CHARACTERIZATION, INSECTICIDAL AND MOSQUITO REPELLENT PROPERTIES OF ESSENTIAL OILS FROM HYPTIS SUAVEOLENS LEAVES

ABSTRACT

Essential oils were extracted from the leaves of Hyptis suaveolensby hydro-distillation,Mosquitocidal effect was done through an experiment devised for the purpose of this research. The effect of the essential oils against mosquito larvae (larvicidal), mosquito repellency effectand physicochemical properties were determined using standard methods. Gas Chromatography-Mass Spectrometry (GCMS) and Fourier Transform Infra-Red (FTIR) were both done on the essential oils. Percentage yield of the essential oils was gotten as 0.05, for the larvicidal activity dose dependent mortality of the larvae was observed; there was low mortality rate at lower dosage and not significantly different from each other e.g. 6.25 and 12.50ppm had same LC₅₀ and LC₉₀ of 25.21 and 302.67 respectively, 25ppm had LC₅₀ and LC₉₀ of 9.58 and 352.46 respectively, while 50-1000ppm had LC₅₀ and LC₉₀ of 0.55 and 0.85 respectively. It was observed that the amount of air that enters and leaves the cage affect the rate at which mosquitoes were repelled. In a group of mosquitoes kept in a cage with all sides open (ASO) 65% of the mosquitoes were repelled within 30 minutes while for one side open (OSO) and all sides closed (ASC), 73% and 85% of the mosquitoes were repelled within the same time frame. In a second experiment, 100% of mosquitoes were repelled from the surface of rats with shaven skin where essential oil was applied. The essential oil also demonstrated dose dependent mosquitocidal activity with LC₅₀ and LC₉₀ values of 6 and 21ppm respectively. The essential oil has the following physicochemical properties; Iodine value 23.59+0.12g/100g, saponification 100.18+0.8mgKOH/g, Peroxide value 40.00+0.02meq/kg, Acid value 3.37+0.01mgKOH/g, Ester value 34.30+1.00mg/g and free fatty acids 0.15+0.57%. GC-MS analysis revealed Terpenes to be the major organic compound present in the essential oil which was confirmed by FTIR with the O=C-O-C stretch functional group indicating the presence of terpenes. Thus, the presences of terpenes in    Essential    oil    of   Hyptis    suaveolens    may    have    contributed   to    its    insecticidal    and mosquitocidal repellency properties.

TABLE OF CONTENTS

Title page

ABSTRACT

TABLE OF CONTENTS

List of Abbreviations

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the study

1.2 Statement of the Problem

1.3 Justification

1.4 Aim and Objectives

1.4.1 Aim

1.4.2Specific objectives

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Plants

2.1.1Mechanisms employed by plants to repel mosquitoes (Insects)

2.1.2 Advantages of plants origin (non-synthetic) insecticides

2.1.3 Role of medicinal plants in the treatment of malaria

2.2 Mosquito: Causative vector for Malaria

2.2.1Life cycle of mosquito

2.3 Malaria: Disease caused by Mosquito bite

2.3.1 The malaria parasite

2.3.2 Requirement for the prevention of malaria

2.4 Synthetic Insecticides

2.4.1 Types of synthetic insecticides

2.4.2 Side effects of synthetic insecticides

2.4.3 Toxicity of synthetic insecticides

2.4.4 Remedy

2.5Larvicides

2.6 Biological (Non-synthetic) Insecticides

2.6.1 Hyptis suaveolens (Labiatae Poits)

2.6.1.1 Composition ofHyptis suaveolens

2.6.1.2 Uses ofHyptis suaveolens

2.7Essential Oils

2.8 Monoterpenes

2.9 Sesquiterpenes

2.10 Repellents

2.11 Mechanisms employed by insects against repellents (essential oil components

2.12 Gas Chromatography-Mass Spectroscopy (GC-MS)

2.13 Fourier Transform Infra-Red (FTIR)

CHAPTER THREE

3.0 MATERIAL AND METHODS

3.1 Materials

3.1.1Plant material

3.1.2 Other materials

3.1.3 Chemicals and reagents

3.1.4 Equipment

3.2 Methods

3.2.1 Plant material collection and identification

3.2.2 Extraction of essential oil from Hyptis suaveolens leaves

3.2.3 Mosquito Breeding

3.2.4 Determination of mosquitocidal capability of the essential oil

3.2.5 Determination of mosquito repellency effect of essential oil from Hyptis suaveolens leaves. Experiment one (1)

3.2.6 Determination of mosquito repellency activity of essential oils of Hyptis suaveolens leaves on Rats. Experiment two (2)

