Acknowledgment

We would like to thank the Almighty GOD for giving us the strength and wisdom to successfully
complete our task for his protection and strength, and an ever present helps in the entire
Situation and challenge that we face. Moreover, we would like to express our heartfelt
appreciation and thank to our lecturers, Mr.Girma and Dr. Anuradha for their sustainable and
appreciable guidance, skill, experience sharing of Knowledge by teaching us how to write
successful project proposal.

We would also like to thank the Chemical Engineering Laboratory staff members especially
Ms.Demeku for her contribution by giving information about the availability of necessary
chemicals and equipment’s in the laboratory. Finally, we acknowledge our friends with the
deepest gratitude who encouraged us to strive toward academic-excellence.

i
Table of Contents

Acknowledgment ........................................................................................................................ i

Table of Contents ....................................................................................................................... ii

List of table .................................................................................................................................v

Abbreviations ............................................................................................................................ vi

Abstract ................................................................................................................................... vii

1. Introduction .........................................................................................................................1

1.1. Statement of the problem ..............................................................................................3

1.2. Significance of the study ...............................................................................................3

1.3. Objectives .....................................................................................................................3

1.3.1. General objective ...................................................................................................3

1.3.2. Specific objectives .................................................................................................4

2. Literature review .....................................................................................................................5

2.1. Maize production ..............................................................................................................5

2.2. Maize storage mechanism in Ethiopia ...............................................................................5

2.3. Maize weevils and its control mechanisms ........................................................................6

2.3.1 Cultural Control ..........................................................................................................7

2.3.2. Chemical Control .......................................................................................................8

2.3.3. Biological control ......................................................................................................8

2.3.4. Botanical control........................................................................................................9

ii
 2.2. Vernonia amygdalina plant ............................................................................................. 10

2.3. Bioactive Compounds in Vernonia amygdalina .............................................................. 12

2.3.1. Flavonoids ............................................................................................................... 12

2.3.2. Sesquiterpene lactones ............................................................................................. 13

2.3.3. Triterpenoids ........................................................................................................... 13

2.4. Bioactivities of V. amygdalina ........................................................................................ 14

2.4.1. Pesticidal or Insecticidal activity .............................................................................. 14

2.4.2. Antioxidant .............................................................................................................. 14

2.4.3. Antimicrobial Effect ................................................................................................ 15

2.4.4. Antifungal Effect ..................................................................................................... 15

2.4.5. Antibacterial Effect .................................................................................................. 15

2.4.6. Anti-inflammatory Effect ......................................................................................... 16

2.5. Composition of essential oil of V.amygdalina ................................................................. 16

3. Materials and Methods ...................................................................................................... 17

3.1. Materials ........................................................................................................................ 17

3.1.1. Raw materials .......................................................................................................... 17

3.1.2. Chemicals ................................................................................................................ 17

3.1.2. Laboratory equipment and devices ........................................................................... 17

3.2. Methods ......................................................................................................................... 17

3.2.1. Plant materials collection and preparation for oil extraction ..................................... 17

iii
 3.2.2. Extraction of essential oil ......................................................................................... 18

3.2.3. Analysis of essential oil ........................................................................................... 18

3.2.6. Progeny assessment assays....................................................................................... 19

3.2.7. Damage assessment assays....................................................................................... 20

3.2.8. Repellency ............................................................................................................... 20

3.3. Data analysis .................................................................................................................. 21

4. Cost break down.................................................................................................................... 22

5. Schedule and work plan......................................................................................................... 24

Reference .................................................................................................................................. 26

iv
List of table

Table 1: Different local name of V.amygdalina ......................................................................... 11

Table 2: Cost break down .......................................................................................................... 22

Table 3: Schedule and work plan ............................................................................................... 25

v
 Abbreviations

BHT Butylated Hydroxytoluene

CSA Central Statistical Authority

CSA ASS Central statistical agency Agricultural Sample Survey

DPPH α-diphenyl- β-picryl-hydraz

FAO STAT Food and Agriculture Organization Corporate Statistical Database

FDRE Federal democratic republic of Ethiopia

GC Gas Chromatography

GC-MS Gas Chromatography - Mass Spectrometry

H2SO4 Sulfuric Acid

HCl Hydrochloric Acid

MMT million metric tonnes

S Sitophilus

V Vernonia

vi
 Abstract

85% of Ethiopian people engage in agriculture. They produce a various food crops such as
cereals, pulse, oilseeds, vegetables, root crops, fruit crops, stimulant crops and sugar cane. From
the total grain crop, cereals crop covers 87.48% and maize is 27.43% of the cereals crop
produced by Ethiopian people. Maize in Ethiopia cultivated mostly at mid to lower altitude and
sub humid agro-ecologies along the country’s western, southwestern and eastern borders.
According to the Centre for Regional Agricultural Trade Expansion Support (RATES, 2003),
80 percent of the total national production is used for household consumption and seeds, while
the remaining 20 percent is for sale. It is mainly attacked by weevils called Sitophilus zeamais.
Eticha (1999) reported an annual maize grain loss in Ethiopia in the range between 2 and 30%. In
Jimma Zone, quantitative average maize grain storage loss of 41.0 to 80.0% and on average
64.5% of grain damage was estimated in 2004 from fifty traditional farm stores within three to
six months of which maize.

The efficient and effective control of storage insects like S. zeamais has centered mainly on the
use of synthetic insecticides. However, the use of these chemicals is hampered by many
attendant problems. Therefore botanical insecticides are used as grain protectants as they have
insecticidal properties against stored grain insect pests as well as being safer for human health
and the environment. The main aim of this study is to produce non-toxic and environmental
friendly maize weevils protect by extracting essential oil from locally available vernonia
amygdalina plant.

