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PHYTOCHEMICAL AND BIOLOGICAL
EVALUATION OF DEFATTED SEEDS OF
JATROPHA CURCAS
By
MUHAMMAD NISAR-UL-HAQ
M.Phil. Thesis
DEPARTMENT OF BOTANY
UNIVERSITY OF SCIENCE AND TECHNOLOGY BANNU
Session: 2013- 2015
PHYTOCHEMICAL AND BIOLOGICAL
EVALUATION OF DEFATTED SEEDS OF
JATROPHA CURCAS
The thesis submitted to the Department of Botany University of Science and
Technology Bannu in partial fulfilment of requirements for the Degree of Master
of Philosophy in Botany.
SUBMITTED BY
MUHAMMAD NISAR-UL-HAQ
M. Phil Scholar
RESEARCH SUPERVISOR
DR. SULTAN MEHMOOD WAZIR
Professor Department of Botany,
UST Bannu
CO-SUPERVISOR
Dr. FAIZAN ULLAH
Assistant Professor
Department of Botany
UST Bannu
DEPARTMENT OF BOTANY
UNIVERSITY OF SCIENCE AND TECHNOLOGY BANNU
Session: 2013- 2015
II
AL-QURAN
The example of those who spend their wealth in the way
of Allah, is like (that) grain out of which seven ears shoot
forth(and then) each ear bears a hundred grains(i.e. they
are rewarded seven hundred times) and multiplies (still
more) for whom He Likes and Allah is Infinite, AllKnowing.
(Al -Baqara, Verse, 261)
III
DEDICATION
To my dear parents, teachers, friends and especially to my
late grandfather
Mr. Fazal-haq.
IV
TABLE CONTENTS
S.NO
CONTENTS
PAGE
NO
1
LIST OF TABLES
vi
2
LIST OF FIGURES
vii
3
ACKNOWLEDGEMENTS
4
ABSTRACT
xi
5
INTRODUCTION
1-7
6
REVIWE OF LITERATURE
8-13
7
METHOD AND MATERIAL
14-20
8
RESULTS AND DISCUTION
21-30
9
CONCLUSION
31
10
REFERENCES
32-40
viii-ix
V
LIST OF TABLES
S/O
Tittle
Qualitative Analysis of J. curcas defatted seed
Page N/O
1
2
Quantitative Analysis of J. curcas defatted seed
30
29
VI
LIST OF FIGURES
S.NO
TITLE
PAGE NO.
1. Antibacterial activity of the J. curcas defatted seed. The data represent mean of
three replicates. ……………….………....……..……
22
2. Antifungal activity of the J. curcas defatted seed. The data represent mean of
three replicates ………………………
23
3. DPPH Free radical scavenging activity by the J. curcas defatted seed. The data
represent mean of three replicates…………………………………
24
4. Effect of methanolic extract of Jatropha curcas seed on (a) seed germination (%)
(b) Germination index (c) Germination rate Index (d) Relative water content.
Means sharing common English letters are statistically
similar.………………………………………………………………... 26
5. Effect of methanolic extract of Jatropha curcas seed on (a) shoot length, (b) root
length, (c) seedling fresh weight and (d) seedling dry weight. Means sharing
common English letters are statistically similar…………….. 27
VII
ACKNOWLEDGEMENT
I bow my head before Almighty Allah, The Omnipotent, the Omnipresent, the
Merciful, the Most Gracious, the Compassionate, the Beneficent, Who is the entire and
the only source of every knowledge and wisdom endowed to mankind and who blessed
me with the ability to fulfill these requirements of this challenging task and His Prophet
Hazrat Muhammad (Sallallahu Alaihe Wasallam) who gave us the spirit to learn. With
the blessings of Almighty Allah and His Prophet (Sallallahu Alaihe Wasallam) my
efforts have been crowned with success.
I would like to take this opportunity to convey my cordial gratitude and appreciation to
my Supervisor, Dr. Sultan Mehmood Wazir (Professor Department of Botany UST
Bannu) for shaping this project. His experience and intelligence is the brain, behind this
work. I am also grateful to my Co-supervisor Dr. Faizanullah Wazir (Assistant
Professor Department of Botany UST Bannu) for his continuous co-operation and
guidance. I would like to offer bundles of thanks and pray to God for long lives of my
parents and siblings for their sacrification and co-operation.
I am also thankful to Dr. Rehmat Ali (Assistant Professor Department of Biotechnology
UST Bannu) and Dr. Mir Sadiq Shah (Assistant Professor Department of
Biotechnology UST Bannu) for his guidance and cooperation in my Lab. Work. I am
also thankful to all my friends and fellows, Mr. Adnan Khattak, Mr. Haroon Rasheed
and Mr. Nayab Ali Khan and at last all my brothers and sisters who have never left me
to walk alone in this journey of seeking knowledge.
VIII
Above all I would like to thank Almighty GOD for His free gift of life and
enabling me carry out the study.
MUHAMMAD NISAR UL HAQ
M. Phil Scholar
IX
PHAYTOCHEMICAL AND BIOLOGICAL EVOLVATION OF
DEFATTED SEEDS OF JATROPHA CURCAS
BY
Muhammad Nisar Ul Haq
(M.Phil SCHOLAR)
Approved by
Advisor:
…………………………………………
Prof.Dr. Sultan Mehmood Wazir
Department of Botany
UST, Bannu
Co Advisor:
……………………………………………
Dr. Faizan Ullah
Assistant Professor,
Department of Botany
UST, Bannu
Chairman:
………………………………………..
Dr. Sultan Mehmood Wazir
Chairman, Department of Botany
X
Abstract:
In this study, the antimicrobial, antioxidant, phytotoxic, and phytochemical properties
of defatted seeds of Jatropha curcas were evaluated. A crude methanolic extract of
defatted seeds was tested against three fungal strains—Aspergillus niger, Aspergillus
flavus, and Aspergillus fumigatus–and five bacteria: Escherichia coli and Klebsiella
pneumoniae (gram-negative), and Micrococcus luteus, Bacillus subtilis, and
Staphylococcus aureus (gram positive). The methanolic extract was diluted in
dimethylsulfoxide to final concentrations of 1, 2, 3, 4, and 5 mg/10 ml. The largest
zones of inhibition against K. pneumoniae, M. luteus, and B. subtilis were achieved
using the concentration of 5 mg/10 ml. The concentration of 1 mg/10 ml was most
effective against S. aureus and E. coli. In a 1, 1-diphenyl-2-picrylahydrazyl (DPPH)
radical scavenging assay, the 5 mg/10 ml concentration of the Jatropha seed extract
showed the strongest activity. Higher concentrations of the Jatropha seed extract (10
mg/50 ml and 5 mg/50 ml) significantly inhibited the germination of radish seeds, and
had negative effects on radish seedling relative water content, shoot length, root length,
seedling fresh weight, and seedling dry weight (p<0.05). Phytochemical analyses of the
defatted seeds detected alkaloids (7.3%), flavonoids (0.39%), and soluble phenolics
(mg gallic acid equivalents/g extract). Based on these results, it was inferred that J.
curcas seeds contain active ingredients that are effective against pathogenic microbes,
and therefore could be used to formulate drugs to treat various diseases.
