ABSTRACT
Clearing agents are among the most noxious and hazardous chemicals found in histology laboratories. Xylene has probably been the most commonly used chemical in the histology laboratory despite its hazards. There has been several laboratory tests to replace xylene as clearing agent but most of these commercially available substitutes in some cases are less effective, more expensive, slightly biodegradable and are not readily available than xylene itself. The present study was aimed at evaluating the clearing abilities of some nontoxic concentrated hydrophobic liquids commonly called essential oils namely citrus oil, citrodora oil, and neem oil during histological processing of some selected organs of adults Wistar rats. The three essential oils were used alongside xylene to clear livers, kidneys, Brains, testes and intestinal tissues of Wistar rats. The citrus oil was extracted from the peels of citrus oranges by hydro distillation, while the citrodora oil was extracted from the leaves of eucalyptus plants by hydro distillation method. The neem oil was extracted from the seeds of neem tree by Cold n-hexane method. Physical properties, the phytochemical and Gas Chromatography-Mass Spectroscopy (GC-MS) studies of the oils were carried out using the methods of Siddiqui and Alli, (1997); Tresae, (1989) and Ladan, (2011) respectively. The tissues were histologically processed using the method of Bancroft and Stevens (2008). The result showed that citrus oil and xylene were colorless while citrodora oil was pale yellow and neem oil was dark brown. The densities of the oils are 0.80 g/ml for citrus oil, 0.80g/ml for citrodora oil, and 0.89 g/ml for neem oil when compared to 0.84 g/ml of xylene. The viscosity of neem oil was 36cp when compared to that of citrus oil which was 0.35cp, citrodora oil 0.34cp and xylene 0.45cp. It was also demonstrated that neem oil has the highest value of flash point at 108 0C while citrus and citrodora oils had similar flash points at 340C and 350C respectively and xylene had flash point at 390C. The oils had refractive indexes of 1.46,
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1.46, 1.47 and 1.50 for citrus, citrodora, neem oil and xylene respectively. Histological observation showed that the cytoplasmic and nuclear staining ability appeared to give good contrast in citrodora oil followed by citrus oil when compared with xylene. Significant changes in the section and staining ability were observed in the neem oil cleared sections when compared to xylene, citrus and citrodora oil (p˂0.05). The result of the present study showed that the three essential oils used in this work can clear tissues substantially well. Citrodora oil appeared to be better followed by citrus oil while the neem oil had the least ability to clear tissue properly.
TABLE OF CONTENTS
Cover page …………………………………………………………………………………………………..i Fly Page ………………………………………………………………………………………………………ii Title Page …………………………………………………………………………………………………….iii Declaration …………………………………………………………………………………………………..iv Certification …………………………………………………………………………………………………v Acknowledgment ………………………………………………………………………………………….vi Table of Contents ………………………………………………………………………………………….vii List of Tables ………………………………………………………………………………………………xii List of Figures ………………………………………………………………………………………………xiii List of Plate ………………………………………………………………………………………………….xiv Abstract ……………………………………………………………………………………………………….xvi CHAPTER ONE 1.0 Introduction …………………………………………………………………………………………..1 1.1 Statement of the research problem …………………………………………………………6 1.2 Justification …………………………………………………………………………………………..7 1.3 Significance of the Study ………………………………………………………………………..7 1.4 The Aim of the Study ……………………………………………………………………………..7 1.5 The Objectives of the Study …………………………………………………………………..7 1.6 Hypothesis ……………………………………………………………………………………………..8 CHAPTER TWO 2.0 Literature review …………………………………………………………………………………..9 2.1 Xylene ……………………………………………………………………………………………………10 2.2 Production and properties of Xylene ………………………………………………………11
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2.3 Applications of Xylene ……………………………………………………………………………13
2.3.1 Solvent and general applications …………………………………………………………….13
2.3.2 Laboratory uses of xylene ………………………………………………………………………13
2.3.3 Health effects (Toxicity of xylene) ………………………………………………………….14
2.3.4 Xylene substitutes for clearing tissues ……………………………………………………..15
2.3.5 Limonene Reagents ……………………………………………………………………………….15
2.3.6 Aliphatic Hydrocarbon…………………………………………………………………………..15
2.3.7 Aromatic Hydrocarbon Mixture (BTX) ……………………………………………………15
2.3.8 Mineral Oil Mixture ………………………………………………………………………………16