Essential oil will be extracted from V.Amygdalina leafs by using steam distillation.
Characterization, identification and determination of relative amounts of components of the
essential oil will be done through Gas-Liquid Chromatography (GC), Gas Chromatography -
Mass Spectrometry (GC-MS), and GC Co injection of the essential oil with authentic standards.
Adults of S.zeamais will be collected from Melkassa Agricultural Research Center and will be
cultured. The Mortality, damage, Progeny and repellency action of essential oil against maize
weevils will be assessed.

vii
 1. Introduction

Higher percent of Ethiopian people engage in agriculture (farmers). These farmers produce the
food crops that grouped into cereals, pulses, oilseeds, vegetables, root crops, fruit crops,
stimulant crops and sugar cane. Stimulant crops consist of Chat, coffee and hops. Out of the total
grain crop produced by Ethiopian people 87.48% was under cereals. Maize, teff, wheat and
sorghum made up 27.43% (83,958,872.44 quintals), 17.26% (52,834,011.56 quintals), 15.17%
(46,429,657.12 quintals) and 16.89% (51,692,525.40 quintals) of the grain production
respectively. They hold 76.75 % of cereals produced by Ethiopian people (FDRE CSAASS,
2018). Maize, wheat and sorghum containing the larger amount of Ethiopian grain crop products
are easily attacked by maize weevils (Sitophilus zeamais) in store, but it mainly attacks maize.

Maize in Ethiopia cultivated mostly at mid to lower altitude and sub humid agro-ecologies along
the country’s western, southwestern and eastern borders. As the crop is grown by a large number
of smallholders (8.7 million), it is critical to small holder livelihoods in Ethiopia. Maize in
Ethiopia cultivated mostly at mid to lower altitude and sub humid agro-ecologies along the
country’s western, southwestern and eastern borders. According to the Centre for Regional
Agricultural Trade Expansion Support (RATES, 2003), 80 percent of the total national
production is used for household consumption and seeds, while the remaining 20 percent is for
sale. Weevil has different family and species. From those the Sitophilus zeamais mainly attack
maize but it also able to develop on all cereals (E.F. Asawalam1 et al., 2006). Eticha (1999)
reported an annual maize grain loss in Ethiopia in the range between 2 and 30%. In Jimma Zone,
quantitative average maize grain storage loss of 41.0 to 80.0% and on average 64.5% of grain
damage was estimated in 2004 from fifty traditional farm stores within three to six months of
which maize (Dubale Befikadu1 et al, 2015).It is capable of inhibiting reserved breeding grounds
near the threshing floors that are normally full of plant residues, where the population builds up
in before moving to granaries. Adult females chew grains creating a small hole in which they lay
eggs and then seal the hole with a secretion. The optimum temperature for oviposition is around
25oC and at grain moisture contents of over 10 percentages. Larvae tunnel in grains and are
responsible for most of the damage. Pupation takes place inside the grain and adults chew their
way out through the outer layer of the grain. Adults live for 5-6 months depending on the
temperature and humidity of grains (R. Suleiman, K.A. and Rosentrater, 2015).

1
The efficient and effective control of storage insects like S. zeamais has centered mainly on the
use of synthetic insecticides (E.F. Asawalam1 and A. Hassanali, 2006). However, the use of
these chemicals is hampered by many attendant problems such as the development of insect
resistant strains, toxic residues in food and humans, workers safety and high cost of procurement
(E.F. Asawalam and A. Hassanali, 2006). These problems have necessitated research on the use
of alternative eco-friendly insect pest control methods amongst which are the use of botanical
pesticides (awoke yohanis et al,2014 ; E.F. Asawalam and A. Hassanali,2006; Emeribe E.O,2018
).

Studies in some African countries suggest that extracts of locally available plants can be
effective as crop protectants (Emeribe E.O, 2018). Natural products are excellent alternatives to
synthetic pesticides as a mean to reduce negative impacts on human health and the environment.
Essential oils of aromatic plants among the various kinds of substances have received particular
attention as natural agents for insect management. Plant essential oil are renewable non-
persistent in the environment and relatively safe to natural enemies, non-target organisms and
human beings (Karim Saeid and Hadi Shahab-Ghayoor, 2015). Therefore botanical insecticides
are used as grain protectants as they have insecticidal properties against stored grain insect pests
as well as being safer for human health and the environment.

Vernonia amygdalina is one of those locally available plant used for a multipurpose and rapid
regenerating plant. This plant is available in our country (Etefa Gurmesa, 2017). V. amygdalina
produces a variety of flavonoids and bitter sesquiterpene lactones which contribute to the
bioactivities of this plant (Swee Keong Yeap et al ,2010). The bioactivities of this plant are
antifungal, antibacterial, antimalaria, Antiparasite activity, Antihelmintic activity,antiviral
activity, Anticancer and cytotoxic effect, Antimutagenic acitivity, Antifertility activity,
antioxidant, Antidiabetic effect and Pesticidal or Insecticidal activity (Swee Keong Yeap et al
,2010). Nevertheless, the usefulness of V. amygdalina was not only beneficial for medical and
healthcare purposes but it also serves as a low cost, environmental friendly, readily available and
non-toxic natural inhibitor for the corrosion of mild steel in HCL and H2SO4, as well as
aluminium alloy in HCL and HNO3 (Swee Keong Yeap et al ,2010).Therefore the main goal of
this study is to extract essential oil from vernonia amygdalina plant for maize weevils (Sitophilus
zeamais) protection.

2
 1.1. Statement of the problem

Above 85% of Ethiopian people engage in agriculture. Ethiopian farmer produce cereals such as
maize, teff, sorghum and wheat in higher amount. They also produce rice, peas, beans, lentils
and other cereals in smaller amount. All those grain crops are attacked by maize weevil
(Sitophilus zeamais), but this type of weevil mainly attack the maize which is the higher grain
crop produced by Ethiopian farmers. Currently the Ethiopian farmers use weevils protect which
is not environmentally friendly, toxic to human and not easy to use these crops after weevils
protect (pests) is added. Therefore botanical insecticides are used as grain protectants as they
have insecticidal properties against stored grain insect pests as well as being safer for human
health and the environment. The main aim of this study is to produce non-toxic and
environmental friendly maize weevils protect by extracting essential oil from locally available
vernonia amygdalina plant.

