LARVICIDAL POTENTIAL OF EXTRACTS OF PERSEA AMERICANA SEED AND CHROMOLAENA ODORATA LEAF AGAINST AEDES VITTATUS MOSQUITO

ABSTRACT

The larvicidal activity of various solvent (ethanol, ethyl acetate and n-hexane) extracts of

Persea americana seed and Chromolaena odorata leaves against Aedes vittatus mosquito was analysed. The most potent solvent (n-hexane) extracts of both plants were fractionated using column chromatography and most effective fractions isolated and identified using Gas Chromatography Mass Spectrometry and Fourier Transform Infrared techniques. Phytochemical screening revealed the presence of steroids, cardiac glycosides and terpenoids in all the extracts. The larvicidal bioassay of Persea americana seed gave LC₅₀ values of 0.827ppm, 1.799ppm and 2.732ppm for n-hexane, ethanol and ethyl acetate extracts respectively, while, Chromolaena odorata leaf extract had LC₅₀ values of 1.835ppm, 3.314ppm, and 5.163ppm for n-hexane, ethanol and ethyl acetate respectively. Column chromatographic fractionation of most potent n-hexane (crude) extracts of both plants, showed increased activity in some of the fractions of Persea americana (nHPa6) and Chromolaena odorata (nHCo6) which showed higher mortality, with LC₅₀ values of 0.486ppm and 1.308ppm respectively. GC/MS analysis of components in nHPa6 and nHCo6 showed oleic acid as the most abundant, in fractions of both plants. The FTIR analyses of nHPa6 and nHCo6 showed absorption bands of the functional groups present, which included; alcohol, alkane, alkene, alkyl halide, aldehyde, carboxylic acid and carbonyl ester, thus, supporting the GCMS result. The n-hexane, ethanol and ethyl acetate extracts of P. americana seed and C. odorata leaves have shown good larvicidal activity and should therefore be further exploited for the control of mosquito larvae.

