S. N. Malviya et al.

, ARPB, 2011; Vol 1(1) ISSN 2250 - 0774

(Research paper)

VOL -1(1) ESTIMATION AND CHARACTERIZATION OF

JULY-SEPTEMBER 2011 PROTEIN PRESENT IN SEED EXTRACT OF
JATROPHA CURCAS

*S. N. Malviya, R. Malakar, M. Yadav, A. Mishra and

A. Tiwari
University Institute of Technology, Rajiv Gandhi Proudyogiki
Vishwavidyalaya Bhopal (M.P.) India

ABSTRACT:

The amino acid composition of Jatropha seed is excellent.

The Jatropha press cake contains 24–28% protein on dry
basis. The protein extracted from press cake proteins had a
Received on 15/09/2011
solubility of about 90% above pH 9. At present, Jatropha
Revised on 23/09/2011
seed meal, however, cannot be used as an animal feed
Accepted on 8/10/2011
ingredient because of the presence of several anti-nutrients
*Corresponding Author and a toxic factor and is hence put back into the soil. In the
present study, estimation of protein and characterization has
Mr. S. N. Malviya been performed by Biuret method for simple protein and by
School of Biotechnology, Lowry’s method for tyrosine, tryptophan derivatives
University Institute of (aromatic residues) of protein. Result revealed that protein
Technology, estimation by Biuret method for simple protein showed the
Rajiv Gandhi Proudyogiki absorbance 0.669 and 0.695nm, the concentration of protein
Vishwavidyalaya is 68-70 mg/ml and by Lowry’s method, tyrosine and
Bhopal (M.P.) India tryptophan derivatives showed the absorbance 1.707 and
Email: 1.809 nm, the concentration of protein is 100-110 mg/ml.
malviyasatyam@yahoo.com

KEYWORDS: Solubility, Anti-Nutrients, Tryptophan,

Jatropha

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INTRODUCTION contains approximately 24.60% of crude

protein, 47.25% of crude fat, and 5.54% of
Jatropha curcas, a member of the
moisture contents8. The Jatropha press
Euphorbiaceae family, is a versatile tree of
cake contains 24–28% protein on dry
economic importance because of its
basis. The protein extracted from press
several industrial and medicinal uses
cake proteins had a solubility of about
Jatropha curcas is a drought resistant
90% above pH 9. They suggested that the
tropical tree and the oil from its seeds has
detoxified J. curcas seed cake has
been found useful for medicinal and
potential to be exploited as a novel source
veterinary purposes, as insecticide, for
soap production and as a fuel substitute1,2. of functional protein for food
9
The seed which is black and oval in shape applications .
is rich in fixed oil2. The amino acid Botanical Features
composition of Jatropha seed protein is It is a small tree or shrub with
also good. At present, Jatropha seed meal,
smooth grey bark, which exudes whitish
however, cannot be used as an animal feed
colored, watery, latex when cut. Normally,
ingredient because of the presence of
it grows between three and five meters in
several anti-nutrients and a toxic factor
height, but can attain a height of up to
and is hence put back into the soil3. The
eight or ten meters under favorable
Jatropha kernel contains 400–600 gm/Kg
conditions. It has been estimated that the
oil, and the cake obtained after oil
life of the plant is up to 50 years.
extraction is rich in protein. The use of
Jatropha cake/meal in food or feed is Leaves
limited owing to the presence of toxic and It has large green to pale-green
antinutritional constituents4. The latex of leaves, alternate to sub-opposite, three-to-
Jatropha curcas contains an alkaloid five lobed with a spiral phyllotaxis10.
known as "Jatrophine" which is believed Flowers
to have anti-cancerous properties. It is also
The petiole length ranges between
used as an external application for skin
6-23 mm. The inflorescence is formed in
diseases and rheumatism and for sores on
the leaf axial. Flowers are formed
domestic livestock5. In addition, the tender
terminally, individually, with female
twigs of the plant are used for cleaning
flowers usually slightly larger and occur in
teeth, while the juice of the leaf is used as
the hot seasons. In conditions where
an external application for piles. Finally,
continuous growth occurs, an unbalance of
the roots are reported to be used as an
pistillate or staminate flower production
antidote for snake-bites6. The fatty acid
results in a higher number of female
composition and the positional distribution
flowers11.
of the fatty acids of individual
phospholipids were also reported. The Fruits
composition of Jatropha curcas oil from Fruits are produced in winter when
Egypt consists of many fatty acids such as the shrub is leafless, or it may produce
Palmitic 18.22 %, stearic 5.14 %, Oleic several crops during the year if soil
28.46 % and Linoleic 48.18%7. Akintayo, moisture is good and temperatures are
2004 was reported that Jatropha oil sufficiently high. Each inflorescence yields