1.2. Significance of the study

This study will contribute a significant method of maize weevils (Sitophilus zeamais) protection
by extracting essential oil from v.amyhdalina plant. In our country the farmers produce maize,
sorghum and wheat in larger amount. The primary goal of these farmers is to store these grain
crops and use for food and for other purpose when it is needed. However, these grain crops are
easily attacked by maize weevils. In addition the farmers fear to use the current synthetic
chemical weevil protection due to they think about its toxicity to human and environmental
pollutant. This study will contribute for our country that it is better to use essential oil extracted
from V.Amyhdalina than synthetic chemical to protect weevils in the way of not to pollute the
environment and to reduce the toxicity to the human.

1.3. Objectives

1.3.1. General objective

The goal of this proposed research project is to extract essential oil from Vernonia
amygdalina for maize weevil (Sitophilus zeamais) protection.

3
 1.3.2. Specific objectives

The specific objectives of this study are:

 To analyze the chemical composition of V. amygdalina leaves and to identify the active
chemical constituents of the essential oil
 To evaluate the effect of the essential oil of V. amygdalina leaves on mortality,
reproduction (on number of F1 progeny) and repellency of S. zeamais;
 To determine the effects of the essential oil on the weight loss of stored maize grains.

4
2. Literature review

2.1. Maize production

Maize is the most widely grown cereal crop. In the global production of cereal crops, maize
ranks first. Maize production and consumption vary greatly throughout Africa. In large parts of
eastern and southern Africa, maize is the principal staple food produced and consumed by most
farming households. Maize is produced in Africa on over 20 million hectare, which is 16% of the
total world area under maize production. The total annual production is about 25 million tones
and this represent 6% of the total world production. The eastern and southern African countries
produce 64% (16.2 million tones) of all Africa maize. Ethiopia is the second most important
producer next to Kenya in east Africa (dawit kidane, 2005). The crop is widely produced in
western, southern, south western, north western and eastern part of the country. Maize is
extremely an important crop grown in Ethiopia. At present, it is the first in total production and
yield per hectare among cereals. According to the Federal Democratic Republic of Ethiopia
Central Statistical Agency (FDRE CSA, 2018) Maize covers 27.43% of the cereals crops
production in country.

2.2. Maize storage mechanism in Ethiopia

In sub-Saharan Africa to estimate maize grain losses in traditional storage practices have shown
that the losses are generally high. From harvest to consumer market, maize grain postharvest
losses in Africa are estimated to range 14 to 36%. In Ghana, about 15 % of maize grains
harvested are lost annually due to attacks by maize weevils. One in five kg of grain produce in
sub-Saharan Africa is estimated to be lost by pest infestations and associated grain decays of
maize grain loss remained the highest In Nigeria 10 to 12% loss of maize grains stored in
traditional storage containers similar to Gombisa due to insect pests. Loss of about 18% was also
in other African countries for maize grain stored in polypropylene sacks for the storage periods
of six months.

In Ethiopia traditionally maize grain is stored both in- and outdoors by Ethiopian farmers for
consumption and to sell in the later months of the year depending on the quantity produced per
household. Survey conducted in three major grain producing areas of Ethiopia viz. Hetosa, Ada

5
and Bako indicated that majority of farmers (93.3%) are using various traditional grain storage
containers that expose their stored grains to be attacked by storage pests and other factors that
contribute to deteriorations where by per house hold grain 12% was estimated losses from the
total grain produced (Abebe and Bekele, 2006). Eticha (1999) reported an annual maize grain
loss in Ethiopia in the range between 2 and 30%. In Jimma Zone, quantitative average maize
grain storage loss of 41.0 to 80.0% and on average 64.5% of grain damage was estimated in 2004
from fifty traditional farm stores within three to six months of which maize (Dubale Befikadu1 et
al, 2015)

Today, more than ever, safe grain storage and prevention of post-harvest losses by farmers has
become more a necessity than a rule to overcome shortage of grain and to tackle starvation and
hunger in Ethiopia. This is crucial to ensure food security and to feed the ever-increasing
population of the country where more than 85% of the broad mass of the population earns its
livelihood directly or indirectly from agriculture. Minimizing the post-harvest grain losses due to
destructive agents such as storage fungi, pests need due attention by researchers, grain managers,
farming community and consumers. The need for maintenance of the grain nutritive and other
inherent qualities through improved storage methods is unquestionable and timely.

2.3. Maize weevils and its control mechanisms

The maize weevil, Sitophilus zeamais is one of the most destructive stored product pests of
grains, cereals, and other processed and unprocessed stored products in sub-Saharan Africa. S.
zeamais causes qualitative and quantitative damage to stored products, with grain weight loss
ranging between 20 to 90% for untreated stored maize, and the severity of damage depends on
factors which include storage structures and physical and chemical properties of the produce
(James Adebayo Ojo1 and Adebayo Amos Omoloye, 2016).

Maize weevil is widely distributed throughout the warm humid areas of the world and can attack
a wide range of cereals although it is particularly a pest of maize. It is well established in tropical
countries including Ethiopia. Maize weevil and rice weevil are found in all warm and tropical
parts of the world. These pests are carried all over the world in grain shipments and can establish
themselves wherever there is food and where grain moisture and temperature are favorable. In
various locations, one species may be more common than the other. The female weevil chews a

6
minute hole in the grain in which the eggs are deposited. Eggs, larval and pupal stages are all
found within tunnels and chambers bored in the grain and are not seen. Because larval stages
feed in the internal parts of the grain, it is difficult to detect infestations early. Damage caused by
this insect becomes noticeable when the adult insect makes holes that reach approximately 1mm
in size in the grain and deposits its eggs within the hole. The insect then closes up the
developmental stage of the insect takes place within the grain after which the adult weevil bores
its way out, leaving a characteristic emergence hole on the grain. Adults emerge from the grain
and can be seen walking over the grain surfaces. The life span (from egg to adult) is about 30
days, which is slightly longer than that of rice weevil. Mating takes place within a few hours
after the adult weevils emerge. Adult maize weevil is an active flier resulting in many field
infestations in areas adjacent to infested storage (Yeshiwork Alemne,2017).

