LARVICIDAL ACTIVITY OF Picralima nitida - University Of

LARVICIDAL ACTIVITY OF Picralima nitida,

AN APPROACH IN MALARIAL VECTOR CONTROL.

BY

NWABOR, OZIOMA FORSTINUS

PG/M.Sc./11/58608

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

NOVEMBER, 2014

TITLE PAGE

LARVICIDAL ACTIVITY OF Picralima nitida,

AN APPROACH IN MALARIAL VECTOR CONTROL.

BY

NWABOR, OZIOMA FORSTINUS

PG/M.Sc./11/58608

A DISSERTATION SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES,

UNIVERSITY OF NIGERIA NSUKKA, IN PARTIAL FULFILMENT OF THE

REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE (M.Sc.)

DEGREE IN ENVIRONMENTAL MICROBIOLOGY

SUPERVISOR: REV. SR DR M. E. U. DIBUA

MARCH, 2014

CERTIFICATION

Mr. Nwabor, Ozioma Forstinus (Reg.No. PG/M.Sc./11/58608), a postgraduate student in the

Department of Microbiology, majoring in Environmental Microbiology, has satisfactorily

completed the requirements for course work and research for the degree of Masters in Science

(M.Sc.) in Microbiology. This work is embodied in his original project and has not been

submitted in part or full for either diploma or degree of this university or any other university.

CERTIFIED BY

……………………………….. …………………………………

REV. SR DR M.U.E. DIBUA DATE

PROJECT SUPERVISOR

………………………………… …………………………………… PROF. A. N. MONEKE DATE HEAD OF DEPARTMENT

DEDICATION

To my siblings, Chinonso, Chinwendu, Amarachi,

Chimankpam, Tochukwu and Onyedikachi.

ACKNOWLEDGEMENT

I am most grateful to my heavenly Father, by whose grace I was able to overcome all the trials

and difficulties that assailed me, and whose mercies saw me through this program.

My sincere gratitude goes to my parents, Mr. Matthias Uche Nwabor and Mrs. Ugonnia Comfort

Nwabor. Your sacrifice is the greatest lesson I have learnt. May God bless you.

To my supervisor Rev. Sr Dr M.U.E. Dibua, my earnest prayer is that God will bless you

abundantly and grant you good health. Thank you sister, I appreciate your effort.

I also want to thank all my lecturers, Prof. Mrs. I.M. Ezeonu, Prof. A.N. Moneke, Prof. I.N.S.

Dozie, Prof. C.U. Anyanwu, Dr C.N. Eze, Dr A.C. Ike, Dr E.A. Eze, Dr S. N. Enenmuo, Dr

Vincent Chigor and others. God bless you all. I am also grateful to Dr. Goddy Ngwu of the

Department of Zoology for his assistance and advice and to the Department of Pharmaceutics for

granting access to their laboratory.

Certainly am not forgetting my family, The Catholic Association of Post Graduate Students

(CAPS), and my friends, Ezeokwuora Chioma, Dickson I. Dickson, Illoanya Ulomma, Osita

Odiachi, Onyenma, Ngozi and many more. Thank you for making me the better person I am

today. May God bless you all.

TABLE OF CONTENTS

Title page i

Certification iii

Dedication iv

Acknowledgement v

Table of Content vi

List of Figures ix

List of Tables x

List of Acronyms xi

List of Appendices xiii

Abstract xiv

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1.1 Introduction 1

1.1.1 Aim and objectives 2

1.2 Literature Review 3

1.2.1 Botanical description of the plant Picralima nitida 3

1.2.2 Classification 3

1.3 Uses of the Plant “Picralima nitida” 4

1.4 Biology of mosquito 5

1.5 Ecology of Mosquitoes 6

1.5.1 Habitat productivity 7

1.5.2 Spatial and temporal distribution 8

1.5.2 Gonotrophic cycle 9

1.6 Malaria Prevention and Control 9

1.6.1 Drug treatment 10

1.6.2 Indoor residual spraying (IRS) 10

1.6.3 Mosquito nets 11

1.6.4 Larval control 12

1.6.5 Chemical larvicides 13

1.6.6 Microbial insecticides 13

1.7 Phytochemicals as Larvicidal Agents 14

1.8 Major Phytochemicals from Plants 15

1.9 Extraction Procedures 20

1.9.10 Mode of action of phytochemicals in target insect body 22

1.10 Review of Synthetic Insecticides 23

1.10.1 Insecticide resistance 24

1.11 Classes of Insecticides 24

1.12 Mechanism of Resistance 26

1.13 Insecticide Resistance Detection Techniques 27

CHAPTER TWO: METHODOLOGY 2.1 Collection of Plant Materials 30

2.2 Sample Preparation and Extraction 30

2.3 Phytochemical Screening 31

2.3.1 Test for alkaloids 31

2.3.2 Test for saponins 32

2.3.3 Test for phenols/tannins 32

2.3.4 Test for anthraquinones 32

2.3.5 Test for glycosides 32

2.3.6 Test for flavonoids 33

2.3.7 Test for steroids and terpenoids 33

2.4 Raising of Anopheles gambiae larvae 33

2.5 Larvicidal Bioassay 34

2.6 Determination of LC50 and LC95 35

CHAPTER THREE: RESULTS

3.1 Larvicidal Effect of Plant Extracts on Anopheline Larva 36

3.2 Relative Median Potency 49

3.3 Median Lethal Concentration 58

3.4 Median Lethal Time 67

3.5 Percentage yield 69

3.6 Phytochemical Characteristics 71

CHAPTER FOUR: DISCUSSION AND CONCLUSION 73

REFERENCES 78

APPENDICES

LIST OF FIGURES

Figure Title

Page

1.1: Plant Parts used in the Study. (A) Seed, (B) Leaf, (C) Pulp, (D) Fruit

4

1.2: The life cycle of mosquitoes

6

1.3: Structure of Some Alkaloids Found in P. nitida

18

2.1: The Fourth Instar Anopheles Larva following incubation

34

3.1: Effect of Concentrations of Aqueous Leaf Extract of P. nitida on A. gambiae

37

3.2: Effect of Concentrations of Methanolic Leaf Extract of P. nitida on A. gambiae

38

3.3: Effect of Concentrations of Aqueous Seed Extract of P. nitida on A. gambiae

40

3.4: Effect of Concentrations of Methanolic Leaf Extract of P. nitida on A. gambiae

41

3.5: Effect of Concentrations of Aqueous Pulp Extract of P. nitida on A. gambiae

43

3.6: Effect of Concentrations of Methanolic Pulp Extract of P. nitida on A. gambiae

