Applied Energy 93 (2012) 245250

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Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Experimental assessment of toxic phorbol ester in oil, biodiesel and seed cake
of Jatropha curcas and use of biodiesel in diesel engine
Lalit Prasad a, Subhalaxmi Pradhan b, L.M. Das a,, S.N. Naik b
a
b

Centre for Energy Studies, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India
Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India

a r t i c l e

i n f o

Article history:
Received 9 February 2011
Received in revised form 10 June 2011
Accepted 27 November 2011
Available online 5 January 2012
Keywords:
Biodiesel
Phorbol ester
Dehulled cake
Transesterication
Performance study

a b s t r a c t
The present study deals with estimation of toxic phorbol esters in Jatropha curcas oil, cake and biodiesel
and performance emission of different blends of biodiesel in diesel engine. The jatropha seed was collected from Chattishgarh, India and oil content of the seed kernel was 56.5%, determined by soxhlet apparatus. The oil was subjected to biodiesel preparation by twin step method of acid esterication followed
by alkali transesterication. The total conversion of jatropha oil methyl ester (JOME) after reaction was
96.05% from proton nuclear magnetic resonance (1H NMR) studies. The phorbol esters content of oil, cake
and biodiesel was determined by high performance liquid chromatography (HPLC, Waters). The phorbol
esters content of the oil was more (2.26 0.01 mg/g) than the cake (0.6 0.01 mg/g) but no phorbol
esters peak was detected in biodiesel. The performance and emission study of the fuel blends (JB2, JB5
and JB10) with conventional diesel were tested for their use as substitute fuel for a single cylinder direct
injection diesel engine at constant speed (1500 rpm). The emissions such as CO, HC and smoke opacity
decreased whereas NOx and BSCF increased with biodiesel blends.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Biofuels offer prominent alternative to fossil fuels, but a consistent scientic framework is needed to ensure policies that maximize the merits and minimize the demerits of the utilization of
biofuels. Many countries are moving towards the partial and gradual replacement of fossil fuels with biofuels. The biofuels as an
alternate to petroleum fuels is spurred by global political, economical and environmental events, especially rising crude oil prices.
Biodiesel is gaining importance as an alternative fuel for diesel
engine due to its biodegradability, non-toxicity, renewability and
carbon neutrality. Biodiesel is the monoalkyl ester of long chain fatty
acids derived from transesterication of vegetable oils and animal
fat with short chain mono-hydric alcohols in presence of catalyst.
Due to the food verses fuel problem, use of edible oil as a fuel
source for transportation/industry may not be viable. Non-edible
oils such as Jatropha (Jatropha curcas), Karanja (Pongamia pinnata),
Castor (Ricinus communis), animal fat, waste oil and tallow can
replace vegetable oils as fuel sources. Hence the use of sustainable
non-edible oil with low cost for biodiesel feedstock can serve as an
effective alternative to reduce the cost of raw materials [1].
J. curcas is a potential source for the production of biodiesel;
however the overall economic return from jatropha-derived
Corresponding author. Tel.: +91 11 26591260; fax: +91 11 26591121.
E-mail address: lmdasgroup@gmail.com (L.M. Das).
0306-2619/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2011.11.076

biodiesel production is limited. The economic return greatly improved through a purication of nutrient-rich jatropha seed cake
into an excellent animal feed. Such processes would enhance the
value and sustainability of jatropha as a renewable energy source.
At present it is only used as a fertilizer or for biogas production [2].
The barriers to use of jatropha oil and meal as a feed is the high
toxicity; that makes them unsuitable for human or animal consumption. The toxic and anti-nutritive compounds which have
been identied include trypsin inhibitors, curcin (a lectin), avonoids, vitexine and 12-deoxyl-16-hydroxyphorbol ester [3]. Jatropha meal cannot be utilized as a feed component unless toxic
and anti-nutritional compounds such as phorbol esters, trypsin
inhibitors, and phytates are effectively removed. Phorbol esters
are the major impediment to the wide commercial use of jatropha
seeds as a feedstock. Phorbol esters have been found to be responsible for skin irritant effects and tumor promotion since they activate protein kinase C (PKC) [3,4]. The oil and isolated toxic phorbol
ester fraction showed haemolytic activity, disrupting red blood
cells in rabbits and severely irritant reaction followed by necrosis
in mice [5]. The seed oil is purgative and contains small amount
of irritant curcanoleic acid, which is related to ricinoleic acid and
crotonoleic acid, the principle active ingredients of castor oil and
croton oil respectively [6]. Biodiesel fuel exhaust emissions are
suspected of having acute adverse health effects, such as irritation
of the eyes and upper respiratory tract and the induction and
enhancement of allergic response [7]. The particulate matter