The common control methods for this pest are the use of chemical insecticides, biological
control, and botanical insecticides among others. There cannot be a realistic success in applied
ecology and pest monitoring and management without a better understanding of the phenology
and dynamics of insects’ life cycle (Dawit Kidane,2005).

2.3.1 Cultural Control

Farmers use a range of traditional methods of control or devise strategies to cope with insect
infestation, which are selected from one generation to the next. The most common traditional
treatments to limit insect activity are mixing inert materials and organic materials with stored
grain. The success of protection depends upon the effect of the preservative on the grain, the
rapidity of its action, the period of storage and proper mixing (Dawit Kidane, 2005; ).

A similar effect can also be achieved through treatment with wood ash, collected from burnt tree
wood or a farmers stove. Some farmers may also add fine sand to hinder the pest activity, in
which the high proportion of the quartz damage to sensitive cuticle of the newly hatched insect
(yeshiwork alemne, 2017). In an experiment in Bako Research Center, heating maize grain at 70-
80oC in an oven for 1 hour, maize plus dockage, and exposure to the sun showed comparable
results to the standard insecticide pirimiphos-methyl at 10ppm in protecting maize grain from the
maize weevil (Ahmed Ibrahim and Aschalew Sisa, 2012).

7
Exposure to sunlight or exposure followed by sieving of the grains usually at monthly interval is
also well known technique among farmers and create an unfavourable environment for weevils
(dawit kidane, 2005). Other traditional methods include winnowing, shaking, restaking grain
sand mixing grains with small sized grains (teff) reduce insect activity (yeshiwork alemne, 2017;
dawit kidane, 2005).

2.3.2. Chemical Control

Many small scale farmers adopted the use of insecticides for limiting the population of storage
insect pests. Chemicals that are used to treat grain stores and grain must possess the correct blend
of biological activity and either low mammalian toxicity or short residual life. Dusting and
fumigation of grains are the most commonly used chemical methods (dawit kidane, 2005).

Dusting is an easy applied method, can be implemented with very cheap tools such as small
containers or cloth bags with punctured holes in the lid. For small amounts of grains, dust can be
mixed with grains using a shovel. Dust should be mixed throughlly and distributed evenly all
over the produce. Dusts can also be applied on floors, flat surfaces and around the bottom of
storage containers. The most commonly used insecticide dusts among farmers are malathion,
deltamethrin, permethrinandprimiphos-methyl (dawit kidane).

Fumigants are low molecular weight chemicals, highly toxic and volatile and are, therefore self-
dispersing and non-persistant. Fumigation is a widely used method all over the world particularly
for large scale storage. Fumigants have an ability to kill all insect stages residing in the grains,
but do not protect grain from new attacks. Fumigants must be used in air tight containers. The
most widely spread fumigants in use are Phosphine (PH3) and Methyl bromide (CH3Br) (dawit
kidane, 2005; yeshiwork alemne, 2017).

2.3.3. Biological control

Biological control involves using biological means, as opposed to chemical and physical means,
to control pest species. Traditionally, biological controls have involved the introduction of an
exotic species such as predators, parasites and diseases. However, the efficacy of biological
control using natural enemies depends on a complex but delicate relationship between natural
enemies and their insect pest hosts whose balance can be offset by a changing climate. It is well

8
known that temperature fluctuations are the major factors affecting insect biology, activity and
distribution of natural enemies in agro-ecosystems (B. Paparatti and S. Speranza, 2005).

It may provide a useful safe alternative for the control of crop pests. Various instances are well
documented with regard to the possibilities of using natural enemies to suppress the stored grain
insects (dawit kidane, 2005). However, the use of biological control against stored product is still
limited though recently gaining emphasis due to environmental and health concern (B. Paparatti
and S. Speranza; Dawit kidane,2005; yeshiwork alemne, 2017).

2.3.4. Botanical control

Botanicals are typically pesticides, working in the same fashion as commercial synthesis.
However, botanical have several modes of action. Toxicity against insect may be expressed by
direct killing particular life stages of the insect, interfering with mating or suppressing
reproduction, acting a repellent or affecting host finding and selection in a way that prevents
infestation, reducing or preventing feeding (Asawalam, E F.and Hassanali,A., 2006). The other
characteristics shared by most botanicals are their persistence in the environmental is less; they
act very quickly to stop feeding by plant insects that is they cause immediate mammalian toxicity
and not phytotoxic (Dawit kidane,2005; Asawalam, E F. andHassanali, A.,2006).

Plant products can be obtained either from the whole plant or from a specific part by extraction.
The most common way of using plants in post-harvest protection is the admixture of powders,
oils and more purified insecticides including use of essential oils and organic solvent extracts
(Asawalam, E F and Hassanali, A., 2006; Swee Keong Yeap et al.,2010).

Use of plant products or insecticides is one of the important approaches of insect pest
management and it has many advantages over synthetic insecticides. Plant materials with
insecticidal properties provide small-scale farmers with locally available, biodegradable and
inexpensive method of pest control for storage (Karim Saedi and Hadi Shahab.Ghayoor, 2015).
Farmers, in Ethioipia, use local herbs by mixing with grain to reduce infestation in stored grains (
Dawit kidane, 2005).

9
2.2. Vernonia amygdalina plant

Vernonia amygdalina is a multipurpose and rapid regenerating soft wooded shrub of 2 to 10 m

tall with petiolate leaves of around 6 mm in diameter. This plant has been named differently by
different ethnics around the world. It is a species under the genus Vernonia Schreb which
contains about 1000 species. More than 500 of these Vernonia plants are distributed in Africa
and Asia, approximately 300 in Mexico, Central and South America and around 16 can be found
in the United States. However, the taxonomic ranking of this genus at all levels; ranging from its
species up to the generic term can be a real challenge. Even taxonomy of V. amygdalina from
different geographical area (Ethiopia and Cameroon) could be different. One of the proposed
solutions is through classification of the active chemical compounds, isolated from each species
(Swee Keong Yeap et al., 2010).