TABLE OF CONTENTS

ABSTRACT

CHAPTER ONE

1.0 INTRODUCTION

1.1 Statement of Research Problem

1.2 Justification

1.3 Aims and Objectives

1.3.1 General Aim

1.3.2 Specific Objectives

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Mosquito

2.1.3 Life Cycle of Aedes

2.1.4 Mosquito Morphology and Feeding Habits

2.1.5 Mosquito Born Diseases

2.1.6 Mosquito Control Methods

2.1.7 Active Ingredients in Plants Responsible for Larval Toxicity

2.1.8 Mechanism and mode of action of insecticide/larvicide

2.1.9 Toxicity Response Determinant and Variation of plant derived larvicides

2.1.10 Scope for isolation of toxic larvicidal active ingredients from plants

2.2 Chromolaena odorata

2.2.1 Classification of C. odorata

2.2.2 Origin and Distribution

2.2.3 Traditional Uses of C. odorata

2.2.4 Phytochemical Composition of C. odorata

2.2.5 Medicinal Values of C. odorata

2.2.6 Antibacterial effect of C. odorata

2.2.7 Toxicity of C. odorata

2.3 Persea americana

2.3.1 Classification of Persea americana

2.3.2 Biological activities of  Persea americana constituents

2.3.3 Phytochemical composition of avocado seed

2.3.4 Tradomedicinal Uses of Avocado Seed

2.3.5 Larvicidal and antimicrobial activities

2.3.6 Toxicity of avocado seed

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Chemicals

3.1.2 Plants Collection and identification

3.2 Methods

3.2.1 Preparation of extracts

3.2.2 Mosquito Larvae culture

3.3 Phytochemical Analysis

3.3.1 Test for Saponins

3.3.2 Test for tannins

3.3.3 Test for flavonoids

3.4.4 Test for sterols

3.4.5 Test for Terpenoids

3.4.6 Test for Anthracenes

3.4.7 Test for cardiac glycosides

3.4.8 Test for alkaloids

3.4 Preparation of Stock Solutions

3.4.1 Preparation of Test Concentrations For Bioassay

3.5 Larvicidal Bioassay

3.5.1 Determination of Lethal Concentrations

3.6 Thin Layer Chromatography (TLC)

3.6.1 Column Chromatography

3.7 Characterization of Larvicidal Compounds In The Bioactive Fraction

3.7.1 Fourier Transform Infra-Red Spectroscopy(FTIR)

3.7.2 Gas Chromatography/Mass Spectroscopy (GC/MS)

3.8 Statistical Analysis

CHAPTER FOUR

4.0 RESULTS

4.1       Phytochemical Constituents of Extracts of Persea americana Seed and Chromolaena odorata Leaf

4.2 Larvicidal activity of different solvent extracts of Persea americana seed against          Aaedes vittatus mosquito

4.3 Larvicidal Activity of Different Solvents Extract of Chromolaena odorata Leaf Against Aedes vittatus Mosquito

4.4 Larvicidal activity of chromatographic fractions of n-hexane extracts  of Persea americana seed  against Aedes vittatus

4.5 Larvicidal activity of chromatographic fractions of n-hexane extracts of Chromolaena odorata leaf against Aedes vittatus mosquito

4.6 GC/MS Characterisation of Most Potent Chromatographic Fraction (nHPa6) of P. americana

4.7 GC/MS characterisation of Most Potent Chromatographic Fraction (nHCo6) of C. odorata

4.8 Functional group Identification of nHPa6

4.9 Functional groups Identification of nHCo6 fraction

CHAPTER FIVE

5.0 DISCUSSION

CHAPTER SIX

6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS

6.1 Summary

6.2 Conclusions

6.3 Recommendations

REFERENCES

APPENDICES

Abbreviations

LC = Lethal Concentrations

GCMS = Gas Chromatography Mass Spectrometry FTIR = Fourier Transform Infra-Red

n-Hexane = Normal Hexane

TLC = Thin Layer Chromatography

DMSO = Dimethyl Sulfoxide

JE = Japanese Encephalitis

WHO = World Health Organisation

Cx = Culex

An = Anopheles

Ae = Aedes

CHIKV = Chikungunya Virus

Bti = Bacillus Thuriengiensis

Bs = Bacillus Sphaericus

DDT = Dichloro-Diphenyl-Trichloroethane

IGR = Insect Growth Regulator

CSI = Chitin Synthesis Inhibitor

CNS = Central Nervous System

ACh = Acetyl Choline

AChE = Acetyl Choline Esterase

GABA = Gammaamino Butyric Acid

ATP = Adenosine Triphosphate

PTTH = Prothoracictropic Hormone

CCl₄ = Tetrachloromethane

NMR = Nuclear Magnetic Resonance

CHAPTER ONE

1.0 INTRODUCTION

Insect-transmitted diseases remain a major cause of morbidity and mortality worldwide. Mosquito species belonging to genera; Anopheles, Aedes and Culex, are vectors (Redwane et al., 2002) for the transmission of malaria, dengue fever, yellow fever, filariasis, schistosomiasis and Japanese encephalitis (JE), transmitting diseases to more than 700 million people annually (Oyewole et al., 2010; Govindarajan, 2009). Mosquitoes also cause allergic responses in humans which include local skin irritation and systemic reactions such as angioedema. Aedes spp are generally regarded as a vector responsible for transmission of yellow fever and dengue fever, which is endemic to Southeast Asia, the Pacific island area, Africa, Central and South America.

The World Health Organization (W.H.O. 2012) has recommended vector control as an important component of the global strategy for preventing insect-transmitted diseases. The most commonly employed method for the control of mosquito-borne diseases involve the use of chemical-based insecticide, though it is not without numerous challenges, such as human and environmental toxicity, resistance, affordability and availability (Ghosh et al., 2012).

Extracts from plants has been good sources of phytochemicals as mosquito egg and larval control agents, since they constitute an abundant source of bioactive compounds that are easily biodegradable into non-toxic products. In fact, many researchers have reported on the effectiveness of plant extracts or essential oils against mosquito larvae. They act as larvicides, insect growth regulators, repellents, and oviposition attractants (Pushpanathan, 2008; Samidurai et al., 2009; Mathivanant et al., 2010).

Persea Americana is an ever green tree belonging to Lauraceae family and its fruits are commonly known as avocado pear or alligator pear. The plant originates from Central America but it has shown easy adaptation to other tropical regions, thus widely cultivated in tropical and subtropical regions. The various parts (leaves, fruits and seed) of this plant have numerous uses from edible pulp as source of nutrients to the seed preparation as remedy (Arukwe et al., 2012).

