1

University of Calicut
"STUDIES ON PESTICIDAL EFFICACY OF THE
PLANT-ERYNGIUM FOETIDUM AGAINST STORED
GRAIN PEST-SITOPHILUS ORYZAE "

Dissertation submitted to Christ College (Autonomous)

in partial fulfillment of the requirements for the degree of
BACHELOR OF SCIENCE IN CHEMISTRY

Submitted by:

SREERAG K S [Reg No: CCASSCH036]

Under the supervision of

Dr. TOM CHERIYAN
Assistant Professor, PG & Research Department of Chemistry
Christ College, Irinjalakuda-680125
Thrissur, Kerala, India
May -2021
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Studies on pesticidal efficacy of the plant-eryngium foetidum against stored

grain pest-sitophilus oryzae

Bonafide record of work done by

SREERAG K S [Reg No : CCASSCH036]

Literature review submitted in partial fulfillment of the requirement for the degree
of
BACHELOR OF SCIENCE
In Chemistry
CALICUT UNIVERSITY
May 2021

…………………
………………
Dr. TOM. CHERIYAN Dr. V.T. JOY
Faculty Guide Head of the Department

Certified that the candidate was examined in the viva voice examination held on

………………………… ……………………….
(Internal Examiner) (External Examiner)
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Certificate

This is to certify that this review entitled Studies on pesticidal efficacy of the plant-eryngium
foetidum against stored grain pest-sitophilus oryzae , submitted towards the partial fulfillment for
the award of degree of Bachelor of science in chemistry at Christ College, Irinjalakuda represents
original research carried out by, SREERAG KS [Reg No : CCASSCH036] under the supervision of
Dr. TOM CHERIYAN during the academic year 2020-2021.

Student Supervisor
SREERAG K S Dr. TOM CHERIYAN
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Acknowledgement
We would like to express our deep sense of gratitude to our guide Dr. Tom Cheriyan, for the
invaluable guidance, constant support and constructive suggestions for the evolution of the project.
The way he mentored our team, helping us to make improvisations in the work, has motivated us
greatly. We would wish to express our sincere obligation to Dr. Jolly Andrews CMI, Principal,
Christ College (Autonomous), Irinjalakuda. We express our heartfelt gratitude to Dr. V T Joy, the
Head of the Department of Chemistry, for all the valuable support concerned with the betterment
of our project. We are thankful to our class teacher Dr. Jibin A K, and all the faculties of the
Department of Chemistry, for their valuable encouragement. The combined efforts of our team
have served as the driving force towards the completion of this project. We are deeply grateful to
our parents and friends for their moral and technical support in helping us finalize the project.
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CONTENTS

CHAPTERS PAGE NO:

1. INTRODUCTION 1-10

1.1 Pests of stored product

1.2 Rice weevil
1.3 Chemical pesticides
1.4 Botanicals as alternative to synthetic pesticides
1.5 Plants description
1.6 Significance of the study
1.7 Objectives
1.8 Review of literature
2. MATERIALS AND METHODS 11-15

2.1 Study area

2.2 Plant material
2.3 Experimental pest
2.4 Preparation of plant extracts
2.5 Crude extract dilution
2.6 Treatment-Pesticidal activity
2.7 Data analysis
3. RESULT AND DISCUSSION 16-20

4. CONCLUSION 21

REFERENCES 22-23
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CHAPTER –1 INTRODUCTION AND OVERVIEW

For global food security, the agricultural sector of the world economy must achieve a
production level that ensures adequate food supply to feed the increasing population as well as
provides raw materials for the industries (Riebeiro BM et al 2003). This is particularly so as the
energy sector is vigorously pursuing research into the use of grains and root crops as sources of
starch for Conversion into bio-fuels (White NDG 1995). Coincidentally, these crops (maize, rice,
millet, sorghum, soybeans, cowpeas, sugarcane, groundnuts, etc.) are the stable foods in most parts
of the developing countries of the world such as Africa, South America and Asia (Hug etal 1997).
In addition to the above new development in the industrial utilization of these crops, they are
frequently and vigorously attacked by herbivorous insects and other pests such as phytopathogens
and molluscs. In fact the loss due to pests and diseases is about 35% on the field and 14% in
storage, giving a total loss of about 50% of agricultural crops annually (Zetter JL et al 1990). Thus
the world food production is adversely affected by insects and pests during crop growth, harvest
and storage (Isman M.B 2006). Apart from the farm environment insects and pests constitute
serious means in the home, gardens and bodies of water, and transmit a number of diseases by
acting as hosts to some disease-causing parasites. Thus elimination of these insects and pests or
mitigation of their activities will go a long way in reducing world food crisis as well as improve
human and animal health (Aslam I et al 2005).The ancient man had deployed different methods of
control, including sprayers, magic spells, cultivation systems, mechanical practices as well
asapplication of organic and inorganic substances to protect his crops from the attack of weeds,
diseases and insect pest (Avyaz A et al 2008).
1.1 PESTS OF STORED PRODUCT
Prevention of food losses during postharvest storage is of paramount economic importance
(Brattsten LB1983). The major pests of stored grain are classified in two groups namely, primary
pest: Those which are capable of penetrating and infesting intact kernel of grain and secondary
pest: which cannot infest the whole grain but feed on as broken kernels. High moisture weeds,
seeds and grain damaged by primary pests. The important primary pest are rice weevil (Sitophilus
oryzae), Granary weevil (Sitophilus granaries), lesser grain borer (Rhyzoper- thadominica) etc.
secondary pests are mites (Acarina:Tetranychidae), Rusty grain beetle(Crypt- olestes
ferrugineus),saw-toothed grain beetle(Oryzaephilus surinamensis)etc (Kim SI et al 2003).