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a bunch of approximately 10 or more process for characterization of protein is

ovoid fruits. A three, bi-valved cocci is SDS-PAGE technique have been the main
formed after the seeds mature and the approaches in this work.
fleshy exocarp dries12.
MATERIALS AND METHODS
Seeds In this study we were estimating
The seeds become mature when the the total protein, found in seed extract of
capsule changes from green to yellow, Jatropha curcas followed by their
after two to four months from fertilization. characterization. We have used various
The blackish, thin shelled seeds are oblong techniques, instruments and glassware.
and resemble small castor seeds13. Following method and protocol were used
in this research.
Varieties of Jatropha curcas
There a number of varieties of Preparation of seed extract
Jatropha. Best among these is Jatropha The seeds of Jatropha curcas
curcas. It is the most primitive form and were collected from the energy park of
has the potential to be cultivated for Rajiv Gandhi Proudyogiki
biodiesel and medicinal properties14. Vishwavidyalaya, Bhopal, planted for
Some of the others are biodiesel production purpose. Mature and
sun dried seeds of Jatropha curcas were
• Jatropha curcas (nontoxic)
collected. Soon after the collection, the
• Jatropha integrerrima seeds were cleaned by hand using a paper
• Jatropha gossypifolia towel, cracked individually without
damage, and the kernels were stored in a
• Jatropha glandulifera
plastic container at room temperature. The
• Jatropha tanjorensis dried seed kernels were well grinded to
• Jatropha multifida fine powder using a blender. Exactly
300gm of seed kernel powder was
• Jatropha podagrica measured into container and extracted with
Jatropha curcas is a very important methanol and ethanol by soxhlet apparatus
beneficial plant to the human. Its for 48 hrs and then filtered16. 23 gm and
phytoconstituents of different parts shows 26 gm semisolid mass was obtained after
different activities like antimicrobial, evaporation of 300 gm of each extract of
fungicidal, medicinal and pesticidal. methanol and ethanol and two solutions of
Jatropha seed contain rich in protein and each were prepared by 10mg/ml for
lipid that’s utilized in production of bio- protein estimation and characterization
diesel15. Some species of Jatropha shows process.
harmful effect due to the presence of Protein estimation
phorbol ester. The Jatropha protein curcin
shows the antitumor and anticancer The Jatropha protein were
properties and phytoconstituents shows the successfully extracted and estimated from
different activities. Various methods for the corresponding seeds using the principle
estimation of these proteins from the seeds of Biuret method and Lowry’s method.
have been investigated. The suitable Their isolation and characterization is done