44

3.7: Comparative Activity of the Extracts on A. gambiae at 72 hours

46


47


48

3.10: Median Lethal Time of Samples

68

LIST OF TABLES

Table Title Page

3.1: Relative Median Potency Estimates for 24 hrs Aqueous Extracts

50


51


53

3.4: Relative Median Potency Estimates for 24 hrs Methanolic Extracts

54


56


57

3.7: LC50 and LC95 of Aqueous Leaf Extracts at Varying Time Intervals

59

3.8: LC50 and LC95 of Methanolic Leaf Extracts at Varying Time Intervals

60

3.9: LC50 and LC95 of Aqueous Seed Extracts at Varying Time Intervals

62

3.10: LC50 and LC95 of Methanolic Seed Extracts at Varying Time Intervals

63

3.11: LC50 and LC95 of Aqueous Pulp Extracts at Varying Time Intervals

65

3.12: LC50 and LC95 of Methanolic Pulp Extracts at Varying Time Intervals

66

3.13: Percentage Yield of Samples

70

3.14: Phytochemical Characteristics of Picralima nitida Samples

72

LIST OF ACRONYMS

AChE Acetylcholinerase

ACTs Artemisinin-based Combination Therapies

Bs Bacillus sphaericus

Bt Bacillus thuringiensis

Btk Bt serovar kurstaki

CDC Center for Disease Control

CDPH California Department of Public Health

CI Confidence interval

DDT Dichloro-diphenyl-trichloroethene

DMSO Dimethylsulphoxide

EPA Environmental Protection Agency

FeCl3 Ferrous Chloride

GABA Gamma-aminobutyric acid

GCMS Gas Chromatography and Mass Spectroscopy

H2SO4 Sulphuric Acid

ICMR Indian Council of Medical Research

IMM Integrated Mosquito Management

IPM Integrated Pest Management

IR Infra Red

IRAC Insecticides Resistance Action Committee

IRS Indoor Residual Spraying

ITNs Insecticide-treated nets

IVM Integrated Vector Management

kdr Knock-down resistance

LC50 Median Lethal Concentration

LD50 Median Lethal Dose

LLINs Long-lasting insecticidal nets

LT50 Median Lethal Time

NEA National Environment Agency,

NMR Nuclear Magnetic Resonance

OPs Organophosphates

PPM Parts Per Million

RBM Roll Back Malaria

SD Standard Deviation

SP sulfadoxine pyrimethamine

TLC Thin Layer Chromatograph

UNICEF United Nations Children’s Fund

UV Ultraviolet

WHO World Health Organisation

LIST OF APPENDICES

Appendix Title

Page

1: Effect of aqueous leaf extract of on An. gambiae at various time intervals

91

2: Effect of methanolic leaf extract of on An. gambiae at various time intervals

91

3: Effect of aqueous seed extract of on An. gambiae at various time intervals

92

4: Effect of methanolic seed extract of on An. gambiae at various time intervals

92

5: Effect of aqueous pulp extract of on An. gambiae at various time intervals

93

6: Effect of methanolic pulp extract of on An. gambiae at various time intervals

93

7: Probit Estimate for methanolic leaf

94

8: Probit Estimate for methanolic seed

94

9: Probit Estimate for aqueous seed

95

10: Probit Estimate for aqueous leaf

95

11: Probit Estimate for methanolic pulp

96

12: Probit Estimate for aqueous pulp

96

13: Median Lethal Time for Methanolic Leaf, Aqueous Leaf and Methanolic Seed

97

ABSTRACT

Mosquitoes constitute a serious Public Health menace, resulting in millions of death worldwide each year. Emergence of insecticide resistant strains of the mosquitoes poses a serious threat and hence calls for alternative control measures. This study assessed the larvicidal efficacy of the methanolic and aqueous extracts of different parts of Picralima nitida against the 4th instar larvae of the malaria vector Anopheles gambiae. Larvicidal activities of the leaf, seed and pulp of the plant were therefore studied on laboratory reared larvae of A. gambiae at concentration ranges of 0.5 mg/ml to 5.0 mg/ml. The LC50 and LC95 values were obtained from probit analysis using SPSS version 16.0, at 95% confidence limit (CL). The Median Relative Potency of the extracts was also obtained using probit analysis at (P ≤0.05). Results of the study indicated that the LC50 and LC95 values of the aqueous leaf extracts were 3.14 mg/ml and 42.15 mg/ml at 24 h, 0.35 mg/ml and 4.73 mg/ml at 48 h and 0.16 mg/ml and 2.20 mg/ml at 72 h. A lower larvicidal activity was however observed with the methanolic leaf extract: LC50 value of 48.38 mg/ml, 15.82 mg/ml and 0.33 mg/ml at 24 h, 48 h and 72 h respectively. Methanolic seed extract on the other hand, exhibited a higher degree of potency compared with the aqueous seed extract with a low LC50 value of 0.87 mg/ml, 0.21 mg/ml and 0.15 mg/ml at 24, 48 and 72 h respectively; and an LC95 values of 0.74 mg/ml, 0.18 mg/ml and 0.12 mg/ml at same time interval. A higher efficacy of activity was exhibited by the aqueous pulp extract than the methanolic pulp extract; with lowest LC50 of 2.79 mg/ml at 72 h, and LC95 value of 10.40 mg/ml. The relative potency estimate of the aqueous extract at 24 h gave the following result: aqueous leaf extract was 5.48 times more potent than the aqueous seed; the aqueous pulp 2.20 times greater than the aqueous seed whereas the aqueous leaf was 2.50 times more potent than the aqueous pulp. Similar trend was also observed at 48 and 72 h. Comparatively, at 24 h the methanolic seed extract was 269.76 times more potent than the methanolic leaf extracts; the methanolic pulp extract 2.40 times more potent than the methanolic leaf while the methanolic seed gave a potency of 112.49 times more than the methanolic pulp. However at 48 and 72 h, there was a reversal in trend with the methanolic leaf extract showing a relative potency of 2.73 and 11.51 times that of the methanolic pulp. The LT50 (Median Lethal Time) of the extracts, evaluated at concentration of 1.0mg/ml with P = 0.05, was the following LT50; 28h, 3.6 h and 57 h for aqueous leaf extract, methanolic seed extract and methanolic leaf extract respectively. Similarly, comparative evaluation of the overall efficacy of the various extracts showed that the methanolic seed extract exhibited the highest degree of activity (P<0.05), followed by the aqueous leaf extract and methanolic leaf extract. Conclusively, aqueous seed, methanolic pulp and aqueous pulp extract showed a relatively lower activity and an LT50 value of 300 hrs and above at a concentration of 1.0 mg/ml. The results of this research therefore underscores the efficacy of the plant and further suggest the use of methanolic seed, aqueous leaf and methanolic leaf extracts of P. nitida as an eco-friendly alternative in malaria vector larviciding.

CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Mosquitoes are vectors of disease causing agents found within almost all tropical and subtropical

countries. They are responsible for the transmission of pathogens causing some of the life

threatening and debilitating diseases of man, such as; malaria, yellow fever, dengue fever,

chikungunya, filariasis, encephalitis, etc (Chandra et al., 2008; ICMR, 2003). Beatty et al., 2007,

reported that fifty-five percent (55%) of the world’s population are at risk of mosquito borne

diseases in 124 countries. According to WHO/UNICEF (2005) first comprehensive report on the

Roll Back Malaria partnership, malaria is endemic in 117 countries with some 3.2 billion people

living in risk areas all over the world. It further states that each year, there are about 350-500

million clinical cases of malaria worldwide with over 1 million death. About 59% of all clinical

cases occur in Africa, 38% in Asia, and 3% in the Americas. Malaria mortality is also highest in

Africa with 89% of all deaths whereas 10% occurs in Asia and less than 1% in the Americas. Of

all malaria cases caused by Plasmodium falciparum, the most deadly human malaria species,

74% are in Africa, 25% in Asia and 1% in the Americas. Anopheline mosquitoes are the vectors

responsible for the transmission of deadly malaria etiological agent “plasmodium” (Wendy et al.,

2012). Despite several efforts in the field of vector control, the medical and economic burden

caused by vector-borne diseases continues to grow as current control measures fail to cope

(Radhika et al., 2011). There is therefore an urgent need to identify new control strategies that

will remain effective, even in the face of growing insecticide and drug resistance (Achs and

Malaney, 2002).