3.2.7 Test for larvicidal effect of the essential oil (WHO, 2005) guideline

3.2.8 Characterization of essential oil

3.2.8.1 Gas Chromatography-Mass Spectroscopy (GC-MS)

3.2.8.2 Fourier Transform Infra-Red Spectroscopy (FTIR) Spectroscopic Analysis

3.2.8.3 Determination of Physicochemical Properties

i. Determination of Iodine value

ii. Determination of Saponification value

iii. Determination of Acid value

iv. Determination of Peroxide value

v. Determination of Ester value

vi. Determination of Free Fatty Acid value

3.2.8.4 Statistical analysis

CHAPTER FOUR

4.0 RESULTS

4.1 Yield of essential oils from Hyptis suaveolens leaves

4.2 Mosquitocidal capability of Essential oil from Hyptis suaveolens leaves

4.3 Repellency Effect of Essential oil from Hyptis suaveolens leaves

4.3.1Mosquito repellent potential of Hyptis suaveolens essential oil. Experiment one

4.3.2 Mosquito repellent potential of Hyptis suaveolens Essential oil. Experiment two

4.4Larvicidal effect of essential oil from Hyptis suaveolens leaves

4.5Physicochemical properties of Essential oil from Hyptis suaveolens leaves

i.Iodine value

ii.         Saponification value

iii.  Acid value

iv.  Peroxide value

v.         Free Fatty Acids value

vi.  Ester value

4.6Gas Chromatography- Mass Spectroscopy (GC-MS)

4.6.1 Characterization of the Essential oils from Hyptis suaveolens by GC-MS

4.7Fourier Transform Infra-Red Spectroscopy (FTIR)

4.7.1 Characterization of the Larvicidal Compound(s) in the bioactive Fraction by (FTIR)

CHAPTER FIVE

5.0 DISCUSSION

CHAPTER SIX

6.0 CONCLUSION, SUMMARY AND RECOMMENDATIONS

6.1Conclusion and Summary

6.2Recommendations

REFERENCES

List of Abbreviations

Acronym                            Full meaning

ACT                                      Artemisinin derivative- based combination therapy

ACTs                                   Artemisinin-based combination treatments

AMA                                   American Medical Association

Amu                                     Atomic mass unit

ASC                                     All sides closed

ASO                                     All sides open

DDT                                     Dichloro diphenyl trichoroethane

DEET                                  Diethyl-meta-toluamide

EM                                        Electromagnetic

EO                                        Essential oil

EPA                                     Environmental Protection Agency

FTIR                                    Fourier transform infra-red

GC-MS                               Gas Chromatography-Mass Spectroscopy

IR                                          Infrared

IUGR                                  Intrauterine growth retardation

LC                                         Lethal concentration

L. poits                               Labiatae points

OSO                                     One side open

Ppm                                      Part per million

RDT                                     Rapid diagnostic test

WHO                                   Who health organization

AchE                                   Acetylcholinesterase

CHAPTER ONE

1.0  INTRODUCTION

1.1  Backgroundto the study

Plants have always served as food and medicine to man since the beginning of life. Their nutritional and medicinal potentials have been attributed to the phytochemicals and other chemical constituents contained in them. Despite their importance, it has been reported that out of the 250,000 to 500,000 species of existing plants on earth, only about 300 species are utilized in the food, pharmaceutical, cosmetics and perfume industries. Traditionally used medicinal plants produce a variety of compounds of known therapeutic properties (Umedum et al., 2014).

