MAKERERE UNIVERSITY

COLLEGE OF NATURAL SCIENCES

DEPARTMENT OF CHEMISRTY

Formulation of a botanical pesticide from lemongrass

YEAR OF STUDY : III

NAME : Lubwama Kenneth

REG NO : 09/U/2708/PS

STUDENT NO : 209005757

PROGRAM : Bachelor of Science in industrial chemistry

COURSE CODE : ICH 3235

University supervisor : Dr. John Wasswa

A project report submitted for the partial fulfillment for the award of the bachelor’s

degree of science in industrial chemistry to the college of Natural sciences, Makerere

University Kampala

June 2012

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DECLARATIONI LUBWAMA KENNETH declare that the information presented in this report, as a partial

fulfillment for the requirements of Bachelor’s degree in Industrial Chemistry is my original

work. The information in this report has never been presented anywhere for academic

qualifications.

Student Signature

………………………………………………..

LUBWAMA KENNETH

Supervisor’s Signature

………………………………………………..

DR. WASSWA

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ACKNOWLEDGEMENTFirst, let me thank the Almighty GOD who has enabled me finish my project successfully. I

highly thank Him for the gift of life, wisdom and knowledge which enabled me to complete the

project well.

I also take my sincere appreciation to the entire NTEGE family for all the support given to me. I

thank the laboratory technicians of Makerere University who have helped me in extraction and

analysis of the essential oil. I also thank my supervisor Dr. John Wasswa and Dr Maud

Kamatenensi for their guidance towards the completion of this project. I appreciate all my

fellow students with whom I have been able to share the knowledge and discussion, which has

led to the completion of this project successfully.

ii

DEDICATION

To my parents, brothers, sisters, children to be and friends with immense love

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Table of ContentsDECLARATION..............................................................................................................................iACKNOWLEDGEMENT...............................................................................................................iiDEDICATION...............................................................................................................................iiiACRONYMS AND SYMBOLS....................................................................................................viLIST OF FIGURES, TABLES AND GRAPHS...........................................................................vii

FIGURES..................................................................................................................................viiTABLES....................................................................................................................................viiGRAPHS...................................................................................................................................vii

CHAPTER I. INTRODUCTION.....................................................................................................11.0. lemongrass.......................................................................................................................11.1. Background of study.......................................................................................................21.2. Problem statement..........................................................................................................21.3. Objectives.........................................................................................................................3

1.3.1. General objective.....................................................................................................31.3.2. Specific objectives....................................................................................................3

1.4. Scope of Study.................................................................................................................32.0. CHAPTER II. LITERATURE REVIEW.................................................................................4

2.1. Pesticides..............................................................................................................................42.1.0. Definition.......................................................................................................................42.1.1. History of a pesticide.....................................................................................................42.1.2. Uses of pesticides..........................................................................................................52.1.3. Benefits..........................................................................................................................5

2.2. Botanical pesticides..............................................................................................................62.2.1. Definition of botanicals.................................................................................................72.2.2. Characteristics of Botanicals.........................................................................................72.2.3. Advantages of botanicals...............................................................................................82.2.4. Disadvantages of botanicals..........................................................................................82.2.5. Some common botanicals..............................................................................................9

2.3. Lemongrass.........................................................................................................................122.4. Essential oil.........................................................................................................................13

2.4.1. Definition.....................................................................................................................132.4.2. Physical Properties of Essential Oils...........................................................................132.4.3. Chemical Properties of Essential Oil...........................................................................142.4.4. USES OF ESSENTIAL OIL:......................................................................................152.4.5. METHODS OF EXTRACTION OF THE ESSENTIAL OIL....................................16

2.5. Density................................................................................................................................202.5.1. Calculation of density..................................................................................................202.5.2. Uses of density.............................................................................................................20

2.6. Refractive index..................................................................................................................202.6.1. Measuring of refractive index......................................................................................202.6.2. Uses of the refractive index.........................................................................................21

3.0. CHAPTER III METHODOLOGY.........................................................................................22

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3.1. Extraction of the essential oil.............................................................................................223.1.0. Materials and Equipment used.....................................................................................223.1.1. Collection, Identification and characterization of the Lemmon grass.........................223.1.2. Material preparation and extraction.............................................................................223.1.2. Experimental Set up.....................................................................................................23

3.2. Determination of the refractive index.................................................................................233.3. Determination of the density..............................................................................................233.4. Pesticide making.................................................................................................................24

3.4.1. Materials......................................................................................................................243.4.2. Procedure.....................................................................................................................24

4.0. CHAPTER IV RESULTS AND DISCUSSION....................................................................254.1. Results.................................................................................................................................254.2. Discussion of results...........................................................................................................254.3. Significance of study..........................................................................................................27

5.0. CHAPTER V. RECOMMENDATION AND CONCLUSION.............................................285.1. Recommendation................................................................................................................285.2. Conclusions.........................................................................................................................28

REFERENCES..............................................................................................................................29

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ACRONYMS AND SYMBOLSDDT; dichlorodiphenyltrichloroethane