10
Table 1: Different local name of V.amygdalina

Country Local name(s)

Ethiopia Grawa, ibicha

Cameroon Ated nkol, Suwaaka

Ghana Awonoo, Awonwene, Jankpantire

Uganda Labwori, Omubirizi, Ekibirizi

Nigeria Onugbo,Ewuro, Etidot Ityuna, Oriw

China Ikaruga

Congo Mpasi nyioso

English Bitter leaf

Source:(Oluwaseun Ruth Alaraa et al.,2017)

Water is the key factor for the growth of its leaves. Thus, high yield can be obtained during rainy
season. V. amygdalina is a short cycle crop which can be harvested twice per month for up to
seven years. Planting V. amygdalina can be easy. The presence of bitter principles also protects
V. amygdalina from most of the animals, insects and microbes (Swee Keong Yeap et al.,
2010).In Ethiopia this plant has been widely used as fuel wood, stakes, construction of poles,
fencing of agroforestry buffer zone and as ingredient for compost. Due to its bitterness, it also
can be used as a bittering agent, a hop substitute and for the control of microbial contamination
in beer brewing without affecting the quality of malt (Etefa Gurmesa, 2017). It is also used to

11
make honey wine called Tej in country. The traditional practices of V. amydalina in Ethiopia are
Stomach disorder, skin wound, diarrhea, scabies, hepatitis, ascarasis, tonsillitis, fever, mastitis,
tapeworm and worms infection, Stomach ache (worm expulsion) . Besides its use as a single
herbal ingredient, V. amygdalina has also been incorporated as an important ingredient in
traditional polyherbal formulations to treat various diseases (Swee Keong Yeap et al., 2010).

2.3. Bioactive Compounds in Vernonia amygdalina

Several studies had been done in isolating and characterizing some bioactive compounds from V.
amygdalina. The phytochemical studies had resulted in the isolation of flavonoids, saponins,
alkaloids, tannins, phenolics, terpenes, steroidal glycosides, triterpenoids, and several types of
sesquiterpene lactones. Sesquiterpene lactones (vernodalinol, vernolepin, vernomygdin,
hydroxyvernolide, vernolide and vernodalol) had been reported to inhibit breast cancer cell
growth, possessed antitumoral and antimicrobial properties, and exhibited a significant
bactericidal activity against gram bacteria. Isolated vernoniosides from V. amygdalina leaves
exhibited anti-inflammatory property and used in the treatment of gastrointestinal disorder when
tested with murine macrophage cell line and wild chimpanzees, respectively. Flavonoids,
tannins, saponins, and triterpenoids had been studied to possess antioxidant and hypolipidaemic
effects (Oluwaseun Ruth Alara et al, 2017).

2.3.1. Flavonoids

Flavonoids are a group of natural compounds found in plants with variable phenolic structures.
They are present virtually in all parts of the plant. Flavonoids are responsible for the taste,
colour, protection of vitamins and enzymes, and prevention of fat oxidation. The pharmacology
importance of flavonoids, including, antioxidant, hepatoprotective, antibacterial, anti-
inflammatory, anticancer, and antiviral activities are indispensable in the human body. V.
amygdalina possesses flavonoids which can be extracted from the leaves using organic solvents.
Different flavonoids had been isolated from the crude extracts of V. amygdalina leaf. In the early
study, three flavones (luteolin, luteolin 7-O-βglucuronoside and lutelin7-O-β-glucoside) were
identified. The identified flavones showed strong antioxidant activities. Luteolin exhibited
stronger antioxidant activity in comparison to the synthetic butylated (BHT) at 15 mg/l. Besides
this, luteolin 7-O-glucuronoside (the most abundant compound) and luteolin 7-O-glucoside also

12
possess similar antioxidant activity but significantly lower as compared to luteolin. This may be
attributed to the blockage of glycosides with glucose or uronic acid linked at the 7-O position
compared with the unsubstituted 5, 7- OH of luteolin. Higher antioxidant inhibition was
observed in the ethanolic extraction of luteolin than aqueous extract. This may contribute to the
higher antioxidant activity of ethanol extract compared to water extract (Oluwaseun Ruth Alara
et al, 2017).

2.3.2. Sesquiterpene lactones

Sesquiterpene lactones are another group of phytochemicals isolated from V. amygdalina. They
possess pharmacology properties like antibacterial, anaesthetic, antifungal, anti-inflammatory,
antiprotozoal, and antimicrobial. Most of the isolated sesquiterpene lactones from V. amygdalina
were from leaf, stem, and root. The isolated compounds were vernolide, vernolepin, vernodalin,
hydroxyvernolide, vernodalol, vernomygdin, vernomenin and 11,13-dihydrovernorodeline.
Vernodalin and vernolide had been reported to exhibit potent activity, while the activity of
hydroxyvernolide and vernodalol was weak. The lower activity of hydroxyvernolide, vernolepin
and vernodalol could be explained by the loss of hydrophobicity in the acyl moiety.

The antifeedant activity was noticed from the isolated 11,13-dihydrovernorodeline, this can resist
insects from feeding on the plant but does not result in lethality. Vernolepin had also been
reported to show antiplatelet property against ADP, arachidonic acid and collagen-induced
platelet aggregation in rabbits. Similar to other isolated sesquiterpenes lactones, vernomenin
inhibited L-1210 cancer cell lines and P-388 leukaemia mouse (Oluwaseun Ruth Alara et al,
2017).

2.3.3. Triterpenoids

Triterpenoids have been isolated from the leaf, root and stem of V. amygdalina. They can be
evaluated for use in new functional foods, cosmetics, foods, and healthcare products. They
possess diverse pharmacological properties including anticancer, anti-inflammatory,
hepatoprotective, antioxidant, antibacterial, antileukaemia, analgesicand anti-nociceptive. Some
of the isolated triterpenes are thiamine, ascorbic acid, pyridoxine, glycine, cysteine, casein

13
hydrolysate, eucalyptol, beta piene, myrtenal, and alpha-muurolol (Oluwaseun Ruth Alara et al.,
2017).

2.4. Bioactivities of V. amygdalina

V. amygdalina had been reported to possess several bioactivities like Pesticidal or Insecticidal
activity, antimicrobial, antimalarial, antithrombotic, antioxidant, anti-diabetic, laxative,
hypoglycemic, antihelmintic, anti-inflammatory, cathartic, anticancer, antifertility, antifungi,
antibacterial, and among others (Oluwaseun Ruth Alara et al., 2017; Swee Keong Yeap et
al.,2010).