XI
Phytochemical and Biological evaluation of defatted seeds of Jatropha curcas
Introduction
From time unmemorable plants have been used in the cure of different ailments caused
by pathogenic microbes. Plant extracts are sources of a number of biologically active
compounds (Khan et al., 2011). Plants are the most significant sources of medicine.
Plant derivative compounds (phytochemicals) have been attracting much attention as
natural alternatives to synthetic compounds (Kalimuthu et al., 2010). Extracts of plants
were utilized for the cure of numerous infections and these procedures are the basis for
all traditional systems of medicine (Kalimuthu et al., 2010).
Natural products obtained from different plant sources are of remarkable medicinal
value and are used to cure various diseases. Researchers have attempted to extract the
most effective antimicrobial agents from different kinds of resources such as folk
medicinal plants (Khan et al., 2011). The freeze-dried fruit powder of mulberry has
hypolipidemic and anti-oxidant effects (Yang et al., 2010). The use of Allium sativum
preparations have great effect in cardiovascular patients having higher low density
lipids (LDL) cholesterol levels (Ried et al., 2013). From Artemisia japonica artemisene
is extracted which is used as anthelmintic in animals, another product known as
artemitrene is used as anti-malarial especially effective against Plasmodium falciparum
(Hayat et al., 2009). The significance of natural antioxidants for cosmetic and food
uses has been emphasized by various works, (Spigno and De Faveri et al., 2007).
The seeds of Guava plant are reported to contain a large number of biologically active
compounds such as phenolic compounds (Packer et al., 2010). Various seeds that were
reported to contain a greater number of phenolic compounds include apricot kernel
(Yiğit et al., 2009), mango seed (Maisuthisakul et al., 2008) and citrus seeds (Bocco et
al., 1998)
The members of family Annonaceae, (custard apple family) is a family of flowering
plants containing of shrubs, trees or rarely lianas (Slik, 2003). Bioactive compounds
1
extracted from the family members showed exciting biological characteristics like anticancer, anti- HIV effects (De Quan et al.,
1999), insecticidal effect (Ashok Kumar et
al., 2010) and beneficial cytotoxic constituents (Kingston et al.,
1992). Mature fruit is
delicious, cooling, respectable tonic and tranquillizing, supplements blood, improves
muscular stregth, and cuts burning feeling, bent to nausea and queasiness. Leaf extract
could be used for the treatment of lice (Morton et al., 1987). The leaves are also used
as styptic, anthelmintic, insecticide and the chemical constituent acetogenins existing
in the leaves are accountable for exact cytotoxic to sevral human cancers (Xu et al.,
1992).
The
bark
is
a
commanding
astringent
and
is
used
as
vermifuge
and
antidysentric. Root bark and stem of these plants have isoquinoline alkaloids (Nadkarni
et al., 2002). Phytochemical components from the plants are roemerine, anonaine,
norcorydiene,
crystalline
norisocorydine,
alkaloids
–
carvone,
muricine,
corydine,
muricinine,
linalool,
squamocin-B,
samoquasine
squamocin-I,
A,
two
motrilin,
kaurenoic acid, squamocenin, phenolic and nonphenolic alkaloids, (2,4-cis and trans)squamolinone bullacin Betc, (2, 4-cis and trans)-9-oxoasimicinone,.( Li et al., 1990;
Chopra et al., 2002).
Monoterpenoid
indole
alkaloids
characterize
a
great
class
of
natural
products
surrounding a wide range of pharmacological uses. They are mostly produced by plants
belonging to families such as Rubiaceae, Loganiaceae, Nyssaceae and Apocynaceae
and, currently, their biosynthesis is under desperate investigation (De Luca, 2011; StPierre et al., 2013). Amongst the Apocynaceae family, plants of the genus Rauvolfia
have been mainly studied for their alkaloid content (Ganapaty et al., 2001).
Reactive oxygen species (ROS), occasionally called as active oxygen species, are
numerous systems of actuated oxygen, which contain free radicals like hydroxyl
radicals (OH.) and superoxide ions besides non-free radical species for example
hydrogen peroxide (H2O2). (Yildrim, et al., 2001). Those ROS show significant role in
degenerative or pathological routes, such as coronary heart diseases, aging, cancers,
inflammations, atherosclerosis, neurodegenerative disorders cataracts
and Alzheimer’s
disease. (Huang, et al., 1998)
2
Living organisms require sufficient amount of oxygen for their metabolism and energy
production. But, free radicals are formed during the energy production process (Packer,
et al., 1999).
The role of free radical reactions in disease pathology is well-known and is recognized
to be elaborate in many severe and prolonged illnesses in human beings, such as neurodegeneration, immunosuppression, aging, atherosclerosis and diabetes (Harman D et
al. 1998). A difference among ROS and the characteristic antioxidant volume of the
body, directed the usage of nutritional and /or therapeutic additions especially during
the disease attack. Studies on medicinal plants, fruits, and vegetables have showed the
existence
of
antioxidants
such
as
flavonoids,
phenolics,
proanthocyanedens
and
tannins. The antioxidant contents of medicinal plants may contribute to the safety they
provide from infections. The absorption of natural antioxidants has been oppositely
related with injury and death rate from deteriorating disorders (Gulcin et al., 2012).
Hepatic diseases continue a thoughtful health issue. Free radicals cause cell destruction
by the process of covalent binding and lipid peroxidation with following tissue injury.
Antioxidant means of natural source have concerned distinct concern as of their free
radical scavenging capacities (Osawa T et al., 1990).
Jatropha curcas
Jatropha curcas is the member of family Euphorbiaceae commonly named as Purging
nut, Physic nut, and Barbados nut, is a bush or small tree like plant which reaches the
height up to five meters (5m). It is generally used as anti-parasitic, anti-coagulant, antitumorous, wound healing activities, insecticidal, used for abortion of pregnancy and
anti-inflammatory (Ejelonu et al., 2010). Jatrophin extracted from the latex is used in
cure of various skin diseases, rheumatism, wound healing and cough (Uche and
Aprioku et al., 2008). Moreover, the seed oil of Jatropha is highly feasible in the
production of biodiesel fuel so that is why it is also called as biodiesel plant in all over
the world (Okujagu et al., 2006; Belewu, 2008).
