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

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

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1098 R. S. Susarla et al. Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101

2.2 Methods converted to fatty acid methyl ester for the GC analysis of
the total extract. Thus ethyl acetate soluble extract was deriv-
2.2.1 Estimation of residual oil in karanja expelled cake atized to methyl esters by the treatment of 10% BF3-etherate
in methanol for 1.0 h under reflux condition. The derivatized
The residual oil content in the expelled karanja cake was extract was solubilized in ethyl acetate and washed with
determined using SER 148 Solvent Extractor (VELP water. The ethyl acetate-soluble was dried over anhydrous
Scientifica, Germany). The residual oil was extracted from Na2SO4, concentrated in the rotary evaporator and then
expelled cake (5.0 g) with hexane (75 mL) for 4.0 h. The injected in GC–MS and GC. GC–MS was carried out with
extractions were conducted in duplicate from the same batch Agilent 6890 N gas chromatograph connected to Agilent
of expelled cake. The mean oil content in the expelled cake 5973 mass spectrometer (Palo Alto, USA) and GC on
was found to be 12.8%. HP 6850 Series gas chromatograph equipped with a FID
detector. The GC–MS detection was performed at 70 eV
2.2.2 Extraction of karanjin from karanja expelled cake (m/z 50–550; source at 2308C and quadruple at 1508C) in
the EI mode attached with HP-1 ms capillary column
For the extraction of karanjin from expelled cake, a digitally (30 m
0.25 mm
0.25 mm). The oven temperature
controlled overhead stirrer attached with four bladed paddle was programmed for 2 min at 808C, raised to 3008C at
type stirrer (Heidolph, Germany) and reflux condenser was 108C/min, and finally maintained at 3008C for 15 min.
used. Expelled karanja cake (300 g) containing 12.8% of The carrier gas, He flows at 1.0 mL/min, and the split ratio
residual oil was extracted using three different organic sol- is maintained at 50:1. Structural assignments were based on
vents at RT (308C) over a period of 8 h using digitally interpretation of mass spectrometric fragmentation. The
controlled overhead stirrer. The contents were filtered and GC was performed in HP1 capillary column (30 m

the organic solvent was removed from the filtrate to obtain the 0.25 mm
0.5 mm) and the oven temperature program-
crude extract. The crude extract was further extracted thrice ming is similar to that of GC–MS with the injector and
with 10% aqueous methanol (3
150 mL). The solvent was detector temperature maintained at 300 and 3258C. The
removed from the extract using rotary evaporator and dried carrier gas, N2 flows at, 1.5 mL/min, and the split ratio
under reduced pressure to get the crude polar extract. The was 50:1.
crude polar extract was solubilized in ethyl acetate (200 mL)
and washed with water. The ethyl acetate-soluble extract was 2.2.4 Statistical analysis
dried over anhydrous sodium sulfate. The organic solvent was
removed from the extract using rotary evaporator and dried The data, presented as mean  SD, were analyzed by a
under reduced pressure to get karanjin-rich extract, which on paired Student’s t-test to evaluate the level of statistical sig-
crystallization from ethyl acetate produced karanjin, as white nificance. A p-value of less than 0.05 was considered
solid having melting point in the range of 161–1638C. The significant.
structural confirmation of karanjin was based on NMR, IR,
and mass. 1H and 13C NMR spectra were recorded on 300 3 Results and discussion
and 75 MHz (Varian, Palo Alto, USA) spectrometer,
respectively, and IR on a Perkin Elmer (Model: Spectrum Karanjin is the principal furanoflavonoid present in the kar-
BX) FT-IR spectrometer. Mass spectra was recorded on anja seed. It is known to possess many potent biological
Agilent 5973 mass spectrometer (Palo Alto, USA) in the activities, important among them are insecticidal and mos-
EI mode and are given in mass units (m/z). The spectral quito repellant activities [9, 10]. Most of the applications of
data matched well with those reported in the literature karanjin are accomplished either as oil or as cake in utilizing it
[12]: IR (cm1, KBr): n 1640–1620, 1580–150, 1160– as natural biopesticide and also for their mosquito larvicidal
1150; 1H NMR (CDCl3; 200 MHz): d 3.94 (s, 3H), 7.16 activity [13–15]. Isolation of karanjin from oil and roots are
(d, J ¼ 2.3 Hz, 1H), 7.52 (m, 4H), 7.75 (d, J ¼ 2.3 Hz, reported in the literature [2–4, 16]. However, all these
1H), and 8.15 (m, 3 H); 13C NMR (75 MHz; CDCl3): d methods require a column chromatographic purification at
60.4, 104.5, 110.2, 117.2, 119.9, 122.0, 126.4, 128.6, 128.9, some stage. Moreover, there is no report of its isolation from
130.4, 130.9, 142.0, 146.0, 150.1, 155.0, 158.3, 175.3; expelled cake. As karanja oil being identified as ingredient for
EI–MS (m/z): 292, [M]þ, 291 [M-1]þ. the preparation of biodiesel in India, it is important to add
value to all of its major and minor byproducts.
2.2.3 Estimation of karanjin in ethyl For every ton of production of karanja-based biodiesel,
acetate-soluble extract about 2 tons of expelled karanja cake will be generated. In
general, edible oilseed cake has applications as a source of
Karanjin was analyzed qualitatively using GC–MS and quan- protein for human consumption, as animal feed, and also as
titatively by GC. TLC profile of ethyl acetate soluble extract fertilizer. All these applications of edible oilseed cake cannot
showed the presence of free fatty acids and needs to be be extended to nonedible oilseed cakes such as karanja, due to