Current vector control strategies include chemical-based control measures, non-chemical-based

control measures and biological control agents (Poopathi and Tyagi, 2006). Chemical-based

control measures have dominated over other strategies over the years. Radhika et al. (2011),

reported that repetitive use of man-made insecticides for mosquito control disrupts natural

ecosystems and the biological control systems, and lead to reemergence of, and increase in

mosquito populations. In their studies, Das et al. (2007) and Zhang et al. (2011) also pointed out

that the continuous use of chemical-based insecticides has resulted in the development of

resistance, detrimental effects on non-target organisms and human health problems.

Consequently, they suggested the need for alternative control measures. This leaves biological

control as a viable alternative. The use of biological control agents such as predatory fish

(Legner, 1995), bacteria (Becker and Ascher, 1998), protozoa (Chapman, 1974), fungus

(Murugesan et al., 2009), nematodes (Kaya and Gaugler, 1993) and plant products (Mathur,

2003) had shown promising results in the control of mosquito populations.

The emergence of insect vectors that are resistance to available insecticides coupled with the

environmental and economic burden arising from continuous use of insecticides and the health

problems caused by these vectors calls for urgent alternative control measures. In ancient times,

herbal products were used as natural insecticides before the discovery of synthetic insecticides

(ICMR, 2003). Phyto-products, on account of their minimal hazardous effect on the environment

and availability may serve as alternatives in the control of mosquitoes. Reports have shown that

various phytochemicals are potent adulticidal and larvicidal agents (Roark, 1947; Sukumar et al.,

1991). Roark, (1947) described approximately 1,200 plant species having potential insecticidal

value, while Sukumar et al. (1991) listed and discussed 344 plant species that only exhibited

mosquitocidal activity. In view of the discovery that plants from various families have shown

some degree of activity against various developmental stages of insect vectors, there is a need to

evaluate local Nigeria plants for their possible activity. It was therefore important to carry out

this research.

1.1.1 Aim and Objectives

The aim of this study therefore was to evaluate the larvicidal effect of the plant “Picralima

nitida” against the fourth instar larva of the vector “Anopheles gambiae”. The specific objectives

of the study were:

i. To determine the phytochemical constituents of the crude methanolic and aqueous leaf,

seed and pulp extracts of Picralima nitida.

ii. To determine the larvicidal efficacy of these extracts on An. gambiae larvae within 72 h.

iii. To determine the median lethal concentration (LC50), the 95% lethal concentration (LC95)

and the median lethal time (LT50) of the various extracts on An. gambiae within 72 h.

iv. To determine the relative potency ratio of the extracts.

1.2 Literature Review

1.2.1 Botanical description of the plant Picralima nitida Picralima nitida, family Apocynaceae was first described and characterized by T. H. Durand in

1909 (Meyer et al., 2006). The plant is commonly called the Akuamma plant and is referred to as

Osu-Igwe in Igboland, south-east Nigeria (Nkere and Iroegbu, 2005). However, this same plant

is called “Ntos” in Oguta LGA, Imo State, Nigeria, where samples for this research were

collected. The tree is about 15 to 20 meters high, with circumference of about 50 centimeter. At

maturity, the leaves are pinnate (Figure 1 B) with about 14 to 18 leaflets. The thick bark of on

average a centimeter is granular and yellow-orange hue punctuated. It is used in indigenous

pharmacopoeia to treat malaria and stomach ache (amoeba). Berries have an ellipsoid form, with

large size and green in color (Figure 1 D). During the fall on the ground, they turn to yellow and

the seeds germinate on the ground with many seedlings. In Nigeria, this plant is prominent in the

south west, southeast and south-south regions of the country.

1.2.2 Classification

Kingdom Plantae

Family Apocynaceae

Subfamily Rauvolfioideae

Genus Picralima

Species Picralima nitida

Figure 1.1: Plant Parts used in the Study. (A) Seed, (B) Leaf, (C) Pulp, (D) Fruit.

1.3 Uses of the Plant “Picralima nitida” The plant, Picralima nitida, has provided drugs used in the treatment of many diseases

(Nwakile and Okere, 2011). According to Adjanohoun et al. (1996), berries of the plant are used

in traditional medicines for treating typhoid and fight against muscular pain. Extracts of the plant

have been used in the treatment of pathogenic diseases (Ubulom et al., 2011), protozoan

infections (Okokon et al., 2007) and non pathogenic diseases (Kouitcheu et al., 2006). Diabetes

mellitus is a major endocrine disease that is treated with the extracts of the plant (Inya-Agha et

al., 2006). The Larvicidal and Antifungi properties of leaf samples of P. nitida has been

investigated by Ubuloma et al. (2012), and was reported to show a significant larvicidal effect on

A. gambiae. The methanolic and aqueous extracts of the leaf, seed and pulp has been reported to

exhibit certain level of larvicidal effect (Dibua et al., 2013; Nwabor et al., 2014).

In addition to the above description of the species, Meyer et al. (2006) assessed the medicinal

composition of P.nitida from laboratory analysis, and found that the active component of P.

nitida is formed by more than 10 alkaloids present in different tree parts (from bark, leaves,

roots). Their names derive from the local name "Akuamma" (Okunji et al., 2006).

According to Meyer et al. (2006), these alkaloids play several biological roles such as:

• Anti-inflammatory (pseudo-akuammigine),

• Anti-fever (erythrocytic phase inhibition of P. falciparum with use of roots, stem bark

and fruit skin),

• Antimicrobial (against Gram bacteria and fungi with use of root bark),

Hypoglycemic control (with use of roots and fruits) and,

• Anti-malaria (with fruits), and anti-leishmaniasis (with roots).

1.4 Biology of Mosquito Mosquitoes undergo complete metamorphosis, having egg, larval, pupal and adult stages (Figure

1.2). There are generally six immature stages during mosquito development; the egg stage, four

larval stages and the pupal stage. Mosquito larvae are commonly referred to as “wrigglers” and

pupae as “tumblers”. There are two subfamilies in the mosquito family (Culicidae): Anophelinae

(gambiae, funestus, arabiensies) and Culicinae (quinquefasciatus, pipiens, tarsalis, salinarius

etc). Most larvae in the subfamily Culicinae hang down just under the water surface by the

siphon, whereas anopheline larvae lie horizontally just beneath the water surface supported by

small notched organs of the thorax and clusters of float hairs along the abdomen (Aymere and

Laikemariam, 2006). Anopheline larvae have no prominent siphon. The larvae of An. gambiae

breathe atmospheric oxygen through two ‘spiracular openings’ on the eighth segment of their

abdomen and feed by moving brushlike structures on their mouthparts that create a current of

water (Merritt et al., 1992). They filter out microorganisms, particulate organic matter or detritus

and biofilm (Clements, 2000; Mutuku et al., 2006). However, there are a few species of

mosquitoes whose larva capture and eat that of other species (Wendy et al., 2012). The larvae

undergo four molts (each successively larger), the last of which results in the pupal stage. The

pupal stage of mosquitoes does not feed. Pupae give rise to adult mosquitoes in 2 to 4 days. The

emergence process begins with splitting of the pupal skin along the back. An emerging adult

must dry its wings and groom its head appendages before flying away (Wendy et al., 2012).

Accordingly, this is a critical stage in the survival of mosquitoes. If there is too much wind or

wave action, the emerging adult may fall over, becoming trapped on the water surface to die.

