Eur. J. Lipid Sci. Technol.

2012, 114, 1097–1101 1097

Short Communication
A method for isolation of karanjin from the expelled cake of
Pongamia glabra

Radha S. Susarla1, Marthanda M. Murthy2, Bhamidipati V. S. K. Rao1, Pradosh P. Chakrabarti1,

Rachapudi B. N. Prasad1 and Sanjit Kanjilal1

1
Centre for Lipid Research, Indian Institute of Chemical Technology, Hyderabad, India
2
Division of Organic Chemistry II, Indian Institute of Chemical Technology, Hyderabad, India

Due to the renewed interest in the production of biodiesel from nonconventional oils like karanja
(Pongamia glabra), huge quantity of expelled cake will be generated in near future. However, due to
the presence of several antinutritional components, expelled karanja cake cannot be used as feed for
poultry and livestock. It needs detoxification and the first step during detoxification is the removal of
karanjin, the major bioactive constituent. The present study aimed at isolation of karanjin from expelled
cake. Accordingly, a simple and scalable process was developed for the isolation of karanjin with purity in
the range of 95–97% from the expelled cake of karanja. Different solvents were screened by varying
temperature, time, and amount of extracting solvents to extract karanjin from the expelled cake and
dimethyl carbonate (DMC) was found to be the best among them. DMC extraction of 1.0 kg of expelled
cake yielded 3.6 g (0.36% with respect to expelled cake) of karanjin of 97% purity.

Keywords: Biodiesel / Expelled cake / Isolation / Karanjin / Pongamia glabra

Received: January 10, 2012 / Revised: February 29, 2012 / Accepted: April 16, 2012
DOI: 10.1002/ejlt.201200016

1 Introduction expelled cake is important. Effective utilization of various

Karanja is a medium sized glabrous tree found throughout
India and belongs to the species Pongamia glabra Vent [1].
The plant is traditionally used in the Indian system of medicine.
Karanja seed contains 27–39% oil, 20–30% protein, and a
group of furanoflavonoids that constitute 5–6% by weight of
the oil [1]. There are many review articles reported on P. glabra
[2–5] covering the chemistry and biological activities. Karanjin,
the principal furanoflavonoid constituent (1.25%, oil basis) was
first crystalline compound isolated from the seed oil [2].
Being nonedible, karanja oil has been identified as most
suitable oil for biodiesel preparation in India [6, 7]. As a
result, huge quantities of expelled cake will be produced in
near future. This biodegradable waste, if handled properly
would maintain the natural balance of essential elements and
thereby promote more harvests from nature. Moreover, to
improve the economics of karanja-based biodiesel pro-
duction, value addition to its by-product, especially the
Correspondence: Dr. Sanjit Kanjilal, Centre for Lipid Research, Indian
Institute of Chemical Technology, Hyderabad 500 007, India
E-mail: sanjit@iict.res.in
Fax: þ91 40 27193370
Abbreviations: DMC, dimethyl carbonate; IPA, isopropanol; TOL, toluene
major and minor bioconstituents in the expelled cake would
certainly help the economics of biodiesel. Presently, karanja
cake is being used as manure, fungicide, and insecticide.
However, expelled karanja cake cannot be used as feed for
poultry and livestock due to the presence of several antinutri-
tional constituents, such as karanjin, pongamol, glabrin, phy-
tate, tannins, and trypsin inhibitor. Utilization of deoiled
karanja cake is possible only after complete removal of all
these antinutritional components [8]. Karanjin being the
major furanoflavonoids having important biological activities
[5, 9–11], its effective utilization is utmost important for the
karanja-based biodiesel industry. This is possible only if there
is a scalable process for the isolation of pure karanjin from
the expelled cake, which has not been attempted earlier. The
present work was pursued with the objective of developing a
process for the isolation of pure karanjin from the expelled
karanja cake using simple solvent partitioning approach.
2 Materials and methods
2.1 Materials
Expelled karanja cake was collected from a local industry.
All solvents were purchased from M/s SD Fine Chem
(Mumbai, India).