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Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101 Karanjin from expelled karanja cake 1099

the presence of several antinutritional constituents [8]. On protocol is shown in Scheme 1. As karanjin is soluble in
the other hand, there is a need to improve the economics of oil, the solvent which has better oil extractability will extract
biodiesel production. Expelled cake is the major by-product more karanjin. This has necessitated the determination of oil
of the karanja-based biodiesel industry. During expelling, extractability of these three solvents, which was done by
karanjin gets enriched in deoiled cake. It is worthwhile to estimating the oil content in the left-over cake.
have an appropriate process for the isolation of pure karanjin A comparative study of extraction of expelled cake
from the expelled cake in order to have its effective utilization (300 g) containing 12.8% residual oil using all three solvents
as natural insecticide. Hence the present study reports (1.0 L) were conducted at 308C for a period of 8 h.
a simple solvent partitioning approach for the isolation of Extractions were conducted in triplicates and the results
karanjin from the expelled cake avoiding column chromato- are shown as average of three extractions (Table 1). There
graphic separation at any stage. During expelling, about is no significant difference in the weight of crude extract
8–15% of oil gets retained in the cake depending upon obtained using the three solvents. However, the oil content
the type and efficiency of expeller unit. It is necessary to in the left-over cake was found to be 3.4, 4.9, and 5.0%,
estimate the oil content in the expelled cake in order to assess respectively, when the extractions of expelled cake were car-
the extractability of different solvents for oil as well as ried out using DMC, TOL, and IPA. Thus, out of 12.8% of
karanjin. The expelled cake in the present study was found residual oil present in the expelled cake, DMC has extracted
to contain 12.8% of residual oil. 9.4%, followed by 7.9% by TOL and 7.8% by IPA. This
As the furanoflavonoids are oil soluble, the prime objec- indicates that DMC has better oil extractability compared to
tive of the extraction was to extract as much residual oil as TOL and IPA. Thus oil extracted, as given in Table 1 is
possible from the expelled cake using a solvent which solu- determined by subtracting the oil content in the left over cake
bilize both oil and karanjin. Accordingly, three different from the residual oil in the expelled cake, which can also be
solvents, namely isopropanol (IPA), toluene (TOL), and obtained from the weight of aqueous methanol-insoluble
dimethyl carbonate (DMC) were chosen. After extraction, extract.
the organic solvent was evaporated to get the crude extract, The amount of water-soluble extract was determined
which was further extracted with 10% aqueous methanol to theoretically by subtracting the combined weights of oil
separate the major nonpolar oil from other minor polar com- extracted and ethyl acetate-soluble extract from the crude
ponents including karanjin. The methanol-soluble extract extract. Results obtained from Table 1 indicated that amount
may contain many water-soluble polar components other of water-soluble components are negligible during DMC
than karanjin depending upon the extractability of initial extraction compared to those obtained during IPA and
solvent chosen for the extraction of expelled cake. TOL extraction. Moreover, the quantity as well as purity
Evapoartion of methanol, solubilizing in ethyl acetate, and of karanjin obtained during DMC extraction is better than
the subsequent water work-up will remove those water-solu- those obtained during TOL and IPA extraction. Purity of
bles. This will eventually enrich karanjin in the ethyl acetate karanjin is in the range of 82–86% in case of TOL and IPA
soluble extract. A schematic representation of extraction 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

Scheme 1. Schematic representation of the extraction of karanjin Values are mean  SD, n ¼ 3; values in the same row with common
from the expelled karanja cake. superscripts are not significantly different at p<0.001.