2.4.1. Pesticidal or Insecticidal activity

Methanol extract of V. amygdalina leaf induced 50% of mortality on Spodoptera litura through
deterrent of feeding activity at 2 mg/ml and induced 50% of mortality on Culex pipiense pallense
at 10 µg/ml (Ohigashi et al., 1991b). V. amygdalina powder possessed pesticidal activity against
Sitophilus zeamais (LC50 = 0.3 g) and Callosobruchus maculates (LC50 = 0.39 g). The principle
of this effect may be due to the presence of hydrocyanic acid and oxalic acid. However,
concentrated extract was recommended to give better response. One to five grams of V.
amygdalina dry leaf powder was able to control the growth of S. zeamais and Sitophilus oryzae
on stored maize grains and rice (Swee Keong Yeap et al., 2010).

According to E.F. Asawalam and A. Hassanal, essential oil extracted from v.amygdalina can also
protect S. zeamais (maize weevils). (E.F. Asawalam and A. Hassanal, 2006).

2.4.2. Antioxidant

The presence of flavonoids in the V. amygdalina extracts had been attributed to their antioxidant
property. V. amygdalina ethanol extract was shown to possess antioxidant activity from DPPH
(2,2-diphenyl-1-picrylhydrazyl) radical scavenging test. Total flavonoid and phenolic contents
was found to be correlated positively with total antioxidant activity of the plant (Oluwaseun Ruth
Alara et al., 2017; Swee Keong Yeap et al., 2010). At 250 µg/ml, ethanol extract of
V.amygdalina showed highest reducing capacity and DPPH radical scavenging activity effect as
compared to the same concentrations of its isolated compounds vernodalol and vernolide. This

14
result suggested that the comparatively higher antioxidant activity of V. amygdalina ethanol
extract may be the result of synergistic effect brought about by both the compounds. Comparing
DPPH radical scavenging activity of different extracts from the root of V. amygdalina, ethanol
was found to be the best extraction which can inhibit 77% of scavenging activity followed by hot
water (63%) and cold water (49%) at 1 µg/ml. This plant contains natural antioxidants against
aqueous radicals and reactive species ions (Asuzu and Chinwe, 2018; USwee Keong Yeap et al.,
2010).

2.4.3. Antimicrobial Effect

The aqueous and ethanol extracts of V. amygdalina leaves had shown antimicrobial effects
against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and
Candida albicans with the MIC ( minimum inhibitory concentration) values ranged between 12.5
and 50 mg/ml (Oluwaseun Ruth Alara et al., 2017;).

2.4.4. Antifungal Effect

Sesquiterpene lactones had been known to be highly antifungal. The presence of vernodalol and
vernolide which belongs to sesquiterpene lactones in the V. amygdalina leaves extracts made
them exhibit higher antifungal effect against Penicillium notatum and Aspergillus flavus with
LC50 (lethal concentration) values of 0.4 mg/ml each (Swee Keong Yeap et al., 2010).

2.4.5. Antibacterial Effect

Sesquiterpene lactones from V. amygdalina leaves had exhibited antibacterial against five gram-
positive bacteria with the MIC value of 0.25 mg/ml, but lack efficacy against gram-negative
bacteria (Swee Keong Yeap et al., 2010). Likewise, the ethanolic stem extracts inhibited
antibacterial effect against Staphylococcus aureus with 50 mg/ml concentration. The aqueous,
ethanolic and methanolic extracts of the leaves had been reported to exhibit strong potency
against clinical bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli
(Swee Keong Yeap et al.,2010; Oluwaseun Ruth Alara et al., 2017; ).

15
2.4.6. Anti-inflammatory Effect

V. amygdalina leaves extracts had been reported to possess anti-inflammatory activity when
applied to the ear of rat suffering from inflammation (Swee Keong Yeap et al.,2010; Oluwaseun
Ruth Alara et al., 2017). It produced a significant reduction when compared with the application
of acetylsalicylic acid. The percentage of inhibition of leaves extracts was higher than roots
extracts (Oluwaseun Ruth Alara et al., 2017).

2.5. Composition of essential oil of V.amygdalina

According to E.F. Asawalam and A. Hassanal studies total of 17 compounds were identified in
the essential oil of V. amygdalina by GC - MS and GC Co - injection with authentic standards.
Constituents present in >0.94% were the ones identified. The oils are a mixture of monoterpenes
and sesquiterpenes. 1, 8 Cineole occurred in the largest quantity (25.11%). This is followed by
Beta pinene (14.54%), Myrtenal (6.52%), Transpinocarveol (6.24%), Alpha pinene (4.93%) and
Linalool (4.28%) (E.F. Asawalam and A. Hassanal, 2006).

16
3. Materials and Methods

3.1. Materials

3.1.1. Raw materials

The main raw material that we must have to conduct our research is v.amygdalina leaf.

3.1.2. Chemicals

 n-hexane/ethanol
 water/steam

3.1.2. Laboratory equipment and devices

 Leaf cutter
 bag
 Anhydrous sodium sulphate containing filter paper
 Small amber- coloured vials.
 Funnel
 GC, GC-MS
 Grinder
 Steam distillery
 1-litre glass jar
 Oven
 30x10 cm nylon mesh tube

3.2. Methods

3.2.1. Plant materials collection and preparation for oil extraction

V. amygdalina leaf will be collected from around Addis Ababa. Leafs will be air dried in a well-
ventilated area for five days. To increase the contact area between leaf and steam the leafs will

17
be grinded by grinder at AAiT school of chemical and bioengineering laboratory before
extraction procedure.

3.2.2. Extraction of essential oil

The essential oil will be extracted by steam distillation using Clavenger apparatus at AAiT
School of chemical and bioengineering lab. The condensing oils will be collected in n-hexane
solvent and the solution will be filtered with a filter paper containing anhydrous sodium sulphate
in a funnel to remove any remaining traces of water. Hexane will be then removed by distillation
at 60 ºC from essential oil. When condensation stopped, the oil will be collected and weighed
into small amber- coloured vials.