This is the reason why little if any mosquito breeding occurs in open water, but occurs at the

water’s edge among weeds. With optimal food and temperature, the time required for

development from larva to adult can be as short as 7 days (CDC, 2004). Adult mosquitoes of

both sexes obtain nourishment for basic metabolism and flight by feeding on nectar (Wendy et

al., 2012). In addition, females of most species need a blood meal from birds, mammals, or other

vertebrates for egg development. They suck blood via specialized piercing-sucking mouthparts.

Figure 1.2: The life cycle of mosquitoes (source: Aymere and Laikemariam, (2006))

1.5 Ecology of Mosquitoes

Larvae and pupae of mosquitoes are always found in water. Breeding sites may be anything from

water in discarded automobile tyres and the axils of plants, to pools, puddles, swamps, and lakes.

It is very important to note that mosquito species differ in their breeding habits, biting behavior,

flight range, and so forth. Typical habitats of An. arabiensis and An. gambiae are puddles,

shallow ponds, burrow-pits, brick-pits, tyre tracks, ditches, human foot and animal hoof prints

which are often created by the activities of humans or domestic animals (Koenraadt et al., 2004).

These habitats are open, containing no, little or low (grass) aquatic vegetation (Mwangangi et al.,

2007a) and are often of a transient nature, as their availability corresponds to precipitation

(Koenraadt et al., 2004). An. gambiae can colonize a breeding habitat within a few days after the

site is created (Minakawa et al., 2005). Besides temporary habitats, An. arabiensis is also found

in market garden wells (Robert et al., 1998) and water storage tanks. Another typical

characteristic of breeding sites of An. gambiae is their shallow nature. Gimnig et al. (2001)

showed that water bodies inhabited by An. arabiensis were on average 18.0 (95% CI ± 3.5) cm

deep, by An. gambiae 29.4 (± 10.7) cm and by both species 9.7 (± 4.1) cm on the average. In

another field study, average depths of 6.2 (±5.3 SD) and 10.6 (± 7.2) cm were recorded in dirt

tracks and in ditches, respectively (Koenraadt et al., 2004).

Despite the dogma that An. gambiae is most often found in turbid water collections, various

studies that examined the characteristics of larval habitat or larval population dynamics, failed to

give a clear relationship between the presence of immatures and the clarity of breeding sites. It is

known that dark substrates receive more eggs than light ones and moist substrates more than dry

ones (Huang et al., 2007). Minakawa et al. (1999) concluded that An. gambiae preferred turbid

water over clear water. This was supported by Gimnig et al. (2001) who observed that An.

gambiae and An. arabiensis were associated with habitats that were high in turbidity and that

both species increased in larval densities with increasing water turbidity. In contrast, Munga et

al. (2005) found that An. gambiae preferred clear rainwater over natural water from forests and

natural wetlands, which contained more impurities and was supported by Sattler et al. (2005)

who showed a preference of An. gambiae to breed in rather clear water bodies. Other factors that

may play an important role in habitat selection are volatile compounds that are produced by

microbial populations in the breeding site (Sumba et al., 2004), chlorophyll a content in the

breeding site (Mwangangi et al., 2007a) or the presence of conspecific larvae or aquatic

predators (Munga et al., 2006). Some studies reported no effect of turbidity on the occurrence of

An. gambiae (Mwangangi et al., 2007b). However, An. arabiensis and An. gambiae are often

found to share larval habitats (Edillo et al., 2002). A clear difference in requirements for the

larval environment of the two species has not been observed, but is subject of discussion. Several

studies suggest the requirements are similar (Gimnig et al., 2001), others think they differ, but

were unable to show that explicitly (Minakawa et al., 1999).

1.5.1 Habitat productivity Mosquito breeding site productivity, estimated in terms of the numbers and size of mosquitoes

produces over time depends, not only on the initial number of eggs that are deposited, but on the

growth, development rate and survival of the mosquito immatures. Larval developmental rate,

survival and adult size affect the transmission of malaria. The time to develop from an egg into

an adult, combined with larval survivorship, determines the numbers of emerging mosquitoes

over time. The size of the emerging adults is of importance, as larger females have been found to

survive longer and have a greater fecundity (Ameneshewa and Service, 1996). Smaller and

virgin females on the other hand require a second or third blood meal in order to develop mature

eggs, prolonging the time to their first oviposition (Lyimo and Taken, 1993). Intermediate-sized

mosquitoes were found to be more infectious to humans (Lyimo and taken, 1993). Besides size,

various biotic and abiotic factors also affect the growth, development and survival of the

immature mosquitoes and consequently affect habitat productivity (Paaijmans, 2008).

Under laboratory conditions, where larvae were exposed to constant temperatures, Bayoh and

Lindsay, (2003) showed that larvae took 9.8 to 23.3 days to develop into adults, depending on

the temperature. Another laboratory study investigated the duration between oviposition and

pupation and reported a time period between 7 and 27 days (Chen et al., 2006). In another field

study, it was shown that the duration of the immature lifetime of Anopheles gambiae ranges from

8 to 22 days in habitats of different size (Gimnig et al., 2002). Eggs hatch within one day, larvae

grow into pupa within 6-19 days and the pupal stage lasts 1-2 days. A similar field study by

Gimnig et al. (2002) observed a shorter time range of the development from egg to adult, which

was 8.4-11.5 days. Service, (1977) observed that larvae, newly hatched from the eggs, took on

average 11.8 days to develop into adults, in small ponds and pools, ditches and rice fields. The

mortality observed among the immature stages of An. gambiae in the field is extremely high. In

all, only a small fraction (2-8%) of the larvae eventually survives to the adult stage (Okogun,

2005).

It is highly likely that many biotic and abiotic variables, interact and a combination of these

factors affect the productivity of a breeding site (Robert et al., 1998). In general it is believed

that; nutrition, larval densities and water temperature are the principal contributing factors that

affect growth and development of mosquito immatures (Mutuku et al., 2006).

1.5.2 Spatial and temporal distribution

Mosquito species differs in their distribution within the environment. Among the specie

Anopheles, An. gambiae is usually the predominant species in wet environments with high

humidity whereas An. arabiensis is more common in hotter zones with less rainfall (Kirby and

Lindsay, 2004). However, both species occur sympatrically across a wide range of tropical

Africa (Coetzee et al., 2000). Breeding of An. gambiae is mostly restricted to the rainy seasons

with larval and adult densities increasing rapidly and the species predominating over An.

arabiensis, and An. funestus which are more dominant species during the dry periods (Gimnig et

al., 2001; Minakawa et al., 2002). The distance between oviposition site and blood host may

affect the oviposition choice (Munga et al., 2006). Minakawa et al. (2002) showed that

immatures of An. gambiae would be found in breeding sites closer to houses and further away

from cowsheds and a study by Charlwood and Edoh (1996) showed that significantly more

larvae of An. arabiensis than An. gambiae were collected in pools close to cattle and suggested

that species distribution may be explained to a large extent by the presence of suitable hosts

instead of breeding site availability.

1.5.3 Gonotrophic cycle The gonotrophic period or gonotrophic cycle is defined as the time period between two

ovipositions. This period includes the search for a host, the ingestion and digestion of a blood

meal, the maturation of the ovaries and the search for a suitable aquatic breeding site to deposit

the mature eggs. Each gonotrophic cycle lasts about 2-4 days for An. gambiae (Toure et

al.,1998), but its length will depend on factors such as breeding site availability (Gu et al., 2006),

number of previous gonotrophic cycles and temperature (Afrane et al., 2005; Maharaj, 2003) . In

the field only a small percentage of females of An. gambiae survive for more than three or four

gonotrophic cycles (Charlwood et al., 2000). Although a small percentage was found to survive

for over ten cycles (Gillies and Wilkes, 1965).

