Kaffir Lime) Leaves Oil

Home , Hydroxycitronellal

ACTIVATING EFFECT OF AROMATHERAPY INHALATION WITH Citrus hystrix (KAFFIR LIME) LEAVES OIL

ALISA STEPHANIE RASION

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THESIS SUBMITTED IN FULFILLMENT FOR THE DEGREE OF MASTER OF SCIENCE

FACULTY OF SCIENCE AND NATURAL RESOURCES UNIVERSITY OF MALAYSIA SABAH 2018 UNIVERSITI MALAYSIA SABAH

BORANGPENGESAHAN STATUS TESIS

JUDUL: ACTIVATINGEFFECT OF AROMATHERAPYINHALATION WITH Citrus hystrix (KAFFIR LIME) LEAVESOIL

IJAZAH: SARJANA SAINS (KIMIA INDUSTRI)

Saya ALISA STEPHANIE RASION, sesi 2016-2018, mengaku membenarkan tesis Sarjana ini disimpan di Perpustakaan Universiti Malaysia Sabah dengan syarat-syarat kegunaan seperti berikut: - "

1. Tesis ini adalah hak milik Universiti Malaysia Sabah 2. Perpustakaan Universiti Malaysia Sabah dibenarkan membuat salinan untuk tujuan pengajian sahaja. 3. Perpustakaan dibenarkan membuat salinan tesis ini sebagai bahan pertukaran antara institusi pengajian tinggi. 4. Sila tandakan (/): üýtºi6r: iýi:: f 11 Hi1ýc11 Uia VIYL, 'ýi ý

SULIT (Mengandungi maklumat yang berdarjah keselamatan atau kepentingan Malaysia seperti yang termaktub di dalam AKTA RAHSIA 1972)

TERHAD (Mengandungi maklumat TERHADyang telah ditentukan oleh organisasi/badan di mana penyelidikan dijalankan

x TIDAK TERHAD

DisahkanOleh,

NURULAINBINTI ISMAIL PUSTAKAWAIWANAN ABAH

ALISA STEPHANIERASION (TandatanganPustakawan) MS1521001T

Tarikh :V October 2018 (Prof. Madya. Dr. How Siew Eng) Penyelia DECLARATION

I declarethat this thesis and the work presentedin it are my own and has been generatedby me as the result of my own original research.

27t'' September2018

Alisa Sianie Rasion MS1521001T

11 CERTIFICATION

NAME ALISA STEPHANIE RASION MATRICNO. MS1521001T TITLE ACTIVATING EFFECT OF AROMATHERAPY INHALATION WITH Citrus hystrix (KAFFIR LIME) LEAVES OIL DEGREE : MASTER OF SCIENCE (INDUSTRIAL CHEMISTRY) VIVA DATE 9THAUGUST 2018

CERTIFIED BY;

SIGNATURE SUPERVISOR

PROF. MADYA.DR. HOW SIEW ENG "nýýö ACKNOWLEDGEMENTS

First and foremost, praisesand thanks to the God,the Almighty, for His showersof blessingsthroughout my research work to complete the research successfully.I would liketo expressmy sinceregratitude to my advisorProf. Madya.Dr. How Siew Eng for the continuoussupport of my master study and research,for her patience, motivation,and immense knowledge. Her guidance helped me in all the time of researchand writing of this thesis and finally I am extremelygrateful to my parents and to my friends for providing me with unfailing support and continuous encouragementthroughout my years of study and through the process of researchingand writing this thesis. This accomplishmentwould not have been possiblewithout them. Thank you.