AOAC; Association of Official Analytical Chemists

USEPA; United States Environmental Protection Agency

BC; Before Christ

NCSU; North Carolina State University

UV; Ultra Violet

PBO; Piperonyl Butoxide

SCO2; Supercritical Carbon dioxide

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LIST OF FIGURES, TABLES AND GRAPHS

FIGURESFigure 1…………………………………………1

Figure 2…………………………………………9

Figure 3………………………………………….10

Figure 4…………………………………………11

Figure 5…………………………………………11

Figure 6………………………………………….12

Figure 7………………………………………….21

Figure 8………………………………………….23

TABLESTable 1………………………………………….15

Table 2………………………………………….23

GRAPHSGraph 1…………………………………………26

Graph2………………………………………….26 2

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CHAPTER I. INTRODUCTION

1.0. lemongrass

Lemongrass is scientifically called cymbopogon citratus. It belongs to kingdom plantae, order

poales, family poaceae and genus cymbopogon. Lemongrass is native to India and tropical Asia.

Lemon grass is found in many parts of Uganda. Lemongrass oil contains 70–80 percent citral.

Citral is a pale yellow liquid with a strong lemon odour. It is a non polar terpene aldeyde

compound. Its chemical name is 3, 7-dimethylocta-2, 6-dienal. (C10H10O,

(CH3)2C=CHCH2CH2C(CH3)=CHCHO). The mass fractions are 78.9% carbon, 10.6% hydrogen

and 10.5% oxygen. Citral undergoes isomerism to form a cis form (citral b) and a trans form

(citral a)

Citral has got the structural diagram shown below,

Figure 1

It’s mainly grown as a food crop used in herbal tea because of its sharp lemon flavor. However;

lemongrass has many other uses, such as: ornamental plant, perfume in soap and a medicine to

treat various health ailments including acne, athlete’s foot, flatulence, muscle aches and scabies

(Athens, 2002). Further, bioactivity studies have indicated that the various components of

lemongrass essential oil contains antimicrobial, antifungal, antibacterial and mosquito repellent

properties (Schaneberg and Khan, 2002; natural resources industries (NRI), 2001), and that citral

isolated from this oil is used in the industrial manufacture of vitamin A.

Because of these important uses, lemongrass oil is of great use in the agricultural sector, most

especially for the protection of stored food crops such as maize.

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Loss of stored maize due to insect pest damage is a very big problem in Africa, including

Uganda. The most dangerous and damaging storage insect pest is Larger Grain Boer

(Prostephanus truncates). This borer is believed to have originated from Central America and it

was transported to Africa through maize import in the late 1990s. This insect does not only affect

maize but also cassava chips. Losses of up to 60% were reported at household level in Tanzania

(Golob and Hodges. 1982; Hodges et al, 1983; Keil, 1988).

To protect stored food crops from insect pest damage, Uganda has to import all her pesticide

requirements that are worth millions of dollars. Although synthetic pesticides are effective in

controlling many storage insect pests, they also have many undesirable attributes to humans,

animals and the environment. This is mainly through direct contact, inhalation, water, and soil

and air pollution. Further, many insect pests have shown that they develop resistance to the

continued use of the same pesticide for a long time (Aggarwal et al., 2001) and this situation

leads to high insect pest population rather than low insect pest population. Hence the need to

investigate the use of botanical pesticides to control insect pests on stored food crops.

1.1. Background of study

Botanical pesticides have been used by local communities in Uganda for time immemorial. Local

farmers have traditionally used botanical pesticides either as powdered plant material or as an

aqueous extract to control insect pests on stored food products. In both of these formulations, the

concentration of the active ingredients is not known and it may be present in low concentrations.

Although botanical pesticides are used in Uganda, there is still need to develop them to a more

modern formulation.

It is against this background of reducing the importation of synthetic pesticides and formulation

of a locally manufactured botanical pesticide that is safe and environmental friendly that this

research project was conducted.

1.2. Problem statement

Many synthetic pesticides used to prevent pest damage to stored food crops are expensive and

dangerous to humans, animals and the environment so there is need to get a natural (botanical)

pesticide that is safe, environmental friendly and also cheap economically.

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1.3. Objectives

1.3.1. General objective

To formulate a cheap human and environmental friendly pesticide from cymbopogon citratus

oil

1.3.2. Specific objectives

To extract essential oil from locally grown cymbopogon citratus.

To determine the refractive index and density of the oil extracted at different

temperatures.

To get a cheaper method of controlling food crop pests.

1.4. Scope of Study

The scope of study will involve collecting lemongrass samples of cymbopogon citratus from a

Makerere University Kampala, preparation of the sample for extraction, extraction of essential

oil from the lemongrass of cymbopogon citratus by hydro-distillation, determining the refractive

index and density of the oil extracted at different temperatures and formulation of a pesticide

using the extracted essential oil.

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2.0. CHAPTER II. LITERATURE REVIEW

2.1. Pesticides

2.1.0. Definition

A pesticide is a chemical used to prevent, destroy, or repel pests. (USEPA) Pesticides are also

defined as substances or mixture of substances intended for preventing, destroying, repelling or

mitigating any pest (Wikipedia).

