Separation and Purication Technology 116 (2013) 265270

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Separation and Purication Technology

journal homepage: www.elsevier.com/locate/seppur

Simultaneous extraction and separation of phorbol esters and bio-oil

from Jatropha biomass using ionic liquidmethanol co-solvents
Godwin Severa a, Guneet Kumar b, Mike Troung c, Gregory Young c, Michael J. Cooney a,
a
Hawaii Natural Energy Institute, University of Hawaii, Honolulu, HI 96816, United States
b
Suganit Systems Inc., Reston, Virgina, United States
c
Department of Chemical Engineering, San Jose State University, CA 95192, United States

a r t i c l e i n f o a b s t r a c t

Article history: This work evaluates the applicability of an ionic liquidmethanol co-solvent system to extract phorbol
Received 31 January 2013 esters from jatropha biomass. The extracted phorbol esters readily solubilized into the co-solvent due
Received in revised form 30 May 2013 to a shared amphiphilic nature. Methyl sulfate anion had superior performance relative to acetate anion,
Accepted 2 June 2013
extracting and solubilizing higher quantities of phorbol esters. Compared to traditional methods which
Available online 7 June 2013
only extract phorbol esters, the co-solvent system simultaneously extracted and separated the bio-oil
to its own separate immiscible phase and with no appreciable loss of protein the majority of protein
Keywords:
remaining with the biomass and only a small residual amount (1 mg/ml) solubilizing into the co-sol-
Phorbol esters
Ionic liquids
vent. The optimum co-solvent concentration for simultaneous extraction of phorbol esters and bio-oil
Jatropha was determined to be 30 wt% IL and 70 wt% methanol. With respect to the extracted kernel biomass,
Bio-oil the high proportion of protein (6870%) and low phorbol esters (0.15 mg/g kernel) content suggested a
Solvent extraction high suitability as an animal feed.
2013 Elsevier B.V. All rights reserved.

1. Introduction detoxied de-lipied jatropha seed cake through solvent extraction

Jatropha biomass has long been promoted as a useful source of
renewable bio-oil feedstock for production of biodiesel. Similar to
all oil-seed feed stocks the development of a successful bio-oil
from oil seed platform depends upon having a viable value added
market for the de-lipied biomass. The soybean industry, for
example, is heavily subsidized by the sale of the de-lipied seed
cake for consumption by humans or animals [1]. Similar use of
de-lipied jatropha biomass is limited, however, by the presence
of toxic components such as phorbol esters and lectins [2,3]. Phor-
bol esters are toxic compounds that cannot be deactivated by heat
treatment [4,5] and cause a variety of biological effects including
tumor promotion and inammation, even at very low concentra-
tions. Consequently they must be removed or deactivated before
the biomass can be consumed [68]. This limitation has fostered
arguments against the development of a bio-oil from jatropha oil-
seed market, which is unfortunate given the high nutritional con-
tent of jatropha biomass (the de-lipied seed kernel meal contains
about 60% proteins). More, the anti-pesticidal activity of the phor-
bol esters gives added potential for their use as natural pesticides
[5,7,9].
There have been several studies on the detoxication of jatro-
pha biomass. For example, Martinez-Herrera et al. has partially
Corresponding author. Tel.: +1 (808) 956 7337; fax: +1 (808) 956 2336.
E-mail address: mcooney@hawaii.edu (M.J. Cooney).
with a combination of alcohols, NaHCO3 and heat treatment [5].
Haas and Mittelbach reduced the phorbol esters in jatropha oil by
use of strong bases and bleaching agents [10]. Unfortunately these
treatments have not materialized in commercial settings mostly
because the treatments do not fully reduce active phorbol esters
concentrations to non-toxic levels. The multiple steps involved in
the processing of the biomass up to the detoxication stage are
also costly. In this work we address these limitations by presenting
the application of an ionic liquidmethanol co-solvent system that
both extracts and solubilizes the phorbol esters with the simulta-
neous separation of the extracted bio-oil to a separate immiscible
phase.
While co-solvents mixtures of room temperature ionic liq-
uids (RTILs) diluted with polar covalent molecules such as
methanol have been previously investigated for their ability to
extract and auto partition bio-oil from a variety of biomass
sources [1113], to our best knowledge this is the rst report
of its kind on the co-solvents application to the extraction of
phorbol esters from bio-oil rich biomass. Specically, we report
on the co-solvents capacity to extract phorbol esters from jatro-
pha biomass and on the extent to which the properties of the
co-solvent system can be ne-tuned to optimize the co-extrac-
tion of both the phorbol esters and bio-oil into separate phases
in a single step. Finally, the protein content of the detoxied
biomass is also measured to characterize its potential as an ani-
mal feed.