Some of the common forms among them are the following: (Niber BT 1994).
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✓ Sitophilus oryzae : pest of stored rice, wheat, maize ETC.

✓ Triboliumcastaneum: pest of almost all grains and grain products, particularly the flour of
rice, wheat, and Bengal gram.
✓ Rhizoperthadominica : pest of unpolished rice.
✓ Trogodermagranarium(wheat weevil): major pest of stored wheat, rice, maize, oats, barley
Etc.
✓ Araecerus fasciculatus : pest of dried tapioca, ginger, arecanut etc.
Among the cereals, rice is the most important staple food supplying energy requirements for most
of the world population (Arthur F.H.1996). However during storage a loss of about 10- 20% rice
grains occurs due to stored grain pests (Sarac A and Tunc I 1995). Stored rice seeds and milled
rice is prone to be infested by rice weevils, Sitophilus oryzae causing heavy economic losses (Lee
SE et al 2003). Repellents are considered as the best source of protection against pest attack upon
stored product. Adult weevils feed on rice grains and lay eggs within the grain surface and the
larvae develop inside the grain thus feeding preferentially on the germ of the grain (Cetin H et al
2006). Because both larvae and adults feed on germ of the grain, the grain is completely damaged
beyond any use (Park C 2000). Prevention of food loss during post harvest storage without creating
environmental problems is therefore, of paramount economic importance (Li WQ et al 2010). 1.2
SITOPHILUS ORYZAE (Rice weevil)

Scientific classification
Kingdom : Animalia
Phylum : Arthropoda Class :
Insecta

Order : Coleopteran
Family : Curculionidae
Genus : Sitophilus

Species : S.oryzae

It is commonly called the rice weevil. It is the most common and the most destructive pest of stored
gains (Neghban M et al 2007). It enjoys worldwide distribution and is found in stored gains such
as rice, wheat barley, bajra, etc. which attack economically important crops (Shayaet al 1991).
Adult weevils are somewhat cylindrical and reddish-brown or brownish- black, with prolonged
snout or rostrum and ridged elytra. But most of the time they remain inside the grains for feeding.
But mating usually takes place outside the grains. Soon after mating, female weevil makes pits or
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cavities in the kernel of the grain, deposit eggs in them at the rate of one egg in each cavity and
seals their mouth with a plug of gelatinous secretion (Tunc I et al 2000). A female may lays an
average of 4 eggs per day and may live for 4 -5months. In about 6 or 7 days, eggs hatch out into
soft, white and legless larvae. They bore into the grains and actively feed on the interior of the
grain, hollowing it out. After 3 or 4 weeks larvae change to pupae, initially, pupae are dirty white.
But later on they become dark-brown. Pupa life lasts for 6-14 days. Adult weevils come out
through irregular holes made in the grain. The whole life cycle is completed inside the grain
passing through a period of 4 -7 weeks (Hodges RJ 1986).
1.3 CHEMICAL PESTICIDE
Between 500 BC and the 19th century a number of substances classified as pesticides and defined
as “Any substances or mixture of substances intended for preventing, destroying, repelling or
mitigating any pest” were used to control pests (Makanjuola WG 1989). Control of stored product
pests relies heavily on the use of synthetic insecticides and fumigants, which has led to problems
such as environmental disturbances, increasing cost of application, pest resurgence, pest resistance
to pesticides and lethal effects on non target organisms (birds, animals, fish) in addition to direct
toxicity to users (Niber BT 1994). Fumigation plays a very important role in insect pest elimination
in stored products. Fumigant mainly depends on methyl bromide or phosphates. However, methyl
bromide was banned in many countries starting in 2004 because of its ozone depleting
properties(Hansen and Jensen 2002).Many alternatives have been tested to replace methyl bromide
fumigation for stored product and quarantine uses (Hatil and Kamal 2009).
There is an urgent need to develop safe alternatives that have the potential to replace the toxic
fumigants, yet are effective, economical and convenient to use (Ayvazet al.2008).Biological
control may be an effective strategy for stored product pest management. Botanical pesticides have
the advantage of providing novel modes of action against insects that can reduce the risk of cross
resistance as well as offering new leads for design of target specific molecules (Hasantet al 2002).
Natural products from plants have attracted researchers in recent years as potential sources of new
pesticides. The folkloric use of higher terrestrial plants by the natives of various parts of the world
as pesticidal and antimicrobial materials has been well known (Rahman TR et al 2007).
1.4 BOTANICALS AS ALTERNATIVE TO CHEMICAL PESTICIDE
Tens of thousands of natural products have been identified from plants and hundreds of thousands
are yet to be isolated and screened for their bioactivities. This large reservoir of organic chemicals
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is largely untapped or under-tapped for use as pesticides (Radmila Almas et al 2008). Higher plants
are a rich source of novel natural substances that can be used to develop environmental safe
methods for insect control (Arnasonet al.1989). Insecticidal activity of many plants against several
insect pests has been demonstrated (Jilaniand Su1983, IsmanM.B2000). The deleterious effects of
plant extracts or pure compounds on insects can be manifested in several manners including
toxicity, mortality, anti feed–ant, growth inhibitor, suppression of reproductive behavior and
reproduction of fecundity and fertility (Rajendran and Sriranjini 2006).
Plants have developed effective morphological and chemical defense mechanisms that ensure
survival under rough environmental conditions and in the presence of natural enemies. These
chemical defenses either may produce mortality of the insect by acting as an insecticide or may
affect common biochemical and physiological functions and act as repellants, anti-feedants or
inhibitors (Mathelaet al 1989). In many developing countries, utilization of locally available
materials to protect stored products against pest damage is common practice in traditional farm
storage system (Fernando and Karunaratne 2013). These plant materials were not only used as
insecticides but also as insect repellants and insect anti feedant. Compared to conventional
pesticides crude or botanical pesticides tend to have broad spectrum activity, and are relatively
specific in their mode of action, easy to process by the small scale farmers and would be safe for
the higher organisms (Viglianco et al; 2008). Also these pesticides do not contribute to resistance
development or pest resurgence, nor do they cause negative effects on non-target organisms and
also they do not affect food quality (Soon KM et al 2003).
A detailed screening of different plant species were carried out before beginning of the study.
About two species of plants were screened. Name of the plant species which were screened is
noted here. They are: Hetropogoncontortus, Sebestianachamaelea and Calotropis procera. On the
basis of the results of pesticidal screenings, it was found that Calotropis procera shows more
pesticidal activity against Sitophilus oryzae.
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1.5 PLANT DESCRIPTION- Eryngiumfoetidum