A pesticide is also defined as any substances or mixture of substances intended for preventing,

destroying, repelling or mitigating any pest. (JOURNAL OF PESTICIDE REFORM/SUMMER

1999 VOL.19, NO.2)

2.1.1. History of a pesticide

Even before 2000BC, humans have utilized pesticides to protect there crops. The first know

pesticide was elemental sulfur dusting used in ancient Sumer about 4500 years ago in ancient

Mesopotamia. The Rig Veda, which is about 4000 years old, mentions the use of poisonous

plants for pest control (Indian Journal of Plant Protection). By the 15th century, toxic chemicals

such as arsenic, lead and mercury were being applied to crops to kill pests. In 1950s, arsenic-

based pesticide became dominant (Ritter SR. 2009. Pinpointing Trends in Pesticide Use in 1939.

C&E News). PAUL MULLER discovered that DDT was a very effective insecticide.

Organochlorines like DDT were dominant, but they were replaced in the US by organophosphate

and carbamates by 1975. The first legislation providing federal authority for regulating pesticides

was enacted in 1910 (Goldman, L.R. (2007). "Managing pesticide chronic health risks :)

however, decades later during the 1940s manufacturers began to produce large amounts of

synthetic pesticides and their use became widespread (Daly H, Doyen JT, and Purcell AH III

1998). Some sources consider the 1940s and 1950s to have been the start of the "pesticide era.”

(Graeme Murphy December 1, 2005) Seventy-five percent of all pesticides in the world are used

in developed countries, but use in developing countries is increasing (Miller GT 2004).

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In the 1960s, it was discovered that DDT was preventing many fish-eating birds from

reproducing, which was a serious threat to biodiversity. Rachel Carson wrote the best-selling

book Silent Spring about biological magnification.

The agricultural use of DDT is now banned under the Stockholm Convention on Persistent

Organic Pollutants, but it is still used in some developing nations to prevent malaria and other

tropical diseases by spraying on interior walls to kill or repel mosquitoes (Lobe, J Sept 16, 2006).

In the 17th century, nicotine sulfate was extracted from tobacco leaves for use as an insecticide.

The 19th century saw the introduction of two more natural pesticides, pyrethrum, which is

derived from chrysanthemums, and rotenone, which is derived from the roots of tropical

vegetables (Miller, GT 2005). Since then, pyrethrum compounds have become the dominant

insecticide (Ritter SR.2009).

2.1.2. Uses of pesticides

Pesticides are used to control organisms that are considered to be harmful to crops and humans

(Wikipedia)

2.1.3. Benefits

There are two levels of benefits for pesticide use, primary and secondary. Primary benefits are

direct gains from the use of pesticides and secondary benefits are effects that are more long-term

(Pimentel, David).

2.1.3.1. Primary benefits

1. Controlling pests and plant disease vectors

Improved crop/livestock yields

Improved crop/livestock quality

Invasive species controlled

2. Controlling human/livestock disease vectors and nuisance organisms

Human lives saved and suffering reduced

Animal lives saved and suffering reduced

Diseases contained geographically

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3. Prevent of control organisms that harm other human activities and structures

Drivers view unobstructed

Tree/brush/leaf hazards prevented

Wooden structures protected

2.1.3.2. Secondary benefits

1. Community benefits

Farm and agribusiness revenues

Nutrition and health improved

Food safety and security

2. National benefits

Workforce productivity increased

Increased export revenues

National agriculture economy

3. Global benefits

Assured safe and diverse food supply

Less greenhouse gas

Reduced civil unrest

2.2. Botanical pesticides

The use of synthetic pesticides during the last half-century has often been careless and

indiscriminate, and has led to a number of well-known problems. Synthetic pesticides have

caused contamination of the environment with toxic residues in many regions around the world,

as well as side-effects on non-targeted insects and other organisms, and an increase in the

number of pest species resistant to pesticides (Zalom et al., 1992; Kumar et al., 2002). The

Geneva-based World Health Organization reports three people are poisoned by pesticides every

minute around the world.  All in all, about 10,000 die annually because of pesticides. Reports

show that 62% of pesticides sold in the Philippines are insecticides. Of these, 46% are applied to

rice and 20% to vegetables. 

6

Biologically based technologies have been employed because they are environmental friendly,

cheap and they provide lasting, highly selective and effective pest control and these technologies

include the use of botanicals.

2.2.1. Definition of botanicals

Botanical are naturally occurring chemicals extracted from plants. Natural pesticide products are

available as an alternative to synthetic chemical formulations but they are not necessarily less

toxic to humans (NCSU). Botanicals are also defined as pesticides derived from plants

(Oklahoma Cooperative Extension Fact Sheet).

2.2.2. Characteristics of Botanicals

Botanicals have been used for centuries and were widely used in the United States until the

1940s and 50s when synthetic pesticides were introduced. The synthetics pesticides quickly

became popular because they did not break down as quickly. But insecticides that last longer can

potentially leave residues in the soil and water supply, and on food.

As with any insecticide, there are advantages and disadvantages associated with botanicals.

• Botanicals degrade rapidly from sunlight, air, and proper moisture, which generally makes

them less toxic to the environment, but may also require them to be applied more often,

applied correctly, and with more precise timing.

• Botanicals act quickly to stop feeding of insect pests and often cause immediate paralysis or

cessation of feeding, but they may not cause the insect’s death for hours or days.