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1098 R. S. Susarla et al.
2.2 Methods
2.2.1 Estimation of residual oil in karanja expelled cake
The residual oil content in the expelled karanja cake was
determined using SER 148 Solvent Extractor (VELP
Scientifica, Germany). The residual oil was extracted from
expelled cake (5.0 g) with hexane (75 mL) for 4.0 h. The
extractions were conducted in duplicate from the same batch
of expelled cake. The mean oil content in the expelled cake
was found to be 12.8%.
2.2.2 Extraction of karanjin from karanja expelled cake
For the extraction of karanjin from expelled cake, a digitally
controlled overhead stirrer attached with four bladed paddle
type stirrer (Heidolph, Germany) and reflux condenser was
used. Expelled karanja cake (300 g) containing 12.8% of
residual oil was extracted using three different organic sol-
vents at RT (308C) over a period of 8 h using digitally
controlled overhead stirrer. The contents were filtered and
the organic solvent was removed from the filtrate to obtain the
crude extract. The crude extract was further extracted thrice
with 10% aqueous methanol (3
150 mL). The solvent was
removed from the extract using rotary evaporator and dried
under reduced pressure to get the crude polar extract. The
crude polar extract was solubilized in ethyl acetate (200 mL)
and washed with water. The ethyl acetate-soluble extract was
dried over anhydrous sodium sulfate. The organic solvent was
removed from the extract using rotary evaporator and dried
under reduced pressure to get karanjin-rich extract, which on
crystallization from ethyl acetate produced karanjin, as white
solid having melting point in the range of 161–1638C. The
structural confirmation of karanjin was based on NMR, IR,
and mass. 1H and 13C NMR spectra were recorded on 300
and 75 MHz (Varian, Palo Alto, USA) spectrometer,
respectively, and IR on a Perkin Elmer (Model: Spectrum
BX) FT-IR spectrometer. Mass spectra was recorded on
Agilent 5973 mass spectrometer (Palo Alto, USA) in the
EI mode and are given in mass units (m/z). The spectral
data matched well with those reported in the literature
[12]: IR (cm1, KBr): n 1640–1620, 1580–150, 1160–
1150; 1H NMR (CDCl3; 200 MHz): d 3.94 (s, 3H), 7.16
(d, J ¼ 2.3 Hz, 1H), 7.52 (m, 4H), 7.75 (d, J ¼ 2.3 Hz,
1H), and 8.15 (m, 3 H); 13C NMR (75 MHz; CDCl3): d
60.4, 104.5, 110.2, 117.2, 119.9, 122.0, 126.4, 128.6, 128.9,
130.4, 130.9, 142.0, 146.0, 150.1, 155.0, 158.3, 175.3;
EI–MS (m/z): 292, [M]þ, 291 [M-1]þ.
2.2.3 Estimation of karanjin in ethyl
acetate-soluble extract
Karanjin was analyzed qualitatively using GC–MS and quan-
titatively by GC. TLC profile of ethyl acetate soluble extract
showed the presence of free fatty acids and needs to be
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Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101
converted to fatty acid methyl ester for the GC analysis of
the total extract. Thus ethyl acetate soluble extract was deriv-
atized to methyl esters by the treatment of 10% BF3-etherate
in methanol for 1.0 h under reflux condition. The derivatized
extract was solubilized in ethyl acetate and washed with
water. The ethyl acetate-soluble was dried over anhydrous
Na2SO4, concentrated in the rotary evaporator and then
injected in GC–MS and GC. GC–MS was carried out with
Agilent 6890 N gas chromatograph connected to Agilent
5973 mass spectrometer (Palo Alto, USA) and GC on
HP 6850 Series gas chromatograph equipped with a FID
detector. The GC–MS detection was performed at 70 eV
(m/z 50–550; source at 2308C and quadruple at 1508C) in
the EI mode attached with HP-1 ms capillary column
(30 m
0.25 mm
0.25 mm). The oven temperature
was programmed for 2 min at 808C, raised to 3008C at
108C/min, and finally maintained at 3008C for 15 min.
The carrier gas, He flows at 1.0 mL/min, and the split ratio
is maintained at 50:1. Structural assignments were based on
interpretation of mass spectrometric fragmentation. The
GC was performed in HP1 capillary column (30 m

