MAKERERE

UNIVERSITY

COLLEGE OF NATURAL SCIENCES DEPARTMENT OF CHEMISRTY

Formulation of a botanical pesticide from lemongrass

YEAR OF STUDY NAME REG NO STUDENT NO PROGRAM COURSE CODE University supervisor

: : : : : : :

III Lubwama Kenneth 09/U/2708/PS 209005757 Bachelor of Science in industrial chemistry ICH 3235 Dr. John Wasswa

A project report submitted for the partial fulfillment for the award of the bachelors degree of science in industrial chemistry to the college of Natural sciences, Makerere University Kampala June 2012

DECLARATION I LUBWAMA KENNETH declare that the information presented in this report, as a partial fulfillment for the requirements of Bachelors 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 Supervisors Signature .. DR. WASSWA

ACKNOWLEDGEMENT First, 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.

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

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Table of Contents
DECLARATION..............................................................................................................................i ACKNOWLEDGEMENT...............................................................................................................ii DEDICATION................................................................................................................................iii Table of Contents............................................................................................................................iv ACRONYMS AND SYMBOLS....................................................................................................vi LIST OF FIGURES, TABLES AND GRAPHS...........................................................................vii FIGURES...................................................................................................................................vii TABLES....................................................................................................................................vii GRAPHS....................................................................................................................................vii CHAPTER I. INTRODUCTION.....................................................................................................1 2.0. CHAPTER II. LITERATURE REVIEW.................................................................................4 2.1. Pesticides...............................................................................................................................4 2.1.0. Definition.......................................................................................................................4 2.1.1. History of a pesticide.....................................................................................................4 2.1.2. Uses of pesticides...........................................................................................................5 2.1.3. Benefits .........................................................................................................................5 2.2. Botanical pesticides..............................................................................................................6 2.2.1. Definition of botanicals..................................................................................................7 2.2.2. Characteristics of Botanicals ........................................................................................7 2.2.3. Advantages of botanicals...............................................................................................8 2.2.4. Disadvantages of botanicals...........................................................................................8 2.2.5. Some common botanicals..............................................................................................9 2.3. Lemongrass.........................................................................................................................12 2.4. Essential oil.........................................................................................................................13 2.4.1. Definition.....................................................................................................................13 2.4.2. Physical Properties of Essential Oils .........................................................................13 2.4.3. Chemical Properties of Essential Oil ..........................................................................14 2.4.4. USES OF ESSENTIAL OIL: .....................................................................................15 2.4.5. METHODS OF EXTRACTION OF THE ESSENTIAL OIL.....................................16 2.5. Density................................................................................................................................20 2.5.1. Calculation of density..................................................................................................20 2.5.2. Uses of density.............................................................................................................20 2.6. Refractive index..................................................................................................................20 2.6.1. Measuring of refractive index......................................................................................20 2.6.2. Uses of the refractive index.........................................................................................21 3.0. CHAPTER III METHODOLOGY........................................................................................22 3.1. Extraction of the essential oil..............................................................................................22 iv

3.1.0. Materials and Equipment used.....................................................................................22 3.1.1. Collection, Identification and characterization of the Lemmon grass.........................22 3.1.2. Material preparation and extraction ............................................................................22 3.1.2. Experimental Set up.....................................................................................................23 3.2. Determination of the refractive index.................................................................................23 3.3. Determination of the density...............................................................................................23 3.4. Pesticide making.................................................................................................................24 3.4.1. Materials......................................................................................................................24 3.4.2. Procedure.....................................................................................................................24 4.0. CHAPTER IV RESULTS AND DISCUSSION....................................................................25 4.1. Results.................................................................................................................................25 4.2. Discussion of results...........................................................................................................25 4.3. Significance of study ..........................................................................................................27 5.0. CHAPTER V. RECOMMENDATION AND CONCLUSION..............................................28 5.1. Recommendation................................................................................................................28 5.2. Conclusions.........................................................................................................................28 REFERENCES..............................................................................................................................29

ACRONYMS AND SYMBOLS DDT; 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

FIGURES
Figure 11 Figure 29 Figure 3.10 Figure 411 Figure 511 Figure 6.12 Figure 7.21 Figure 8.23

TABLES
Table 1.15 Table 2.23

GRAPHS
Graph 126 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 7080 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. (C 10H10O, (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 Its 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, athletes 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.

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. 2

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.

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, arsenicbased 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).

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 5

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 insects 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 withboth good and bad insects.

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.

2.2.5. Some common botanicals (1) PyrethrinsPyrethrum 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 insects 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 leaffeeding 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.

Figure 3

(3) Neema 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 limitednew 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. 13

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 MYRCENE Linalool Borneol Neral (citral A) Geranial (citral B) Geranyl acetate Table 1 CONCENTRATION (%) 2.64-12.03 (Addae- Mensah et al., 1995) 0.43-0.78 (Addae- Mensah et al., 1995) 0.34-0.47 (Addae- Mensah et al., 1995) 27.05-30.03 (Schaneber and Khan 2002) 29.95-42.41 (Schaneberg and Khan 2002) 0.18-0.83 (Addae- Mensah et al., 1995)

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 persons response to an aromatic stimulus (D. Pandey and P.S.Rao Virendra).

Importance of Essential Oil in pharmaceutics Essential Oils have versatile applications in pharmaceutics. Some of the applications are listed 15

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 (Lessence 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. 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 16

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 120 oF 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 oils 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)

Super Critical CO2 Extraction: Supercritical CO2 extraction (SCO2) involves the heating of carbon dioxide at 87OF and pumping 17

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 200C or higher temperatures. In the presence of steam or boiling water, however, these substances are volatilized at a temperature close to 100C (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. 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 18

steam. The essential oil forms a film on the surface of the water since its 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). Its 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. = 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, n1sin1= n2sin2, where 1 and 2 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.

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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.

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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, 22

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.

3.1.2. Experimental Set up The extraction of the essential oil was carried out using experiment Clevengers 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. 23

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, = m/v Where; is the density of the sample, m is the mass of the oil sample and v is the volume of the oil sample.

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

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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) 10 20 30 40 50 60 Table 2 Refractive Index 1.47775 1.47382 1.46922 1.46503 1.46029 1.45788 Volume of Mass of Density oil (cm3) 0.26 0.32 0.44 0.54 0.62 0.68 oil (g) 0.223 0.272 0.370 0.450 0.514 0.557 (gcm-3) 0.858 0.850 0.841 0.833 0.829 0.819

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 25

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.

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.

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Graph 2

4.3. Significance of study As an agricultural country, Uganda appreciates the urgency of developing new and productionincreasing 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.

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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.

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