Alisa Stephanie Rasion 27thSeptember 2018

iv ABSTRACT

Citrus hystrix leaves oil has a scent with unique nuances because it is rich in natural constituent citronellal which possesses soothing properties and has the potential to be used as aromatherapy. However, there have been relatively few published about the C. hystrix leaves extract and its efficacy as aromatherapy. This research was aimed to investigate the effects of C. hystrix leaves oil on human behavioural parameters after inhalation. C. hystrix essential oil was obtained from fresh leaves by steam distillation and the oil yield obtained was 0.14% with pale yellow in colour while the chemical profile of C. hystrix leaves volatile oil was analysed using the gas cromatography-mass spectrometry (GC-MS). It was found that the citronellal is the highest percentage (85.00%) followed by citronellol (4.75%), citronellyl propanoate (2.74%), delta-3-carene (1.15%), geranyl acetate (0.84%), (E)-ß-ocimene (0.69%), linalool (0.42%), terpinolene (0.40%), myrcene (0.35%), camphene (0.25%), ß- pinene (0.24%), and a-Phellandrene is the lowest percentage (0.05%). 3-(4,5- dimethylthiazol- 2yl)-2,5-Diphenyl tetrazolium bromide (MTT) test was used to measure cytotoxicity of the oil and from this research it was found that the essential oil of C. hystrix (leaves) showed less toxic to the normal human liver cells (WRL 68) with the highest value of ICso (40.23±14.86 ug/mL) where it required a higher concentration of the C. hystrix (leaves) oil to inhibit the activity of the cells by 50% while the low dose concentration of paclitaxel reduced the percentage viable cells (0.0016±0.0003 ug/mL). There were 40 respondents participated this survey which were randomly assigned into two groups, control group (treated with virgin coconut oil (VCO) since this oil has no scent and it is typically used as a carrier oil) and the C. hystrix oil group and each group consists of 20 respondents. Visual analog scale (VAS) was used for this survey for the subjects to rate their emotional condition in order to assess subjective behavioural arousal in terms of relaxation, vigour, calmness, attentiveness, mood and alertness after inhalation. Data obtained in this study were analyzed using Mann-Whitney-U-Test, Wilcoxon Signed Rank Test, and Spearman's Rho Correlation. Based on the Mann-whitney U Test result, it showed hystrix that C. oil gave arousal effects in relaxation, vigorous and attentiveness and this data was supported by Wilcoxon signed rank test data for beginning and ending time data where the of C. hystrix oil category were significantly different in score of effects at the beginning and ending observation data for relaxation, vigorous and attentiveness rated by respondents. From this finding, C. hystrix oil gave arousal effects in relaxation, vigorous and attentiveness and these were supported by the Spearman's Rho Correlation test where relaxation had positive medium correlation with variable vigorous, r=0.47, n= 20, p<0.05, and variable vigorous had positive strong correlation with variable attentiveness, r=0.74, n= 20, p<0.05. The C. hystrix leaves has been oil experimentally shown to have a positive effect on relaxation, vigorous and attentiveness than subjects in the control group and these results provide evidence for its use in aromatherapy for the relief of depression and uplifting mood in humans and this might be due to higher content of citronellal in C. hystrix leaves oil and it is safe to be used.

V melegakankemurungan dan menaikkanmood manusiadan ini mungkin disebabkan o%h kandungancitronellal yang tinggi di dalam daun minyakpati C. hystrix dan is selamat digunakan.

vii TABLE OF CONTENTS

Page

TITLE i DECLARATION 11 CERTIFICATION III ACKNOWLEDGEMENTS iv ABSTRACT v ASSRRAK VI TABLE OF CONTENTS viii LIST OF TABLES X LIST OF FIGURES XII LIST OF ABBREVIATIONS/ SYMBOLS XIII LIST OF APPENDICES xv

CHAPTER 1: INTRODUCTION 1 1.1 Introduction 1 1.2 Significanceof the Study 4 1.3 Scopeof the Study 5 1.4 ResearchObjectives 5

CHAPTER 2: LITERATURE REVIEWS 6 2.1 Aromatherapy 6 2.2 EssentialOil 8 2.3 Compositionof EssentialOil 11 2.4 Actions of Essential Oil On the Body 12 2.5 Aroma-therapeuticEffect of EssentialOil on HumanBehavioural and 14 EmotionalChanges 2.6 OlfactionPhysiology 15 2.7 PsychologicalEvaluation of FragranceEffects Using a Visual Analog 17 Scale(VAS)

viii 2.8 Genus Citrus and Its Therapeutic Benefits 18 2.9 Kaffir Lime 21 2.10 Steam Distillation 26 2.11 Citronellal 27 2.11.1 Source of Citronellal 28 2.11.2 Biological Benefits of Citronellal 29 2.11.3 Toxicity of Citronellal 33 2.11.4 Method to Analyse the Toxicity of Citronellal 35