1383-5866/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2013.06.001
266 G. Severa et al. / Separation and Purication Technology 116 (2013) 265270
2. Experimental
2.1. Materials and reagents
Jatropha seeds were obtained from University of Hawaii,
Department of Tropical Plant and Soil Science. The seeds were
de-shelled and the kernels ground up in a blender prior to being
treated with co-solvent. 1-Ethyl-3-methylimidazolium methyl sul-
fate ([C2mim][MeSO4]), 98% (Aldrich), 1-ethyl-3-methylimidazo-
lium acetate ([C2mim][Ac]), 97% (Aldrich), phorbol 12-myristate
13-acetate (Sigma), methanol (Fisher scientic), acetonitrile HPLC
grade (Fisher scientic), dichloromethane (SigmaAldrich), hexane
(Fisher scientic), modied Lowry protein assay kit (Thermo scien-
tic) , Biuret total protein reagent (Sigma), Bradford protein assay
kit (Thermo scientic) and total Kjeldahl nitrogen reagent set
(Hach) were used as received from manufacturer. Deionized, l-
tered water was used to make aqueous solutions, resistivity
18 MX cm (Barnstead E-pure water purication system).
2.2. Co-solvent extraction process
The co-solvent extraction of phorbol esters and bio-oil was per-
formed on biomass using the solvent technique of Young et al. [11].
The ionic liquid ([C2mim][MeSO4] or [C2mim][Ac]) was mixed
with methanol at different weight ratio to a nal total mass of
6.6 g. At least 0.4 g of biomass was added to the co-solvent to ini-
tiate the extraction process (usually performed at 64 C unless
otherwise stated). All biomass-co-solvent mixtures were agitated
for 22 h in a sealed test tube. Thereafter the extraction mixture
was centrifuged for 10 min at 6000 g to leave three layers; a top li-
pid phase, a middle co-solvent phase, and a bottom solid biomass
phase. The combined top lipid and middle co-solvent phases were
then separated from the bottoms phase by decanting and then
washed three times with 5 ml of hexane to produce a top lipid en-
riched hexane phase and a lower lipid-free co-solvent phase. The
lower lipid free co-solvent phase were pooled together and set
aside at room temperature until further analysis. The lipid en-
riched hexane phases were pooled together and then rinsed once
with 5 ml of water to remove any residual polar compounds. The
water washed lipid enriched hexane phase was then set aside until
further analysis. The bottom biomass phase was washed three
times with 5 ml of water and lastly with 5 ml of methanol to re-
move any traces of ionic liquid. The washed biomass was then
dried at 80 C and stored until further analysis.
2.3. Phorbol esters assay
2.3.1. Controls
Two methods to determine the absolute amount of phorbol es-
ters in the untreated jatropha kernel (i.e. control) were evaluated:
solvent extraction into methanol or dichloromethane [14,15].
Dichloromethane has been widely used in literature for phorbol es-
ter extraction. The methanol extraction process involved reuxing
1 g of the ground untreated kernel in 8 ml of methanol at 64 C for
22 h in sealed glass tubes, after which the samples were ltered
through 0.2 lm lters to recover the phorbol esters ltrate. The
phorbol esters in the untreated kernel were also extracted into
dichloromethane using a variation of the method reported by Mak-
kar et al. [6]. Specically, the ground kernel-dichloromethane mix-
ture was reuxed at 45 C for 3 h as opposed to being ground in a
mixture of sand and dichloromethane. The mixture was then vac-
uum-ltered over Whatman lter paper and the ltrate recovered.
The residual phorbol esters remaining in the biomass residue