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by Column Chromatography and Sodium silica gel was prepared in 1:1:1 ratio of
dodecyl sulphate polyacrylamide gel Acetone: Hexane: Methanol solvent
electrophoresis (SDS-PAGE). Biuret system. The slurry was prepared into the
method is based on copper ions binding to column for settling the silica gel. After
peptide bonds of protein under alkaline settlement of silica gel more solvent was
conditions to give a violet or purple color. added to top of the column, repeat this
The intensity of the charge transfer enough times so that all of the silica gel is
absorption bond resulting from the Cu- wet and it begins dripping out the bottom.
protein complex is linearly proportional to About 5 ml of seed extract of Jatropha
the mass of protein present in the solution. was loaded on the column, 2ml of solvent
The chromophore or light-absorbing center was added drop wise in the column. Elute
seems to be a complex between the peptide of the column was collected at every 5
backbone and cupric ions. The Folin assay minutes time intervals. The TLC was
(Lowry method, 1951) is dependent on the performed for separation and identification
presence of aromatic amino acids in the of proteins presents in the seed extract.
protein. First, a cupric/peptide bond
Determination of molecular weight of
complex is formed and then this is
Protein by SDS-PAGE of the partially
enhanced by a phosphomolybdate complex
purified Protein:
with the aromatic amino acids. The
phenolic group of tyrosine and tryptophan SDS-PAGE was performed by
residues (amino acid) in a protein will the method of Laemlli18, 1970 in order to
produce a blue purple color complex, with determine the molecular weight of
maximum absorption in the region of 600 partially purified protein. SDS is an
nm wavelength, with Folin- Ciocalteau anionic detergent which denatures protein
reagent which consists of sodium tungstate by wrapping around the polypeptide
molybdate and phosphate. Thus the backbone. On an average one SDS
intensity of color depends on the amount molecule binds for every two amino acid
of these aromatic amino acids present and residue. SDS confers a negative charge to
will thus vary for different proteins. Most the polypeptide in proportion to its length,
proteins estimation techniques use Bovin i.e., the denatured polypeptide becomes
Serum Albumin (BSA) universally as a rods of negatively charged clouds with
standard protein, because of its low cost, equal charge or charge densities per unit
high purity and ready availability17. length. Samples to be run on SDS-PAGE
are firstly boiled in sample buffer
Isolation of Protein by Column containing β-mercaptoethanol and SDS.
Chromatography The mercaptoethanol reduces any disulfide
Chromatography is based on bridge present that is holding together the
mobile phase and stationary phase. It is a protein tertiary structure, and the SDS
separation technique in which a mobile binds strongly, and denatures the protein.
phase carrying a mixture is caused to move In denaturing SDS-PAGE separation,
in contact with a selectively absorbent therefore migration is determined not by
stationary phase. intrinsic electrical charge of the
The column of chromatography polypeptide but by molecular weight.
was plugged with cotton. The slurry of

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SDS-PAGE gel electrophoresis 540nm respectively (Table-1). The

was performed on resolving and stacking concentration of protein was calculated to
gel at 12.5% and 4% concentrations of be 68-70 mg/ml in seed extract of
acrylamide: bis-acrylamide respectively. Jatropha (Fig.1).
The MW (Da) of protein was determined
Table 1: Spectrophotometric absorbance
by using standard molecular weight
of Biuret method
markers. 6µl of the Laemmli dye or
sample buffer added to 12µl of partially Tube BSA Absorbance
purified protein, MW markers and No. (mg/ml) (540nm)
standard protein separately and heated for 1 0 0.102
5 min at 95oC in a water bath. 9µl of each 2 20 0.206
mixture was injected to the stacking gel.
3 40 0.398
The electrophoresis unit (Banglore GeNei)
was filled with the 1x running buffer and 4 60 0.416
the electrophoresis was carried out at 5 80 0.797
200V. Gels were placed in the staining
6 100 0.988
solution for 2-3 hr at 37oC with agitation,
followed by its destaining in the destaining 7 Test 0.669
solution with agitation, overnight. The 8 Test 0.695
destained gels were immediately
photographed.
RESULTS AND DISCUSSION Lowry,s Method:

The present study was focused on The aromatic amino acid of

estimation and characterization of proteins Jatropha protein react with Folin-
from the seed extract of Jatropha curcas. Ciocalteau reagent (Lowry,s reagent) and
The Jatropha protein were successfully form a complex which is blue purple color
extracted and estimated from the complex17. In Lowry’s method the
corresponding seeds using the principle of Jatropha extract showed the absorbance at
Biuret method and Lowry’s method and 1.707 and 1.809 nm (Table-2). The
their characterization is done by Column concentration of protein is 100-110 mg/ml
Chromatography and Sodium dodecyl (Fig. 2). This suggests that, the folin-
sulphate polyacrylamide gel ciocalteau reagent react with aromatic
electrophoresis (SDS-PAGE). residues of protein and yields a blue colour
which is turn in red. The study conducted
The seed extract of Jatropha by Becker et al., 2009 the simple protein
contains high concentration of protein. and tyrosine-tryptophan derivatives,
These proteins show different medicinal proteins are present in similar percentage,
activity19. In our study we found that and similar results were observed in our
tyrosine, trytophan derivative of protein study. The protein composition of
and simple protein are present in seed Jatropha kernel was 68-70 mg/ml in
extract of Jatropha. By the Biuret method Biuret method and 100-110 mg/ml in
the Jatropha extract showed the Lowry’s method of protein estimation. The
absorbance at 0.669 and 0.695 nm at most of the parameters of Jatropha curcas