Medicinal plants are used in traditional treatments to cure variety of diseases. In the last few decades there has been an exponential growth in the field of herbal medicine. Natural products have been a source of drugs for centuries (Dinet al., 2011). Traditional medicines (plants source) has been used for thousands of years for the treatment of malaria and are the source of two main groups (artemisinin and quinine derivatives) of modern antimalarial drugs (Kazembe et al., 2012).

Repellents are substances applied to the skin, which prevent insects from biting such surface (Traoré-Coulibalyet al., 2013).An insect repellent is a substance that causes an organism to move away from the odour source, insects perceive thevolatile repellents by smell (Luts et al., 2014).

Essential oils are volatile natural complex secondarymetabolites characterized by a strong odour and have agenerally lower density than that of water (Arun et al., 2009). They are natural volatile mixtures of hydrocarbons with a diversity of functional groups, and their repellent activity has been linked to the presence of monoterpenes and sesquiterpenes (Moreiraet al., 2010;Chaubey 2012; Traoré-Coulibalyet al., 2013). Essential oils are plant products obtained by hydro-distillation or other methods (Luts et al., 2014). This complex of compounds are produced by plants, giving them their characteristic smell and taste, and are usually composed of 20–80 or more substances. Their main components are monoterpenes (C10) and sesquiterpenes (C15) derived from isoprene (Luts et al., 2014).Several monoterpenes have been reported as insect repellents (Luts et al., 2014).There are 17,500 aromaticplant species among higher plants andapproximately 3,000 essential oils are known out of which300 are commercially important for pharmaceuticals,cosmetics and perfume industries. Apart from insecticidal potential they are lipophilic in nature and interfere with basic metabolic, biochemical, physiological and behavioural functions of insects. They are also used as flavour in food products, odorants in flagrances, pharmaceuticals (antimicrobial) and as insecticides (Moreiraet al., 2010).

Hyptis suaveolens (L.Poit) is one of the important traditional medicinal plants belonging to family lamiaceae (Umedum et al., 2014). It is commonly called Bush mint, Bush tea, Pignut, or Chan. It is known generally as Vilayati Tulsi in Hindi, Konda Thulasi in Telugu, Bhustrena in Sanskrit, Daddoya-ta-daji ( family name) and specifically Sarakuwan sauro in Hausa, Efiri (family name) in Yoruba, Nchuanwu (family name) in Ibo, and Tanmotswangi-eba in Nupe (Umedum et al., 2014; Ghafari et al., 2014).

Leaves of Hyptis suaveolens have been traditionally used as a stimulant, antisplasmoidic, against colds and diarrhoea (Shaikat et al., 2012). It has been and still in use traditionally to repel mosquitoes by burning the leaves (Singh et al., 2011; Vongsombothet al., 2012).

The repellent activity of the whole Hyptis suaveolens essential oil and single major constituents against adults of S. granarius has been evaluated (Benelli et al., 2012). Amongst the several methods available to evaluate the repellence of natural products, the filter paper tests in Petri dish is one of the most commonly used bioassays (Benelli et al., 2012). Several authors reported a large variability in the composition of this family due to genetic, geographical and seasonal factors. Since the biological activities of essential oils are composition-dependent, it is apparent that it is very important to fully characterize these mixtures from the chemical point of view (Benelli et al., 2012).

Malaria is a major public health problem with an estimated two million children worldwide dying of it yearly. Regardless of the fact that it is one of the oldest recorded diseases, malaria remains one of the world‘s most deadly infectious diseases. It is arguably, the greatest menace to modern society in terms of morbidity and mortality. Though preventable, treatable and curable, there is no known vaccine against the disease. This makes it an efficient and unrepentant killer. Several centuries after its discovery, malaria still remains a devastating human infection, resulting in 300-500 million clinical cases and three million deaths every year. Malaria is endemic throughout Nigeria (Olurishe et al., 2007; Nigeria Malaria Indicator Survey 2010; Okafor and Oko-Ose 2012; Etusim et al., 2013; Odey et al., 2013).