(recovered from the Whatman lter paper) was extracted using
ultrasonic treatment in 50 ml of dichloromethane for 3 min. This
mixture was then vacuum ltered over Whatman lter paper
and the biomass residue (recovered from the Whatman paper)
rinsed once with 5 ml of dichloromethane. The recovered ltrate
was then combined with the ltrate from the previous extraction.
The dichloromethane was then removed under vacuum at 40 C
using rotor evaporator. The recovered oily product was then
washed (i.e. vortexed at 2000 rpm for 1 min) four times with an
equivalent mass of methanol. After each wash the methanol phase
was removed and pooled with the other methanol washes. The
combined methanol solution was then ltered using a 0.2 lm
syringe lter before injection on HPLC to determine the mass of
extracted phorbol esters.
2.3.2. Samples
The quantity of phorbol esters in the top bio-oil phase, the mid-
dle co-solvent phase, and the bottom biomass phase was measured
as follows. The top bio-oil phase (Section 2.2) was rst washed four
times with an equivalent amount of methanol (w/w) and the
washes pooled together [10] and the methanol ltrate then con-
centrated under rotavap to approximately 1.5 ml (because of smal-
ler initial sample sizes). The middle co-solvent phase (Section 2.2)
was analyzed directly. The dried bottom biomass phase (Sec-
tion 2.2) was treated exactly like the controls (Section 2.5.1) except
for the case of methanol ltrate which was concentrated under rot-
avap to less than 1 ml. All nal solutions were ltered using 0.2 lm
syringe lter before injection on HPLC.
2.3.3. HPLC analyses of phorbol esters
All solutions were injected onto a reverse phase Eclipse XDB
C18 column (125  4.6 mm, 5 lm particle size) maintained at
room temperature. The carrier solvent was a mixture of 80% aceto-
nitrile and 20% deionized water pumped isocratically at a ow rate
of 1 ml min 1. Detection was measured in absorbance mode at
280 nm using a variable wavelength detector (Cole Parmer,
DVW-10 with D2/W lamp and path length of 7 mm). The quantity
of phorbol esters per gram of sample was expressed as the equiv-
alent of pure phorbol 12-myristate 13-acetate standard [10]. The
phorbol esters peaks had retention times between 9 and 14 min
and the phorbol esters standard had a retention time of 28 min.
2.4. Bio-oil yield assay
The bio-oil yields from the jatropha kernel was calculated by
weighing the amount of bio-oil recovered from the pooled hexane
washes of the top lipid and the middle co-solvent phases (Sec-
tion 2.2) [11].
2.5. Protein assays
Protein assays were executed on untreated biomass, the middle
co-solvent phase, and the treated bottom biomass phase. To deter-
mine the most appropriate assay for each sample, several were ini-
tially performed and evaluated for accuracy. These included
Kjeldahl, Bradford, Biuret and Lowry assays [16]. As the Lowry
and Biuret assays were found to interact with the ionic liquid (data
not shown) the Bradford and Kjeldahl assays were selected for the
co-solvent and treated bottom biomass phases, respectively. The
protein content in the untreated biomass was measured using
the Kjeldahl method.
2.5.1. Protein analyses of untreated biomass and bottom solid phase
Approximately 0.1 g of either untreated or dried bottoms bio-
mass was rst digested using sulfuric acid and hydrogen peroxide
in a Kjeldahl digestion apparatus as per the modied Nessler meth-
od (Hach Company, Model 23130-20). During the digestion the
nitrogen bound in the protein and other organic molecules is rst
 G. Severa et al. / Separation and Purication Technology 116 (2013) 265270 267