0.25 mm
0.5 mm) and the oven temperature program-
ming is similar to that of GC–MS with the injector and
detector temperature maintained at 300 and 3258C. The
carrier gas, N2 flows at, 1.5 mL/min, and the split ratio
was 50:1.
2.2.4 Statistical analysis
The data, presented as mean  SD, were analyzed by a
paired Student’s t-test to evaluate the level of statistical sig-
nificance. A p-value of less than 0.05 was considered
significant.
3 Results and discussion
Karanjin is the principal furanoflavonoid present in the kar-
anja seed. It is known to possess many potent biological
activities, important among them are insecticidal and mos-
quito repellant activities [9, 10]. Most of the applications of
karanjin are accomplished either as oil or as cake in utilizing it
as natural biopesticide and also for their mosquito larvicidal
activity [13–15]. Isolation of karanjin from oil and roots are
reported in the literature [2–4, 16]. However, all these
methods require a column chromatographic purification at
some stage. Moreover, there is no report of its isolation from
expelled cake. As karanja oil being identified as ingredient for
the preparation of biodiesel in India, it is important to add
value to all of its major and minor byproducts.
For every ton of production of karanja-based biodiesel,
about 2 tons of expelled karanja cake will be generated. In
general, edible oilseed cake has applications as a source of
protein for human consumption, as animal feed, and also as
fertilizer. All these applications of edible oilseed cake cannot
be extended to nonedible oilseed cakes such as karanja, due to
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Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101
the presence of several antinutritional constituents [8]. On
the other hand, there is a need to improve the economics of
biodiesel production. Expelled cake is the major by-product
of the karanja-based biodiesel industry. During expelling,
karanjin gets enriched in deoiled cake. It is worthwhile to
have an appropriate process for the isolation of pure karanjin
from the expelled cake in order to have its effective utilization
as natural insecticide. Hence the present study reports
a simple solvent partitioning approach for the isolation of
karanjin from the expelled cake avoiding column chromato-
graphic separation at any stage. During expelling, about
8–15% of oil gets retained in the cake depending upon
the type and efficiency of expeller unit. It is necessary to
estimate the oil content in the expelled cake in order to assess
the extractability of different solvents for oil as well as
karanjin. The expelled cake in the present study was found
to contain 12.8% of residual oil.
As the furanoflavonoids are oil soluble, the prime objec-
tive of the extraction was to extract as much residual oil as
possible from the expelled cake using a solvent which solu-
bilize both oil and karanjin. Accordingly, three different
solvents, namely isopropanol (IPA), toluene (TOL), and
dimethyl carbonate (DMC) were chosen. After extraction,
the organic solvent was evaporated to get the crude extract,
which was further extracted with 10% aqueous methanol to
separate the major nonpolar oil from other minor polar com-
ponents including karanjin. The methanol-soluble extract
may contain many water-soluble polar components other
than karanjin depending upon the extractability of initial
solvent chosen for the extraction of expelled cake.
Evapoartion of methanol, solubilizing in ethyl acetate, and
the subsequent water work-up will remove those water-solu-
bles. This will eventually enrich karanjin in the ethyl acetate
soluble extract. A schematic representation of extraction
Scheme 1. Schematic representation of the extraction of karanjin
from the expelled karanja cake.
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Karanjin from expelled karanja cake 1099
protocol is shown in Scheme 1. As karanjin is soluble in
oil, the solvent which has better oil extractability will extract
more karanjin. This has necessitated the determination of oil
extractability of these three solvents, which was done by
estimating the oil content in the left-over cake.
A comparative study of extraction of expelled cake
(300 g) containing 12.8% residual oil using all three solvents
(1.0 L) were conducted at 308C for a period of 8 h.
Extractions were conducted in triplicates and the results
are shown as average of three extractions (Table 1). There
is no significant difference in the weight of crude extract
obtained using the three solvents. However, the oil content
in the left-over cake was found to be 3.4, 4.9, and 5.0%,
respectively, when the extractions of expelled cake were car-
ried out using DMC, TOL, and IPA. Thus, out of 12.8% of
residual oil present in the expelled cake, DMC has extracted
9.4%, followed by 7.9% by TOL and 7.8% by IPA. This
indicates that DMC has better oil extractability compared to
TOL and IPA. Thus oil extracted, as given in Table 1 is
determined by subtracting the oil content in the left over cake
from the residual oil in the expelled cake, which can also be
obtained from the weight of aqueous methanol-insoluble
extract.
The amount of water-soluble extract was determined
theoretically by subtracting the combined weights of oil
extracted and ethyl acetate-soluble extract from the crude
extract. Results obtained from Table 1 indicated that amount
of water-soluble components are negligible during DMC
extraction compared to those obtained during IPA and
TOL extraction. Moreover, the quantity as well as purity
of karanjin obtained during DMC extraction is better than