2.4 Essential oils ………………………………………………………………………………………….16 2.4.1 Brief History of Essential Oils ………………………………………………………………..18 2.4.2 Production of Essential Oils……………………………………………………………………19
2.5 Health benefit of essential oils …………………………………………………………………23
2.5.1 Therapeutic use of Essential Oils …………………………………………………………….23
2.5.2 Effect of Essential oils on Culinary (Flavouring)/ Ingestion ……………………….24
2.5.3 Industrial effect of Essential oils……………………………………………………………..24
2.5.4 Toxicity of essential oils ………………………………………………………………………..25
2.6 List of some essential oils and the part of plant they are gotten from ……….27
2.7 Eucalyptus oil ……………………………………………………………………………………….28 2.7.1 Origin of eucalyptus oil …………………………………………………………………………28 2.7.2 Citodora……………………………………………………………………………………………….29 2.7.3 Cultivation and Extraction of Eucalyptus oil …………………………………………….30 2.7.4 Chemical Properties and Composition of Eucalyptus Oil …………………………..31 2.8 Origin and history of orange …………………………………………………………………..34 2.8.1 Origin and History of Essential Oils ………………………………………………………..34
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2.8.2 Physical and Chemical Characteristics of Orange Oil ………………………………..35 2.8.3 Composition of Orange Oil …………………………………………………………………….37 2.8.4 Propagation and growth of Oranges ………………………………………………………..39 2.8.5 Harvesting, Storage and Extraction of Oranges Oil ……………………………………39 2.8.6 Uses of orange oils ………………………………………………………………………………..39 2.8.7 Warnings and Precautions when using orange essential oil ………………………..40 2.8.8 Orange peel consumption ………………………………………………………………………40
2.9 Neem oil (Azadirachta indica) ………………………………………………………………….40
2.9.1 Origin and History of Neem …………………………………………………………………..41
2.9.2 The physical, Chemical Properties and Composition of Neem Oil ………………41
2.9.3 Physical and Chemical Properties of Neem Oil …………………………………………42
2.9.4 Chemistry of Neem ……………………………………………………………………………….44
2.9.5 Cultivation and Production of Neem Oil ………………………………………………….48
2.9.6 Storage of Neem Oil ……………………………………………………………………………..49
2.9.7 Extraction of Neem Oil ………………………………………………………………………….49
2.9.8 Uses of Neem Oil ………………………………………………………………………………….49
2.9.9 Therapeutic uses of Neem Oil…………………………………………………………………50
2.9.10 Toxicity of Neem Oil …………………………………………………………………………..51
2.9.11 Safety precautions and warnings when using Neem …………………………………51
CHAPTER THREE 3.0 Materials and methods …………………………………………………………………………..52 3.1 Reagents and materials …………………………………………………………………………..52 3.2 Plant materials for the oil ……………………………………………………………………….52 3.2.1 Collection and Identification of Plant ………………………………………………………52 3.2.2 Extractions of the Essential Oils ……………………………………………………………..53
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3.2.3 Eucalyptus oil and Citrus oil …………………………………………………………………..53 3.2.4 Neem Oil ……………………………………………………………………………………………..53 3.2.5 Determination of the percentage yield of the Oils ……………………………………..53 3.2.6 Phytochemical Studies of the Essential Oils ……………………………………………..54 3.2.7 Physical Properties of the Essential Oils ………………………………………………….56 3.2.8 Gas Chromatography–Mass Spectroscopy (GC-MS) Studies of the Oils ……..60 3.3 Experimental animals …………………………………………………………………………….61 3.3.1 Experimental Protocol …………………………………………………………………………..61 3.3.2 Animals Sacrifice and Fixation ……………………………………………………………….62 3.3.3 Method of tissue processing……………………………………………………………………66 3.4 Tissue processing ……………………………………………………………………………………66 3.4.1 Dehydration………………………………………………………………………………………….68 3.4.2 Clearing ……………………………………………………………………………………………….68 3.4.3 Infiltration ……………………………………………………………………………………………70 3.4.4 Embedding …………………………………………………………………………………………..70 3.4.5 Sectioning ……………………………………………………………………………………………70 3.5 Staining and mounting ………………………………………………………………………….70 3.6 Photomicrograph …………………………………………………………………………………..71
3.7 Evaluation criteria and method ………………………………………………………………71
3.8 Average time, volume and cost of the oils ………………………………………………..72
3.9 Statistical analysis ………………………………………………………………………………….72
CHAPTER FOUR 4.0 Results…………………………………………………………………………….73 4.1 Result of phytochemical screening of the oils ……………………………………………..73 4.2 Physical properties of the oil……………………………………………………………………..75