Scientific classification
Kingdom : Plantae

Class : Insecta

Order : Apiales

Family : Apiacea

Genus : Eryngium

1.5.1 OTHER NAMES FOR Eryngiumfoetidum

Common name :Long coriander,Sawleafcoriander,Bandhaniyah,Fitweed,Mexican coriander.
English : Mexican coriander
Hindi : Ban-dhaniya
Malayalam : African Malli
1.5.2 MORPHOLOGY OF Eryngiumfoetidu
Botanical name : Eryngiumfoetidum
Family : Apiaceae
Synonyms : Eryngium antihystericum Rottler
Medicinal uses : The roots and leaves of the plant are used to prepare tea,which will stimulate
appetite,improvedigestion,soothe stomach pains.It has
anticlastogenic,anticarcinogenic,antidiabetic.
MORPHOLOGICAL CHARACTERISTICS
It is an annual to biennial herb pungently scented grow to 15 to 45 cm tall with the taproot.In India
it reported to be perennial.The leaves are arranged in a basal rosette form,leaf blades are lanceolate
to oblanceolate or spatulate in shape, margins spinous toothed. Flowering shoots divaricately
branched, bear more deeply toothed leaves. Flowers are white in cylindrical heads, subtended by
a whorl of about 2 to 3 cm long, 5 to 6 unequal brackets resembling the upper leaves. Calyx teeth
ridged and acute. Petals white or greenish, fruits are ellipsoid or subglobose and greenish. Seeds
are semiterete.(1)
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1.5.3 USES OF Eryngiumfoetidum