1.6 Malaria Prevention and Control Despite the huge investment and intensive research in the development of malaria vaccine,

science is yet to record a break through. However, a number of effective preventive methods are

currently utilized to combat malaria. The policies and prevention strategies used are defined by

the available resources and epidemiological setting of the diseases (Nejla, 2007).

Environmentally, to prevent these diseases, the mosquito population must be kept at a low level

at all times. The most effective way to control the mosquito population is to get rid of their

breeding sources (National Environment Agency, 1995). As far as possible, stagnant waters

should be removed permanently by good and regular housekeeping practices such as filling up

ground depressions, disposing discarded containers properly and clearing choked drains and roof

gutters. For those mosquito breeding habitats that cannot be removed permanently, a competent

pest control operator should be engaged to look out for them within premises and treat them with

insecticides to prevent breeding. Prevention of malaria encompasses a variety of measures that

may protect against being bitten by the disease vector or against the development of disease in

infected individuals (WHO, 1992). Full coverage and access to prevention methods is the means

to reducing malaria incidence and eradicating the disease. There are three primary prevention

strategies that are currently being utilized by 107 malarious countries. The first is drug treatment,

the second is indoor residual spraying to eradicate mosquitoes, and the third, is mosquito nets to

prevent bites (WHO, 2005).

1.6.1 Drug treatment Given the increasing incidence of resistance to previous drugs used in malaria therapy, current

malaria drug treatment focuses on combination drug therapies as recommend by the World

Health Organization. The synergistic effect of these drugs are employed as the resistance of the

disease to conventional drug therapies, such as chloroquine, sulfadoxine pyrimethamine (SP) and

amodiaquine, has increased. Artemisinin-based Combined Therapies (ACTs) are the most

effective drug treatments currently. They produce a very rapid therapeutic response to malaria.

Since 2001, 42 malaria-endemic countries have started using ACTs (WHO, 2005; Roll Back

Malaria, 2006).

1.6.2 Indoor residual spraying (IRS) IRS is a highly-effective strategy for combating malaria and may provide a lasting impact in

areas of intense transmission. Unfortunately, the availability of low-risk and cost-effective

insecticides is diminishing due to increasing mosquito resistance and little development of new

compounds over the past 20 years. Approximately 50% of African nations use IRS in malaria

control (WHO, 2005; Roll Back Malaria, 2006). However, despite the use of IRS, malaria

remains a major Public Health problem in Africa. To date, IRS has only been implemented in

Nigeria in a limited fashion. However, according to the National Malaria Strategic Plan 2009--

2013, the objective was to gradually scale up spraying to cover 20% of households nationwide

(or almost seven million households) by 2013.

1.6.3 Mosquito nets Mosquito nets, particularly insecticide-treated nets, are a highly recommended strategy for the

prevention of malaria. Mosquito nets serve as the principal prevention strategy against malaria

because they are cost-effective, efficacious, and more available than other strategies. Long-

lasting insecticide nets have recently been developed and provide protection for up to five years.

Most of the mosquitoes that carry the malaria parasite bite individuals during the night, hence

bed nets protect individuals from the mosquitoes during this time by preventing contact and thus

reducing the risk of malaria. Furthermore, if treated with the insecticide, the net repels

mosquitoes and shorten the life of the mosquito (WHO, 2005). The use of mosquito nets has

consistently shown a reduction in malaria cases and overall mortality related to malaria (Roll

Back Malaria, 2006).

All of these physical prevention methods require the availability of health infrastructure and

education campaigns to effectively implement strategies and educate populations on the need for

malaria control. Current malaria vector control, using either insecticide-treated nets (ITNs) or

indoor residual spraying (IRS) relies on the continued susceptibility of Anopheles mosquitoes to

a limited number of insecticides. Long-lasting insecticidal nets (LLINs) and indoor residual

spraying (IRS) are the mainstay of malaria vector control programme because they are highly

effective, have a relatively low cost, and their manufacture and distribution can be rapidly scaled

up. Other interventions such as environmental management and larviciding can be useful but

only under certain conditions, depending on the target vector and the local situation (WHO,

2012). Vector control is a critical facet of malaria control today and is expected to continue to be

so. Vector control remains the single largest category of spending for malaria control by donors.

Twelve insecticides from four classes (organochlorines, organophosphates, carbamates and

pyrethroids) have been recommended for IRS (Kelly-Hope et al., 2008; Najera and Zaim, 2002),

but only pyrethroids have been approved for treating bed nets. Since the mid-1950s, there have

been numerous reports of reduced Anopheles susceptibility to DDT, malathion, fenithrotion,

propoxur and bendiocarb, and resistance to all four classes of insecticides has been found in

Anopheles species in different parts of Africa (Awolola et al., 2002 ; N’Guessan et al., 2003). A

much more recent development is that of pyrethroid resistance with cross-resistance to DDT,

first reported in Anopheles gambiae from Côte d'Ivoire (Elissa et al., 1993) and now widespread

in West Africa. Pyrethroid-DDT cross-resistance presents a major challenge for malaria vector

control in Africa because pyrethroids represent the only class of insecticides approved for

treating bed nets and DDT is recommended for use in IRS (WHO, 2006).

1.6.4 Larval control

Larval control is the foundation of most mosquito control programs. Whereas adult mosquitoes

are widespread in the environment, larvae must have water to develop. Control efforts therefore

can be focused on aquatic habitats. Minimizing the number of adults that emerge is crucial to

reducing the incidence and risk of disease. The three key components of larval control are

environmental management, biological control, and chemical control. Larviciding is a general

term for killing immature insects by applying agents, collectively called larvicides, to control

larvae and/or pupae stages of these insects (Flourida Mosquito Control, 2009). This is an

evolving control measure that targets the larva stage of the mosquito. Many people think that the

best time to begin a mosquito control program is when the numbers of biting female mosquitoes

reach an intolerable level. Contrary to this believe, the best time to begin a mosquito

management program is before the adult mosquitoes emerge. Control efforts should begin

immediately after the mosquito eggs have hatched, the breeding site should be inspected, and the

numbers of larvae present quantified to determine whether or not the use of an insecticide is

justified (CDC, 2004). Mosquitoes are most efficiently and economically destroyed when they

are in the larval stage and are concentrated in their breeding site. Preventing the larvae from

becoming adult mosquitoes minimizes the area that would have to be treated. It also prevents the

development of an annoyance or health problem and it reduces the potential environmental

impacts of the adult mosquito control program (CDC, 2004). Larviciding can reduce overall

insecticide use in a mosquito control program by reducing or eliminating the need for ground or

aerial application of insecticides to kill adult mosquitoes (CDC, 2004). Nandita et al. (2008)

considered mosquitoes in the larval stage an attractive target for pesticides because they breed in

water and, thus, are easy to deal with in this habitat whereas Ubulom et al., (2012) opinioned that

larviciding is a preferred option in vector control because larvae occur in specific areas and can

thus be more easily controlled. Treatment of mosquito breeding sites provides control before the

biting adults appear and disperse from such sites.

1.6.5 Chemical larvicides Chemical pesticides are rarely used to control mosquito larvae. Organophosphate larvicides are

used infrequently because of their potential non-target effects and label restrictions. Temephos is

currently the only organophosphate registered for use as a larvicide in California (CDPH, 2010).