CHAPTER 3: RESEARCH METHODOLOGY 37 3.1 Sample Preparation 37 3.2 Extraction Method 38 3.3 Analysis of Volatile Compound 39 3.4 Toxicity Test 40 3.5 Subjects 42 3.6 Data Analysis 43

CHAPTER 4: RESULT AND DISCUSSION 45 4.1 Chemical Component of C. hystrix Volatile Oil 45 4.2 Toxicity of C. hystrix Oil 53 4.3 The Stimulating Effect of C. hystrix(Leaves) Oil Aroma on Human 54 4.3.1 Comparisonof Controland Treatment Relatedto Six Variables 55 4.3.2 ComparisonBeginning and Ending 56 4.3.3 RelationshipAmong Variables 59

CHAPTER 5: CONCLUSION 63 5.1 Conclusion 63 5.2 Future Research 64

REFERENCES 65

APPENDICES 81

ix LIST OF TABLES

Page

Table 2.2 Percentagecomposition of leaves 22 oils of Kaffirlime

Table3.1 GC-MSconditions for extractedoil 39 analysis

Table3.2 Definitionof relaxation,vigorous, 43 calmness,attentiveness, mood and alertness

Table4.1 Chemicalcomposition of C.hystrix 45 leavesoil usingGC-MS

Table 4.2 Comparisonof chemical 46 compositionof Citrus hystrix leaves oil betweenthe present data and previousdata of the existing literature (%)

Table4.3 In vitro cytotoxicityof essentialoil 54 of C. hystrix(leaves)

Table4.4 Comparisonof mean rank and sum 55 of rank betweencontrol and C. hystrix leavesoil categoryfor the effects of relaxation,vigorous, calmness,attentiveness, mood and alertness

X Table4.5 Rating mean (±standard deviation) 57 and comparison of rating at the beginning and ending observation time of six variables rated by 20 respondents for the effects of Control

Table 4.6 Ratingmean (±standard deviation) 57 and comparisonof rating at the beginningand ending observation time of six variablesrated by 20 respondentsfor the effects of Citrus hytrix leavesoil

Table 4.7 Spearman's Rho Correlation 60 between six variables for the effects of Citrus hytrix leaves oil

XI LIST OF FIGURES

Page

Figure2.1 The flow of air inhalation 13 into the nostrils

Figure 2.2 Structure of the 27 Citronellal

Figure 2.3 Structure of the 29 Citronellol,Citronellal and Hydroxycitronellal

Figure 3.1 Kaffir lime leaves 37

Figure 3.2 Steam distillation (40L) 38

Figure 4.1 The chromatogram of C. 49 hystrix oil components

Figure4.2 The chromatogramof 50 standard citronella)

Figure4.3 The chromatogramof C. 51 hystrix oil confirmedwith the citronellal

XII LIST OF ABBREVIATIONS/ SYMBOLS

Rpm Revolutions per minute

9 Gram

Kg Kilogram

L Liter mL Mililiter

Nm Micrometer mg Miligram m Meter mm Milimeter eV Electronvolt

N9 Microgram

NL Microlitre

% Percentage

oc DegreeCelsius

CO2 Carbondioxide

XIII nm Nanometre

WRL68 The human hepatic cell line

DMEM Dulbecco'sModified EagleMedium

PSG Penicillin-Streptomycin-Glutamine

FBS Fetal bovine serum

NMDA N-methyl-D-aspartate (receptorthat is used to controlthe synaptic plasticityand memory function)

ICso Inhibitory concentration by 50%

L-NAM E N(uo)-nitro-L-arginine methyl ester (induced hypertension)

Ca2+ Calciumions

f Standarddeviation

> Greaterthan or equal to

Lessthan

> Greaterthan

XIV LIST OF APPENDICES

Page

Appendix A The mass spectrum of each of the 81 identified compounds in C. hystrix leavesoil

Appendix B Library of GCMS 88

Appendix C GCMS(C, hystrixoil components) 90

Appendix D GCMS(C. hystrixoil confirmed 91 with the citronellal)