3
After the extraction of oil seed meal is obtained as a byproduct which can be a rich
source of various phytochemicals with higher biological activity. Earlier studies have
conveyed that it contains bioactive components which possibly will cure sexually
communicated diseases, mouth odour and jaundice, and as antiseptic during child birth
(Igbinosa et al., 2008; Namuli et al., 2011). (Rug and Ruppel 2000) also stated the
molluscicidal and larvicidal actions of the seed extracts.
Recently, (Oskoueian et al.
2011; James et al. 2011) showed that the methanolic extracts of the latex, roots, stem
bark, and leaves showed antimicrobial, cytotoxic, wound healing, antioxidant, and antiinflammatory activities. Different parts of the plant have been reported to contain
glycosides,, alkaloids tannins saponins flavonoids,, and phenolics (Thomas et al., 2008;
Oskoueian et al., 2011) and its bioactive components are thought to have potentials as
antioxidan, anticancer, anti-inflammatory, and antimicrobial t principles (Rathee et al.,
2009).
Antimicrobial Assay;
The antimicrobial assay is performed to determine the anti-bacterial and anti-fungal
activity of plant extracts (Khan et al. 2011).
The most common anti-bacterial assay is
agar well diffusion method whereas agar tube dilution process is followed to determine
antifungal test (Khan et al., 2012)
The hunt for antibacterial through natural sources have established more consideration
and struggles are put
to detect plant constituents that could perform like appropriate
antibacterials to change artificial . Phytochemicals obtained from plant constituent
assist as a sample to develop fewer poisonous and additional effective drugs in
.
suppressing the development of microbe These constituents have important salutary
usages
against
human
pathogens
encountering
bacteria,
virus
or
fungi.
Frequent
investigations were directed on chemical constituents of many plants, conducting
antibacterial activity as well as for the detection of novel antibacterial, antifungal
constituents.
So,
neutralceuticals
medicinal
and
nutrition
plants
are
Supplements.
finding
their
Though,
for
way
some
into
pharmaceuticals,
periods,
there
was
increasing attention in plant usages and the discovery of their ingredients with
4
antimicrobial
assets.
Numerous
efforts
were
made
to
extract
novel
antimicrobial
compounds from many types of sources. One of most significant source of traditional
medicinal plants and take out portions of medicinal plants. Methodical screening of
phytochemicals might end in detection of new effective constituents. Conferring to
WHO intelligences, 40% losses of human lifves were happened through communicable
germs in developing countries. In addition to infections, conservation of food is
becoming an additional difficult problem, through introduction of novel products in
market which need more safety against pathogenic microorganisms (Marino et al.,
2001).
Many antifungal agents are presently available for the cure and control of fungal
infections and diseases. The usage of these medicines as therapeutic agents however is
inadequate. This is due to various contests such as toxicity, adsorption, stability, and
drug solubility (Cedric et al., 2004). In addition, some of these drugs are costly and
usually unobtainable to citizens of developing countries, mainly those living in the
rural areas (Sule et al., 2011). The shortages in the use of chemotherapeutic agents as
control agents in fungal diseases, further boosts the use of plants as a form of alternate
medicine.
By the development in Science and Technology, exceptional advancement are made in
drugs filed with the inventions of numerous plant based and artificial drugs (Preethi et
al., 2010). Antibiotics are definitely most significant therapeutic detections of the 20
th
century that had success against severe bacterial effects. But, only 1/3rd of the
communicable infections recognized are cured these products. This is since for the
development of tough pathogens that were elsewhere doubted for the concern of years
of common unselective use, nonstop and mismanagement of antibiotics (Enne et al.,
2001, Westh et al., 2004). Antibiotic resistance was enlarged significantly in current
decades and is pretense an always growing therapeutic problem. Some approaches to
decrease
the
resistance
to
antibacterial
through
means
of
antibacterial
resistance
inhibiting constituents of plants (Kim et al., 1995, Alagesaboopathi et al., 2011). Plants
produce a diversity of complexes to protect their own selves in contradiction of a good
range of pathogens. It is probable that plant constituents showed targeted places but
5
these consumed by antibacterial would be vigorous contrary to
drug unaffected
pathogens (Ahmad et al., 2001). Plants used as outdated treatments for abundant human
infections
from
prehistoric
times
and
in
various
portions
world.
Therefore,
investigators in recent time’s salaried care to harmless phytomedicines and bioactive
constituents separated from plant types used in herbal medications with appropriate
beneficial directory for progress of novel drugs (Pavithra et al., 2010).
Phytotoxicity/Allelopathy;
The word Allelopathy could be defined by means of the intrusion of a plant in the
development and formation of other (comprising microbes) by mean of the release of
chemical complexes in the surroundings (Rice et al., 1984). Usually, allelochemicals
perform through stimulating cellular and metabolic variations, comprising alterations
in sheaths functionality, the absorbance of nutrient and water, the respiratory and
photosynthetic events, enzyme activity and protein synthesis, and in the hereditary
substantial
stimulating DNA
and
RNA
changes
(Inderjit
et
al.,
2006).
In
this
background, various agronomic research work has applied to the detection of new
materials through phytotoxic action initiated from plants with allelopathic prospective,
pointing the switch of weeds and plant pathogens (Ferreira and Aquila et al., 2000).
By the Lemnas essay, it was detected that natural antitumor complexes could prevent
Lemna
development.
It
was
being
revealed
that
certain
materials
rouse
leaf
propagation, and the essay might be valuable to identify novel plant development
stimulating substance. Commercial value for such natural, recyclable, herbicides and
plant development stimulating substance might soon be occupied by natural products
discovered through humble and
suitable
Lemna
bioassay (Atta-ur-Rahman
et
al.,
1991).
Numerous researchers have stated phytotoxicity of many medicinal plants (Khan et al. 2002)
stated, Abroma augusta kernel oil stoped development of Lemma equinoctials. (Allan and Adkins
et al., 2005) described, Alphitonia excelsa, Chamaesyce hyssopifolia, Ageratum conyzoides
Acacia farnesiana, and Melaleuca quinquenervia presented phytotoxicity in contradiction of
Lemna aequinoctialis. (Hussain et al. 2009) described, chloroform, n-butanol, n-hexane and
6
aqueous extracts of Nepeta juncea exposed inconsequential phytotoxicity effect in contrast to L.
minor. (Khuda et al. 2012) informed the phytotoxic activity of chloroformic extracts of
Achyranthes aspera and ethyl acetate extracts of Duchesnea indica
7
Aims and objective;
Investigation will be carried out to achieve the following objectives.
