalline embelin
in good quantities (Table ll). Embetin
isolated with diethyl ether and chloroform
by simple extraction confirmed to all
physicaland spectraldata available in the
Iite ratu re 8, a. The c rystal n e mate rial showed
1
I i

embelin at R, 0.8 on TLC in the mobile

phase toluene: ethyl acetate: formic acid
(5:5: 0.5, Vn/A/). However, only embelin
Fig.3: lR Spectrum of Embelin lsolated with Chloroform isolated with diethyl ether/chloroform
!
showed single spot without any tailing,
I
whereas in other cases there was tailing,
suggesting the compounds are not
i
completely free from impurities.
i

I
UV spectrum of embelin isolated with
chloroform and diethyl ether exhibited l.,ax
at 305 nm, (Fig. 2). lR spectrum showed
I

I bands at 3302.13cm-1 (O-H stretching),

i 2916.37cm-1 (asymmetric stretching),
2846.93cm 1 (symmetric stretching)
I
1612.49 (C=O stretching) 1458.18cm-1
(C-H bending of -CH, group) 856.39cmi
I

(C- H out of plane). (Fig. 3)The mass

spectrum showed an M*, mlzpeakat 296.
I

The purity of embelin was f urther conf irmed

I
by the 1H-NMR (400 MHz, CDCI') which
showed shifts at 6r: d 7.75 (1H, brs)
t
Fig.4: Mass Spectrum of Embelin lsolated with Chloroform 6.02(1H, s, H-6), 2.48 (2H, f, H-1'), 1.48

26 TNDTAN DRUGS 53 (02) FEBRUARY 2016

L
b

Fig.5: NMR spectrum of Embelin lsolated with chloroform
(2H, m, H-2'), 1 .31 (2H, brs, H-3,), 1 .27 (1 4H, b rs, H-4, I H-
'10') and
0.91 (3H, t, H-11'). (Fig. 4)
DISCUSSION
Several methods for isolation of embelin have
been reported which involve multiple steps such as
extractionsfora long period of time, purification bycolumn
chromatography, re-crystallization etc. This leads to high
cost of production. Diethyl ether extraction gives pure
embelin in good yields. But use of this solvent is attendant
with high risks of inflammation and health hazards.
Moreover, the recovery of the solvent is not feasible. The
method of extraction with chloroform followed by petroleum
ether wash of the dried extract proved to be effective in
isolating embelin with good yield and high purity. Hence,
we recommend simple cold extraction in chloroform for
the isolation of embelin.
CONCLUSION
A simple maceration with chloroform was found to be
most idealfor isolation of embelin. Our method completely
eliminates the need for any column chromatography as
repofted in literature. Hence, it is economical and has the
potential for scale up.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Manish D. Shah, Vice
President, Lok Jagruti Kendra for his encouragement
and for providing the facitities; to Dr. Ditip Maheshwari,
Mr. Darshil Shah, Dr. Jignesh Shah and Miss Krupa Thula
of the Department of Quality Control, L.J. lnstitute of
Pharmacy for taking UV and lR spectra; M/s Synzeal
TND|AN DRUGS 53 (02) FEBRUARY 2016
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Research Labs, Ahmedabad for taking the
mass spectra; National Facilities for Drug
Discovery Centre, Saurashtra University,
Rajkot, for taking the lH NMR spectra;
Dr. Arun Parikh, former Head of deparlment
ol Chemistry, Saurashtra University for
interpreting the spectra.
REFERENCES
1. Anonymous. Quality Standards of lndian
Medicinal Plants 2003,lndian Council of
Medical Research, New Delhi, Vol-lV:
1 30-1 36.
2. Jha N.K. and Pandey L K... Embelia ribes:
Vidanga (Feature Article), phytopharm.,
2008,3-14.
3.
Radhakrishnan N. and Alam M.:Antifertility
activities of embelin in albino rats, lndian
J. Exp. Biol., 1975, 13(1) 70-73.
4. Krishnaswamy M. and Purushothaman K.K.: Antiferlility
properties ot Embelia ribes: (Embelin), lndian J. Exp.
Biol., 1980, 18(1 1) 1359-1362.
5. Chitra M., Sukumar E., Suja V. and Shyamala Devi C.S.:
Antitumor, anti-inflammatory and analgesic property of
embelin, a plant product, Chemother., 1994, 40(2)109_
1 13.
6. Handa S. S., Chawla A. S. and Sharma A.K.: plants with
anti-inflammatory activity, Fitoter., 1992, 63(1) 3-31 .
7. Chitra M., Shyamala Devi C.S. and Sukumar E.: protective
action of embelin against lipid peroxidation in tumour-bearing
rats, Fitoter., 1994, 65(4) 317-321.
Daniel 8., Avijit M. and Kaleab A.: ln vitro antimicrobial
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Pharm. J., 201 4, 30 (1 ) 50-56.
9. lndian Herbal Pharmacopoeia. Revised New Edition.
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10. Sekar M., Shrishailappa B.R., Boreddy S.T. and Veeresh
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Chem. Pharm.Buil.,2011,-A structure-activity
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K.
and Banerjee S.: Colorimetric determination of embelin in
Embelia ribes, lndian Drugs, 1997, 34(10) S9O_592.
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Ministry of Health and Family Welfare, Govt. of lndia.. New
Delhi 2001. 1 (1)163-165.
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z/