AppendixE VisualAnalog Scalefor the effect 92 of aromatherapyassessment

AppendixF Rating(Meant SD) for the 99 stimulatingeffect of controland C. hystrix aroma at the beginning and ending observationtime

AppendixG Researchpermission letter 111

AppendixH Consentform 113

xv CHAPTER 1

INTRODUCTION

1.1 Introduction

Aromatherapy is defined by Sense of Smell Institute (SSI) as the therapeutic effects of aromas on physical conditions such as digestive problems and menstrual disorders as well as psychological conditions such as chronic depression (Balchin, 2006). In a modern aromatherapy, both essential oils and their aromas are used for balancing, harmonizing and promoting the health of body, mind and spirit where the effects of an aroma can be instantaneous including both direct and indirect psychological effects even thinking about a smell may have a similar effect to the smell itself. However, some evidences showed that inhaled or dermal applied essential oils enter the blood stream and in relevant molecular, cellular or animal models, exert measurable psychological effects, indicates that the effects are primarily cabenefits.

Extraction obtained from mostly basic plant parts such as stems, seeds, flowers, resins, leaves,barks, roots, or fruit rinds producedessential oils which are highly concentratedsubstances (Rao and Pandey,2006). Essentialoils flavour and their therapeutic or odoriferous properties often used in foods, medicines, and cosmetics.There are three ways essentialoils canbe absorbedincluding through the olfactory and respiratorysystems (vapour inhalation),transdermally through lotions or compressesinvolving massage and during bathing,or orally, through consumption of essentialoils in capsulesor as additives to food or medicinal preparations,for examplebut it is more to the herbal medicinethan aromatherapy(Perry, and Perry, 2006). Citrusessential oil is the good source of aromatherapyand it was the most popular source of perfume and fragrance essencesbecause of its good sour and sweet tastes, pleasant and refreshing aroma, as well as extensive growing areas worldwide.

1 There are several plants of Citrus rich in essential oil often used in the anxiety treatment and among these plants are orange, Citrus sinensis (Faturi et al., 2010), Citrus aurantium, bergamot (Carvalho-Freitas and Costa, 2002; Pultrini etat., 2006), Citrus Amon, lemon (Ceccarellia et al., 2004) and these essential oils are often used depression as aromatherapy agents for relieving and for reducing stress and which have been used topically for their antibiotic and antiviral properties for over the years (Setzer, 2009; Cooke and Ernst, 2000). Since the genus Citrus essential oil showed positive emotional effects in studies with humans and demonstrated anxiolytic as well as sedative effects in experimental models of anxiety and hence, it could be a great be alternative (Pimenta et al., 2012). Hence, C. hystrix(Kaffir lime) leaves will used leaves for this research which is from the same genus Citrus and the C. hystrix that have a strong fragrant lemon scented. It was previously reported that heart disease, dizziness and indigestion, as well as physical nourishment, could be treated by C. hystrix which used as herbal medicine (Abdul, 2013). C. hystrix leaves have antimicrobial properties and commonly been used for cooking and its essential oil or oleoresin for aromatherapy (Sid et al., 2016).

C. hystrix is an indigenous plant in Southeast Asia including Malaysia, Indonesia and Thailand. C. hystrix from Rutaceae family has it's own aroma and commonly used as spices in cooking and medicinal herbs. C. hystrix has a bumpy fruit with a rough skin while the leaves are an aromatic herb which used broadly in adding a distinctive aroma and flavour to food. There are two types of essential oil made from C. hystrix, one from the peel and the other from leaves. The C. hystrix leaves contain oleoresin such as citronellal which give it aroma. The major component in the C. hystrixleaves is citronellal (Kawiji, eta/., 2015). Basically,the higher content of geraniol, citronellal hydrate, citronellal, linalool, linalyl acetate, the more delicate its aroma (Kawiji et al., 2015). The major component of the ethyl acetate extract from leaves of C. hystrixis citronellal (73.08%) and that from hydrodistillation is also citronellal (100%) while the major components of the hydrodistillated essential oils from C. hystrix peels are limonene (16.85%) and cironellal (3.57%) whereas citronellal (2.80%), limonene (0.31%), and pinene (0.13%) were major components of essential oil of the ethyl acetate extract (Megawati, 2009). Exposure to C. hystrix