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L. Prasad et al. / Applied Energy 93 (2012) 245250

derived from biodiesel and fossil diesel fuel exhausts have cytotoxic effect [8]. To use jatropha, oil and other related by-products
requires high safety level that has not been fullled so far. To overcome these problems, estimation of toxic constituents of jatropha
oil, biodiesel and its derived products is required.
Production of biodiesel from non-edible oil seeds and its utilization in diesel engine is being actively pursed in Engine and
Un-conventional Fuel Laboratory at Indian Institute of Technology Delhi [914]. The objective of the present study is extraction
and estimation of toxic phorbol esters in J. curcas oil, cake and
biodiesel and use of biodiesel in diesel engine. Performance
and emission study of biodiesel blends such as JB2 (2% of Jatropha biodiesel + 98% Conventional diesel), JB5 (5% of Jatropha biodiesel + 95% Conventional diesel), and JB10 (10% of Jatropha
biodiesel + 90% Conventional diesel) were compared with conventional diesel in single cylinder diesel engine at constant
speed (1500 rpm).
2. Materials and methods
2.1. Procurement of sample
Jatropha seed was collected from Chattishgarh, India for the
study. The sample was cleaned manually to remove all foreign
materials. The cleaned seeds were sun dried and then dried in
hot air oven at 80 C till free from moisture. The seeds were decorticated manually to obtain kernel.
2.2. Oil content determination and characterization of oil
About 50 g of kernel was grinded for 1 min, sieved through a
2 mm sieve. For oil content determination the grinded kernel
was extracted in soxhlet apparatus using petroleum ether (boiling
point 6080 C) [15]. The extract was concentrated in rotavapor;
the residual oil was cooled and weighed. The physicochemical
properties (acid value, saponication value, unsaponiable matter
and iodine value) of the oil were determined by using ASTM methods (D-1980-67; D-1959-67; D-1962-69).
2.3. Preparation and characterization of biodiesel
JOME was prepared from seed oil by two step procedure (acid
catalyzed reaction and alkali catalyzed reaction) due to high acid
value of the oil [14]. The reaction mixture was separated in a separating funnel. The upper biodiesel layer was washed with hot distilled water (7080 C) until the resulting water from washing
become neutral. The moisture was removed from biodiesel by drying at 80 C under vacuum and passing the ester layer over anhydrous sodium sulfate. The conversion of jatropha oil to
corresponding methyl ester was determined by 1H NMR spectroscopy on a Brucker 300 MHz instrument (Brucker DPX 300, Rheinstetten, Germany). The yield of transesterication reaction was
calculated from NMR results as given in the following equation:

Percentage conversion of methyl ester C



2  AME
100 
3  ACH2

where C is percentage conversion of triglycerides to corresponding

methyl ester while AME, integration value of the protons of the
methyl esters (the strong singlet); and ACH2 , integration value of
the methylene protons respectively. The factors 2 and 3 derived
from the fact that the methylene carbon possesses two protons
and the alcohol (methanol derived) carbon has three attached protons as described by Gelbard et al. [16]. The pure biodiesel was analyzed in HPLC for detection of toxic phorbol esters.