The seed extracts of Persea americana has many vital application in traditional medicine, for the treatment of diarrhoea, dysentery, tooth ache, intestinal parasites, skin infection (mycoses) and management of hypertension and the leaves have been reported to have anti- inflammatory and analgesic activities (Adeyemi et al., 2002; Ozolua et al., 2009). Phytochemical screening of avocado seed shows the presence of fatty acids, Triterpenes, anthocyanin, flavonoids and abcissic acids (Leiti et al., 2009).

Chromolaena odorata is a weed which belongs to Asteraceae family. It is found in tropical and subtropical areas, extending from west, central and southern Africa to India, Sri Lanka, Bangladesh, Laos, Cambodia, Thailand, southern China, Taiwan, and Indonesia. The weed goes by many common names including; Siam weed, devil weed, French weed, communist weed (Vaisakh and Pandey, 2012). In Nigeria, the Chromoleana odorata is referred to as

―Obu inenawa‖ by the Igbo and ―ewe awolowo‖ by the Yoruba. This plant is exploited traditionally for its medicinal properties, especially for external uses as in wounds, inflammation and skin infections. Some studies also demonstrate the efficacy of its leaf extract, as antioxidant, anti-inflammatory, analgesic, anti-microbial and cytoprotective agent (Ajao et al., 2011). The oil from C. odorata also had been exploited as insecticide, ovicide and larvicide (Noudogbessi et al., 2006). Previous phytochemical studies of the leaf extracts of C. odorata show the presence of alkaloid, cardiac glycosides, anthocyanin, tannin, and flavonoids (Ngozi et al., 2009).

1.1              Statement of Research Problem

An estimated 3.3 billion people are at risk of malaria globally, with populations living in sub-Saharan Africa having the highest risk (WHO, 2012) and two-fifths of the world‘s population is at risk of dengue fever (WHO, 2003). Malaria alone accounts for about 50 per cent of out-patient consultation, 15 per cent of hospital admission, and also among the top three causes of death in the country.

In recent years, the use of many synthetic insecticides in mosquito control programme has been limited, due to many challenges such as, high cost of synthetic insecticides, environmental sustainability, toxic effect on human health (immune suppression), and other non-target organisms, environmental persistence, higher rate of biological accumulation and magnification through ecosystem, as well as increasing insecticide resistance on large scale (Srivastava and Sharma, 2000; Raghvendra and Subbarao, 2002). These challenges have resulted in an urge to search for environmentally sustainable, biodegradable, affordable and target selective insecticides against mosquito species (Saxena and Sumithra, 1985; Kumar and Dutta, 1987; Chariandy et al., 1999; Markouk et al., 2000; Tare et al., 2004).

Consequently, the application of eco-sustainable alternatives such as biological control of vectors has become the main focus of the control programme to replace the synthetic chemical insecticides (Gosh et al., 2012). One of the most effective alternative approaches under the biological control programme is to utilise the plants biodiversity as a reservoir of safer insecticides of botanical origin as a simple, affordable and sustainable method of mosquito control.

1.2              Justification

Mosquito larvae is the easiest stage to target in its life cycle and several studies have documented the efficacy of plant extracts as a reservoir pool of bioactive toxic agents against mosquito larvae. Furthermore, evolution of the resistance to plant-derived compounds has rarely been reported (Sharma et al., 2006).

However, the main reasons for the failure in laboratory to field utilisation of bioactive phytochemicals are poor characterization and inability to determine the active toxic components responsible for larvicidal activity (Ghosh et al., 2012). Hence, there is a need for the characterisation, of various plant extracts to determine the active (larvicidal) components of locally available plants for mosquito control. This will help to reduce dependence on expensive and mostly imported products, and stimulate local efforts to enhance the general public health.

1.3              Aims and Objectives

1.3.1        General Aim

The aim of this study was to investigate the larvicidal potential of extracts of Persea americana seed and Chromolaena odorata leave against Aedes vittatus larvae

1.3.2        Specific Objectives

a.       Phytochemical analysis (qualitative) of the crude extracts of persea americana seed and Chromolaena odorata leaves.

b.      Determination  of the most  potent solvent extracts  with larvicidal  activity against

Aedes vittatus larvae