2 aroma producedthe effect of reducing"disguise iritation" which is evaluatedbased on cognitive evaluation and it indicates that exposure to the C. hystrix aroma producesa positive emotive responsewhich may impact clarity of mind and health (Megawati, 2009). C. hystrix could be a great alternative and has the potential in researchand commercializationfor the solution in insect repellent, aromatherapy, and spa practices, making shampoo, beauty product as well as antioxidants compound(Nurhani etal., 2013).

Since Citrus hystrix leaves oil has a scent with unique nuances because it is rich in natural constituent citronella) which possesses soothing properties and has the potential to be used as aromatherapy. Steam distillation was used in this research because steam distillation produces more citronellal from the leaves than the other methods where previous results reported that citronellal was the major component in C. hystrix leaves oil with the highest percentage (80.04%) (Srisukh et al., 2012) compared to other techniques such as solvent extraction, hydrodistillation, supercritical fluid extraction, pressurised liquid extraction (PLE), and maceration extraction where the results report that the percentage of citronellal is 74.8%, 66.85%, 1.41%, 25.87%, and 25.66% respectively (Tinjan and Jirapakkul, 2007; Loh etal., 2013; Haiyee and Winitkitcharoen, 2012; Kawiji etal., 2015).

Citronellal gives therapeutic benefits including antiviral, antimicrobial, tonic, vasodilators, a hypotensive, calming, antipyretic, sedative, and spasmolytic (Bouayed and Bohn, 2012) and at the various activity levels, C. hystrix leaves oil has excellent activity against many respiratory bacteria (Srisukh etal., 2012). Thus, it can be used as air purifying to clean the air naturally using the air purifier. Steam distillation is used to extract essential oil for this research because it is economy for small and medium scale industry, cheap for a basic level process, as well as there is no changing properties of oils produced by this method. The oil never spoil in this method since the steam reduces the boiling point of a particular component of the oil and this method also faster than other methods (Kumar, 2010). Steam distillation is the oldest

3 method and simplest method to produce essential oil using leafy materials for commercial purpose.

Before the essential oil of C. hystrix can be tested on humans, toxicity test should be conducted first. Safety tests begin with acute toxicity testing where a single dose of the test compound is given to animals and this test is focusing on determining the range between the two possible doses which is no adverse effect and the dose that is life-threatening. Toxicity divided into two types which are acute or chronic and these types are based on the number of exposures to a poison as well as the time it takes for toxic symptoms to develop. Short-term exposure with a high dose causes acute toxicity while the repeated or long-term exposure with low doses causes chronic exposure. MTT test is commonly used for cytotoxicity testing because of their rapid, standardized and sensitive in determining if a material contains significant quantities of hazard components. The MTT (3- [4,5-di methyl th iazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay determines the cells activity where it is based on the conversion of MTT into formazan crystals by living cells (Meerloo et al., 2011). MTT test can be extrapolated the results to toxicity to mammals since it uses tissue culture from cell (Tunjung et a/., 2015). MTT assay is more suitable technique for the application to toxicity test for human and because for this reason MTT test is used for this research to determine the C. hystrix oil toxicity.

1.2 Significance of the Study Hecticdaily tasks of modern life can give a stressful life and people nowadaystend to take drugsof pain killer to stop the pain or the uncomfortablesymptom that linked to depression,anxiety and stress but drugs are toxic to the body and have the side effects compareto the natural moleculessuch as those found in essentialoils. An essentialoil is multi-dimensionaland filled with homeostaticintelligence where it has the capability in restoring the body to healthy balanceand if there are changesin body conditions, the oil will adapting, increasing or reducing blood pressure as needed,stimulating or suppressingenzyme activity as needed,energizing or relaxing

4 as needed (Stewart, 2005). Treatment with essential oils is safer and fewer adverse effects.