The leaves of eryngiumfoetidum, are aromatic and smell like coriander; in view of its origin it is
popularly Mexican coriander. It has many medicinal uses in Central and South America: a root
decoction is taken as sudorific, diuretic febrifuge and stimulant; as a laxative and as a remedy for
colds and fever(2).
1.5.5 TOXICITY OFEryngiumfoetidum
Long used as culinary seasoning, the adverse effect of chronic high dose consumption has not
been evaluated. Study assessed the chronic toxicity of EF in mice. Results suggest daily
consumption of E. foetidum for 24 weeks at doses higher than 0.8% EF diet (35 times of human
consumption) may cause adverse effect on kidney function in mice.
1.5.6 PROPERTIES OFEryngiumfoetidum
(1) ANTIBACTERIAL PROPERTY
The objective of the study was to evaluate antibacterial activity of aqueous and methanol extract
of Eryngiumfoetidum against E. coli, Staph aureus and Ps. aeruginosa. Phytochemical screening
was done followed by antibacterial activity by disc diffusion method. Three different
concentrations of the plant extracts were taken and for comparison ciprofloxacin, ceftriaxone,
ampicillin and amikacin were taken as standards. The zone of inhibition was measured for their
antibacterial activity. The methanol extract of Eryngiumfoetidum showed mild to moderate
antibacterial activity against Staph. aureus whereas there was no antibacterial activity shown by
aqueous extract. Phytochemical screening of the plant revealed the presence of tannins, saponins
and triterpenoids. The results of this study concludes that methanol extract of Eryngiumfoetidum
possess antibacterial activity against gram positive bacteria and further studies are required to
confirm the phytoconstituents responsible for its antibacterial activity.
(2)ANTIOXIDANT PROPERTY
Study evaluated hydro-distilled oils of E. foetidum to GC-MS analysis for radical scavenging
activity using DPPH and FRAP assays. DPPH assay yielded IC50 values of 56, 46, and 54.5
µg/mL for leaf, stem and root oils, respectively. In FRAP assay, highest reducing potential was
seen in the leaf oil. Study suggests a potential source. Study investigated the concentration of
natural bioactives and antioxidant capacity in various solvents of E. foetidum leaf extracts.
Bioactive compounds viz., polyphenol, tannin, anthocyanin, flavonoids, carotenoids, and ascorbic
acid varied in different solvents and were highest with acetone and methanol extracts.
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Study showed antioxidant activity with potential use by food and pharmaceutical industry.
(3) ANTICLASTOGENIC PROPERTY
Study evaluated the anticlastogenicity and clastogenicity of E. foetidum leaf using in vivo mouse
peripheral blood erythrocyte micronucleus assay. Results showed EF has no clastogenicity, but
possesses anticlastogenic potential against both direct (MMC)- and indirect DMBA)-acting types
of clastogen in mice of natural antioxidants.
(4) ANTI-INFLAMMATORY AND ANALGESIC PROPERTY
Study evaluated a decoction of leaves for anti-inflammatory and analgesic properties. Results
showed a dry residue from the decoction given orally inhibited carrageenan-induced edema in rat
paw. Topical application also inhibited swelling of mouse ear caused by TPA. Analgesic activity
was evidenced by potent inhibition of abdominal writhings provoked by acetic acid as pain
stimulant.The anti-inflammatory and antioxidant activities of E. foetidum leaf extract on
LPSactivated murine macrophages. Pre-treatment with the leaf extract inhibited the elevation of
IL-6, TNF-α, iNOS and COX-2, along with cognate mRNAs in a dose-dependent manner. Effects
were due to inhibition of LPS-induced phosphorylation of JNK and p38 as well as IkB. The extract
yielded bioactive compounds viz., lutein, ß-carotenes, chlorogenic acid, kaempferol and caffeic
acid.
(5) ANTI MICROBIAL PROPERTY
 Study evaluated the antimicrobial activities of B. pilosa, G. ciliata, and Eryngiumfoetidum
against 6 clinical strains of Helicobacter pylori in vitro and in vivo. The lowest MIC value (64
µg/mL) and best spectrum of bactericidal effect (MBC/MIC=1) was seen in the methanol extract
of E. foetidum. The antibacterial and antifungal activity of leaves of Eryngiumfoetidum. An ethyl
acetate extract of leaves showed the highest antimicrobial activity against the four bacterial strains
( B. subtilis, S. aureus, E. coli, P. aeruginosa) and Candida albicans.
6) LEISHMANICIDAL ACTIVITY
 Study evaluated the in vitro leishmanicidal and cytotoxic activities of isolated compounds from
defatted aerial parts of E. foetidum. Bioassay-guided fractionation yielded two compounds, a
daucanesesquiterpene and a terpene aldehyde ester derivative. Compound 1, lasidiol
pmethoxybenzoate inhibited growth of both L. tarentolae and L. donovani with IC50s of 14.33 and
7.84 µM, respectively, with no cytotoxicity.
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(7) BIOSORBENT PROPERTY