those obtained during TOL and IPA extraction. Purity of
karanjin is in the range of 82–86% in case of TOL and IPA
extraction, but the same in case of DMC extraction is in the
range of 90–92%. Thus the extractability of oil as well as
karanjin of DMC from the expelled cake is comparatively
better than those of IPA and TOL extraction. At the same
time, DMC did not extract any water soluble components,
which makes the extraction process cleaner and environmen-
tally friendly. Thus, DMC is found to be the best solvent for
the extraction of karanjin from the expelled cake.
Table 1. Comparative study of extraction of karanjin from the
expelled cake (300 g) using three different extracting solvents
(1.0 L)
Extract (g) TOL DMC IPA
Crude extract from cake 40.4  2.1a 38.9  1.8a 40.7  1.2a
Oil extracted 23.4  0.8a 28.2  1.2b 23.4  1.0a
Ethyl acetate soluble extract 7.0  3.2a 9.9  0.8a 7.3  2.1a
Crystallized karanjin 0.8  0.2a 1.2  1.0a 0.73  0.8a
Values are mean  SD, n ¼ 3; values in the same row with common
superscripts are not significantly different at p<0.001.
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1100 R. S. Susarla et al.
Accordingly, different extraction conditions such as
temperature, the amount of DMC and extraction time were
varied to maximize the extraction of karanjin. All extractions
during optimization were conducted in duplicate and the
results are shown as average of two extractions. The optim-
ization results are based on residual oil extractability, amount
of ethyl acetate solubles, and the karanjin content in ethyl
acetate solubles (as per GC). For the optimization of
temperature, extractions were carried out at different tem-
peratures (30, 50, 70, and 908C) for 8.0 h using 1.0 L of
DMC on 300 g of expelled karanja cake containing 12.8%
residual oil. Effect of temperature on the extractability of oil
as well as karanjin from the expelled cake was found to be
negligible. Henceforth, the extraction was conducted at RT,
i.e., at 308C.
In a similar manner, the quantity of DMC needed for
efficient extraction of karanjin was also optimized by con-
ducting the extraction for 8 h at the same scale as mentioned
above and maintaining the optimum temperature of 308C,
but varying the volume of DMC. Though increasing the
volume of extracting solvent should have increased the effi-
ciency of extraction of karanjin, but no appreciable differ-
ences were observed (Fig. 1). Accordingly, the optimum
volume of DMC is found to be 1.0 L for 300 g of expelled
cake under the studied extraction conditions.
The extraction time was also optimized by carrying out
the extractions at the same scale mentioned above at the
optimized temperature (308C) and volume of extracting sol-
vent (1.0 L), but varying the extraction time. All three
studied parameters showed increasing trend with the increase
in extraction time. However, such increase is nominal beyond
8 h of extraction time. Thus the results obtained in Fig. 2
indicate that the optimum extraction time is 8 h for efficient
extraction of karanjin.
Residual oil extractability (g)
35 Ethyl acetate solubles (g)
Karanjin content in ethyl acetate solubles (g)
30
25
Amount (g)
20
15
10
5
0
0.5 1.0 1.5 2.0 2.5 3.0
Solvent quantity (L)
Figure 1. Optimization of solvent quantity for the extraction of
karanjin from expelled cake.
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Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101
Residual oil extractability (g)
35 Ethyl acetate solubles (g)
Karanjin content in ethyl acetate solubles (g)
30
25
Amount (g)
20
15
10
5
0
6 8 10 12 14 16
Time (h)
Figure 2. Optimization of extraction time for the extraction of kar-
anjin from the expelled cake.
Based on these optimized results, large scale extraction of
expelled cake was carried out. Briefly, expelled karanja cake
(1.0 kg) was extracted with 3.5 L of DMC at RT (308C)
using digital overhead stirrer over a period of 8 h. The
amount of ethyl acetate-soluble extract was found to be
40 g, which is about 4.0% with respect to expelled
cake. GC analysis of this extract (after derivatization)
showed the presence of 19–20% karanjin along with
karanja FAME (48–52%), lanceolatin (6–7%), and other
furanoflavonoids (22–26%). The extract on crystallization
from ethyl acetate produced 3.6 g of karanjin of purity
97%. The large scale extraction was conducted in duplicate.
Results obtained indicate that DMC extracts 0.3–0.4% of
karanjin with respect to expelled cake of purity in the range
of 95–97%.
4 Conclusions
A scalable process was developed for the extraction of
karanjin from the expelled karanja cake. Different solvents
as well as extraction conditions were optimized for the effi-
cient extraction of karanjin. DMC is found to be the best
among the studied solvents. The optimized extraction time
is 8 h at ambient temperature (308C) with 3.5 L of DMC
for 1.0 kg of expelled cake containing 12.8% residual oil.
The extraction yielded 3.6 g of karanjin of 97% purity. The
reported process can be easily adopted by the karanja-based
biodiesel industry. The left over cake can be taken up for the
removal of other antinutritional components. The process
will add value not only to karanjin but also to deoiled karanja
cake.
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Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101
Authors gratefully acknowledge the financial grant received from
the Department of Science and Technology, Govt. of India under
Technology System Development (TSD) Program.
The authors have declared no conflict of interest.
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