 To determine phytochemical composition of Jatropha curcus defatted seed meal obtained
as byproduct of biodiesel.
 To determine antimicrobial, antioxidant, cytotoxic and phytotoxic potential of the crude
methanolic extract of Jatropha curcus defatted seed meal.
8
9
LITERATURE REVIEW
(Igbinosa et al., 2009) Investigated invtro anti-microbial activity of crude, methanolic
ethanolic and aqueous extracts of bark of stem of J curcas. The extracts displayed
antimicrobial activities through regions of inhibition stretching from 8 - 20, 5 - 12 and
0 - 8 mm for methanol, ethanol and aqueous extracts correspondingly. The least
inhibitory concentration (MIC) of the ethanolic extracts were between 0.5 - 6.25mg/ml
but that of methanolic extracts varied from 0.5 to 10 mgml-1. The minimum
bactericidal concentration (MBC) for ethanol extracts varied concerning 2.0 and 12.50
mg/ml, whereas methanolic varied from 2.0 - 20 mg/ml. Yet over entirely the extracts
presented considerable activity in contradiction of all fungal species observed. Zones of
inhibitions displayed via extracts in contradiction of assessment fungal types varied
among 15 - 18, 15 - 20 and 5 - 10 mm for ethanolic, methanolic and aquas extracts
correspondingly.
Phytochemical
screening
presented
the
occurrence
of
steroids,
saponins, flavonoids, alkaloids, tannin and glycosides in extracts. The capability of
crude stem extracts of J. curcas to inhibit the development of fungi and bacteria is a
sign of its wide-range antimicrobial capacity which might remain active in groups of
microbial infections.
(Kalimuthu et al., 2010) studied antimicrobial activity compair to six microorganisms.
At extreme range (20 and 23mili meter width in inhibition) the fungicidal activities of
leaf extract in vivo were noteworthy. Though, the methanolic extracts of leaf derivative
lump of Jatropha curcas presented advanced fungicidal activity through concomitant
rise in concentrations. (Sharma et al. 2005) studied the efficacy of Jatropha curcas on
inactivation
of
some
microorganisms
i.e.
Staphylococcus
aureus,
fluorescens,
Escherichia coli, Pseudomonas, Bacillus subtilis and aeruginosa. It was assumed that
the ethanolic extract
fluorescens,
Escherichia
presented antimicrobial
activity in
coli, Pseudomonas aeruginosa
contrast
and
to
Pseudomonas
Staphylococcus
aureus.
But no antimicrobial activity was found compare to Bacillus subtilis. Ethanolic extract
of Jatropha leaves presented the largest inhibition zones (i.e. 11mm.)
Compair to E.
coli.
10
(Oskoueian et al., 2011) worked on defatted Jatropha curcas L. (J. curcas) seeds
contained a great fraction of basic protein (61.8%) and comparatively slight acidic
cleaner fiber (4.8%) and impartial cleaner fiber (9.7%). Spectrophotometric study of
the methanolic extract presented the existence of saponins, flavonoids and phenolics
with values of 3.9, 0.4 and 19.0 mg/g DM, correspondingly. Great presentation liquid
chromatography (HPLC) studies displayed the occurrence of pyrogallol (phenolics),
gallic acid, myricetin (flavonoids), rutin and daidzein (isoflavonoid). The quantity of
phorbol
esters
in
the
methanolic
extract
projected
by
High
Performance Liquid
Chromatography PLC was 3.0 ± 0.1 mg/g DM. Other metabolites identified by GC-MS
include:
propanediol,
β-sitosterol,
2-furancarboxaldehyde,
5-(hydroxymethy)
(hydroxymethy),
and
2-furancarboxaldehyde,
acetic
acid
furfural
2-(hydroxymethyl)-2
in
the
methanolic
(2-furancarboxaldehyde)
nitro-1,
3-
extract;
and
5-
acetic
acid in warm H2O extract. Methanolic and hot aquas extracts of seed exhibited
antimicrobial activity in difference to individually Gram +ve and Gram -ve disease
causing bacteria (inhibition range: 0–1.63 cm) at the concentrations of 1 and 1.5
mg/disc. Methanolic extract showed antioxidant activities that are greater than hot
aquas extract and similar to β-carotene.
(Srinophakun et al., 2011) invested the kernel material of Jatropha curcas as organic
fertilizer for chineeskal, tomato and potato. The aforementioned stayed reasonable that
the combination of synthetic manure and Jatropha seed cake (1,600 kg/rai) provided
the maximum plant performance.
(Sachdeva et al., 2011) Described the classification, botanical description of the plant,
its distribution and ecological requirement of Jatropha curcas L. They also studied
information about the presence of different chemicals including toxins in different parts
of the plant. They summarized the potential of plant for various pharmacological
activities. They collected and discussed information about the toxins and detoxification
methods. (Rahman et al. 2014) reported that extracts from seeds and leaves of Jatropha
curcas inhibit the mycelium growth of Colletotrichum musae that causes anthracnose
11
disease in bananas. The extracts J. Curcas also have molluscidal, insecticidal and
fungicidal properties.
(Malviya et al., 2011) said that amino acid composition of Jatropha seed is tremendous.
The Jatropha press cake possesses 24–28% protein on dry basis. The protein extracted
from press cake proteins had a solubility of about 90% above pH 9. They proposed,
estimation of protein performed by Biuret method for simple protein and by Lowry’s
method for tyrosine, tryptophan derivatives (aromatic residues) of protein. They said
that protein estimation by Biuret method for simple protein exhibited the absorbance
0.669 and 0.695nm, the concentration of protein is 68-70 mg/ml and by Lowry’s
method, tyrosine and tryptophan derivatives exhibited the absorbance 1.707 and1.809
nm, the concentration of protein is 100 -110 mg/ml.
(Safi et al., 2012) studied the biological activities of methanol extract of the root of
Jatropha
curcas
(Family:
Euphorbiaceae)
like
antimicrobial
and
free
radical
scavenging activities. The antimicrobial activity was determined by disk diffusion
method compair to some gram +ve and gram -ve bacteria and fungi. Chloroform
soluble fraction exhibited potent antimicrobial activity in terms of zone of inhibition
(9.33 - 13.33 mm) and spectrum of activity compared to the reference standard
kanamycin
(21
-
26.33 mm).