The management of severe malaria remains challenging, mainly due to the fact that it does not only depend on the use of effective anti-malaria drugs but also the use of effective parenteral anti-malaria drugs. Other factors that make management of malaria challenging are; cost-intensive supportive measures, unavailability of highly skilled personnel, insufficient functional referral systems, lack of blood transfusion services, lack of good infrastructure and inadequate organization of hospital services. Parenteral quinine had been the first-line treatment for severe malaria in Nigeria until June 2011 when the policy was revised to intravenous artesunate as a first-line antimalarial drug (Odey et al., 2013).

Malaria caused byPlasmodium falciparum is the most dangerous form of the disease resulting in life threatening complications such as anaemia and cerebral malaria. The pattern of exposure to malaria infection, the type of treatment and the degree of compliance with the anti-malaria regimen, local drug resistance patterns, individual‘s age and genetic makeup all tend to influence the severity of the disease (Etusim et al., 2013).

A synthetic insecticide is a poisonous chemical or mixture of chemicals that is intended to prevent, repel, or kill any insect or pest. However, synthetic insecticides present hazardous impacts far beyond their intended targets. Insecticides have inherent toxicity because they are designed to kill living organisms that are considered "pests", that is any unwanted insect. Many insecticides are known to pose significant, acknowledged health risks to people— including birth defects, damage to the nervous system, disruption of hormones and endocrine systems, respiratory disorders, skin and eye irritations and various types of cancers (Agency for Toxic Substances and Diseases Registry, 1994).

The synthetic insecticide Dichloro diphenyl trichoroethane (DDT) was widely used in urban aerial sprays to control urban mosquito and other insects. DDT has caused chronic effects on the nervous system, liver, kidneys, immune systems,tremors, increased blood levels of liver-produced enzymes, changes in the cellular chemistry in the central nervous system, hind leg paralysis, convulsions and subtle effects on neurological development and decreased in thyroid function in experimental animals.Teratogenic, mutagenic, carcinogenic effects and sterility were also observed in experimental animals(Agency for Toxic Substances and Diseases Registry, 1994).

Chemical repellents are important in protecting people from blood-feeding insects and may therefore also reduce transmission of arthropod borne diseases. N,N-diethyl-3-methylbenzamide (DEET) is one of the most well-known arthropod repellents. DEET is generally safe for topical use if applied as recommended, although adverse effects such as serious neurologic effects have been reported. It does not readily degrade by hydrolysis at environmental pHs and has been identified as aubiquitous pollutant in aquatic ecosystems (Abagli and Alavo 2011).

1.2  Statement of the Problem

^Ø  Paré Toéet al.,(2009) have shown that people‘s motivation to use mosquito nets considerably decreased less than a year after the campaigns and people prefer to spend night without insecticidal nets. Therefore, use of mosquito net is not sufficient to effectively control malaria vectors, especially in the West African countries where urbanization promotes the proliferation of mosquitoes.

^Ø  To achieve successful vectors control and reduce substantially the prevalence of malaria and other vector-borne diseases, an integrated management of these vectors must be adopted.

^Ø  Insecticides have inherent toxicity and are known to pose significant health risks to people.

^Ø  Globally, the malaria situation is serious and still deteriorating, hence the need to search for solution.

^Ø  Malaria predominantly affects the poor and underprivileged hence the need to search for affordable means of eliminating the disease.

1.3  Justification

^Ø  Traditionally,Hyptis suaveolens has been used and is still in use to repel mosquito but the scientific basis behind this practice has not been validated.

^Ø  Mosquitoes have become the most important single group of insects well-known for their public health importance (Singhet al., 2011).

^Ø  Considering the use of the plant to repel mosquitoes, it is worthwhile exploring the repellency properties of the plant essential oil and larvicidal property for a possible mosquito control strategy (Singh et al., 2011).

^Ø  On the possible eradication of malaria, Arigbabuwo opined that prevention is better than cure (Okafor and Oko-Ose 2012). Hence the search for possible eradication of mosquito right at the larvae stage.

^Ø  To the best of my knowledge, there are no sufficient data available on the physicochemical properties of essential oil of Hyptis suaveolens leaves, hence looking at its physicochemical properties for possible use in making soap, cream or perfume for use to repel the mosquito.