 Study evaluated three plants from the Apiaceae family viz., parsley, coriander, and culantro to
remove lead from aqueous solution. Results showed the adsorbents have a very low economic
value and can be used for removing lead ions from contaminated waters.
(8)ANTIPROLIFERATIVE PROPERTY
 Study investigated the in vitro antioxidant and antiproliferative activities of Eryngiumfoetidum
leaves. The leaf showed high antioxidant potential by DPPH assay. The cytotoxic effect of leaf
essential oil showed highest CTC50 value against PC-3 cell lines.
1.6 SIGNIFICANCE OF THE STUDY
Insects are a problem in stored grain throughout the world. Stored product pest causes serious
losses in weight and quality of the stored products. Sitophilus oryzae can be a major pest in storage
of grain based products, which cause severe damage to cereal grains throughout the world. This
species has a long association with human stored food and has been found in association with a
wide range of commodities including grain, peas, nuts and dried fruits. Control of stored product
pest relies heavily on the use of synthetic insecticides and fumigants, which has lead to problems
such as environmental disturbances, increasing cost of application and pest resistance to pesticides
and lethal effects on non-target organisms, in addition to direct toxicity to users. Biological control
may be an effective strategy for stored product pest management. Botanical pesticides have the
advantage of providing novel modes of action against insects that can reduce the risk of cross
resistance as well as offering new leads for design of target specific molecules.
Plant material have been shown to posses potential for development as new fumigants and they
may have the advantage over conventional fumigants in terms of low mammalian toxicity, rapid
degradation and local availability. Compared to conventional pesticides crude or botanical
pesticides tend to have broad spectrum activity, and are relatively specific in their mode of action,
easy to process by the small scale farmers and would be safe for the higher organisms. Also these
pesticides do not contribute to resistance development or pest resurgence, nor do they cause
negative effects on non-target organisms and also they do not affect food quality. The main
significance of the study is described below:
✓ It is an easy method to protect stored grain from Sitophilus oryzae.
✓ Biological pesticide does not cause any damage or pollution to the environment.
✓ It can be adopted by the farmers to protect storage grain in the storage facility.
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✓ This study will improve the agriculture field by maintaining food quality and quantity.
1.7 OBJECTIVES
✓ To screen the pesticidal activity of Eryngium foetidum
✓ To do standard extraction of the plant which are found having pesticidal activity after
preliminary screening.
✓ To do bioassay to determine mortality rates of Sitophilus oryzae with different
concentration of plant extract.
1.8 REVIEW OF LITERATURE
Plant-based pesticides (botanic pesticides or botanicals) have been in use as pesticides for over 150
years. It was only very recently that the synthetic insecticides effectively became the prominent
agrochemicals for controlling all forms of agricultural pests and have assumed a very important
position in the marketplace. However, in the past three decades so much has been reported in
literature in respect of natural products that were identified with potent pesticidal activity such as
feeding detergency and toxicity to insects in laboratory assay.
Several studies have been carried out in Sitophilus oryzae. And some of those studies have been
described below.
Tiwari (1993) tested plant powder of Acoruscalamus as a grain protectant against Sitophilus oryzae
in wheat grains. The product of Acoruscalamus, significantly reduced the kernel infestation and
F1progeny of the weevil. And it was also proved to be effective at a concentration of 0.05%. In
1995 Xieet al studied the repellency and toxicity of neem extracts against Sitophilus oryzae. In
1997 Paneru studied the pesticidal effect of Eucalyptus oil against Sitophilus oryzae.
Ahmed (2000) studied the efficacy of Castor-seed extract, as an insecticide, against Sitophilus
oryzae . He found that it inhibit cholinesterase and peroxidase enzymes in the insect body. And the
enzyme inhibition levels varied differently in adult insects, at different exposure time and with the
extracts taken with different solvents.
In 2002 A.B.M.S. Hasnat, S.N. Reza, M.S. Ullah and Masum Ahmad studied the efficacy of
nishinda (VitexnegundoL.) and bitter gourd leaf (Momordicacharantial) extract was assessed on
the basis of toxicity, repellency and antifeedant effects by rice weevil. Both the botanicals had
repellent and antifeedant effects but Nishinda leaf extract showed more toxic effect on the
mortality (21.22%) for rice weevil.
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In 2003Soon Kim (et.al) studied the pesticidal activities of aromatic plant extracts and essential
oils against Sitophilus oryzae. Using direct contact and fumigation method. The plant species
studied were, Aloysia polystachia (Griseb) Moldenke (Verbenaceae); Solanum argentinum and
bitter etlillo (solanaceae).
In 2006 A.U.R. Saljoqiet al studied the effect of six plant extracts on Sitophilusoryzae in the stored
wheat grains. Ethanol extracts of six plants materials i.e. bakain drupes (Meliaazdarach), habulas
leaves (Myrtuscommunis), mint leaves (Menthalongifolia), bakain leaves, harmal shoots and seeds
(Pegnum harmala) and lemon grass roots (Cymbopogon citrates) against rice weevil, Sitophilus
oryzaeL were tested to determine their insecticidal properties. The results revealed that all of the
tested materials with some variations had repellent and lethal effects against the pest as compared
with the untreated check. Considering the % mortality of the insect as a main index, bakain drupes
proved to be the most effective of these six plant materials. In 2007 M. M. M. Ruppaet al studied
the toxic effect of plant extract on adults of Sitophilus oryzae. they studied the effectiveness of
extracts of Pepper nigrum and essential oils of Ocimum basillicum and eucalyptus globules against
Sitophilus oryzae and found that the extracts of Pepper nigrum did not control in an effective way
the S.oryzae. But other essential oils provide quite promising results.
In 2008 Radmila Almasi(et.al) studied the insecticidal activities of different doses of plant extracts
obtained from Piper nigrum, Carumcarvi and Sesamum indicum against rice weevil Sitophilus
oryzae L. The extract of P. nigrum was found to be the most efficient causing the highest mortality
rate.
In 2011 Mumtaz Akhtar studied the repellent effects of certain plant extracts against Sitophilus
oryzae. In the present study, ethanol extract of Psidiumguajava, Citrus reticulata, Citrus limon,
Citrus sinensus and Azadira chtaindica were evaluated for their repellent affects against rice
weevil, S. oryzae. Results indicated that P. guajavawas most effective while A. Indicaas least
effective among all tested treatments. For all treatments, repellent effects increased in dose
dependent manner.
In 2013 Fernando investigate the potential of powdered leaf extracts of Olaxzeylanica in different
solvents such as methanol, ethanol and hexane as repellant against the Sitophilus oryzae. And he
found that methanol extract of leaves produced highest repellent effect on weevil. In March 2014
M. Bhubaneshwari (et.al) investigate the effect of botanical plant powders of indigenous botanical
plants in Manipur, India. Melia azadarach, Perthenium hysterophorus, Phlogocanthus
 16

thyrsiflorus, Vitextrifolia, Zanthoxylum acanthopodium and Azadira chtaindica was tested for their
efficacy on mortality, rate of adult emergence, grain damage effect against rice weevil, Sitophilus
oryzae on rice grain. The plant powder Azadirchta indica was found highly effective in prohibiting
the adult emergence and reduction in grain damage per cent over other treatment. There is no doubt
that a number of plants possess pesticidal activity and investigations by various research groups in
different parts of the world have confirmed this. On the basis of the results of pesticidal screenings
it has been established that a number of plants have broad pesticidal activity and those commonly
used in traditional agricultural applications in many parts of the developing countries, particularly
in the tropical areas.