Methanolic
crude extract
also
exhibited
moderate
antimicrobial activities compair to the tested organisms. In the evaluation of DPPH free
radical scavenging activity, Methanolic crude extract and chloroform soluble fraction
exhibited strong antioxidant activity with IC50 value of 35.62 μg/ml and 43.81 μg/ml
respectively where the standard antioxidant butylated hydroxytoluene (BHT) exhibited
the IC 50 value of 18.31 μg/ml. At the same time, highest amount of phenolic contents
were found in methanolic crude extract and chloroform soluble fraction having TPC
value of 36.37 and 27.01 mg of GAE/ gm of extractive respectively. These results
suggested that bioactivity guided isolation can be carried out to separate the bioactive
principles.
(Mbakwem-Aniebo et al., 2012) reported that the capacity of the crude stem extracts of
J. curcas to inhibit the growth of fungi. It indicated that J. curcas possesses broad
12
spectrum fungicidal prospective which may be employed in the controlling of fungal
diseases.
(Rachana et al., 2012) suggested the effectiveness of Jatropha curcas Fruit compair to
some selected microorganisms which are known to cause diseases in human beings and
the relative reading of degree of antimicrobial possessions of numerous fruit portions
of
Jatropha
curcas
specifically,
Seed
Covering,
Fruit
Pericarp.
Cold
water,
Methanolic, Ethanolic, and Ethyl Acetate extracts of dry fruit portions were prepared at
closing strength of 500 mg/ml and tested
compair to infectious microorganisms that is
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using agar well
diffusion method. The Methanolic extracts of the Seed Grain exhibited the maximum
zone of inhibition of 25.5mm.
(Oloyede et al., 2012) worked on phytochemical composition, radical scavenging and
bactericidal activities of aqueous extract of leaves of Jatropha curcas L. They
examined Quantifiable phytochemical studies of phenols, alkaloids, flavonoids
and
tannins contents. They supposed that radical scavenging activity remained measured by
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and H2O2 inhibition assays while Disc
diffusion and Agar well (ditch) diffusion techniques was achieved for bactericidal
activity in contradiction of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus
aureus,
Proteus
and
Klebsiella
pneumonia
species
at
500,250,125and62.5
mg/mL
correspondingly. It also presented bactericidal activity in contrast to Pseudomonas
aeruginosa, Escherichia coli, and Klebsiella pneumonia at 500 and 250 mg/ml (MIC =
125 mg/mL), which was resistant to erythromycin, ampicillin, and cloxacillin and the
extract was sedentary
compair to Proteus species and Staphylococcus aureus at these
concentrations, however all remained sensitive to gentamycin.
(Narayani et al., 2012) studied the phytochemical and bactericidal stuff of crude
chloroform, acetone, methanolic, petroleum ether and aqueous extracts of Jatropha
curcas L. leaves. The extracts of J. curcas shown the occurrence of steroids, alkaloids,
phenolic groups, saponins, and flavonoids. Bactericidal activity of J. curcas displayed
diverse gradation of zone of inhibition in contrast to the tested bacterial pathogens.
13
Chloroform extracts of J. curcas exhibited the comprehensive range of bactericidal
activity and maximum zone of inhibition 10 mm was detected in contrast to S. aureus
and E. coli. Petroleum ether extract exhibited bactericidal activity in contrast to P.
aeruginosa. and Proteus sp through zone of inhibition of 10 and 4 mm correspondingly.
Methanolic extract of J. curcas demonstrated the bactericidal activity compared to 3
pathogens
excluding
S.
aureus.
Acetone
extract
exposed
the
bactericidal
activity
compared to S. aureus only.
(Nisa et al., 2013)
studied the antimicrobial and DPPH free radical scavenging
activities for various dilutions of 5 R. dentatus extracts were examined compared to
various
experimental
Pseudomonas
Escherichia
bacterial
aeruginosa
coli)
and
strains
(Klebsiella
Staphylococcus
fungal
strains
aureus,
(Candida
pneumoniae,
Shigella
Salmonella
typhimurium
kruesie,
Candida
flexneri,
and
parapsilosis,
,Accremonium spp., Candida albicans, Aspergillus flavus, and Aspergillus versicolor).
Between entire extracts, butanolic extract exhibited solid bactericidal activity compared
to Klebsiella pneumoniae 20mm zone of inhibition and aqueous extract exhibited no
activity compare to some strains of bacteria. Although in case of fungal strains, the
extreme fungicidal activity were detected compare to Aspergillus flavus via aqueous
extract. The antioxidant activity exposed that extracts exhibited scavenging effect in
concentration-dependent method on hydroxyl radicals and superoxide anion radicals.
The
phytochemical
exhibited
presence
experiments
of
conducted
terpenoids,
through
flavonoids,
crude
extracts
anthraquinones
of
R.
saponins,
dentatus
alkaloids,
cardiac glycosides and tannins. Total phenolic content of those extracts were projected
quantitatively after
standardization curve of gallic acid and it ranging from 45 µg/mg
in petroleum ether extract to 145 µg/mg in butanolic extract.
(Ojha et al., 2013) evaluated the anti-larval activity of vital oils of Jatropha curcas
were on larva of Aedesa egypti. The anti-larval activity was firmed in terms of LD50
assessment on getting on 3rd or else early 4th instar larva intended on behalf of a
period of twenty four hours. The hexane extract of J. curcas kernel oil exhibited antilarval
effects after twenty four hours of contact; with, the maximum larval mortality
14
was
noted
compare
to
the
fourth-instar
larva
of
standards
LC
50=640
ppm
(0.064%).No mortality was noticed in the control. The results specify the kernel oil
extract of J. curcas be able to be successfully used as potential candidates for
controlling Aedes aegypti and can be considered for eco-friendly vector control plans.
(Kumari et al., 2014) reported that bactericidal activity of Jatropha curcas indicated
various grades of inhibition zone as compared to the examined pathogenic bacteria.
The methanol stem extract exhibited highest zone of inhibition as compared to Bacillus
subtilis (13.3±0.04mm) among bacteria. The chloroform extract was also found to be
effective as compared to pathogenic strains tested. However, aqueous extracts of
Jatropha curcus were found to be inactive
compare to Bacillus subtilis, Escheria coli
but gives the result similar compare to Pseudomonas putida (7.0 ± 0.05mm) this
showed that more active ingredient may not be dissolved in aqueous extract.