converted to ammonia and ammonium salts [16]. The total crude ecules to efciently penetrate and disrupt internal hydrogen bond-
protein was then determined as total Kjeldahl nitrogen (TKN) ing between biomass bers. The marked drop off in extraction
(Hach method number 8075) [17]. To obtain the amount of crude yield as the ionic liquid (IL) concentration exceed 60 wt% have
protein the TKN value was multiplied by 6.25. The nitrogen to pro- been reported to occur because of the increased co-solvent viscos-
tein conversion factor is based on the assumption that a mixture of ity which comes with increasing IL concentration, an event that de-
pure proteins contains 16% nitrogen. It assumes that the non-pro- creases the ability of the co-solvent molecules to penetrate into the
tein nitrogen content in the biomass is negligible and is normally biomass interior [19].
used when the specic conversion factor is unknown as is the case At 45 wt% ionic liquid, the majority ( roughly 71% or 4.5 mg
with jatropha kernel. per gram kernel) of the phorbol esters was extracted into the
[C2mim][MeSO4] co-solvent with only sparing amounts following
2.5.2. Co-solvent layer analyses the bio-oil (0.6 mg per gram oil) or remaining in the biomass
To estimate protein content in the middle co-solvent layer, the (0.3 mg per gram de-lipied kernel). The remaining phorbol es-
co-solvent was rst ltered through an SFCA or PEM 0.45 lm lter. ters were lost to the aqueous phase during the wash steps. The
The co-solvent ltrate was then analyzed for presence of protein amount of phorbol esters remaining in the bottom biomass phase
using the Bradford method [16] using bovine serum albumin ( 0.3 mg per gram of de-lipied kernel or 0.15 mg per gram whole
(BSA) as an external standard for quantication of the protein kernel) is comparable to that found for non-toxic Mexican varieties
content. possessing around 0.11 mg per gram kernel [6], conrming the
effectiveness of the co-solvent to extract phorbol esters. The effec-
tiveness of the co-solvent to extract phorbol esters from kernel bio-
3. Results and discussion mass in a single extraction step also compares well against
traditional methods that sometimes involve several extraction
3.1. Determination of baseline control for phorbol esters analyses steps to reach the required levels of detoxication [9,15]. It is envi-
sioned that the phorbol esters will be recovered from the co-sol-
Two methods to determine the absolute amount of phorbol es- vent through a selective extraction process into a solvent that is
ters in the untreated jatropha kernel (i.e. control) were evaluated: immiscible with the co-solvent. The co-solvent will then be recy-
solvent extraction into methanol or dichloromethane [14,15]. The cled and reused to minimize environmental pollution and reduce
measurement of phorbol esters in untreated jatropha kernel using the high costs of solvent makeup.
methanol method recovered 6.3 mg phorbol esters (PE) per gram of [C2mim][MeSO4] and [C2mim][Ac] have been used for bio-oil
kernel, a value that is consistent with published results reporting extraction and pretreatment of biomass, respectively [20,21].
phorbol esters quantities as high as 6.5 mg per gram of kernel The properties of both anions are well understood and both
[18]. The use of dichloromethane, by contrast, produced only equipment and regulatory regulations exist to handle them. In