3.2.3. Analysis of essential oil

Characterization, identification and determination of relative amounts of components of the

essential oil will be done through Gas-Liquid Chromatography (GC), Gas Chromatography -
Mass Spectrometry (GC-MS), and GC Co injection of the essential oil with authentic standards
at Addis Ababa University, natural science collage department of chemistry lab.

3.2.4. Sitophilus zeamais culture

Adults of S.zeamais will be collected from Melkassa Agricultural Research Center and will be
cultured in Addis Ababa University, Department of Biology Insect Science insectory at 27 ±2 ºC,
60-65% R.H (relative humidity) (E.F. Asawalam, and A. Hassanali, 2006). The food media used
will be whole maize grains. Fifty pairs of S. zeamais will be placed in 1-litre glass jar containing
400 g of maize grains. The jars will be then covered with nylon mesh held in place with rubber
bands. Maize grains used for the study will be purchased locally from the farmers around Addis
Ababa. Grains will be disinfested in the oven at 40 ºC for 4 hours and kept in the laboratory
before use.

3.2.5. Mortality assessment assays

Essential oil will be applied to the grains at the rate of 0.012, 0.06 and 0.3% (30, 150 and 750
mg/250 g of grain dissolved in 10 ml of 95% n-hexane and shaken thoroughly to ensure uniform

18
distribution over grain surface. Treated grains will be kept for 24 h to allow the hexane to
evaporate completely before bioassays will be conducted.

Essential oil extracts will be mixed separately with 250 g of maize grains in glass jars at the three
different dosages indicated above. Two blank controls will run periodically consisting of hexane
treated grain and the untreated grain. Ten pairs of 5-7 day old S. zeamais adults will be
introduced into jars containing the different treated and untreated maize grains. Jars will be
covered with nylon mesh held with rubber bands, and shaken gently for proper admixture. Each
treatment was replicated four times. The experiment will be arranged in completely randomized
design in the laboratory. Number of dead insects in each vial will be counted after 1, 2, 3, 4, 5,
6, and 7 days to estimate maize weevil mortality.

%Mortality=

Data on percentage adult weevil mortality will be corrected using Abbott’s (1925) formula:

PT =

Where:

PT = Corrected mortality (%)

PO = Observed mortality (%)

PC = Control mortality (%)

3.2.6. Progeny assessment assays

In a similar experiment to mortality assessment, 10 pairs of S. zeamais will be introduced into

treated and untreated grains. After 30 days oviposition period, parent adults will be removed.
Insects subsequently emerging will be counted to estimate FI progeny production. Counting was
stopped after 33 days to avoid overlapping of generation.

19
3.2.7. Damage assessment assays

Damage assessment will be carried out on treated and untreated grains. Samples of 100 grains
will be taken from each jar and the number of undamaged and damaged (grains with
characteristic holes) grains will be counted and weighed. Percentage weight loss will be
calculated, using FAO (1985) method as follows:

( )
% Weight loss=

Where:

U = weight of undamaged fraction in the sample

N = total number of grains in the sample

Ua = average weight of one undamaged grain

D =weight of damaged fraction in the sample

3.2.8. Repellency

Repellent action of the essential oil extracts against S. zeamais will be assessed in a choice
bioassay system, consisting of two 1-L glass jars connected together at their rims by means of a
30x10 cm nylon mesh tube. A 5.0 cm diameter circular hole will be cut at the middle of the mesh
for the introduction of test insects. 250 g of maize will be put into the glass jars. Grains in one jar
will be treated with essential oil extracts while untreated grains in the other jar acted as control.
Twenty-five adults of S. zeamais will be introduced into the nylon mesh tube through the circular
hole by means of a 5 cm-diameter funnel. Number of insects present at the control (NC) and
treated (NT) jars will be recorded after 1 hour exposure. All repellency assays will be replicated
four times and will be carried out in the laboratory at 27 ± 2 ºC and 60-65% R.H. Percent
repellency (PR) values will be computed as:

% PR=

Where:

20
% PR=percentage repellency

NC=Number of insects present at the control (untreated jars)

NT= Number of insects at treated jars

3.3. Data analysis

Data analysis will be done by Analysis of variance (ANOVA), Microsoft excel. To find out the
effects of the treatment on %mortality, %number of F1 progeny and % weight loss one-way
analysis of variance will be used.

21
4. Cost break down

By believing the other found in our university laboratories.

Table 2: Cost break down

No. Budget description Unit Quantit Total cost

cost(ETB y (ETB Birr)
birr)

1 Stationery

1.1 Paper 0.5 40 20

1.2 Pen 8 2 16

1.3 Printing 0.75 60 45

Sub total 81 birr

2 Laboratory Experiments

2.1 Chemicals All available in lab.

2.2 Laboratory equipment’s and apparatus

Leaf cutter 50 1 50

Bag (quantal) 10 1 10

Other equipment’s and apparatus Available in lab.

Sub total 60 birr

3 Samples

maize grain 15 4 60

22
 Adult weevils Available at melkassa agriculture
research Centre

Sub total 60 birr

4 Travel expense for sample 200 birr

collection

5 Contingency (10%) 40 birr

Total 441 birr

23
5. Schedule and work plan

Tasks to be done

Task 1: Proposal preparation and approval.

Task 2: Collecting raw material (V.Amygdalina).

Task 3: Raw material preparation before it is allowed into the steam distillation.

Task 4: Extraction of essential oil from V.Amygdalina.

Task 5: Analyze of the chemical composition of V. amygdalina leaves and identification of the
active chemical constituents of the essential oil.

Task 6: Maize weevil (Sitophilus zeamais) culture.

Task 7: Evaluation of the effect of the essential oil of V. amygdalina leaves on mortality,
reproduction (on number of F1 progeny) and repellency of S. zeamais.
Task 8: Determination of the effects of the essential oil on the weight loss of stored maize grains.
Task 9: Report writing (document development).