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1.2

Absorbance Vs Concentration
1 0.988
Absorbance (540 nm)

0.8 0.797

0.66
0.6 9 0.695

0.4 0.398 0.486

0.2 0.206
0.102
0
68&70
0 20 40 60 80 100
mg/ml
Concentration (mg)

Fig.1 Graph Curve of Protein Estimation by Biuret Method

investigated in the present study have been position of the molecular markers on the
compared with the study done by Becker gel. SDS-PAGE of purified protein extract
et al., 2009. Herrera et al. 2006 was revealed bands. The SDS-PAGE was used
reported that the crude protein contents of to determine the molecular weight profile
defatted kernels of Jatropha are 31–35% for the isolated proteins. The SDS-PAGE
in Coatzacoalcos and 65.0% in Castillo de patterns of Jatropha proteins (Fig.3)
Teayo16.The amino acid composition of J. contained one major band indicating the
platyphylla kernel meal was almost similar presence of proteins which is directly
to that in kernel meal of J. curcas. The proportional to the molecular marker of
comparison between the amino acid weight 36 KDa. The molecular weight of
composition of Jatropha meals and SBM the protein subunits were determined by
revealed an almost similar pattern for all comparing the patterns from Jatropha
essential amino acids, except lysine and proteins to those from the 60 KDa
sulphur amino acids. Contents of sulphur molecular weight marker. Becker et al.
amino acids were higher in kernel meals of reported that the determination of the
Jatropha species. molecular weight by using SDS-PAGE
indicates that top band contained proteins
SDS-PAGE for molecular weight
between 30 and 45 KDa19.The partially
determination
purified protein was resolved in between
The molecular weight of the protein was the molecular weight marker of 40 KDa
determined with the help of relative and 30 KDa (fig. 3).

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2
Absorbance Vs Concentration
1.8 1.809
Absorbance(600nm)

1.707
1.6
1.503
1.4
1.2 1.206
1 0.996
0.8
0.6 0.605
0.508
0.4
0.304
0.2
0.102
0
0.5 10 20 40 60 80 100 Test Test
Concentration (mg) (1ml) (1ml)

Fig.2 Graph Curve of Protein Estimation by Lowry’s Method

is approximately 36 KDa which is the

purified protein of Jatropha seed extract.
The seed extracts of Jatropha
curcas contains valuable energy source in
the form of proteins, carbohydrates,
soluble, non-soluble sugar. In this study,
we have estimated and characterised these
important protein in the seed extracts. For
the estimation and characterisation of
these protein molecules, three solvents
were prepared i.e. acetone, hexane and
ethanol (1:1:1) solvents according to their
increasing polarity. Das et al., 2005 used
the solvents hexane, ethyl acetate &
methanol (4:1:1) in column
chromatography for the isolation of
Fig. 3 SDS-PAGE Gel Electrophoresis of protein20.
Jatropha Protein
The results obtained were similar
Lane 1- molecular marker 60 KDa size
to the results of the study done by Das et
Lane 2- SDS-PAGE of partially purified
al., 2005. According to Apiwatanapiwat
protein to determine the MW.
et al., 2009 the total crude protein present
in seed cake of Jatropha curcas was
Thus the molecular weight of this protein 18.98%21. Study conducted by Becker et