1.3 Scope of the Study There have been relatively few published about the C. hystrix leaves oil and its efficacy as aromatherapy. Therefore, the present study of C. hystrix leaves oil is to investigate the potential of inhalation with C. hystrix leaves oil as aromatherapy on healthy human emotional responses where the scope of this research is to study the positive effect of C. hystrix leaves oil on stimulation of human relaxation, vigour, calmness, attentiveness, mood and alertness which linked to individual performance. Stimulating oil gives expected effect on mind and body. The benefits of this research for users are to improve the emotional balance and to improve overall attitude which increases the life quality and health. Users can effectively perform their tasks and make a maximum efficiency.

Research Objectives Objectives of this research are: a) To determine the chemical components of C. hystrix oil using Gas cromatography-mass spectrometry. b) To evaluatethe toxicity of C. hystrix oil. c) To evaluatethe stimulatingeffect of C.hystrix aromaon humanssuch as relaxation,vigour, calmness,attentiveness, mood and alertness.

5 CHAPTER 2

LITERATURE REVIEW

2.1 Aromatherapy Aromatherapy is practicing the use of natural oils and which also referring to essential oils therapy. In aromatherapy, the main therapeutic agents are essential oils which are highly concentrated substances extracted from plants and these oils are good to apply in the treatment of mental and physical balance which give stress relief, revive and revitalize the individual for a next day's duty (Ali et al., 2015). Aromatherapy is the holistic practice of preventative approach and as an active technique to use during the stages of acute and chronic illness. The use of aromatic plants has been around since Neolithic times and these aromatic plants have been used for beauty treatment, perfuming, medicinal practices, cleansing, and ritual bathing as well as religious ceremonies (Stiles, 2017).

In fragrance psychology field, there is a various number of group for study including perfumes (e. g. colognes, fine fragrances,), cosmetic and toiletry products with scent (e. g. hair-care, fragranced skin-care, and hand care products), environmental fragrances (e. g. room fresheners, potpourri) as well as aromatherapy products (essential oils) (Graham, 2000). Fragrance psychology is the perception of individual odour where these odours are likely to affect a various field of human psychological functioning, ranging from perception and mood to cognitive processes and attitude as well as the changes in humans induced by smelling the fragrances of essential oils (Lenochova et at., 2012; Sugawara et at., 2013). The psychology includes the product behavioural effects on the user including others perceptions (personality, the appearance, and projected image) self-perception (moods, self- image, confidence, self-esteem, and attitudes), interpersonal attraction (sexual attraction and liking) and in psychology field also includes motivations or reasons for using or purchasing a product which relating scent to differentiate situation and

6 lifestyles and to link individual fragrances with particular personality aspect and finally it access therapeutic effects including mental and physical particularly to relevant aromatherapy products (Graham, 2000).

Application of single or combined essential oils including diffusing the oils into inhaled air, using in massage oil or adding to bathwater. The inhalation of these essential oils molecules triggers the olfactory nerve which sending the messages to the brain's limbic system including the cognitive, memory, and emotion that are said to stimulate the physiological responses for examples lavender promotes relaxation and eucalyptus relieves congestion. The utilization of extracted aromatic plant essence gives the aromatherapy benefits where the treatment outcome could be achieved by conveying the odours to the central nervous system via the skin and olfactory system. The odours are dispersed in the blood and circulate to different organs which can prevent and treat the central nervous system diseases and for this reason, this application makes the aromatherapy as compatible therapies where the essential oils as the major therapeutic agents can treat several diseases (Lee et al., 2011).

The major methods used in aromatherapy that use the essential oils are including inhalation, local application, and baths. These oils penetrate the human skin surface and once the oils are in the system, they regulate themselves and fix the affected area or malfunction site and this type of therapy uses various permutation and combinations to get relief from numerous illness such as depression, insomnia, headache, indigestion, muscular pain, skin ailments, respiratory problems, urine associated complications, swollen joints and so on (Ali et al., 2015). Aromatherapy can be classified as massage aromatherapy, olfactory aromatherapy cosmetic aromatherapy, medical aromatherapy, and psycho-aromatherapy.