• Most botanicals have low to moderate toxicity to mammals, yet they are still poisons and

pose a hazard to humans or to the environment.

• Most botanicals are not toxic to plants, except insecticidal soaps.

• Botanicals cost more than synthetic insecticides and may not be readily available.

• Potency of some botanicals may differ from one source or batch to the next.

• Botanicals tend to be broad spectrum insecticides, meaning they kill whatever they come in

contact with—both “good” and “bad” insects.

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2.2.3. Advantages of botanicals

1. Plants producing these compounds are known by the farmers because most of the time they

grow in the same general area.

2. Often these plants also have other uses like household insect repellents or are plants with

medicinal applications.

3. The rapid degradation of the active product may be convenient as it reduces the risk of

residues on food.

4. Some of these products may be used shortly before harvesting.

5. Many of these products act very quickly inhibiting insect feeding even though long term they

do not cause insect death.

6. Since most of these products have a stomach action and are rapidly decomposed they may be

more selective to insect pests and less aggressive with natural enemies.

7. Most of these compounds are not phyto toxic.

8. Resistance to these compounds is not developed as quickly as with synthetic insecticides. 

2.2.4. Disadvantages of botanicals

1. Most of these products are not truly insecticides since many are merely insect deterrents and

their effect is slow.

2. They are rapidly degraded by UV light so that their residual action is short.

3. Not all plant insecticides are less toxic to other animals than the synthetic ones.

4. They are not necessarily available season long.

5. Most of them have no established residue tolerances.

6. There are no legal registrations establishing their use.

7. Not all recommendations followed by growers have been scientifically verified. 

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2.2.5. Some common botanicals

(1) Pyrethrins—Pyrethrum comes from a chrysanthemum species, Dendranthema grandiflora,

found in Kenya and Ecuador. Pyrethrins, which are chemical components of pyrethrum, kill

insects by interrupting their nerve impulses.

This insecticide is very fast-acting and causes an immediate “knockdown” paralysis in insects.

However, many insects can break down pyrethrins quickly, and after a brief period they may

recover rather than die.

To overcome this break-down ability, many pyrethrins insecticides contain a synergist such as

piperonyl butoxide (PBO). A synergist enhances insecticidal action by inhibiting the enzymes in

the insect’s body that break down the toxin. PBO is a synthetically produced chemical substance

that is not allowed by some organic certification programs. Before purchasing pyrethrins or other

botanicals, check the label to see if PBO is listed among active ingredients.

Pyrethrins are effective against many sucking and chewing insects as well as flying insects.

Figure 2

(2) Rotenone is extracted from the roots of derris plants in Asia and cube roots in South

America; rotenone is both a contact and stomach poison. It is particularly effective against leaf-

feeding beetles and certain caterpillar pests.

Rotenone is a powerful inhibitor of cellular respiration, the process of converting cell nutrients

into energy. It acts primarily in insects’ nerve and muscle cells, causing them to stop feeding

quickly. Death occurs several hours to a few days later.

Rotenone is extremely poisonous to fish. Mammals detoxify ingested rotenone with enzymes

found in the liver, but it can be quite toxic when inhaled. It is one of the more toxic of the

botanicals.

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Figure 3

(3) Neem—a mixture of leaves, seed, and bark extracts from an evergreen tree native to Asia, the

Azadirachta indica, neem contains a bitter chemical that is both a deterrent to feeding and a

growth regulator. Its bitterness deters insects from feeding.

As a growth regulator, neem is thought to interfere with insect hormone production or reception,

thereby preventing insects from maturing enough to reproduce.

It has extremely low toxicity to mammals, but the seed dust may be very irritating to some

people.

Research has shown that neem has some systemic action in plants, meaning that the plants

absorb it. When applied as a dust to soils, neem can be taken up by the roots of some plants and

transferred to other parts of the plant. Neem may remain active in the soil up to four weeks,

depending on soil conditions.

When neem is applied to plant foliage, its systemic action is limited—new foliage must be

sprayed periodically for adequate protection.

Neem can also control plant diseases, such as powdery mildew, anthracnose, black spot, and

many others.

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(4) Citrus oil (limonene, linalool) are extracts from citrus peels primarily used as flea dips, but

have been combined with soaps as contact poisons against aphids and mites. They evaporate

quickly after application and provide no residual control.

Figure 4

(5) Nicotine concentrate is very poisonous if inhaled. It is derived from tobacco and is

commonly sold as a 40 percent nicotine sulfate concentrate.

Nicotine is a fast acting contact killer for soft bodied insects, but does not kill most chewing

insects. It is less effective when applied during cool weather. Do not spray within 7 days of

harvest. It acts on caterpillars.

Figure 5

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(6) Ryania

This compound is obtained from the roots and stems of a plant native to South America known

as Ryania speciosa (Flacourtiaceae). From this plant many series of alkaloids may be obtained,

of which the most important is ryanodina. This alkaloid is effective as a contact or stomach

poison and directly prevents muscles from contraction, causing paralysis. It has a longer residual

than most botanicals. Toxicity to mammals is moderate. It acts on Japanese beetles, earworms,

codling moths etc.