3.8 mg PE per gram kernel. Given these results the methanol meth- particular, the acetate anion has good properties towards the dis-
od was used as the baseline control against which the co-solvent solution of lignocellulosic biomass [22]. For these reasons we also
extractions were compared. evaluated the efciency of acetate anion to extract phorbol esters.
As seen in Table 1, there was less variation of phorbol esters
3.2. Extraction of phorbol esters with ionic liquidmethanol co- yields with [C2mim][Ac] concentration. However, to our surprise,
solvents the results showed that the co-solvent system with acetate as an-
ion performed less effectively, conrming the signicant inuence
The highest yield of extracted phorbol esters was achieved in of IL anion on extraction efciencies. [19]. The differences be-
pure methanol with only a slight decrease observed as the tween the two co-solvents ability to solvate the phorbol esters
[C2mim][MeSO4] concentration was increased, generally resting is likely due to differences in the hydrogen bond acceptor proper-
at a plateau until the [C2mim][MeSO4] concentration reached ties of the [MeSO4] and [Ac] anions [23]. The [Ac] anion is
60 wt%, after which the extraction yield dropped off dramatically, small, less polarizable, and highly charged and thus strongly
Table 1. The initial high extraction yields are presumed due to hydrogen bonds with the MeOH, thereby reducing its mobility.
hydrogen bonding interactions between the methanol and hydro- This likely reduces the extent to which the co-solvent interacts
xyl groups of phorbol esters, Fig. 1, that remain dominant at with the phorbol esters, especially hydrogen bonding effects with
[C2mim][MeSO4] concentrations below 60 wt%. The high methanol the hydroxyl groups of the phorbol esters. On the other hand, the
concentration are also believed to increase co-solvent permeability [MeSO4] anion is a poor hydrogen bond acceptor since it is large
of the biomass due to the ability of the small sized methanol mol- and polarizable, this can cause poor hydrogen bonding with
methanol and result in highly mobile methanol molecules that
can hydrogen bond effectively with the hydroxyl groups of the
phorbol esters, facilitating its rapid extraction from the biomass
Table 1
[24]. Despite these differences, both co-solvents were able to ex-
Phorbol esters content in [C2mim][MeSO4]MeOH co-solvent after extraction of
jatropha biomass. tract the phorbol esters from the biomass presumably because
the co-solvents are amphiphilic, enabling them to interact
IL concentration (wt% in co-solvent) Extraction yield (mg PE/g kernel)
strongly with the amphiphilic phorbol esters molecules through
[C2mim][MeSO4] [C2mim][Ac] multiple interactions including hydrogen bonding, p-electron dis-
0 6.3 6.3 persion interactions and dipolar interactions. The ionic liquids
10 5.8 3.5 also induce swelling and disrupt hydrogen bonding within the
20 5.7 3.9 biomass matrix enabling smaller solvent molecules such as meth-
25 4.1 3.5
30 4.5 2.9
anol to penetrate cells walls and contact phorbol esters [24]. Gi-
45 4.5 3.7 ven the alignment between these results and previous studies
50 5.4 3.6 that suggest that extraction of target components with ionic liq-
60 5.3 3.5 uids is dependent upon the choice of anion [23], the
70 2.9 3.3
[C2mim][MeSO4]MeOH co-solvent was ultimately chosen as
90 0.8 2.9
the more optimal for phorbol esters extraction.
268 G. Severa et al. / Separation and Purication Technology 116 (2013) 265270