This product can be safely and effectively used to treat temporary water or highly polluted water

where there are few non-target organisms and/or livestock are not allowed access. The efficacy

of temephos may be up to 30 days depending on the formulation (CDPH, 2010).

1.6.6 Microbial insecticides Microbial insecticides are formulated to deliver a natural toxin to the intended target organisms.

Bacteria are single-celled parasitic or saprophytic microorganisms that exhibit both plant and

animal properties and range from harmless and beneficial to intensely virulent and lethal.

Bacillus thuringiensis (Bt), is the most widely used agricultural microbial pesticide in the world,

and the majority of microbial pesticides registered with the Environmental Protection Agency

(EPA) are based on Bt (Florida Mosquito Control, 2009). The Bt serovar kurstaki (Btk) is the

most commonly registered microbial pesticide, and this variety has activity against Lepidoptera

(butterflies and moths) larvae (Florida Mosquito Control, 2009). It was originally isolated from

natural Lepidopteran die-offs in Germany and Japan. Activity of Bt against species of

mosquitoes were reported by Omoya and Akinyosoye (2011). Bt products have been available

since the 1950s. In the 1960s and 1970s, the World Health Organization (WHO) encouraged and

subsidized scientific discovery and utilization of naturally occurring microbes. As a result of

those early studies and a whole body of subsequent work, two lines of mosquito control products

have been developed: crystalline toxins of two closely related gram-positive, aerobic bacteria –

Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus (Bs). Mosquito control agents

based on Bt are the second most widely registered group of microbial pesticides. Highly

successful Bti products have expanded the role of microbial agents into the public health arena

(CDPH, 2010).

1.7 Phytochemicals as Larvicidal Agents

The use of plant products is an evolving alternative for mosquito control. The search for herbal

preparations that do not produce any adverse effects in the non-target organisms and are easily

biodegradable remains a top research issue for scientists associated with alternative vector

control (Redwane et al., 2002). The plant kingdom has shown to be a reservoir of various

chemicals which has been used for various beneficial purposes by man. From the time of

creation, it was recorded that trees were to be used as source of medicine. Today, with the advent

of biotechnology and the discovery of certain potent phyto-components, the scope of usage of

plant has been extended. The phytochemical compounds obtained from the huge diversity of

plant species from the tropical forest are important sources of safe and biodegradable chemicals,

which can be screened for lavicidal activities (Ansari et al., 2000). Many plants from varying

families have proved to be potent against the larva stage of mosquitoes. Kamaraj et al. (2011)

reported moderate larvicidal effect of Annona squamosa, Chrysanthemum indicum, Tridax

procumbens against Anopheles subpictus and Culex spp. Okigbo et al. (2010), also reported the

larvicidal effect of Azadirachta indica (neem tree), Ocimum gratissimum (scent leaf) and Hyptis

suaveolens (pignut weed) against mosquito larvae. Arivoli, et al. (2012), reported the larvicidal

efficacy of plant extracts against the malaria vector Anopheles stephensi. The larvicidal effect of

Lantana Camara Linn against Aedes aegypti and Culex quinquefasciatus was has also been

evaluated by Kumar et al. (2008). Bishnu and Zeev (2005), reported that extracts of Balanites

aegyptiaca (desert date) showed larvicidal effect on Culex pipiens. Studies on the larvicidal

efficacy of aqueous extracts of Striga hermonthica (Delile) Benth and Mitracarpus scaber

(Zucc) on Culex quinquefasciatus (culicidae) mosquito larvae was carried out by Abdullahi et al.

(2011). Ademola and Eloff (2011) studied the Anthelmintic efficacy of cashew (Anarcadium

occidentale L.) on in vitro susceptibility of the ova and larvae of Haemonchus contortus.

Obomanu et al. (2006), studied the Larvicidal properties of Lepidagathis alopecuroides and

Azadirachta indica on Anopheles gambiae and Culex quinquefasciatus. Mgbemena, (2010),

studied the Comparative Evaluation of Larvicidal Potentials of ethanol extracts of A. indica, O.

gratissimium and C. citratus on Ae. aegypti larvae. The larvicidal activities of ethanol extract of

Allium sativum (garlic bulb) against the filarial vector, Culex quinquefasciatus has been reported

by Kalu et al., (2010). Dibua et al. (2013) reported the larvicidal effect of Picralima nitida

against Anopheles gambiae.

1.8 Major Phytochemicals from Plant Phytochemicals are naturally occurring, biologically active chemical compounds in plants. The

prefix “Phyto” is from a Greek word meaning plant. In plants, phytochemicals act as a natural

defense system for host plants and provide colour, aroma and flavour. More than 4000 of these

compounds have been discovered to date and it is expected that scientists will discover many

more. Any one serving of vegetables could provide as many as 100 different phytochemicals.

Phytochemicals are protective and disease-preventing. These natural chemicals have been found

useful to man in various ways.

Phytochemicals have been used both as food and medicine. Their activity against

microorganisms prompts their use as anti-bacteria, anti-fungal and anti-nematode agents. For

instance, Bickii et al. (2006) used an in-vitro test for fifteen crude extracts from the stem bark

and seeds of four medicinal plants (Entandrophragma angolense, Picralima nitida,

Schumanniophyton magnificum and Thomandersia hensii) to check their anti-malarial activity

against the chloroquine-resistant Plasmodium falciparum W2 strain. The results showed that the

extracts of these plants possessed some anti-malarial activity, he however reported that the

methanol extract of P. nitida demonstrated the highest activity in-vitro. Further isolation and

identification of some active compounds from these plants could justify their common use in

traditional medicine for the treatment of malaria or fever in Cameroon. Besides the use of plants

for treatment of ailments, plants and their phytochemicals have long been used from ancient

times as a source of insect repellant or insecticidal agents. Some of the chemicals found in plants

include:

1.8.1 Saponin

Saponins are glycosides with distinctive foaming characteristics. They are natural detergents

found in certain plants. They are found in many plants especially certain desert plants. Saponins

are freely soluble in both organic solvents and water (Hostettman and Marston, 1995). Saponins

have detergent or surfactant properties because they contain both water soluble and fat soluble

components. They are amphipathic compounds, possessing both hydrophilic and lipophilic

portions. They are therefore surface active agents and can be used as emulsifiers. At

concentrations between 200-500ppm, saponins exits as monomers, above 500ppm they aggregate

as micelles with a molecular weight of approximately 100,000Daltons.

1.8.2 Flavonoid Flavonoids are polyphenolic compounds comprising fifteen carbons, with two aromatic rings

connected by a three-carbon bridge. They are the most numerous of the phenolics and are found

throughout the plant kingdom (Harborne, 1993). They are present in high concentrations in the

epidermis of leaves and the skin of fruits and have important and varied roles as secondary

metabolites. In plants, flavonoids are involved in such diverse processes as UV protection,

pigmentation, stimulation of nitrogen-fixing nodules and disease resistance (Pierpoint, 2000).

The main subclasses of flavonoids are the flavones, flavonols, flavan-3-ols, isoflavones,

flavanones and anthocyanidins. Other flavonoid groups, which quantitatively are in comparison

minor components of the diet, are dihydroflavonols, flavan-3,4-diols, coumarins, chalcones,

dihydrochalcones and aurones. The basic flavonoid skeleton can have numerous substituents.

Hydroxyl groups are usually present at the 4′, 5 and 7 positions. Sugars are very common with

the majority of flavonoids existing naturally as glycosides. Whereas both sugars and hydroxyl

groups increase the water solubility of flavonoids, other substituents, such as methyl groups and

isopentyl units, make flavonoids lipophilic (Crozier et al., 2006).