24
Table 3: Schedule and work plan

No Tasks Time in months

2019 2020

8 9 10 11 12 1 2 3 4 5 6 7

1 Task 1

2 Task 2

3 Task 3

4 Task 4

5 Task 5

6 Task 6

7 Task 7

8 Task 8

9 Task 9

25
Reference

Abere Habtamu and Yadessa Melaku (2018). Antibacterial and Antioxidant Compounds from
the Flower Extracts of Vernonia amygdalina.

AdebayoAmosOmoloye and JamesAdebayoOjo (2016):Development and Life History of

Sitophilus zeamais (Coleoptera: Curculionidae) on Cereal Crops.

Ahmed Ibrahim and Aschalew Sisay (2012). Farmers’ Practices and Combinations of Malathion
and Neem Seed Powder Management Options against Sorghum and Maize Insect Pests during
Storage at Bako, West Shoa Zone, Ethiopia. East African Journal of Sciences Vol. 6 (1) 55-64.

Akinola O. Ogunbinua, Guido Flaminib, Pier L. Cionib, Isiaka A. Ogunwandec, and Sunday O.
Okeniyia(2009). Essential Oil Constituents of Eclipta prostrata (L.) L. and Vernonia amygdalina
Delile.

Aliyu A.B, Adeyemi M.M.H, Abdulkadir I, Dambatta M.B, Amupitan J.O, Oyewale A.O.(2014).
Antifeedant activity of Vernonia oocephala against stored product pest Tribolium casteneum
(Herbst).

Asawalam E.F. and Hassanali A. (2006). Constituents of the essential oil of Vernonia
amygdalina as maize weevil protectants. Tropical and Subtropical Agroecosystems 6 : 95 - 10

Asuzu and Chinwe U.(2018). bitter herbs of eastern nigeria (gongronema latifolium, vernonia
amygdalina and vitex doniana). Asuzu, Afr Journal Tradit Complement Altern Med. 15 (3): 47-
56.

Bern.C.J, Rosentrater.K.A, and Suleiman.R, (2015). Evaluation of maize weevils Sitophilus

zeamais Motschulsky infestation on seven varieties of maize. Journal of Stored Products
Research 64:97-102.

Dawit Kidane (2005).Garlic and orange plant materials evaluation for the control of Sitophilus.
spp.(Coleoptera:Curculionidae)andZabrotes subfasciatus (Coleoptera:Bruchidae)inEthiopia.

26
Emsen B.Kordali S. Yildirim E.(2013. Insecticidal effects of monoterpenes on Sitophilus
zeamais Motschulsky.

Etefa Gurmesa (2017). Investigation of Bitter Leaf (Vernonia amygdalina) Extract as a Potential
Substitute for Imported Hops.

Hadi Shahab.Ghayoor and Karim Saeidi (2015). Evaluation of Botanical Insecticides against
Lentil weevil, Bruchus lentis Frolich (Coleoptera: Chrysomelidae: Bruchinae).
agricultural communications,3(4): 23‐27.

Keno T, Azmach G, Wegary D, Worku. M., Tadesse B, Wolde L,Deressa T, Abebe B, Chibsa T.
Suresh M.(2018). Major biotic maize production stresses in Ethiopia and their management
through host resistance. African Journal of Agricultural Research 13(21): 1042-1052.

Kifle Gebreegziabiher, Mulatu wakgari and Muluken Goftishu.(2017).Evaluation of some

botanicals oils for the management of Maize weevil, Sitophilus zeamais Motsch. (Coleoptera:
Curculionidae).

Maribet L.Parugrug and Aurea C. Roxas.(2008). Insecticidal action of five plants against maize
weevil, sitophilus zeamais motsch. (coleoptera: curculionidae).

Nour Hamid Abdurahmana, Olusegun Abayomi Olalerea, Oluwaseun Ruth Alaraa, Siti Kholijah
Abdul Mudalipa (2017). phytochemical and pharmacological properties of vernonia amygdalina:
a review. Journal of Chemical Engineering and Industrial Biotechnology V2:80-96.

Nursuhaili, A.B., Nur Afiqah Syahirah, P.,Martini, M.Y., Azizah, M. and Mahmud,
T.M.M.(2019).Medicinal values, agronomic practices and postharvest handlings of Vernonia
amygdalina.

One kutu, A, Nwosu, L. C and Abakpa, R. E.(2015). Comparative Efficacy of Aqueous Extracts
of Tithonia diversifolia (Asteraceae) and Vernonia amygdalina (Asteraceae) Leaves in the
Management of Sitophilus zeamais Infestation in Stored Maize. IOSR Journal of Pharmacy and
Biological Sciences.

27
Paparatti .B and Speranz.S (2005). Biological Control of Hazelnut Weevil (Curculio nucum L.,
Coleoptera, Curculionidae) Using the Entomopathogenic Fungus Beauveria bassiana (Balsamo)
Vuill. (Deuteromycotina, Hyphomycetes).

Pedro M. Gutierrez Jr.(2016).Insecticidal Activity of Tinospora rumphii Plant Powder Against

Maize Weevil, Sitophilus zeamais. International Research Journal of Biological Sciences,5(4):
37-44.

Sarah K Brown, William S Garver and Robert A Orlando.(2017). 1,8-cineole:An

Underappreciated Anti-inflammatory Therapeutic. Journal of Biomolecular Research &
Therapeutics.

Swee Keong Yeap, Wan Yong Ho1, Boon Kee Beh, Woon San Liang, Huynh Ky, Abdul Hadi
Noaman Yousr and Noorjahan Banu Alitheen (2010).Vernonia amygdalina, an ethnoveterinary
and ethnomedical used green vegetable with multiple bioactivities. Journal of Medicinal Plants
Research Vol. 4(25), pp. 2787-2812.

Udochukwu U, Omeje F.I,Uloma I. S, Oseiwe F. D.(2015). Phytochemical analysis of Vernonia

amygdalina and Ocimum gratissimum extracts and their antibacterial activity on some drug
resistant bacteria.American Journal of Research Communication,3(5):225-234.

Yeshiwork alemnew (2017). Studies on the management of maize weevil (Sitophilus zeamais L.)
using Botanicals on maize grain in storage.

28