Figure 6

(7) Sabadilla is derived from the seeds of South American lilies. It is a broad spectrum contact

poison, but has some activity as a stomach poison. It is most effective against true bugs such as

harlequin bugs and squash bugs. Sabadilla degrades rapidly in air and sunlight, and has little

residual toxicity. It is very toxic to honey bees but least toxic botanical to humans. This one acts

on grasshoppers, moths, blister beetles and army worms.

2.3. Lemongrass

Lemongrass is scientifically called cymbopogon citratus. It belongs to kingdom plantae, order

poales, family poaceae and genus cymbopogon. Lemongrass is native to India and tropical Asia.

It is widely used as an herb in Asian cuisine. It has a subtle citrus flavor and can be dried and

powdered, or used fresh. Lemongrass is commonly used in teas, soups, and curries. It is also

suitable for poultry, fish, beef, and seafood. It is often used as a tea in African countries such as

Togo and the Democratic Republic of Congo and Latin American countries such as Mexico.

Lemongrass oil is used as a pesticide and a preservative. Research shows that lemongrass oil has

anti-fungal properties (Hoffman B.R. 2004).

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2.4. Essential oil

2.4.1. Definition

An essential oil is a concentrated, hydrophobic liquid containing volatile aroma compounds from

plants. Essential oils are also known as volatile, ethereal oils or aetherolea, or simply as the "oil

of" the plant from which they were extracted, such as oil of clove. Oil is "essential" in the sense

that it carries a distinctive scent, or essence, of the plant (www.quinessence.com/oil_testing.htm)

Essential oils are frequently referred to as the “life force” of plants. These "essential" oils are

extracted from flowers, leaves, stems, roots, seeds, bark, and fruit rinds. The amount of essential

oils found in these plants can be anywhere from 0.01 percent to 10 percent of the total. These oils

have potent antimicrobial factors, having wide range of therapeutic constituents. These oils are

often used for their flavor and their therapeutic or odoriferous properties, in a wide selection of

products such as foods, medicine, and cosmetics (www.quinessence.com/oil_testing.htm).

2.4.2. Physical Properties of Essential Oils

Essential oils actually are not oily, unlike the other essential oil extracted from vegetables and

nuts. Some essential oils are viscous; others are fairly solid and most are somewhat watery

(http://www.essentialwholesale.com/aromatherapy.html). Essential oils have a lipid-soluble

molecular structure that allows them to pass through skin easily which is the basis for using them

in massage and aromatherapy. There are about 3000 essential oils available throughout the whole

world yet only 300 essential oils are used generally.

Essential oils are the most concentrated form from any botanical. It is commonly used in

pharmacological because of its nature as an effective remedy for numerous diseases. They are

very volatile and should be kept in a very tight bottle so that they cannot evaporate so easily into

the air.

Essential oils should also be kept in a very small bottle if they are in small amount so that it does

not get exposed to the air inside the bottle.

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Exposure to heat and light also can damage the quality of the essential oils so it must be stored in

a dark place with appropriate temperature.

2.4.3. Chemical Properties of Essential Oil

Essential oils have a very unique chemical property; every single oil normally has more than a

hundred components (http://www.essentialoils.co.zalcomponents.html). These components can

be determined using analytical apparatus such as the gas chromatography and high performance

liquid chromatography. Some of the chemical compounds that can be regularly found in the

essential oils are sesquiterpenes, monoterpenes and phenols.

Sesquiterpenes consists of 15 carbon atoms and has a complex pharmacological action. It has

anti-inflammatory and anti-allergy properties. Due to its nature essential oils that are high in

phenols should be used in low concentration and in a short period of time. This is because they

can lead to toxicity to the body as the liver will be required to work harder to excrete them if the

body has accumulated it over a long period of time.

Another chemical compound regularly seen in essential oils is monoterpene. It can be found

nearly in all essential oils produced from the plant extraction process and have 10 carbons with at

least 1 double bond structure. The 10 carbons are derived from 2 isoprene units and they can

react readily to air and heat sources.

Due to this, the higher the amounts of this compounds in the essential oils, the lesser the time it

will last with high quality.

Research has it that citral (82%) is the main constituent of lemongrass oil. Citral is a pale yellow

liquid with a strong lemon odour. It is a non polar terpene aldeyde compound. Its chemical name

is 3, 7-dimethylocta-2, 6-dienal. (C10H10O, (CH3)2C=CHCH2CH2C(CH3)=CHCHO). The mass

fractions are 78.9% carbon, 10.6% hydrogen and 10.5% oxygen. Citral is a mixture of two

geometrical isomers: neral and geranial. Other compounds found in the oil include linalool,

nerol, geranyl acetate and methyl geranate.

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The table below shows the composition of lemongrass

COMPOUNDS CONCENTRATION (%)

MYRCENE 2.64-12.03 (Addae- Mensah et al., 1995)

Linalool 0.43-0.78 (Addae- Mensah et al., 1995)

Borneol 0.34-0.47 (Addae- Mensah et al., 1995)

Neral (citral A) 27.05-30.03 (Schaneber and Khan 2002)

Geranial (citral B) 29.95-42.41 (Schaneberg and Khan 2002)

Geranyl acetate 0.18-0.83 (Addae- Mensah et al., 1995)

Table 1

2.4.4. USES OF ESSENTIAL OIL:

AROMATHERAPHY: Aromatherapy is a form of alternative medicine that uses volatile plant materials, known as essential oils, and other aromatic compounds for the purpose of altering a person's mood, cognitive function or health. Science has discovered that our sense of smell plays a significant role in our overall health (http://lmkinteriorsltd.wordpress.com).