CHAPTER-2 MATERIALS AND METHODS 2.1 STUDY AREA

Plant leaves were collected from different areas of Iringalakuda in Thrissur district. And
the experimental pests were collected from the local market. The collected study materials were
 17

brought into the laboratory condition. The experiment was conducted in the Zoology and
Chemistry laboratories of Christ College, Irinjalakuda.

2.2 PLANT MATERIAL

Extracts were prepared from plant leaves. The plant used in this study is Eryngium
foetidum. At flowering stage, the plant materials were collected from different areas of named
village. The plants were collected in the middle of January 2015. Leaves were randomly collected
from the plant rinsed with distilled water and shade dried. All the leaf materials were air dried for
10 days. After drying the plant materials were powdered with the help of mixer blender. The leaf
powders of the plant was stored in an air-tight glass bottles without any interference of humidity.
Powders of the leaves were used for extraction in three different solvents. Solvents used for
extraction are Ethanol, Petroleum ether and Ethyl acetate.Fifty gram leaf powder was used for
extraction against three solvents. (Hatil H and Kamali EL 2009)

2.3 EXPERIMENTAL PEST

The adults of Sitophilus oryzae were collected from infested grains purchased from local market.
And it was brought to the laboratory. The pest along with the grains was stored in a plastic
container. Adult insects were sorted out after sieving the grains. The adults were used for
subsequent experiment. They were maintained in containers throughout the study period. Small
holes were provided on the plastic container for proper aeration. (Niber B.T 1994).

2.4 PREPARATION OF PLANT EXTRACTS

Extraction of plant material was carried out with the help of Soxhlet apparatus. (Akhtar Y and
Isman M.B 2004, Niber BT 1994)

2.4 (a) METHOD OF EXTRACION(Harwood et al1989 and Jump UP Soxhlet F1879)

A Soxhlet extractor is a piece of laboratory apparatusinvented in 1879 by Franz von

Soxhlet.It was originally designed for the extraction of a lipid from a solid material. However, a
Soxhlet extractor is not limited to the extraction of lipids. Typically, a Soxhlet extraction is only
required where the desired compound has a limited solubility in a solvent, and the impurity is
 18

insoluble in that solvent. The desired compound has a significant solubility in a solvent then a
simple filtration can be used to separate the compound from the insoluble substance.

Normally a solid material containing some of the desired compound is placed inside a
thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet
extractor. The extraction solvent to be used is taken into a distillation flask and the Soxhlet
extractor is now placed onto this flask. The Soxhlet is then equipped with a condenser.

The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods
into the chamber housing the thimble of solid. The condenser ensures that any solvent vapour
cools, and drips back down into the chamber housing the solid material.

The chamber containing the solid material is slowly filled with warm solvent. Some of the
desired compound will then dissolve in the warm solvent. When the Soxhlet chamber is almost
full, the chamber is automatically emptied by a siphon sidearm,with the solvent running back down
to the distillation flask. The thimble ensures that the rapid motion of the solvent does not transport
any solid material to the still pot. This cycle may be allowed to repeat many times, over hours or
days.

During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many
cycles the desired compound is concentrated in the distillation flask. The advantage of this system
is that instead of many portions of warm solvent being passed through the sample, just one batch
of solvent is recycled.

After extraction the solvent is removed, typically by means of a rotary evaporator, yielding
the extracted compound. The non-soluble portion of the extracted solid remains in the thimble, and
is usually discarded.
 19

Figure 5: Soxhlet apparatus

2.4 (b) Extraction of Eryngium foetidum

Extraction using Petroleum ether as solvent

50g of leaf powder was tightly packed with Whatman No: 4 filter paper. It was then carefully
transferred into Soxhlet extractor for extraction. Petroleum ether (160ml) was used as the solvent
for extraction. Extraction continued until the solvent changes to colourless.
Extraction took about seven hours. Extract was stored at cold temperature.

Extraction using Ethyl acetate as solvent

50g of leaf powder was tightly packed with Whatman No: 4 filter paper. It was then
carefully transferred into Soxhlet extractor for extraction. Ethyl acetate (160ml) was used as the
solvent for extraction. Extraction continued until the solvent changes to colourless. Extraction took
about seven hours. Extract was stored at cold temperature.