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4.3 Refractive index of the oils (RF) at 28 0C……………………………………………………77 4.4 Physical observation of the oils ………………………………………………………79 4.5 Mean time, volume and estimated cost of the oils………………………………81 4.6 GC-MS of the oils ……………………………………………………………………………………83 4.7 Gas chromatography–mass spectroscopy (GC-MS) profile …………………………..86 4.8 Clearing effect and staining quality on sections ………………………………………….91 4.9 photomicrographs of stained tissue sections ………………………………………………..102 CHAPTER FIVE Discussion ……………………………………………………………………………………………………129 Physical properties of the oil…………………………………………………………………………..129 Phytochemical study of the oils ………………………………………………………………………133 Gas chromatograph-mass spectrophotometry (GC-MS) study of the oils ……………..134 Stained tissue sections……………………………………………………………………………………136 CHAPTER SIX 6.0 Summary, Conclusion and Recommendations ……………………………………………139 6.1 Summary ………………………………………………………………………………………………..139 6.2 Conclusion ……………………………………………………………………………………………..140 6.3 Recommendations ……………………………………………………………………………………141 6.4 Limitations ……………………………………………………………………………………………..141 References ……………………………………………………………………………………………………142 Appendices ………………………………………………………………………………………………….151
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CHAPTER ONE
1.0 INTRODUCTION Most Histology and Histopathology Laboratories use either aromatic solvents such as xylene, toluene or aliphatic petroleum distillates for the purpose of clearing and de-waxing in the paraffin histological technique. Paraffin wax is used universally to infiltrate and embed tissues in the final step prior to microtomy (Bancroft and Gamble, 2008). Paraffin wax does not mix with water due to their hydrophobic nature; therefore water present in tissues has to be removed by dehydration using alcohols prior to infiltration and embedding. Following dehydration, the tissues are saturated with alcohol which is also not miscible with paraffin wax (Baker and Silvertons, 1998). Certain agents are used to remove alcohols from the tissue before the tissue can be infiltrated with paraffin wax, these agents are called clearing agents. Clearing agents are sometimes called ―de-alcoholization agents‖ or ante medium. They act as intermediary between the dehydrating and infiltrating solutions. They are miscible with both solutions and have refractive indices similar to proteins with different levels of toxicity (Kiernan, 2010). There are many clearing agents such as: – xylene, toluene, chloroform, acetone, kerosene, diaxane, benzene, petrol, cedar wood oil etc. Most clearing agents are derivatives of aromatic hydrocarbons such as benzene, while others are derived from natural essential oils such as cedar wood oil, olive oil and anicine, etc (Hans et al., 1995).
Bancroft and Stevens (1990), stated that the physical and chemical properties of clearing agents should include the following; high fluidity, miscibility with both ethanol and paraffin, molecular weight not above 350 g/mol, melting point not below 18o C and
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physical and chemical stability. The potential hazard of a compound to health is directly related to its boiling point and vapor pressure, and as such clearing agents should have a very low vapour pressure and high boiling point as well (Baker and Silvertons, 1998). Clearing agents are among the most noxious and hazardous chemicals found in histology laboratories. They are highly inflammable with the exception of olive oil and cedar wood oil. Most of the other clearing agents are harmful to health and constitute health hazards on inhalation or on contact with the skin (Amdur et al., 1991). Xylene has probably been the most commonly used chemical in the histology laboratory despite its hazards. It is expensive but work well for short time clearing of small tissue blocks. Its high solvency factor allows maximum displacement of alcohol and enhancing paraffin infiltration. There is a visual clue given by xylene, it causes an increase in the refractive index of tissue as the dehydrants is removed. Eventually, when completed, the tissue becomes quite translucent, and almost transparent (Carson and Hladik, 2009). Generally only three changes of xylene are required to reach this stage. If the tissue remains opaque it means that either water or ethanol remains in the tissue. If the opacity is very high, then it probably indicates the presence of water (Tardif and Braddeir 1992). Xylene does tend to harden tissues a little, but this does not usually interfere with sectioning qualities. One very distnct defect, though, is that it may reharden decalcified bone excessively. This is not too much of a problem with the softer cancellous bones encountered, but it can make sectioning of decalcified long bones extremely difficult, if not impossible (Stevenson et al., 1998; Kieranan, 2010). Long term immersion of tissue in xylene results in tissue distortions (Visfeldt et al., 1982).