Since ancient times Essential Oils have been used in medicine because of their medicinal

properties, for example some oils have antiseptic properties. In addition, many have an uplifting

effect on the mind, though different essential oils have different properties.

Working of Essential Oil in Aromatherapy: when Essential Oil is inhaled it goes directly from

olfactory system to limbic system of the brain. Brain responds to the particular scent affecting

our emotions and chemical balance. Essential Oils also absorbed by the skin and carried

throughout the body via the circulatory system to reach all internal organs.

We can be benefited by choosing carefully the desired and suitable oils which can promote overall health. Benefits depend upon the unique nature of each person’s response to an aromatic stimulus (D. Pandey and P.S.Rao Virendra).

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Importance of Essential Oil in pharmaceutics

Essential Oils have versatile applications in pharmaceutics. Some of the applications are listed

below.

Antiseptics: The antiseptic properties of Essential Oil make them active against wide range of

bacteria as on antibiotic resistant strains. In addition to this they are also against fungi and yeasts.

The most common sources of essential oils used as antiseptics are: Cinnamon, Thyme, Clover,

Eucalyptus,Culinsavory,and Lavender. Citral, geraniol, linalool and thymol are much more

potent than phenol (D. pandey and P.S.Rao Virendra)

Expectorants and diuretics: When used externally, essential oils like (L’essence de

terebenthine) increase microcirculation and provide a slight local anesthetic action. Till now,

essential oils are used in a number of ointments, cream and gels, whereby they are known to be

very effective in relieving sprains and other articular pains. Oral administration of essential oils

like eucalyptus or pin oils, stimulate ciliated epithelial cells to secrete mucus. On the renal

system, these are known to increase vasodilation and in consequence bring about a diuretic

effect.

Spasmolytic and sedative: Essential oils from the Umbellifereae family, Mentha species and

verbena are reputed to decrease or eliminate gastrointestinal spasms. These essential oils increase

secretion of gastric juices. In other cases, they are known to be effective against insomnia.

2.4.5. METHODS OF EXTRACTION OF THE ESSENTIAL OIL

Extraction is a separation process and the following are the methods of extraction of Essential

Oil.

Solvent Extraction:

In the Solvent Extraction method of Essential Oils recovery, an extracting unit is loaded with

perforated trays of essential oil plant material and repeatedly washed with the solvent. A

hydrocarbon solvent is used for extraction. All the extractable material from the plant is

dissolved in the solvent. This includes highly volatile aroma molecules as well as non-aroma

waxes and pigments. The extract is distilled to recover the solvent for future use. The waxy mass

that remains is known as the concrete.

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The concentrated concretes are further processed to remove the waxy materials which dilute the

pure essential oil. To prepare the absolute from the concrete, the waxy concrete is warmed and

stirred with alcohol (ethanol/methanol).

During the heating and stirring process the concrete breaks up into minute globules. Since the

aroma molecules are more soluble in alcohol than the waxes, an efficient separation of the two

results. This is not considered the best method for extraction as the solvents can leave a small

amount of residue behind which could cause allergies and effect the immune system.

Maceration:

Maceration actually creates more of “infused oil” rather than an Essential Oil. Plant material is

soaked in vegetable oil, heated and strained at which point it can be used for massage. This

method is disadvantageous because it changes the composition of oil.

Cold Pressing:

This method is used to extract the Essential Oils from citrus rinds such as orange, lemon,

grapefruit and bergamot. This method involves the simple pressing of the rind at about 120oF to

extract the oil. The rinds are separated from the fruit, are ground or chopped and are then

pressed. The result is a watery mixture of essential oil and liquid which will separate at a given

time. Little alteration from the oil’s original state occurs. The drawback of this method is, oils

extracted using it have a relatively short shelf life.

Effleurage:

This is one of the traditional ways of extracting oil from flowers. The process involves layering

fat over the flower petals. After the fat has absorbed the essential oils, alcohol is used to separate

and extract the oils from the fat. The alcohol is then evaporated and the Essential Oil is collected

(H. Mukhtar et al)

17

Super Critical CO2 Extraction:

Supercritical CO2 extraction (SCO2) involves the heating of carbon dioxide at 87OF and pumping

it through the plant material at around 8,000 psi, the carbon dioxide is likened to a vapor. With

release of the pressure in this process, the carbon dioxide escapes in its gaseous form, leaving the

Essential Oil behind. The usual method of extraction is through steam distillation.

After extraction, the properties of a good quality essential oil should be as close as possible to the

"essence" of the original plant.

The key to a 'good' essential oil is through low pressure and low temperature processing. High

temperatures, rapid processing and the use of solvents alter the molecular structure, will destroy

the therapeutic value and alter the fragrance (H. Singh, M. Hasan et al.,).