Extraction using Ethanol as solvent

50g of leaf powder was tightly packed with Whatman No: 4 filter paper. It was then
carefully transferred into Soxhlet extractor for extraction. Ethanol (160ml) was used as the solvent
for extraction. Extraction continued until the solvent changes to colourless. Extraction took about
seven hours. Extract was stored at cold temperature.
 20

2.5 CRUDE EXTRACT DILUTION

From each crude extract 15mg, 30mg and 50mg extracts were accurately weighed and serially
dissolved in 1ml of acetone. Then it was made upto 100ml by using distilled water. Thus from
each extract 150ppm, 300ppm, and 500ppm concentration of test solutions were prepared. Test
solutions of three different concentrations of each of the nine extracts were used for testing the
Sitophilus oryzae in three time periods (24hr,48hr,72hr).(Makanjuola WA 1989)

2.6 TREATMENT- PESTICIDAL ACTIVITY

Lab studies have been carried out to ascertain pesticidal property of the plant
extract(Eryngium foetidum) against Sitophilus -oryzae.
DISC METHOD

Figure 6: control used in disc method Figure 7: showing mortality of adults of Sitophilus oryzae

The methodology used for contact assay with impregnated filter paper was proposed Huang et al
(1997). Sheets of filter paper (diameter 3cm) were impregnated with test solution (500ppm,
300ppm, 150ppm) .One ml of each extract solution was spread with the help of pipette over the
filter paper and placed into petri dishes, while control sheets were impregnated by without any
plant extract. The filter paper discs impregnated with extracts were air dried until complete
evaporation. Then they were placed into plate and 25 adults of Sitophilus oryzae were released in
each treatment and control dish, with three replicates per treatment and control plate. The plates
were wrapped in plastic film to prevent escape of pest. Small holes were made on the plastic film
for aeration. Mortality counts were taken after 24hr, 48hr, 72hr intervals of treatment and expressed
 21

as percentages of mortality. Dead insects were counted, death being ascertained by the complete
absence of movement.

2.7 DATA ANALYSIS

The pests Sitophilus oryzaewere collected. Only adults were taken for bioassay. Plant
leaves were sorted and identified to species level. Three plants screened for pesticidal activity was
made. Data were grouped into three tables (table1, table2, table3). Pest mortality was measured in
percentage and if the control mortality was ranged between 5-20%, it was corrected using Abbotts
formula (Abbott 1925).

(% testmortality %controlmortality) 100

Corrected mortality
(100 %controlmortality)

Percentage of pest mortality can be calculated by using the formula

Numberof dead pest

Percentage mortality 100 Total
numberof pest

Standard deviations were calculated for three replicates and recorded for 24hr, 48hr and 72hr.
Graphs were based on time intervals (24hr, 48hr, and 72hr). Microsoft Excel package was used to
prepare percentage mortality graphs against different concentrations of plant extract. Each graph
compares the percentage of mortality of three solvents (petroleum ether, ethyl acetate, ethanol).
 22

CHAPTER-3 RESULTS AND DISCUSSION

Pesticidal activity of the plant extract(Eryngium foetidum) in three different solvents
(Ethanol, Petroleum ether, Ethyl acetate) was tested against adults of Sitophilus oryzae, at different
concentrations (150ppm,300ppm,500ppm). The experiment showed a relation of pest mortality
level among 3 time intervals (24hr, 48hr, and 72hr). The results indicate variation in mortality
among the plant extracts tested against the selected pest. The summary of results, in which the
percentage of mortality of adults of Sitophilus oryzae caused by different plant extracts, are
represented in Table-1.
The results indicate that mortality values significantly increased depending on the
increasing plant extract concentration and with time of exposure. As considering Leaf extracts
ethyl acatate medium Eryngium foetidum is more effective than petroleum ether and ethanol.
Studies were carried out with Eryngium foetidum at the three concentrations in three different time
interval. More over at 500ppm concentration it gives 100%mortality.

The order of increase of mortality rate is: Eryngium foetidum (ethyl acetate) > Eryngium
foetidum (ethanol) > Eryngium foetidum (petroleum ether).

3.1 Pesticidal activity of Eryngium foetidum against Sitophilus oryzae

Table 1: Mortality percentage of sitophilus oryzae treated with plant extracts of Eryngium foetidum
by disc method.
No. of Mortality (%)
Name of the plant Conc.(ppm) insects used
(Solvent) 24(hr) 48(hr) 72(hr)
in the study
(%) (%) (%)
Calotropis procera 500 25 58±0.08 68±0.06 75±0.08
(Petroleum 300 25 50±0.03 63±0.05 68±0.08
ether) 150 25 45±0.08 57±0.09 63±0.07
Control - 25 - - -
Calotropis procera 500 25 67±0.08 77±0.07 84±0.05
(Ethanol) 300 25 60±0.05 72±0.06 79±0.08
150 25 49±0.07 65±0.08 74±0.09
Control - 25 - - -
Calotropis procera 500 25 84±0.02 93±0.05 100±0.03
300 25 78±0.04 89±0.06 95±0.06
(Ethyl acetate) 150 25 70±0.08 83±0.06 90±0.07
Control - 25 - - -
*values are mean percentage of three replication of experiment with 25 insects,±SD
 23

The leaf extract of Eryngium foetidum in three solvents (petroleum ether, ethanol, ethyl
acetate) at three time intervals (24hr., 48hr., 72hr.,) also at different concentrations (150 ppm, 300
ppm, 500 ppm) the more effective one is ethyl acetate extract of Eryngium foetidum leaves.
And most significant result is produced after 72hr treatment i.e. 100% mortality.