1.8.3 Tannin They are general descriptive names for a group of polymeric phenolic substances capable of

tanning leather or precipitating gelatin from solution, a property known as astringency. Their

molecular weight ranges from 500 to 3000KiloDaltons. The tannin compounds are widely

distributed in many species of plants, where they play a role in protection from predation, and

perhaps also as pesticides, and in plant growth regulation (Katie et al., 2006). The astringency

from the tannins is what causes the dry and puckery feeling in the mouth following the

consumption of unripened fruit or red wine (McGee, 2004). Likewise, the destruction or

modification of tannins with time plays an important role in the ripening of fruit and the aging of

wine. Tannins are divided into two groups, hydrolysable and condensed tannins. Hydrolysable

tannins are based on Gallic acid, usually as multiple esters with D-glucose, while the more

numerous condensed tannins (often called proanthocyanidins) are derived from flavonoid

monomers. Tannins may be formed by condensation of flavan derivatives which have been

transported to woody tissues of plants. Alternatively, tannins may be formed by polymerization

of quinine units.

1.8.4 Alkaloids They are natural plant compounds with a basic character and usually contain one or more

nitrogen atom in a heterocyclic ring. They are usually colourless, crystalline, non-volatile solids

which are insoluble in water but soluble in ethanol, ether, chloroform and other organic solvents.

Only very few are liquids which are soluble in water. Most alkaloids have a bitter taste and are

optically active. Most alkaloids are physiologically active while some are extremely poisonous.

The first medically useful example of an alkaloid was morphine isolated in 1805 from papaver

somniferum (Pium). Alkaloid constitutes the major phytochemical of P. nitida (Meyer et al.,

2006). Many of its alkaloids are akuammine derivatives, this probably may be the origin of the

name “akuamma plant”.

Figure 1.3: Structure of Some Alkaloids Found in P. nitida

1.8.5 Glycoside These are compounds that yield one or more sugars upon hydrolysis. A glycoside is composed of

two moieties: sugar portion (glycone) and non-sugar portion (aglycone or genin). Glycosides of

many different aglycones are extensively found in the plant kingdom (Saker and Nahar, 2007).

Many of these glycosides are formed from phenols, polyphenols, steroidal and terpenoidal

alcohols through glycosidic attachment to sugars. Among the sugars found in natural glycosides,

D-glucose is the most prevalent one, but L-rhamnose, D- and L-fructose and L-arabinose also

occur quite frequently. Of the pentoses, L-arabinose is more common than D-xylose and the

sugars often occur as oligosaccharides. The sugar moiety of a glycoside can be joined to the

aglycone in various ways, the most common being via an oxygen atom (O-glycoside). However,

this bridging atom can also be a carbon (C-glycoside), a nitrogen (N-glycoside) or a sulphur

atom (S-glycoside). By virtue of the aglycone and/or sugar, glycosides are extremely important

pharmaceutically and medicinally. For example, digitoxin is a cardiac glycoside found in the

foxglove plant (Digitalis purpurea). Glycosides that exert a prominent effect on heart muscle are

called cardiac glycosides, e.g. digitoxin from Digitalis purpurea. Their effect is specifically on

myocardial contraction and atrioventricular conduction (Saker and Nahar, 2007). Cardiac

glycosides are found only in a few plant families, e.g. Liliaceae, Ranunculaceae, Apocynaceae

and Scrophulariaceae are the major sources of these glycosides. Among the cardiac glycosides

isolated to date, digitoxin and digoxin, isolated from Digitalis purpurea and Digitalis lanata,

respectively, are the two most important cardiotonics. Digitoxin and digoxin are also found in in

Strophanthus seeds and squill (Saker and Nahar, 2007).

1.8.6 Steroids

Steroids are chemical messengers, also known as hormones. They are synthesized in glands and

delivered by the bloodstream to target tissues to stimulate or inhibit some process. Steroids are

nonpolar and therefore lipids. Their nonpolar character allows them to cross cell membranes, so

they can leave the cells in which they are synthesized and enter their target cells (Saker and

Nahar, 2007). Structurally, a steroid is a lipid characterized by a carbon skeleton with four fused

rings. All steroids are derived from the acetyl CoA biosynthetic pathway. Hundreds of distinct

steroids have been identified in plants, animals and fungi, and most of them have interesting

biological activity (Saker and Nahar, 2007). They have a common basic ring structures, three-

fused cyclohexane rings, together the phenanthrene part, fused to a cyclopentane ring system,

known as cyclopentaphenanthrene.

The main feature of steroid, as in all lipids, is the presence of a large number of carbon-

hydrogens that makes steroids nonpolar. The solubility of steroids in nonpolar organic solvents,

e.g. ether, chloroform, acetone and benzene, and general insolubility in water, results from their

significant hydrocarbon components. However, with the increase in number of hydroxyl or other

polar functional groups on the steroid skeleton, the solubility in polar solvents increases.

1.8.7 Terpenoid Terpenoids are compounds derived from a combination of two or more isoprene units. Isoprene

is a five carbon unit, chemically known as 2-methyl-1,3-butadiene. According to the isoprene

rule proposed by Leopold Ruzicka, terpenoids arise from head-to-tail joining of isoprene units.

Carbon 1 is called the ‘head’ and carbon 4 is the ‘tail’ (Saker and Nahar, 2007). Terpenoids are

found in all parts of higher plants and occur in mosses, liverworts, algae and lichens. Terpenoids

of insect and microbial origins have also been found. Terpenoids are classified according to the

number of isoprene units involved in the formation of these compounds (Saker and Nahar, 2007).

1.9 Extraction Procedures

1.9.1 Plant tissue homogenization Plant tissue homogenization in solvent has been widely used by researchers. Dried or wet, fresh

plant parts are grinded in a blender to fine particles, put in a certain quantity of solvent and

shaken vigorously for 5 to 10 minutes or left for 24 h after which the extract is filtered. The

filtrate then may be dried under reduced pressure and re-dissolved in the solvent to determine the

concentration. Some researchers however centrifuged the filtrate for clarification of the extract

(Das et al., 2010).

1.9.2 Serial exhaustive extraction

It is another common method of extraction which involves successive extraction with solvents of

increasing polarity from a non-polar (hexane) to a more polar solvent (methanol) to ensure that a

wide polarity range of compound could be extracted. Some researchers however, employ soxhlet

extraction of dried plant material using organic solvent (Das et al., 2010).

1.9.3 Soxhlet extraction Soxhlet extraction is only required where the desired compound has a limited solubility in a

solvent, and the impurity is insoluble in that solvent. If the desired compound has a high

solubility in a solvent then a simple filtration can be used to separate the compound from the

insoluble substance. The advantage of this system is that instead of many portions of warm

solvent being passed through the sample, just one batch of solvent is recycled. This method

cannot be used for thermolabile compounds as prolonged heating may lead to degradation of

compounds (Nikhal et al., 2010).

1.9.4 Maceration In maceration (for fluid extract), whole or coarsely powdered plant is kept in contact with the

solvent in a stoppered container for a defined period with frequent agitation until soluble matter

is dissolved. This method is best suitable for use in case of the thermolabile compounds (Ncube

et al., 2008).

1.9.5 Decoction This method is used for the extraction of the water soluble and heat stable constituents from

crude drug by boiling it in water for 15 minutes, cooling, straining and passing sufficient cold

water through to produce the required volume (Remington, 2002).