Turbo Distillation Extraction:

Turbo distillation is suitable for hard-to-extract or coarse plant material, such as bark, roots, and

seeds. In this process, the plants are soaked in water and steam is circulated through this plant

and water mixture. Throughout the entire process, the same water is continually recycled through

the plant material. This method allows faster extraction of essential oils from hard-to-extract

plant materials.

Extraction of Essential Oils Using Steam distillation Method:

Steam distillation is a special type of distillation or a separation process for temperature sensitive

materials like oils, resins, hydrocarbons, etc. which are insoluble in water and may decompose at

their boiling point. The fundamental nature of steam distillation is that it enables a compound or

mixture of compounds to be distilled at a temperature substantially below that of the boiling

point of the individual constituent.

Essential oils contain substances with boiling points of up to 200°C or higher temperatures. In

the presence of steam or boiling water, however, these substances are volatilized at a temperature

close to 100°C (the boiling point of water), at atmospheric pressure.

Fresh or sometimes dried, botanical material is placed in the plant chamber of the still and the

steam is allowed to pass through the plant material under pressure which softens the cells and

allows the Essential Oil to escape in vapor form.

18

The temperature of the steam must be high enough to vaporize the oil present, yet not so high

that it destroys the plants or burns the Essential Oils. Essential Oil vapors condense with the

steam. The essential oil forms a film on the surface of the water since it’s less dense than water.

To separate the Essential Oil from the water, the film is then decanted or skimmed off the top.

The remaining water, a byproduct of distillation, is called floral water, distillate, or hydrosol.

It retains many of the therapeutic properties of the plant, making it valuable in skin care for facial

mists and toners (A solution containing chemicals that can change the color of a photographic

print).

In certain situations, floral water may be preferable to be pure essential oil, such as when treating

a sensitive individual or a child, or when a more diluted treatment is required. Rose hydrosol, for

example, is commonly used for its mild antiseptic and soothing properties, as well as its pleasing

floral aroma.

A number of factors determine the final quality of a steam distilled essential oil. Apart from the

plant material, most important are time, temperature and pressure, and the quality of the

distillation equipment. Essential oils are very complex products. Each is made up of many,

sometimes hundreds, of distinct molecules which come together to form the oil's aroma and

therapeutic properties. Some of these molecules are fairly delicate structures which can be

altered or destroyed by adverse environmental conditions. So, much like a fine meal is more

flavorful when made with patience, most oils benefit from a long, slow 'cooking' process.

It is possible that longer distillation times may give more complete oil. It is also possible

however, that longer distillation time may lead to the accumulation of more artifacts than normal.

This may have a curious effect of appearing to improving the odor, as sometimes when materials

that have a larger number of components are sniffed, the perception is often of slightly increased

sophistication, added fullness and character, and possibly, and extra pleasantness.

Advantages of using Steam Distillation

The advantage of Steam Distillation is that it is a relatively cheap process to operate at a basic

level, and the properties of oils produced by this method are not altered. As steam reduces the

boiling point of a particular component of the oil, it never decomposes in this method. This

method apart from being economical, it is also relatively faster than other methods.

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2.5. Density

The density of a material/substance is defined as its mass per unit volume (Wikipedia). It’s

essentially a measurement of how tightly matter is crammed together. The principle of density

was discovered by the Greek scientist Archimedes.

2.5.1. Calculation of density

To calculate the density of a substance, the mass of that substance is divided by its volume.

ῥ = m/v

2.5.2. Uses of density

Density is used to determine how different materials interact when mixed together.

2.6. Refractive index

In optics refractive index of a substance is a number that describes how light or any other

radiation propagates through that medium (Wikipedia)

Encyclopedia Britannica defines refractive index (index of refraction) as the measure of the

bending of a ray of light when passing from one medium into another.

Its most elementary occurrence (and historically the first one) is in Snell's law of refraction,

n1sinθ1= n2sinθ2, where θ1 and θ2 are the angles of incidence of a ray crossing the interface

between two media with refractive indices n1 and n2. Brewster's angle, the critical angle for total

internal reflection, and the reflectivity of a surface also depend on the refractive index, as

described by the Fresnel equations (Eugene Hecht 2002).

2.6.1. Measuring of refractive index

Refractometer; A refractometer is used to measure the extent to which light is bent (i.e.

refracted) when it moves from air into a sample and is typically used to determine the refractive

index of a liquid sample.

20

Figure 7

2.6.2. Uses of the refractive index

Help identify or confirm the identity of a sample by comparing its refractive index to

known values.

Assess the purity of a sample by comparing its refractive index to the value for the pure

substance.

Determine the concentration of a solute in a solution by comparing the solution's

refractive index to a standard curve.

21

3.0. CHAPTER III METHODOLOGY

3.1. Extraction of the essential oil

3.1.0. Materials and Equipment used

• Lemon grass

• Flasks and container

• Clevenger steam distillation apparatus

• Stain steel knife

• Decanting funnel

• Distilled water

• Heat source

• Anhydrous sodium sulphate

3.1.1. Collection, Identification and characterization of the Lemmon grass

Fresh leaves of lemongrass leaves were collected from healthy living plants at Makerere

University Kampala and identified at Makerere University Herbarium.