Several xylene substitutes have been commercially developed in recent years, some being aromatic derivatives of terpene, natural oils and resins produced by some plants and
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animals, and others, hydrocarbons, cyclic monoterpenes and isoparaffinic hydrocarbons, with several trade names, these have been comparatively used in tissue processing (Luna, 1992). As far back as 1978, Maxwell has looked for safer substitutes for xylene in histology. He suggested the use of 1,1,1-trichloroethane as clearing agents. This compound has a high vapour pressure but is non flammable and less toxic than xylene. However, its use as anaesthetic agents precludes it being used as serious clearing agent (Maxwell, 1978).
Histo-Clear is another xylene substitutes that is commercially produce and is regarded to be non toxic. It is claimed to consist of essential oils (isoprene derivatives) produced by distillation of citrus and corn oils. Histo-Clear is excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol. However, the compound is unstable owing to its double bounds. It also has a high vapour pressure (4.5mm Hg at 25oC) and has a penetrating odour (Hans et al., 1995). The least toxic of all the chemicals used in tissue processing are the paraffin. Although the tissue is ultimately embedded in them, paraffin oil has been mentioned in the literature as 1 out of 12 clearing agents suitable for routine use, with clearing time similar to that of chloroform. There have been recent tests on mixing vegetable oils with paraffin wax and even the use of pure paraffin oil as the clearing agent in a single step after dehydration by alcohols (Luna, 1992., Hans et al., 1995).
Vegetable oils consisting mainly of triglycerides are all liable to decompose. The change may arise as a result of several different types of chemical reactions, including hydrolysis of the glycerides and oxidation at double bonds of unsaturated fatty acids. Olive oil and other similar unsaturated oils are not good clearing agents because they easily go rancid (Hans et al., 1995).
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It was reported that Toluene is better at preserving tissue structure and is more tolerant of small amount of water left behind in the tissues than xylene. However, toluene is more expensive and toxic than xylene (Thompson et al., 1978), Isopropyl alcohol has also been recently used as xylene substitute in tissue processing (Falkeholm et al., 2001). It is claimed to be more cost effective, saves time, and improves the laboratory environment. It also causes less shrinkage and hardening of tissue than ethyl alcohol (Dapson et al, 2005). Chloroform has been used in some applications but it has a severe health hazard, acts slowly and may lead to sectioning difficulties (Hans et al., 1995). Other clearing agents include long chain aliphatic hydrocarbon which represent less of a health hazard, but are less tolerated with poorly fixed, dehydrated or sectioned tissues (Hans et al., 1995). Most of these commercially available xylene substitutes are less effective, more expensive, and are not readily available than xylene itself (Gosselin et al., 1984; Amdur et al., 1991; Luna, 1992).
Cedar wood oil is perhaps the most well known natural wood oil for clearing tissues. Cedar wood oil is obtained from juniper and cypress species, both of which are within the general descriptions of being ―cedar‖ trees. For histological processing the major advantage of this oil is that it causes almost no damage to the tissue. However it does take significantly longer, and is significantly more expensive than the usually used alternatives (Gurr, 1962; Bancroft and Gamble,, 2008). Some of the other natural oils, such as clove oil, have also been used histologically but they are usually even more expensive than cedar wood oil and, since there is little advantage to using them in preference to cedar wood oil, they have never been popular (Bancroft and Gamble, 2008). Many natural oils and resins belong to a group of chemicals called terpenes which are produced by
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numerous plants and some animals. The name is derived from ―turpentine‖ which was one of the first to become available. The basic building block of these oils is isoprene. The isoprene units are combined in various ways to produce the individual compounds making up the oils. Some of these natural oils have also been used as mounting media (Canada balsam). As a group they tend to be gentler in action, but they are not as freely miscible with dehydrant agents (Hans et al., 1995). Most essential oils belong to a family of compounds known as terpenes and terpenoids. Terpenes are small organic hydrocarbon molecules; they may be cyclic or acyclic, saturated or unsaturated. Terpenoids are oxygenated derivatives of terpenes, which may contain hydroxyl groups or carbonyl groups. Regardless of their structural diversity, terpenes and terpenoids share certain structural similarities. They contain multiples of five carbon atoms i.e., hemiterpene contains 5 carbon atoms; monoterpene, 10; sesquiterpene, 15; diterpenes, 20; etc. (Crowell, 1999). Terpenes, such as limonene, and terpenoids, such as neral or geranial may be found in abundance in oil sacs located in the outer, colored portion of the rind of many common citrus fruits (Gilles et al., 2010).