15
Material and Methods:
Preparation of defatted seed meal of Jatropha curcas:
Fully matured seeds of Jatropha curcas were collected from the store of the
Department of
Botany University of Science and Technology Bannu. The seeds were
powdered in a Willy Mill (60-mesh size). For the preparation of defatted seed meal,
powdered seed material (100 gm) was treated with n-Hexane (1 L) in a Soxhlet
extractor. The extraction was sustained for 6 h at 60
o
C (AOAC 1960). After this
process the defatted seed meal was dried for 24 hours at room temperature for further
extraction of phytochemicals and biological evaluation.
Extract preparation:
In order to prepare extract 100 gm of defatted seed powder was soaked in 1000ml of
methanol (Merck Co. Darmstadt, Germany) for (3) days through ordinary maceration
process at room temperature 25(±)ºC . The extract was filtered through Whatman No1
filter paper and was concentrated by using rotary evaporator (Panchun Scientific Co.,
Kaohsiung, Taiwan). The obtained thick viscous gummy extract was stored at 4ºC for
further activities. The yield of dry extract (%) was determined in terms of air dried
weight of seed material. The total yield of methanolic extract was 2.5%.
Antibacterial activity:
Agar well diffusion method or cylinder plate Method was used for antibacterial test as
described by (Kavanagh et al., 1963; Leven et al., 1979). Wells were made in seeded
agar and the test sample was then introduced directly in these wells. After incubation,
the diameter of the clear zones around each well was measured and compared against
zone of inhibition of the known concentrations of the standard antibiotics.
16
Preparation of stock solution:
The 5 mg methanolic defatted seed extract was liquefied in 10 ml of dimethyl sulfoxide
(DMSO) in the direction of formulate standard solution which was further diluted to
obtain 4 mg/10 ml, 3mg/10 ml, 2mg/10 ml and 1mg / 10 ml concentrations.
Bacterial strains used
During the assay 2 gram-negative (Escherichia coli, Klebsiella pneumoniae) and 3
gram-positive (Micrococcus leutus, Bacillus subtilis, Staphylococcus aureus) bacterial
strains were used. All the bacterial strains were refreshed in a liquid broth for 24 h and
mixed with physiologically saline and their turbidity was corrected and coordinated by
McFarland 0.5% Barium Sulfate turbidity standard. 4 gram +ve and 5 gram -ve
bacterial verities were used for this test.
Media Preapration and Sterilization
For agar well diffusion technique (Murray et al., 1995 later modified by Olurinola, et
al., 1996) antimicrobial vulnerability was verified on solid (Agar-agar) media in patri
dishes. For antibacterial assay nutrient agar (NA) (40 gm/L) was used for increasing
superficial cluster propagation. The suspension culture, for bacterial cells propagation
remain completed through arranging 2% Lauria Broth (w/v). All the media prepared
was then autoclaved at (121°C) for 20 minutes.
Agar well diffusion technique
Agar well-diffusion technique was used to conduct antibacterial test. Nutrient agar
(NA) were stricken with sterile swabs with 8 hour old - broth culture of individual
bacteria. Bores (10mili meters diameter) were completed in all of those patri dishes by
antiseptic cork borer. Stock solution for every plant extract was made through fractions
of crude of 1 mg/10ml, 2mg/10ml, 3mg/10ml, 4mg/10ml and 5mg/10ml in Dimethyl
sulfa oxide (DMSO). Around 100 micro liter of various fractions of plant solvent
extracts were added sterile syringe to the bores and allowed to diffuse at room
temperature for two hours. Control tests including inoculums deprived of plant extract
were set. The patri dishes remained incubated at 37°C for 18-24 hours for bacterial
17
pathogens. Clarithromycin 1mg/10 was kept as +ve control. The diameter of the
inhibition zone (mm) was measured. Triplicates were sustained and the test was
repeated three times, for every duplicates the readings were taken in three altered
secure guidelines and the mean values were noted.
Antifungal Activity
The Agar tube dilution process was used for antifungal test as described by Choudhary
et al. (1995). For the preparation of inoculums SDA media (saburoud dextrose ager)
was used in order to grow fungus. Different concentrations were prepared from the
stock
viz.
1mg/10ml,
3mg/10ml,
5mg/10ml
in
DMSO.
The
antifungal
agent
Terbinafine concentrations such as 1mg/10ml, 3mg/10ml, and 5mg/10ml were kept as
+ve control and pure DMSO as a -ve control. The tubes remained marked 10 cm and
were filled with 6ml media and 100 µl of stock solutions were kept at slant position
and allowed to solidify at room temperature with a portion of 4mm inoculum taken
from one week old culture of three strains (Aspergillus niger, Aspergillus flavus,
Aspergillus fumigatus). Tubes with that negative and positive control i.e. pure DMSO
and Terbinafine 1mg/10ml were also inoculated and all the tubes were isolated one (1)
week at 28 oC. The % inhibition was calculated as:
% inhibition of fungal growth = [(100 - linear growth in test sample in mm) / (linear
growth
in
control
in
mm)]
×
100…………………………………………………………..….Eq. 1
Antioxidant Activity
The antioxidant activity was carried out as mentioned by (Brand-Williams et al., 1995).
The 1, 1-diphenyl-2-picrylahydrazyl (DPPH) was arranged by desolved 2mg of DPPH
in 50ml of methanol and was kept at 20 ºC till needed. The DPPH stock was further
diluted with methanol in order to get the correct optical density / absorbance of 0.980
±0.02 at 517 nm on a spectrophotometer (Hitachi,s U-510 Tokyo Japan). A 2.8 ml of
DPPH solution and 0.2ml (200µl) of methanolic extract of defatted Jatropha seed
18
(1mg/10ml, 2mg/10ml, 3mg/10ml, 4mg/10ml, 5mg/10ml) were mixed. The tubes were
placed in dark for 15 minutes at room temperature. After that the absorbance was
meassured at 517 nm through spectrophotometer (Hitachi's U-5100 Tokyo Japan). The
scavenging activity was projected on the basis of percentage of DPPH radical
scavenged using the subsequent formula:
Scavenging % = [(control absorbance –sample absorbance) / (control absorbance)
×100]….Eq. 2
The ascorbic acid was kept as positive control.
Phytotoxicity assay
The test for phytotoxicity was carried out as stated by (Atta-ur-Rehman 1991; Arzu
2000). The Raphanus sativus L. seed propagation and various growth limitations were
examined with 5 dissimilar dilutions of Jatropha curcas defatted methanolic seed
extracts (10mg/50ml, 5mg/50ml, 2.5mg/50ml, 1.25mg/50ml and 0.625mg/50ml). The
radish seeds were sterilized by rinsing them with 0.1% mercuric chloride solution for 2
minutes and subsequently washed three times with autoclaved distilled water. The 5 ml
of the each concentration was transferred in autoclaved (sterilized) petri plates having a
sterilized Whatman No. 1 filter paper. The methanol was vacuum evaporated. After
that every petri plate was added with 5 ml sterile distilled water. For the preparation of
negative control, filter paper in petri plate was moistened with methanol (5ml) which
was evaporated and added with 5 ml autoclaved distilled water. For positive control
only 5 ml autoclaved distilled H2O was poured to every patri dish. Ten seeds were kept
per patri dish. The petri dish were kept in dark at 25 oC.