3.1.2. Material preparation and extraction

The leaves of lemongrass were chopped into 15cm long pieces, weighed (170gm) and transferred

into a round- bottomed flask to which was added 200ml of distilled water and steam distilled for

4 hrs using a Clevenger apparatus (AOAC). The total distillation time went up to 5hrs including

an hour for the oil to start distilling. Since the essential oil of lemongrass is less dense than water,

the oil floated on top of the water. Anhydrous sodium sulphate was used to dry the oil and the oil

content was expressed as a percentage.

22

3.1.2. Experimental Set up

The extraction of the essential oil was carried out using experiment Clevenger’s Apparatus setup.

The apparatus consist of a round bottom flask that holds the water and it is connected to another

two way round flask which holds the raw material. The top flask that holds the raw material is

connected to the condenser through the connecter. A heat source is used to heat the water in the

bottom flask. The separating funnel is used for the separation of essential determine.

Figure 8

3.2. Determination of the refractive index

The refractive indices of the oil samples collected at different temperatures were measured using

an ABBE REFRACTOMETER (MODEL AKRUSS OPTRONIC GERMANY) and recorded.

3.3. Determination of the density

The densities of the oil samples collected at different temperatures were calculated as follows;

The volumes were measured using a measuring cylinder and then recorded.

The masses were also measured using a simple electric weighing machine and then

recorded.

The densities were then calculated using the formula below and then recorded,

ῥ = m/v

Where; ῥ is the density of the sample, m is the mass of the oil sample and v is the volume

of the oil sample.

23

3.4. Pesticide making

3.4.1. Materials

sun seed vegetable oil

essential oil (cymbopogon citratus)

Blender

1 stainless steel saucepan

Measuring cylinders

Distilled water

Spray containers

Stirring rod

3.4.2. Procedure

1 liter of Sun seed vegetable oil water was poured into the blender and the blending was done.

Sodium chloride and ethanol were also added to the blender and blending continued. 20ml of

cymbopogon citratus essential oil were also added to the blended mixture and further blending

was done in order to form a homogenous mixture. The mixture was then poured in a stainless

steel saucepan and diluted with 3 liters of distilled water. The dilute mixture was further blended

this time with water for purposes of manual emulsification since water can not homogenously

mix with oil but if blended hardly it can. Packaging was then done by pouring 900 ml of the

pesticide preparation in each spray container

24

4.0. CHAPTER IV RESULTS AND DISCUSSION

4.1. Results

Table showing refractive index, volume, mass and density of oil extracted at different

temperature.

Temperature (OC) Refractive Index Volume of

oil (cm3)

Mass of

oil (g)

Density

(gcm-3)

10 1.47775 0.26 0.223 0.858

20 1.47382 0.32 0.272 0.850

30 1.46922 0.44 0.370 0.841

40 1.46503 0.54 0.450 0.833

50 1.46029 0.62 0.514 0.829

60 1.45788 0.68 0.557 0.819

Table 2

4.2. Discussion of results

Lemongrass oil, which is pale yellow oil, had a refractive index ranging from 1.4775-1.45788

and a density ranging from 0.858-0.819gcm-3 which is compared with those found in literature.

For example, Angus-S et al (1976) reported lemongrass oil refractive index at 20oC to be 1.482

and a density of 0.8863gcm -3 which is almost the same.

Literature has it that the refractive index is directly proportional to the concentration of a

solution.

From graph 1 below, as the temperature of steam distillation increases, the refractive index of the

oil decreases. This is because the volatile components of oil are more volatile than the water and

as the temperature increases, there reaches a time when all the volatile components have

evaporated and only the heavy components remain. This leads to the decrease in the

concentration of the oil in the vapor phase and hence a decrease in the refractive index. This

shows that as the temperature is increased, the concentration of the oil collected decrease and so

if a more concentrated oil sample is needed; less temperature of about 20oC must be used for

distillation.

25

Graph 1

From graph 2 below, the density of the oil collected decreases with any increase in temperature.

This is because as the temperature increase, the volume of the oil extracted increase and yet

density is inversely proportional to the volume.

Since in the formulation of a pesticide the oil is mixed with water homogeneously and yet water

and oil are difficult to mix because of the difference in their densities, so extraction of the

essential oil needed in the formulation of a pesticide should be extracted at temperatures of about

20oC because its at this temperature that the density is near that of water.

Graph 2

26

4.3. Significance of study

As an agricultural country, Uganda appreciates the urgency of developing new and production-

increasing agricultural technologies that utilize local resources. The formulation of a pesticide

using lemongrass oil has important implications for food security, poverty reduction and

environmental protection, the three most important World policy objectives.

27

5.0. CHAPTER V. RECOMMENDATION AND CONCLUSION

5.1. Recommendation

I recommend the following after carrying out the project;

Use of botanical pesticides rather than synthetic pesticides.

Using low temperatures in steam distillation of the essential oil to be used in formulation

of a pesticide.

5.2. Conclusions

The following can finally be concluded after carrying out the above project;

The concentration of the essential oil extracted from lemongrass decreases with increase

in temperature.

The density of the essential oil extracted from lemongrass decreases with increase in

temperature.

When formulating a pesticide, the essential oil should be extracted at around 20oC in

order to get the best pesticide.

28

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