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1100 R. S. Susarla et al. Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101

Accordingly, different extraction conditions such as Residual oil extractability (g)

temperature, the amount of DMC and extraction time were 35 Ethyl acetate solubles (g)
varied to maximize the extraction of karanjin. All extractions Karanjin content in ethyl acetate solubles (g)
during optimization were conducted in duplicate and the 30

results are shown as average of two extractions. The optim-

ization results are based on residual oil extractability, amount 25

of ethyl acetate solubles, and the karanjin content in ethyl

Amount (g)
acetate solubles (as per GC). For the optimization of 20

temperature, extractions were carried out at different tem-

peratures (30, 50, 70, and 908C) for 8.0 h using 1.0 L of 15
DMC on 300 g of expelled karanja cake containing 12.8%
residual oil. Effect of temperature on the extractability of oil 10
as well as karanjin from the expelled cake was found to be
negligible. Henceforth, the extraction was conducted at RT, 5
i.e., at 308C.
In a similar manner, the quantity of DMC needed for 0
6 8 10 12 14 16
efficient extraction of karanjin was also optimized by con-
ducting the extraction for 8 h at the same scale as mentioned Time (h)
above and maintaining the optimum temperature of 308C,
but varying the volume of DMC. Though increasing the Figure 2. Optimization of extraction time for the extraction of kar-
anjin from the expelled cake.
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. Based on these optimized results, large scale extraction of
The extraction time was also optimized by carrying out expelled cake was carried out. Briefly, expelled karanja cake
the extractions at the same scale mentioned above at the (1.0 kg) was extracted with 3.5 L of DMC at RT (308C)
optimized temperature (308C) and volume of extracting sol- using digital overhead stirrer over a period of 8 h. The
vent (1.0 L), but varying the extraction time. All three amount of ethyl acetate-soluble extract was found to be
studied parameters showed increasing trend with the increase 40 g, which is about 4.0% with respect to expelled
in extraction time. However, such increase is nominal beyond cake. GC analysis of this extract (after derivatization)
8 h of extraction time. Thus the results obtained in Fig. 2 showed the presence of 19–20% karanjin along with
indicate that the optimum extraction time is 8 h for efficient karanja FAME (48–52%), lanceolatin (6–7%), and other
extraction of karanjin. 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.
Residual oil extractability (g)
Results obtained indicate that DMC extracts 0.3–0.4% of
35 Ethyl acetate solubles (g)
karanjin with respect to expelled cake of purity in the range
Karanjin content in ethyl acetate solubles (g)
30
of 95–97%.

25 4 Conclusions
Amount (g)

20 A scalable process was developed for the extraction of

karanjin from the expelled karanja cake. Different solvents
15 as well as extraction conditions were optimized for the effi-
cient extraction of karanjin. DMC is found to be the best
10 among the studied solvents. The optimized extraction time
is 8 h at ambient temperature (308C) with 3.5 L of DMC
5
for 1.0 kg of expelled cake containing 12.8% residual oil.
The extraction yielded 3.6 g of karanjin of 97% purity. The
0
0.5 1.0 1.5 2.0 2.5 3.0 reported process can be easily adopted by the karanja-based
Solvent quantity (L) biodiesel industry. The left over cake can be taken up for the
removal of other antinutritional components. The process
Figure 1. Optimization of solvent quantity for the extraction of will add value not only to karanjin but also to deoiled karanja
karanjin from expelled cake. cake.

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Eur. J. Lipid Sci. Technol. 2012, 114, 1097–1101 Karanjin from expelled karanja cake 1101

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the Department of Science and Technology, Govt. of India under 77–84.
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The authors have declared no conflict of interest. Diptera). J. Entomolog. Res. 1990, 14, 44–51.
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