The fuel properties such as caloric value (CV), ash point,

cloud point pour point, density and kinematic viscosity were measured by Bomb Calorimeter, Pensky-Martens Closed Cup Flash
Point apparatus, Cloud Point Pour Point apparatus, Densitometer
and Setavis Kinematic Viscometer respectively.
2.4. Extraction of phorbel ester fraction
Extraction of phorbol esters fraction from jatropha oil was
done by solventsolvent extraction method [5]. The seed oil
was extracted in methanolwater (9:1) mixture using separating
funnel. The combined methanolwater layer was concentrated in
a rotary evaporator and a viscous oily fraction separated from the
aqueous solution after concentration that was again extracted
with diethyl ether. The combined ether layer was washed with
water and evaporated. A brown viscous mass was obtained and
thin layer chromatography (TLC) of that fraction was done for
qualitative determination of toxic constituents.
The phorbol ester fraction from cake was extracted in methanolwater (9:1, v/v) mixture in an orbital shaker for 8 h at
180 rpm and 25 C. The constituents soluble in the methanol
water mixture were extracted in n-hexane by solventsolvent
extraction. The methanolwater layer was concentrated in a rotary
evaporator and a viscous oily fraction was separated from the
aqueous solution after concentration and again extracted with
diethyl ether. The combined ether layer was washed with water,
the lower water layer was discarded and the ether layer was evaporated to obtain crude phorbol esters extract.
The phorbol ester fraction of oil and cake was dissolved in
tetrahydrofuran (THF) for determination of phorbol esters by
HPLC.
2.5. Determination of phorbel ester in oil, cake and biodiesel by HPLC
Phorbol esters content of J. curcas oil, cake and biodiesel [17] was
determined by using high performance liquid chromatography
(Waters 600 HPLC system) equipped with a reverse phase C18 column (Waters Spherisorb, 5 lm, 250 mm  4 mm i.d.) Waters 2998
photodiode array detector, Waters 600 HPLC quaternary pump,
Waters inline degasser and empower software. The column temperature was controlled at 25 C and the ow rate was 1.3 mL min1.
The solvents used were 1.75 ml o-phosphoric acid (85%) in 1L distilled water (A) and acetonitrile (B). All solvents were ltered and
degassed by Waters inline degasser. The gradient used was as follows: 010 min, 60% A and 40% B; 1040 min, 50% A and 50% B;
4055 min, 25% A and 75% B; 5560 min, 100% B and then the column were adjusted to the starting condition (60% A and 40% B).
The phorbol esters (four peaks) appeared between 41 and 48 min.
The peaks were integrated at 280 nm and the results were expressed as equivalent of phorbol-12-myristate 13-acetate (sigma
chemicals), whose peak appeared at 50 min. Each analysis was conducted in three times.
2.6. Fuel performance study of JOME
Jatropha biodiesel was utilized in compression ignition engine
to study its fuel performance and emission characteristics. The test
has been performed with a 4.4 kW single cylinder, air cooled, and
four-stroke compression ignition engine. The test engine was run
at a constant speed of 1500 rpm under steady conditions. Test
was conducted in triplicate to ensure the repeatability in the data.
The test was carried out with jatropha biodieseldiesel blends (JB2,
JB5 and JB10) and compared with base diesel at varying load condition of the engine from 0% to 100% in ve consecutive steps. For
measuring of engine exhaust emission, AVL 437 Smoke meter and
AVL Digas Analyzer were used.

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L. Prasad et al. / Applied Energy 93 (2012) 245250

3. Results and discussions

3.1. Oil content determination and characterization of oil
The oil content of the kernel of the seed collected from Chattisgarh (India) is 56.5% which is within the range of oil content of jatropha seed kernel 4060% which is reported by Makkar et al. [17]. The
physico-chemical properties of the oil were given in Table 1.
3.2. Preparation and characterization of biodiesel
The acid catalyzed pretreatment reduced acid value of oil to an
extent, that the oil is suitable for alkali-catalyzed reaction. Then
the alkali used for alkali-catalyzed reaction compensates the acidity of pretreated oil and remaining part act as transesterication
catalyst. The reaction for preparation of JOME was effectively carried out, which resulted in the desired products biodiesel and glycerol layer. In Fig. 1 the signal due to methylene protons adjacent to
the ester group in triglycerides appear at 2.3 ppm and after the
reaction the methoxy protons of the methyl esters appear at
3.6 ppm. The conversion of oil to methyl ester was 96.05%, calculated from the 1H NMR results.
3.3. Determination of phorbel ester in oil, cake and biodiesel
Phorbol esters were detected in oil and cake but not detected in
biodiesel. The phorbol esters may be degraded during transesterication reaction or removed during washing. Phorbol esters are
unstable and susceptible to oxidation, transesterication and epimerization [18]. HPLC chromatogram of phorbol esters in oil is
shown in Fig. 2. The quantity of phorbol esters was amounted to
be 2.12 0.02 mg/g and 0.6 0.01 mg/g in oil and cake respectively, thus oil bears more phorbol ester than that of cake. The
phorbol esters content was varying from 0.87 to 3.32 mg/g in different varieties of jatropha seeds [17]. It is reported that J. curcas
oil contains 0.31% of phorbol esters [19] and in J. curcas meal the
level of phorbol esters is 0.13 mg/g [20].
3.4. Engine study
The fuel properties of the test blends CV, ash point, cloud
point, pour point and kinematic viscosity were tested before the
engine study and presented in Table 2.
3.4.1. Performance
The performance parameters such as brake thermal efciency
(BTE) and brake specic fuel consumption (BSFC) were measured
and plotted as shown in Figs. 3 and 4. During performance studies,
it was observed that the engine output with biodiesel blends were
comparable with base diesel fuel. However the brake thermal efciency with JB2 was comparatively less whereas specic fuel consumption was higher at all loads.
3.4.1.1. Brake thermal efciency. As load increases the brake thermal efciency for jatropha biodieseldiesel blends were increased
as expected in the base diesel. However all test blends of jatropha
biodiesel shown in Fig. 3 have marginally lower BTE compared to