7 2.2 Essential Oil Essential oils can be defined as volatile substances that are more or less volatile with distillation dry distillation more or less odorous impact, which obtained by steam or but have or by several mechanical treatments. Essential oils are colourless a pleasant smelling liquids with high refractive index. The frequency of essential oils ranges are several times greater than the frequency of herbs and food and thus when apply the the essential oils, the body's overall frequency increase quickly and stabilize form that struggling organ frequency. Since essential oil is a very concentrated possesses nutritive values and healing properties, and thus the use of this oil can improve overall immune function by healing the body, mind, and spirit, while reviving the damaged tissue and bringing oxygen to deprived cells. (Westover, 2012). Essential oils have the ability to bring the body back to normal ranges within seconds and improve overall feelings of well-being (Westover, 2012).

Essential oils are commonly used for scenting purposes including food flavourings and cosmetic fragrances but since aromatherapy is getting popular in recent years, it also leads to the increase uses of essential oils in aromatherapy. (Japan external trade organization, 2011). The properties of essential oil such as antibacterial, anti-infective, antifungal, antiparasitic, anti-tumoral, antiviral, and antiseptic resulted from its unique oxygen or nutrient carrying capacity of essential oil and this oil is highly concentrated, low volume and high value products (Joy etal., 2001). In laboratory studies and clinical trials, essential oils have been shown to destroy and to inhibit tested bacteria and viruses without causing harm (unlike drugs) to cells, organs, tissues, or body (Westover, 2012). Most of the medications (drugs) have potential side effects than the essential oils, for example, morphine which is the prototype of opioid analgesics can exert side effects including sleepiness, respiratory depression, motility, endocrine and autonomic nervous systems disorders, and decreased gastrointestinal nausea (Sarmento-Neto eta/., 2015) while treatment for superficial and deep infections caused by Trichophyton using Ketoconazole which used as antifungal drugs give unpleasant side-effects including abdominal pain and itching, nausea, and the toxicity of these drug limit its therapeutic use in many cases (Shin and Lim, 2004). Rituximab used for the treatment of low-grade or follicular

8 CD20-positive, Hepatitis B and treatment of rheumatoid arthritis (RA) in combination fatal in with methotrexate. However there is potentially complication a patient who blood lymphatic recently received rituximab such as caused allergic reaction, and system disorders, infectious complications, renal disorders, cardiac disorders, adverse pulmonary reactions, neoplastic disorders, gastrointestinal disorders, neurological disorders and developed itching, rash, or urticarial (Kasi eta/., 2012: 4). The agent to treat the primary posttraumatic stress disorder (PTSD) is Benzodiazepines but however it is dangerous in over-dosage and often abused (Bernardy, 2013). Benzodiazepines gave poor sleep overall which leads to increase the risk of fractures and falls, accidents, and give adverse cognitive effects to the elderly where a cognitive "slowness" can be difficult to vary from cognitive reduction (Lader, 2011) as well as causing mild traumatic brain injury (Department of Veterans Affairs, 2010).

Unlike drugs, the long-term effects of essential oil use are considered mild. Essential oil use is non-addictive and proven to provide desirable results (Emerald, 2016). Essential oils, such as Lavender (Lavandula angustifolia), Bergamot (Citrus bergamia) and Ylang Ylang (Cananga odorata), could be implemented into aromatherapy, holistic and massage practice for the potential management of PTSD where the daily use of Lavender oil in aromatherapy with PTSD patients help to decrease depression by 32.7% and to decrease sleep disturbances dramatically, moodiness and overall ill health in 47 PTSDpatients (Uehleke etas., 2012). In multiple studies, Lavender essential oil has anti-anxiety effects caused by modulation of two neurotransmitters, 5-hydroxytryptamine and dopamine (Umezu, 2010). Administration of the Bergamot oil in concentrations of 1.0,2.5, and 5.0% w/w demonstrates a strong anxiolytic effect measured through different tests, and its effects on stress-induced plasma corticosterone has been compared to the effects of diazepam (Saiyudthong and Marsden, 2010). Testing of the Bergamot essential oil effects in experimental settings using different routes of administration shows very promising results for the potential management of a variety of pathological conditions including bacterial and fungal infestation, cardiovalcular conditions and cancer. The main effect of Bergamot essential oil as a neuroprotective in cancer was explained

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