Limonene is another example of clearing agent from terpene. It is the natural component found in the skins of citrus fruits, such as lemons or oranges, and in cooking is usually referred to as lemon or orange zest. The name is derived from Citrus limonum, the lemon tree. Limonene is obtained industrially by the steam distillation of orange peel which is a by product of the orange juice industry. It is a clear, colourless fluid with a distinctly citrus aroma. Although, effective for clearing albeit a little more slowly than xylene, it does have one serious disadvantage. Some people are allergic to it and develop skin rashes when they come in contact with it. When used as the clearant immediately prior to cover slipping there are some reports that the mounting medium, usually dissolved in
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either toluene or xylene, does not mix well with the limonene and flow under the coverslip is affected. In such cases, replacing the limonene with xylene or toluene, or quickly dipping the section in either one just prior to cover slipping should be effective. This does, of course, defeat the purpose of the replacement (Rene, 2000).
Essential oils are also known as volatile or ethereal oil, or simply as the ―oil of‖ the plant from which they were extracted. For instance, eucalyptus oil is from eucalyptus plant and neem oil is from neem plant. Essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants (Pino et al., 2006). An essential oil is a liquid that is generally distilled most frequently by steam or water from the leaves, stems, flowers, bark, roots, or other elements of a plant. 1.1 STATEMENT OF THE RESEARCH PROBLEM Clearing agents are among the most noxious and hazardous chemicals found in histology laboratories. Most of them are synthetic oils of hydrocarbon origin, with different levels of toxicity (Gosselin et al., 1984). For several years, xylene has been widely used as clearing agent of choice inspite its cost, and the health hazard to personnel and the environment (Bush, 1977; Maxwell, 1978; Hans et al., 1995). There have been several attempts to substitute xylene as clearing agent. Recently, xylene substitute as clearing agents was developed by mixing vegetable oils with paraffin wax. Most of these commercially available xylene substitutes are less effective, more expensive, and are not as readily available as xylene (Gosselin et al., 1984; Amdur et al., 1991; Luna, 1992).
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1.2 JUSTIFICATION Several xylene substitutes as clearing agents have been commercially developed in recent years, some being aromatic derivatives of terpene, a natural oils and resins produced by some plants and animals. Other clearing agents are hydrocarbons and most of these substitutes are less effective and more expensive (Maxwell, 1978). Therefore, these essential oils (citrus oil and citrodospra oils) may be used as a substitute to xylene thereby reducing exposure to its hazaeds in histology laboratories. 1.3 SIGNIFICANCE OF THE STUDY Results from this study will extend our knowledge on the uses and availability of some essential oils as clearing agents and as a xylene substitute during histological tissue processing. 1.4 THE AIM OF THE STUDY The aim of the study was to evaluate the clearing potency of some essential oils namely eucalyptus (citrodora) oil, citrus oil, and neem oil using some tissues such as Brain, liver, kidney, intestine and testes of adult Wistar rats. 1.5 THE OBJECTIVES OF THE STUDY The objectives of the research are to study:
i. The effectiveness of essential oils namely citrodora oil, citrus oil, and neem oil as clearing agent in tissue processing.
ii. The effect of these oils on the structures of some tissues of adult Wistar rats.
iii. The effects of these oils on the staining qualities of brain, liver, kidney, intestine and testes of adult Wistar rats.
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iv. The clearing ability of these essential oils with themselves and the standard xylene.
1.6 HYPOTHESIS
i. Essential oils cannot clear animal tissues during tissue processing.
ii. Essential oils can impair the morphology and staining characteristics of animal tissues.
iii. There is no difference between xylene and these essential oils when using them as clearing agents.
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