The germinated seeds were
counted on daily basis until 90% seeds in the control were germinated. The seeds
having 5mm radical was considered as germinated.
After 10 days the plants were
harvested and analyzed for various growth attributes.
Determination of seed germination (%)
Germination is the process by which a plant grows from a seed.
Seed germination (%) was determined as:
Seed
germination
(%)
=
Germinated
seeds
/
Total
seeds
x
100………………………….........Eq.3
19
Determination of seed germination index
Seed germination index = No of seeds germinated at first count + No of seeds
germinated
at
final
count
/
Days
of
first
count
+
days
of
final
count.......................................................................Eq. 4
Germination rate index (GRI)
GRI was calculated as following.
GRI=Germination
index
germination
percentage…………………………………………Eq. 5
Determination of seedling relative water content
Seedling relative water content was determined as:
Seedling relative water content (%): SFW – SDW / STW-SDW X 100
Where SFW- Seedling fresh weight, SDW-seedling dry weight, STW-seedling turgid
weight.
Phytochemicals analysis
Identification of the bioactive chemical ingredients of the defatted J. curcas seed were
carried out using standard procedures. (Krishnaiah et al., 2009; Mattilla et al., 2007)
Qualitative analysis
Test for flavonoids
The methanolic extract (0.5 g) was treated with petroleum ether to remove traces of
fatty materials present. The residue obtained was dissolved in 80% ethanol (20 ml) and
subjected to filtration. The filtrate obtained (3 ml) was mixed with aluminium chloride
(1%) prepared in methanol and the color was observed. The formation of yellow color
was indicator of occurrence of flavonoids.
20
Test for alkaloids
Methanolic extract (0.6 g) was mixed in 1% HCl (8 ml), slightly warmed and filtered.
The filtrate (2ml) was treated separately with Maeyer’s and Dragendorff’s regents and
the turbidity or precipitate formation will be observed for the presence or absence of
alkaloids.
Test for Phenols
The 2 g extract was dissolved in diethyl ether (1 ml) for two hours. The sample
obtained was heated and fumed through 50 ml ether for 15 min. The extract (5 ml) was
taken in a flask and was added with 10 ml distilled water and 2ml ammonium
hydroxide solution. The sample was incubated for 30 minutes at room temperature for
the development of color.
Quantitative analysis
Flavonoids
The 10 g of the defatted seed sample was extracted with 100 ml of 80 % aqueous
methanol. The solution obtained was filtered. The filtrate obtained was evaporated in a
water bath to dryness and weighed to a constant weight (Williamson and Manach et al.,
2005; Mattila and Hellström et al., 2007).
Alkaloids
The defatted seed sample (5 g) was added to 200 ml of 10% CH3COOH in ethanol and
kept for 4h. The mixture obtained was filtered and the filtrate was added with
ammonium hydroxide (concentrated) until the complete formation of precipitate. The
precipitate obtained was washed and diluted with ammonium hydroxide and filtered.
The residue obtained has alkaloids, dried and weighed.
21
Total phenolics content
Folin-Ciocalteau method (Adom and Liu et al., 2002) was used for determination of
total phenolics content of all the extracts used in phytotoxicity assay. The 125µl of the
methanolic extract of was mixed with 500µl distilled water, after that 125µl of FolinCiocalteau reagent was added. The mixture was allowed to stand for six (6) minutes
and the volume was raised to 3 ml by adding 1.25ml of 7% aqueous solution of sodium
carbonate and 1 ml distilled water. The mixture obtained was incubated for 90 min in
dark and the absorbance was measured at 760 nm on a spectrophotometer (Hitachi's U5100 Tokyo Japan, range: 190-1200 nm). The measurements were compared with
different concentrations of Gallic acid standard curve. The concentration of total
phenolics was determined as mg gallic acid equivalents / g sample.
Statistical analysis
The data of phytotoxicity was scrutinized by analysis of Variance (one way ANOVA)
conferring to Steel and Torrie (1980). The assessment between treatment averages was
done by Duncan’s Multiple Range Test (DMRT) and coefficient of correlation among
growth attributes was determined using Statistix (version 8.1 USA).
22
Results and discussion
Anti-bacterial activity of Jatropha curcas defatted seeds;
Various concentrations of Jatropha seed methanolic extract were evaluated for
their antibacterial activity against five bacterial strains (Escherichia coli, Klebsiella
pneumoniae, Micrococcus leutus, Bacillus subtilis, Staphylococcus aureus) using agar
well diffusion method. Results presented in Figure 1 showed that methanolic extract of
Jatropha seed inhibited the growth of tested bacterial strains at all the concentrations.
Highest zone of inhibition against K. pneumonei (20mm), M. leutus (11mm) and B.
subtillis (4mm) was achieved at 5 mg/10 ml of the extract. The susceptibility of S.
aureus and E. coli to the extract was higher at 1 mg/10 ml. All the tested bacterial
stains were highly susceptible to clarathermycin used as positive control and exhibited
maximum zones of inhibitions. The recorded antibacterial activity of the Jatropha seed
extract may be attributed to the presence of several proteins and other bioactive
compounds present in the extract (Idris et al., 2013).
23
Eschrichia coli
Micrococuu leutus
Klebsiella pneumonei
Bacillus subtilis
Staphylococcus aureus
Zone of inhibition (mm)
40
35
30
25
20
15
10
5
0
5mg/10ml
4mg/10ml
3mg/10ml
2mg/10ml
1mg/10ml
Clarathermycin
(1mg/10 ml)
Concentration
Figure 1 Antibacterial activity of the J. curcas defatted seed. The data represent mean of three
replicates.
24
Anti-fungal activity of Jatropha curcas defatted seeds;
Figure 2 showed maximum antifungal activity of A.fumigatus, A.flavous, A.niger was
at 5 mg/10ml.
A niger
A flavous
A fumigatus
120
% Inhibition
100
80
60
40
20
0
Turbinafin 1mg/10ml
Control
jatropha 1mg/10ml
jatropha 3mg/10ml
jatropha 5mg/10ml
Treatment
Figure 2 Antifungal activity of the J. curcas defatted seed. The data represent mean of three
replicates.