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0774
(Research paper)
al., 1998 the total protein are present in antimicrobial activity in future research.
seed extract of Jatropha curcas is 42%19. There should be development of novel
In our study the protein composition of purification system to extract the
Jatropha kernel was 70 mg/ml in Biuret antibacterial compounds present in the
method and 110 mg/ml in Lowry method seed extract of Jatropha curcas. Their
of protein estimation. The most of the appropriate isolation method may raise
parameters of Jatropha curcas the therapeutic index of antibacterial
investigated in the present study have compound present in the seed extract of
been compared with the study done by Jatropha curcas.
Becker et al., 2008. According to their
CONCLUSION
study they were not differentially
estimated the simple protein and tyrosine, The seeds of Jatropha curcas are
tryptophan derivatives (aromatic residues) valuable sources of biodiesel in Asian
of protein. In our study protein estimation countries. The seeds of Jatropha which
by Biuret method for simple protein contain proteins can be used as animal
showed the absorbance 0.669 and meal. The extraction of the protein is a
0.695nm (Table-1). The concentration of low cost process, and most importantly
protein is 68-70 mg/ml (Fig.1) in seed this rich source of protein does not
extract of Jatropha. The protein compete with proteins obtained from
estimation by Lowry’s method for other food crops such as soy and wheat. A
tyrosine, tryptophan derivatives (aromatic different extraction isolation procedure to
residues) of protein the Jatropha extract obtain proteins from the Jatropha seeds
showed the absorbance 1.707 and 1.809 and analysis techniques using like
nm (Table-2).The concentration of protein chromatography, UV-spectrophotometry,
is 100-110 mg/ml (Fig. 2). Both the SDS-PAGE electrophoresis, was applied
method shown the concentration of to confirm the characterization of the
protein is 70-110 mg/ml. proteins from the seed kernel of Jatropha.
In this study, we estimated the total
Akinpelu et al., 2009 was
proteins, their characterization, on the
reported that seed extract of Jatropha are
basis of molecular weight in seed extract
known to be biologically active and
of Jatropha curcas. The protein was
therefore aid the antimicrobial activities
purified from the seed extract of Jatropha
of J. curcas. The present study is clearly
by column chromatography. Till date,
shown that Jatropha seeds have a very
there were no study conducted on the
rich content of basic nutrients like protein.
molecular weight determination of the
Thus it can be used as nutritional
protein present in the seed extract of
supplement. As in previous study done by
Jatropha curcas and hence we have
Makkar et al., 1998 it has been shown that
confirmed the absorbance pattern of our
Jatropha curcas is non-toxic compare to
protein with the absorbance pattern of
some other species of Jatropha so it can
Bovine serum albumin. We found in our
be used for many processes without any
study, the molecular weight of the
hazardous effects22. Certain bioactive
purified protein to be 36 K Da by SDS-
compounds were also found in during PAGE. In this study, the protein partially
study which can be characterized for isolated by column chromatography from
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S. N. Malviya et al., ARPB, 2011; Vol 1(1) ISSN 2250 -
0774
(Research paper)
the seed extract of Jatropha curcas may 4. R.K. Devappa and B. Swamylingappa.
work as a good protein source for food Biochemical and nutritional
system. evaluation of Jatropha protein isolate
This protein may be advantageous prepared by steam injection heating
in improving dietary supplement products for reduction of toxic and
of human for example bournvita, complan antinutritional factors, Journal of
and protein energy product. However the Science Food and Agriculture 88(5):
bioactive compounds showed the 911–919 (2008).
antimicrobial and antibacterial activity 5. H. Usman and J.C. Osuji.
against various clinical isolates. In our Phytochemical and in vitro
study, we have shown the presence of antimicrobial assay of the leaf extract
bioactive compounds in the seed extract of Jatropha curcas, African Journal of
of Jatropha curcas. But there is also need Tradit. Complement. Altern. Med.
to characterize the bioactive components 4(4): 476-480 (2007).
which are responsible for inhibiting the
6. D. A. Akinpelu, O. A. Aiyegoro and
growth of bacteria. There should be a
A. I. Okoh. The bioactive potentials of
development of novel purification system
two medicinal plants commonly used
to extract the antibacterial compounds
as folklore remedies among some
present in the seed extract of Jatropha
tribes in West Africa, African Journal
curcas, their appropriate isolation method
of Biotechnology 8(8): 1660-1664
may raise the therapeutic index of
(2009).
antibacterial compound present in the
seed extract of Jatropha curcas. 7. S. Hawash, N. Kamal, F. Zaher, O.
Kenawi and E.I.G. Diwani. Biodiesel
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