Fig. 1. Chemical structures of phorbol esters in jatropha biomass [28].

3.3. Optimizing co-extraction of both phorbol esters and bio-oil
While the data above would suggest that the traditional ap-
proach of using the highly polar methanol as a pure solvent to ex-
tract phorbol esters is preferred, the co-extraction of both phorbol
esters and bio-oil, however, requires that the highest possible yield
of extracted bio-oil also be achieved. To optimize this two compo-
nent extraction the relative concentration of [C2mim][MeSO4] and
[C2mim][Ac] was increased from 0 to 100 wt% whilst keeping all
other extraction parameters consistent (i.e., extraction time and
 G. Severa et al. / Separation and Purication Technology 116 (2013) 265270 269

temperature identical at 22 h and 64 C, respectively). The results

for [C2mim][MeSO4] are presented in Fig. 2. In general no correla-
tion is observed until the IL concentration reaches 60 wt% after
which both yields gradually decrease with increasing IL concentra-
tion. The key result from Fig. 2, however, is the observation that the
bio-oil yield is highly non-linear, with a maximum achieved at IL
concentrations between 20 and 55 wt%. A similar bio-oil trend
was also observed with [C2mim][Ac], Fig. 3 even though the bio-
oil yields are less than those with [C2mim][MeSO4]. Accordingly,
the [C2mim][MeSO4] concentration range for optimum simulta-
neous extraction of phorbol ester and bio-oil lies between the
range of 3050 wt%, with 30 wt% level preferred as it minimizes
the cost of ionic liquids and lowers the solution viscosity. Although
it has been previously reported that during mechanical extraction
most of the phorbol esters are extracted into the bio-oil, suggesting
that lower phorbol esters yields should have been realized at low
bio-oil extraction yields [9], most of the phorbol esters were recov-
ered in the co-solvent even at the low bio-oil extraction yields. This Fig. 3. Yields of extracted phorbol esters and bio-oil as a function of [C2mim][Ac]
wt%.
result differs from that of mechanical extraction were the quantity
of phorbol esters extracted from the biomass is dependent on the
amount of bio-oil extracted. This is due to the high extractive
and solvation power of the co-solvent for phorbol esters. 1.0 mg per ml of co-solvent. These values are consistent with those
The intriguing non-linear bio-oil yields found for jatropha were reported in literature on protein solubility in pure ionic liquids or
conrmed for additional oil-seed biomass. These results, presented in ionic liquid based aqueous two phase systems which are nor-
in Fig. 4, also showed the same surprising non-linear yield as a per- mally below 1 mg per ml ionic liquid [2527]. Even though these
centage of ionic liquid (relative to methanol) across all biomass reports suggested extraction efciencies as high as 90%, it should
sources. In general the yields began as low as 2.5 wt% in pure be noted that these reports also used samples that contained pro-
methanol with only a mild increase in yield observed as the IL con- tein at starting concentrations as low as 1 mg per ml of solvent. As
centration increased to approximately 25 wt%. Thereafter, further the jatropha biomass source in this study is rich in protein (32 wt%
increases in the ionic liquid concentration resulted in a sharp in- relative to the whole kernel), the low yields of solubilized protein
crease to maximum obtainable yields that remained at their max- actually reect a low solubility of protein in the polar co-solvent.
imum until the ionic liquid concentration approached just under More importantly, our results also show that the bulk of protein re-
60 wt%, at which point the yields steadily decreased with increas- mained with the biomass even after washing with water. It should
ing IL concentration to a nal value of approximately 16 wt% across be noted, however, that any residual ionic liquid remaining in the
all biomass sources. These results are compatible with previous re- biomass after washing with water could result in protein estimates
ports of lower bio-oil yields when using either component of the slightly above their true value. The low solubility of protein in solu-
co-solvent (i.e. IL or methanol) [11]. Molecular modeling studies tion was also conrmed by the Kjeldahl nitrogen analysis of the
(unpublished results) predict that the observed trends in co-sol- bottom biomass phase which revealed that the majority of protein
vent bio-oil extraction of oil seeds is due to solvent aggregation ef- remained with the biomass, Table 2. We believe that the high pro-
fects at different IL concentration. tein content in the de-lipied and de-toxied jatropha biomass,
even after water washes, further enhances the utility of the co-sol-
vent system as a one-step extraction process for jatropha biomass.
3.4. Protein extraction
While it was initially surprising that the medium polarity of the
co-solvent and amphiphilic nature of the ionic liquid would have
Protein analysis was performed to nd out the effect of the co-
better facilitated the extraction and solubilization of greater
solvent extraction process on the nutritive value of the biomass.
Although proteins were found to extract into the middle co-solvent
phase (Table 2) their amounts were minimal at or just below