25
Anti-oxidant activity of Jatropha curcas defatted seeds;
Figure
3
revealed
that
the
DPPH
radical
scavenging
activity
of
the
various
concentrations of the Jatropha seed extract can be ranked as 5 mg/10 ml > 4mg/10
ml>3mg/10 ml> 2mg/10 ml> 1mg/10 ml. The higher antioxidant activity of the
Jatropha seed extract might be due to its high phenolics content.
Extract
Ascorbic acid
80
70
Inhibition (%)
60
50
40
30
20
10
0
5mg/10ml
4mg/10ml
3mg/10ml
Concentration
2mg/10ml
1mg/10ml
Figure 3 DPPH Free radical scavenging activity by the J. curcas defatted seed. The data
represent mean of three replicates.
26
Phytotoxicity of Jatropha curcas defatted seeds;
The phytotoxic effects of the extract were determined on seed germination and early
seedling
growth
of
radish
(Raphanus
sativus
L.).
Phytotoxicity
assay
helps
in
determination of beneficial or harmful effects of natural or synthetic compounds on
growth and development of plants (Badshah et al. 2015). Results presented in (Figure
3 a) showed that methanolic extract at higher concentration (10 mg/50 ml) significantly
inhibited seed germination (%) as compared to control. The other seed germination
indices such as germination index and germination rate index were not significantly
affected by extract at all the concentrations (Figure 3 b, c). The effect of higher
concentration of extracts (10 mg/50 ml, 5 mg/50 ml and 2.5 mg/ 50 ml) on seedling
relative water content was significantly inhibitory as compared to control (Figure 3d).
Higher concentrations of the Jatropha curcas extracts (10 mg/50 ml, 5 mg/50 ml and
2.5 mg/ 50 ml) significantly reduced shoot length, root length, seedling fresh weight
and seedling dry weight as compared to control (Figure 4 a, b, c, d).
The results of the study established that J. curcas seed possess some active ingredients
which inhibited the growth and development of radish plants at higher concentration.
Allelopathy is a phenomenon in which an organism suppresses or stimulates the
growth,
survival
and
reproduction
of
other
organism
through
the
production
of
biochemicals called as allelochemicals (Stamp and Nancy et al., 2003). Allelochemicals
are secondary metabolites of diverse nature which are not compulsory for normal
metabolism and development of the allelopathic organism (Badshah et al. 2015). In
present study the toxic effect of extract was concentration dependent. Only higher
concentrations of the extract exhibited toxic effects. These findings agree with earlier
workers who have reported that toxicity depended on concentration of the extract
(Khan et al. 2011; Admiluyi, 2013; Sher at al., 2014). Allelochemicals reduce the
moisture content in shoots and leaves of susceptible test species. During present
investigation seedling relative water content of radish plants was reduced at higher
concentration of the extract. This decrease in seedling relative water content might be
due the fact the allelochemicals produce drought stress conditions inside the plant. The
susceptible plant fails to absorb sufficient water from the medium, loses its turgidity
27
and exhibits poor growth and biomass production (Barkosky and Einhellig et al.,
2000).
120
3
a
bc
bc
b
ab
Germination Index
Germination (%)
100
c
80
60
40
20
0
2.5
a
a
a
a
a
a
2
1.5
1
0.5
0
Treatments
Germination Rate Index
0.028
a
0.027
a
a
0.026
0.025
b
a
a
a
0.024
0.023
0.022
0.021
Relative Water Content (%)
a
Treatments
120
100
a
a
a
a
a
80
60
b
40
20
0
0.02
d
c
Treatments
Treatments
Fig. 1 Effect of methanolic extract of Jatropha curcas seed on (a) seed germination (%) (b)
Germination index (c) Germination rate Index (d) Relative water content. Means sharing
common English letters are statistically similar.
28
8
a
b
7
ab
7
ab
6
d
5
Root Length (cm)
Shoot Length (cm)
c
6
4
3
2
b
Treatments
Treatments
a
0.8
bc
c
0.6
0.4
0.2
a
Seedling dry weight (gm)
Seedling dry weight (gm)
1.4
1
0
d
Treatments
c
2
0
ab
bc
3
0
ab
bc
4
1
1.2
ab
a
5
1
a
a
c
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
ab
abc
cd
a
d
d
Treatment
Fig. 2 Effect of methanolic extract of Jatropha curcas seed on (a) shoot length, (b) root length,
(c) seedling fresh weight and (d) seedling dry weight. Means sharing common English letters are
statistically similar.
29
Phytochemical composition of Jatropha curcas defatted seeds;
The qualitative analysis of J. curcas defatted seed showed the presence of alkaloids,
flavonoids and phenols (Table 1). The alkaloids and flavonoids were moderately
present whereas phenols were highly present.
Table 1 Qualitative Analysis of J. curcas defatted seed
Sample code
Alkaloids
Flavonoids
Phenols
J. curcas defatted seed
++
++
+++
+ = present, ++ = moderate, +++ = highly present
30
Quantitative analysis
soluble phenolics
showed
that alkaloids
(7.3%), flavonoids
(0.39%)
and
total
(11mg gallic acid eq. / g extract) were present in concentration as
shown in (Table 2).
Table 2. Quantitative Analysis J. curcas defatted seed
Sample Code
Alkaloids (%)
Flavonoids (%)
J. curcas defatted seed
7.3
0.39
Total soluble
phenolics (mg gallic
acid eq./g extract)
11
The phytochemical composition of J. curcas defatted seed exhibits its suitability and
usefulness in drug formulation. Alkaloids are reported for their defensive function in
experimental
animals
(Edeoga
and
Eriata
et
al.,
2001).
They
exhibit
anti-
inflammatory, analgesic activities and improve resistance against diseases (Gupta et
al., 1994). Phenols are reported for their antiviral, antioxidant and antimicrobial tests
(Vasantha et al., 2012). The occurrence of flavonoids, alkaloids and phenols should be
responsible
for
antimicrobial,
antioxidant
and
phytotoxic
activities
of
methanolic
extract of defatted seeds of J. curcas.
31
Conclusion
 The methanolic extract of J. curcas defatted seed exhibited antimicrobial,
antioxidant and phytotoxic activities.
 Higher concentrations of the extract exhibited toxic effects on radish seedling
growth.
 Phytochemicals such as alkaloids, flavonoids and phenols were present in the
defatted seed.
 It was inferred that J. curcas seed possessed active ingredients which were
effective against pathogenic microbes and therefore could be used in the
formulation of drugs in treatment of several diseases.
32
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