Graph-1:Mortality percentage after 24hour treatment of Sitophilus oryzae with Eryngium

foetidum.
90 84
78
80
70
70 67
60 58
60
49 50
50 45
Petroleum ether
40
Ethanol
30 Ethyl acetate
20
10
0
150 300 500
Concentration (ppm)

Here ethyl acetate extract shows higher mortality and petroleum ether extract shows lesser
mortality. Whereas ethanol extract shows medium mortality.

The order of increase of mortality rate is: Eryngium foetidum (ethyl acetate) > Eryngium foetidum
(ethanol) > Eryngium foetidum (petroleum ether).

Graph-2: Mortality percentage atfter 48 hour treatment of Sitophilus oryzae with Eryngium
foetidum.

Here ethyl acetate extract shows higher mortality and petroleum ether extract shows lesser
mortality. Whereas ethanol extract shows medium mortality. The order of increase of mortality
rate is: Eryngium foetidum (ethyl acetate) > Eryngium foetidum (ethanol) > Eryngium foetidum
(petroleum ether).
 24

100 93
89
90 83
80 77
72
68
70 65 63
60 57

50 Petroleum ether
40 Ethanol

30 Ethyl acetate

20
10
0
150 300 500
Concentration (ppm)

Graph-3: Mortality percentage after 72 hour treatment of Sitophilus oryzae with Eryngium
foetidum
120

100
100 95
90
84
79
80 74 75
68
63
60 Petroleum ether
Ethanol
40 Ethyl acetate

20

0
150 300 500
Concentration (ppm)

Here ethyl acetate extract attain 100%mortality, while petroleum ether extract shows lesser
mortality. Whereas ethanol extract shows medium mortality. The order of increase of mortality
rate is: Eryngium foetidum (ethyl acetate) > Eryngium foetidum (ethanol) > Eryngium foetidum
(petroleum ether).
 25

From the analysis of above three graphs, it shows that ethyl acetate extract of Eryngium
foetidum produce higher pesticidal activity than ethanol and petroleum ether extract. Mortality
value increases with concentration of extract and also with time of exposure.

The present work reveals the pesticidal effect of the leaf extracts of Eryngium foetidum in three
solvents (ethyl acetate, petroleum ether, ethanol) against adults of Sitophilus oryzae. The
experiments reveal that ethyl acetate extract of Eryngium foetidum offers higher percentage of
mortality to Sitophilus oryzae. Meanwhile Eryngium foetidum extract in petroleum ether shows
the least mortality rate.

GCMS analysis of Eryngium foetidum from the earlier report show that alkaloids α-Pinene and
Sabinene (Jorge A. Pino et al 1997).are present in higher percentage. The higher mortality rate in
ethyl acetate extract may be due to the presence of α-Pinene and Sabinene.

The toxicity of plant extract to stored product pest, Sitophilus oryzaeis influenced by the
chemical composition of the plant, time of exposure, plant parts used, method of extraction and
concentration of the plant extract.

The results of the present study indicate that, by considering the percentage mortality as a main
index, Eryngium foetidum in ethyl acetate proved to be the most effective plant extract potential to
be use as a grain protectant. These plants have a range of chemicals which can be isolated and used
for pest control. The test plants being medicinal would yield environmentally sound chemicals
having no harmful effects on the non target organisms also these pesticidal properties can be
harnessed cheaply for use in agriculture and related fields.
 26

CHAPTER-5 CONCLUSION

The current study strongly recommends the need of exploring botanical derivatives as pest
control agents. Even though there are differences in concentration of extracts, all solvent extracts
could act as a toxic agent to prevent the proliferation of the test animal.
The toxicity of plant extract to stored product pest, Sitophilus oryzaeis influenced bythe chemical
composition of the plant, time of exposure, plant parts used, method of extraction and
concentration of the plant extract.

The studied plant extracts of Eryngium foetidum leaves in ethyl acetate solvent is highly toxic
against adults of Sitophilus oryzae. Higher doses and exposure time are required to achieve
100%mortality for the adults of Sitophilus oryzae. The ethyl acetate extract of Eryngium foetidum
could be used as a potential grain protectant against Sitophilus oryzae. The use of botanical
materials as insecticides will benefit our agricultural sector. They are not only of low cost, but have
no environmental impact in term of insecticidal hazard. Therefore, the findings of the current
experiments strongly support the use and exploration of botanicals in pest management practices.

The results of pesticidal activity of a number of higher plants based on traditional

knowledge strongly indicate that plants are endowed with pesticidalpropertythat can be harnessed
cheaply for use in agriculture and related fields. The need to useplant-based products arises from
the fact that the synthetic pesticides are harmful to humans, and the entire ecosystem due to high
toxicity and persistence. Also, they are too expensive for the poor farmers in the developing
countries of the world. On the otherhand, plant- based products are cheap and biodegradable and
are therefore environmentally friendly. However, an agricultural programme that depends
essentially onplant-based materials must be backed-up by a vigorous research programme into new
plant sources.
Therefore, the leaf extract of Eryngium foetidum in ethyl acetate solvent,a potentially useful
agent to control Sitophilus oryzae,has to be better explored by doing detailed chemical analysis of
the active compounds present in it and by understanding the mechanism involved in action.
 27

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