Fig. 2. Yields of extracted phorbol esters and bio-oil as a function of Fig. 4. Yields of extracted bio-oil as a function of [C2mim][MeSO4] wt% for the
[C2mim][MeSO4] wt%. various biomass.
270 G. Severa et al. / Separation and Purication Technology 116 (2013) 265270
Table 2
Protein yields from analyses of components of co-solvent extraction.
Kjeldahl protein (wt%) Bradford protein (wt%)
a b a b
Untreated BBP BBP Co-solvent
32 68 66 1.3
BBP denotes bottom biomass phase.
The wt% protein in untreated samples is expressed relative to the untreated biomass whilst the wt% protein in BBP is expressed relative to the dry weight of the BBP phase.
a
[C2mim][MeSO4]MeOH.
b
[C2mim][Ac]MeOH.
amounts of protein, whose outer shells are also amphiphilic, the
limited extraction of protein into the co-solvent is attributed to
several factors. First, there is likely a lack of strong hydrogen bond-
ing between the co-solvent and proteins as the ionic liquids and
methanol are themselves strongly associated through hydrogen
bonding and other electrostatic interactions [23,25]. The pH and
temperature of the co-solvent could also be at values that induce
protein denaturation, conditions that would inhibit solubilization
[25]. Finally, if the proteins are initially stored in a phase associated
with the solid biomass, the conditions described above would sug-
gest good reasons why the proteins do not solubilize into the co-
solvent.
4. Conclusions
In this study, an ionic liquidmethanol co-solvent was shown
for the rst time to effectively co-extract phorbol esters and bio-
oil from jatropha kernel biomass in a single extraction step. Under
optimized conditions for simultaneous extraction of bio-oil and
phorbol esters, 30 wt% [C2mim][MeSO4] and 70 wt% methanol,
nearly all the bio-oil was extracted and auto-partitioned to sepa-
rate immiscible phase and approximately 98% of the phorbol esters
were extracted from the original biomass. The co-solvent did not
extract signicant amounts of protein which remained with the
jatropha biomass even after washing steps. In addition to achieving
effective co-extraction of both bio-oil and phorbol esters, the low
phorbol esters - high protein content of the de-lipied biomass also
possessed high potential as an animal feed.
Acknowledgements
The authors acknowledge nancial support from the Ofce of
Naval Research (N00014-10-1-0310) and SuGanit Systems Inc. for
this work. The authors would also like thank Goro Uehara and Jon-
athan Deenik for donating the jatropha seeds.
References
[1] H.E. Synder, T.W. Kwon, Soybean Utilization, Van Nostrand Reinhold Company,
New York, 1987.
[2] H.P.S. Makkar, K. Becker, F. Sporer, M. Wink, Studies of nutritive potential and
toxic constituents of different provenances of Jatropha curcas, J. Agric. Food
Chem. 45 (1997) 31523157.
[3] J. Lin, X. Zhou, J. Wang, P. Jiang, K. Tang, Purication and characterization of
curcin, a toxic lectin from the seed of Jatropha curcas, Prep. Biochem.
Biotechnol. 40 (2010) 107118.
[4] E.M. Aregheore, K. Becker, H.P.S. Makkar, Detoxication of a toxic variety of
jatropha curcas using heat and chemical treatments, and preliminary
nutritional evaluation with rats, South Pac. J. Nat. Sci. 21 (2003) 5056.
[5] J. Martinez-Herrera, P. Siddhuraju, G. Francis, G. Davila-Ortiz, K. Becker,
Chemical composition, toxic/antimetabolite constituents, and effects of
Protein concentration (mg/ml)
Co-solvent Co-solventa Co-solventb
1.8 0.792 1.108
different treatments on their levels, in four provenances of Jatropha curcas L.
from Mexico, Food Chem. 96 (2006) 8089.
[6] H.P.S. Makkar, A.O. Aderibigbe, K. Becker, Comparative evaluation of non-toxic
and toxic varieties of jatropha curcas for chemical composition, digestibility,
protein degradability and toxic factors, Food Chem. 62 (1998) 207215.
[7] G. Goel, H.P.S. Makkar, G. Francis, K. Becker, Phorbol esters: structure,