base diesel. This is due to the fact that as biodiesel concentration

increases in the blends, the heating value reduces and thereby consumption of the fuel. It can also be seen from the lower caloric
value (38 MJ/kg) of the jatropha biodiesel. This effect leads to decrease of marginal BTE of all blends of jatropha biodiesel.
3.4.1.2. Brake specic fuel consumption. Variation of BSFC with load
for different dieselbiodiesel blends and neat diesel fuel is shown
in Fig. 4. All test blends of jatropha biodiesel exhibit marginally
high BSFC compared to base diesel. This is attributed to low heating value and thereby more quantity of fuel is required for same
quantity of power output. It is observed that trend of variation is
same as base diesel; however BSFC increases as biodiesel content
in the blend increases. It can be due to the fact that engine consumes more fuel with dieselbiodiesel blend than with neat diesel
fuel to develop the same power output due to the lower caloric
value of dieselbiodiesel blends.
Further oxygen improves the efciency of combustion, but it
takes up space in the blend and therefore at lower load there is almost same thermal efciency for JB2, JB5, JB10 and base diesel.
There was slight increase in the apparent fuel consumption rate
observed while operating an engine with at higher load.
3.4.2. Emissions
The exhaust emissions from engine were measured by smoke
meter and digas analyzer. The sensor of the analyzer was exposed
to the exhaust gas and the observations were recorded. The measured emissions were hydrocarbons, carbon monoxide, oxides of
nitrogen and smoke opacity. The respective variation in emissions
with different load conditions can be seen from Figs. 59.
3.4.2.1. Hydrocarbon (HC). The hydrocarbon emissions for jatropha
biodiesel blends are lower at lower and partial loads in comparison
to neat diesel (Fig. 5). This is because of better combustion of biodiesel inside the combustion chamber due to the availability of
oxygen atom in biodiesel. It was observed that at 75% load all
blends of jatropha biodiesel gives almost same hydrocarbon emission. At all loads B10 gives relatively lower HC as compared to
other blends and neat diesel.
3.4.2.2. Oxides of nitrogen (NOx). As NOx emissions are a major concern in a biodiesel fueled engine, the variations of NOx emissions
with respect to load of blends for jatropha biodiesel has been presented in Fig. 6. It was observed that at lower load there are marginal increases in NOx while at higher loads except JB2 and JB5; in
case of JB10 comparatively higher NOx formed with respect to base
diesel. Low NOx emissions are due to existence of less peak temperature as the combustion is shifted from premixed combustion to
mixed controlled combustion due to reduced ignition delay. This
can be attributed to the fact that the combustion efciency of biodiesel is higher than that of petro-diesel [21,22]. It results in lower
combustion chamber temperatures and lower NOx formation. JB5
gives the lowest NOx emissions while JB10 gives the comparatively
higher NOx emissions with respect to base diesel. It can be clearly
seen in the graph that NOx emissions show a rising trend as the
load increases and hence NOx emissions are a direct function of engine loading. This is attributed to the fact that as the load increases

Table 1
Physico-chemical properties of the oil.
Properties

Acid value
(mg KOH/g)

Saponication value
(mg KOH/g)

Unsaponiable matter (%,

w/w)

Iodine value (g/

100 g)

Density (g m/
c)

Viscosity (cSt) at
40 C

Experimental
values

9.76

189.2

1.59

97

0.91

34.3

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L. Prasad et al. / Applied Energy 93 (2012) 245250

Fig. 1. 1H NMR spectrum of jatropha oil methyl ester.

Fig. 2. HPLC chromatogram of phorbol esters extract of oil.