biological activity, and toxicity in animals, Int. J. Toxicol. 26 (2007) 279288.
[8] H.P.S. Makkar, G. Francis, K. Becker, Protein concentrate from jatropha curcas
screw-pressed seed cake and toxic and antinutritional factors in protein
concentrate, J. Sci. Food Agric. 88 (2008) 1542.
[9] R.K. Devappa, H.P.S. Makkar, K. Becker, Optimization of conditions for the
extraction of phorbol esters from jatropha oil, Biomass Bioenergy 34 (2010)
11251133.
[10] W. Haas, M. Mittelbach, Detoxication experiments with the seed oil from
jatropha curcas L, Ind. Crops Prod. 12 (2000) 111118.
[11] G.L. Young, F. Nippgen, S. Titterbrandt, M.J. Cooney, Lipid extraction from
biomass using co-solvent mixtures of ionic liquids and polar covalent
molecules, Sep. Purif. Technol. 72 (2010) 118121.
[12] Y.-H. Kim, Y.-K. Choi, J. Park, S. Lee, Y.-H. Yang, H.J. Kim, T.-J. Park, Y.H. Kim, S.H.
Lee, Ionic liquid-mediated extraction of lipids from algal biomass, Bioresour.
Technol. 109 (2012) 312315.
[13] Q. Huang, Q. Wang, Z. Gong, G. Jin, H. Shen, S. Xiao, H. Xie, S. Ye, J. Wang, Z.K.
Zhao, Effects of selected ionic liquids on lipid production by the oleaginous
yeast Rhodosporidium toruloides, Bioresour. Technol. 130 (2013) 330344.
[14] H.P.S. Makkar, K. Becker, B. Schmook, Edible provenances of Jatropha curcas
from quintana roo state of Mexico and effect of roasting on antinutrient and
toxic factors in seeds, Plant Foods Hum. Nutr. 52 (1998) 3136.
[15] H. Gaudani, M. Gupta, N. Gupta, S. Trivedi, P. Patil, G. Gupta, K. Vamsi, M.P.
Reddy, B.D. Sethiya, M.R. Rathod, Isolation and characterization of phorbol
esters (toxin) from the jatropha curcas L, Int. J. Microbiol. Res. 1 (2009) 17.
[16] S.S. Nielsen, Food Analysis, second ed., Aspen Publishers, Indiana, 1997.
[17] Hach Company, DR/890 Colorimeter: Data Logging Colirimeter Handbook,
ninth ed., Hach company, 2009.
[18] E. Chivindi, B. Kachigunda, F. Fushai, A comparison of the nutrient and anti-
nutrient composition of industrially processed Zimbabwean jatropha curcas
and glycine max meals, Pak. J. Biol. Sci. 8 (2005) 4953.
[19] Z. Wei, Y. Zu, Y. Fu, W. Wang, M. Luo, C. Zhao, Y. Pan, Ionic liquids-based
microwave-assisted extraction of active components from pigeon pea leaves
for quantitative analysis, Sep. Purif. Technol. 102 (2013) 7581.
[20] M.J. Cooney, G. Young, N. Nagle, Extraction of bio-oils from microalgae: a
review, J. Sep. Purif. Rev. 38 (2009) 291325.
[21] G. Brodeur, E. Yau, K. Badal, J. Collier, K.B. Ramachandran, S. Ramakrishnan,
Chemical and physicochemical pretreatment of lignocellulosic biomass: a
review, Enzyme Res. 2011 (2011) 117.
[22] M. Mora-Pale, L. Meli, T. Doherty, R. Linhardt, J. Dordick, Room temperature
ionic liquids as emerging solvents for the pretreatment of lignocellulosic
biomass, Biotechnol. Bioeng. 108 (2011) 12291245.
[23] B. Kirchner, Topics in Current Chemistry, in: B. Kirchner (Ed.), Ionic Liquids,
Springer-Verlag, New york, 2009.
[24] M.G. Bogdanov, I. Svinyarov, Ionic liquid-supported solidliquid extraction of
bioactive alkaloids. II. Kinetics, modeling and mechanism of glaucine
extraction from Glaucium avum Cr. (Papaveraceae), Sep. Purif. Technol. 103
(2013) 279288.
[25] Y. Pei, J. Wang, K. Wu, X. Xuan, X. Lu, Ionic liquid-based aqueos two-phase
extraction of selected proteins, Sep. Purif. Technol. 64 (2009) 288295.
[26] L. Ge, X.-T. Wang, S.N. Tan, H.H. Tsai, J.N.H. Yong, L. Hua, A novel method of
protein extraction from yeast using ionic liquid, Talanta 81 (2010) 18611864.
[27] D.-H. Cheng, X.-W. Chen, Y. Shu, J.-H. Wang, Selective extraction/isolation of
hemoglobin with ionic liquid 1-butyl-3-trimethylsilylimidazolium
hexauorophosphate (BtmsimPF6), Talanta 75 (2008) 12701278.
[28] W. Haas, H. Sterk, M. Mittelbach, Novel 12-deoxy-16-hydroxy phorbol diesters
isolated from the seed oil of Jatropha curcas, J. Nat. Prod. 65 (2002) 14341440.