Fig. 3. Brake thermal efciency for diesel and biodiesel blends.

Table 2
Fuel properties of the test blends.
Fuel
blend

CV
(MJ/kg)

Density
g/cm3 at
15 C

Viscosity
at 40 C
(cSt)

Flash
point
(C)

Cloud
point
(C)

Pour
point
(C)

Diesel
JB100
JB2
JB5
JB10
Test
method

44.60
38.06
44.86
44.65
44.31
ASTM
D420

0.830
0.870
0.831
0.832
0.834
ASTM
D4052

2.86
5.14
2.87
2.90
2.93
ASTM
D445

78
150
84
87
96
ASTM
D93

5
5
4.7
4.3
3.9
ASTM
D2500

9
2
8.5
8.1
7.2
ASTM
D97

in cylinder, temperature increases and subsequent formation of

higher NOx.
3.4.2.3. Carbon monoxide (CO). As the loads increase the carbon
monoxide emissions show a decreasing trend from zero loads to
higher load which can be attributed from Fig. 7. At partial and
higher loads carbon monoxide emissions shows no rising trend
and remain stable up to higher loads. This trend of reduction in

Fig. 4. Brake specic fuel consumption for diesel and biodiesel blends.

carbon monoxide emissions was due to presence of higher oxygen

content in biodiesel which improves combustion and facilitated
better combustion.

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L. Prasad et al. / Applied Energy 93 (2012) 245250

50

Diesel

Smoke (Opacity %)

45

JB2

40

JB5

35

JB10

30
25
20
15
10
5
0

25

50

75

100

Load (%)

Fig. 5. Hydrocarbon for diesel and biodiesel blends.

Fig. 8. Smoke for diesel and biodiesel blends.

Fig. 6. Oxides of nitrogen (NOx) for diesel and biodiesel blends.

Fig. 9. Oxygen in exhaust for diesel and biodiesel blends.

4. Conclusion

Fig. 7. Carbon monoxide (CO) for diesel and biodiesel blends.

3.4.2.4. Smoke. Smoke opacity is shown in Fig. 8, as the loads increase the smoke opacity increases. It is observed that the smoke
opacity for jatropha biodiesel blends are signicantly decreased
comparison to base diesel. This is due to the fact that biodiesel
blends have higher oxygen content which improves the combustion and since biodiesel blends have negligible sulfur content
which reduces soluble organic fraction (SOF) hence low smoke
opacity.
3.4.2.5. Oxygen (O2) in exhaust. The oxygen in the exhaust of biodiesel blends is higher compared to the exhaust of neat diesel. Higher
the percentage of biodiesel blend higher is the presence of oxygen
in exhaust. In Fig. 9 it is observed that the oxygen in exhaust decreases with increase in loads. JB10 shows highest oxygen in exhaust compare to JB2 and JB5 blends; however JB2 and JB5 shows
higher oxygen in exhaust with respect to base diesel.

Biodiesel is now accepted world wide as a clean alternative fuel

for transport sector. J. curcas is a potential oil seed crop for biodiesel production due to its high oil content and good quality of
resulting biodiesel. Although both the oil and cake contain toxic
phorbol esters the biodiesel is free from it. More amount of Phorbol
esters is present in oil (2.12 0.02 mg/g) followed by cake
(0.6 0.01 mg/g). As jatropha oil is a potential source for biodiesel
preparation, huge amount of oil and cake will be generated and
hence need to be handled carefully. Precautions must be taken
when handling the oil to avoid skin contact and ingestion due to
the presence of toxic constituents. Due to the presence of toxic
phorbol ester the oil and other by-products require high safety
level for use.
The different fuel properties of jatropha biodiesel blends were
found to be close to conventional diesel. The jatropha biodiesel
blends shows performance close to diesel therefore its blends can
be used in internal combustion engine. The marginal lower brake
thermal efciency can be attributed to the fact that since caloric
value of jatropha biodiesel (38.00 MJ/kg) is lower than that of diesel
(45 MJ/kg). The substantial decrease in hydrocarbon and smoke
opacity emissions of biodiesel blends is an added advantage in
context to environmental point of view. Due to high oxygen content
and cylinder temperature, oxides of nitrogen are slightly higher for
biodiesel blends. As the load increases, NOx emission increases
which is due to higher in cylinder temperature. Carbon monoxide
(CO) emissions decrease due to better combustion in case of blends.

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