1.9.6 Infusion It is a dilute solution of the readily soluble components of the crude drugs. Fresh infusions are

prepared by macerating the solids for a short period of time with either cold or boiling water

(Remington, 2002).

1.9.7 Digestion

This is a kind of maceration in which gentle heat is applied during the maceration extraction

process. It is used when moderately elevated temperature is not objectionable and the solvent

efficiency of the menstrum is increased thereby (Remington, 2002).

1.9.8 Percolation

This is the procedure used most frequently to extract active ingredients in the preparation of

tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is

generally used. The solid ingredients are moistened with an appropriate amount of the specified

menstrum and allowed to stand for approximately 4 h in a well closed container, after which the

mass is packed and the top of the percolator is closed. Additional menstrum is added to form a

shallow layer above the mass, and the mixture is allowed to macerate in the closed percolator for

24 h. The outlet of the percolator then is opened and the liquid contained therein is allowed to

drip slowly. Finally, the marc is then pressed and the expressed liquid is added to the percolate.

Sufficient menstrum is added to produce the required volume, and the mixed liquid is clarified

by filtration or by standing followed by decanting (Handa et al., 2008).

1.9.9 Sonication The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000

kHz; this increases the permeability of cell walls and produces cavitation. Although the process

is useful in some cases, like extraction of rauwolfi a root, its large-scale application is limited

due to the higher costs. One disadvantage of the procedure is the occasional but known

deleterious effect of ultrasound energy (more than 20 kHz) on the active constituents of

medicinal plants through formation of free radicals and consequently undesirable changes in the

drug molecules (Handa et al., 2008).

1.9.10 Mode of action of phytochemicals in target insect Generally the active toxic ingredients of plant extracts are secondary metabolites that are evolved

to protect them from herbivores (Ghosh et al., 2012). The insects feed on these secondary

metabolites potentially encountering toxic substances with relatively non-specific effects on a

wide range of molecular targets. These targets range from proteins (enzymes, receptors,

signaling molecules, ion-channels and structural proteins), nucleic acids, biomembranes, and

other cellular components (Rattan, 2010). This in turn, affects insect physiology in many

different ways and at various receptor sites, the principal of which is abnormality in the nervous

system (such as, in neurotransmitter synthesis, storage, release, binding, and re-uptake, receptor

activation and function, enzymes involved in signal transduction pathway (Rattan, 2010).

Alkaloids which have been reported to be the major phytochemical of P.nitida has been found to

be a representative of such compounds that interact with ion channels, neurotransmitter

converters such as Acetylcholinerase (AChE) and monomine oxidase, indicating their possible

effects on the nervous system (Devonshire et al., 1992). Rattan (2010) reviewed the mechanism

of action of plant secondary metabolites on insect body and documented several physiological

disruptions, such as inhibition of acetylecholinestrase (by essential oils), GABA-gated chloride

channel (by thymol), sodium and potassium ion exchange disruption (by pyrethrin) and

inhibition of cellular respiration (by rotenone). Such disruption also includes the blockage of

calcium channels (by ryanodine), of nerve cell membrane action (by sabadilla), of octopamine

receptors (thymol), hormonal balance disruption, mitotic poisioning (by azadirachtin), disruption

of the molecular events of morphogenesis and alteration in the behaviour and memory of

cholinergic system (by essential oil), etc. Of these, the most important activity is the inhibition of

acetylcholinerase activity (AChE) as it is a key enzyme responsible for terminating the nerve

impulse transmission through synaptic pathway; AChE has been observed to be

organophosphorus and carbamate resistant, and it is well-known that the alteration in AChE is

one of the main resistance mechanisms in insect pests (Rattan, 2010).

1.10 Review of Synthetic Insecticides Vector control is a central, critical component of all malaria control Strategies (WHO, 2012). It

relies primarily on two interventions: long-lasting insecticidal nets (LLINs) and indoor residual

spraying (IRS). Use of both has increased significantly during the past 10 years as part of a drive

towards universal coverage of all populations at risk, saving hundreds of thousands of lives

(WHO, 2012). In sub-Saharan Africa, insecticide treated nets (ITNs) and indoor residual

insecticide spraying (IRS) are the cornerstones of malaria vector control. The use of chemical

insecticides still remains the predominant means of controlling insect vectors. However, the

emergence of insect resistant species, suggests an alternative replacement to this chemicals.

Currently, pyrethroids are the only class of insecticides approved for treating bednets or curtains

because of their high effectiveness and strong excito repellent effect on mosquitoes, yet low

mammalian toxicity (Etang et al., 2003). However, pyrethroid resistance in An. gambiaen sis.

has been described in West, East and Central Africa (Ranson et al., 2000; Etang et al., 2003).

1.10. Insecticide resistance Insecticide resistance is the term used to describe the situation in which the vectors are no longer

killed by the standard dose of insecticide (they are no longer susceptible to the insecticide) or

manage to avoid coming into contact with the insecticide. The emergence of insecticide

resistance in a vector population is an evolutionary phenomenon (Etang et al., 2004). Insecticide

resistance is widespread, it is now reported in nearly two thirds of countries with ongoing

malaria transmission (WHO, 2012). It affects all major vector species and all classes of

insecticides (WHO, 2012). According to World Health Organisation (2011a), if nothing is done

and insecticide resistance eventually leads to widespread failure of pyrethroids, the public health

consequences would be devastating. Much of the progress achieved in reducing the burden of

malaria would be lost. For example, current coverage with LLINs and IRS in the WHO African

region is estimated to avert approximately 220, 000 deaths among children under 5 years of age

every year (WHO, 2011b). If pyrethroids were to lose most of their efficacy, more than 55% of

the benefits of vector control would be lost, leading to approximately 120 000 deaths not averted

(WHO, 2012).

1.11 Classes of Insecticides

1.11.1 Pyrethroids

Pyrethroids are synthetic chemicals whose structures mimic the natural insecticide pyrethrum.

Pyrethrins are found in the flower heads of some plants belonging to the family Asteracae (e.g.,

chrysanthemums). These insecticides have the ability to knockdown insects quickly. Pyrethrums

can be degraded very easily by ultraviolet light which oxidizes the compounds. In general, this

phenomenon leads to lower environmental risk. Pyrethroids are used for both IRS and LLINs in

the form of α-cypermethrin, bifenthrin, cyfluthrin, deltamethrin, permethrin, λ-cyhalothrin and

etofenprox (WHO, 2006). These have been the chemicals of choice in public health for the past

few decades because of their relatively low toxicity to humans, rapid knockdown effect, relative

longevity (3–6 months when used for IRS) and low cost. They are the only insecticides used

currently in WHO recommended LLINs (WHO, 2006). Pyrethroids have many modes of action

on the mosquito vector. They open sodium channels, leading to continuous nerve excitation,

paralysis and death of the vector (Brown, 2006). They also have an irritant effect, causing an

excito-repellency response, resulting in hyperactivity, rapid knock-down, feeding inhibition,

shorter landing times and undirected flight, all of which reduce the ability of vectors to bite

(Brown, 2006).

1.11.2 Organochlorines

Organochlorines are used in IRS in the form of DDT, which was the insecticide used

predominantly in the eradication campaigns of the 1950s (WHO, 2012). At the Stockholm

Convention on Persistent Organic Pollutants in 2001, use of DDT was banned for all applications

except disease control, because of its environmental effects when used in large volumes in

agriculture. As the number of equally effective, efficient, alternative insecticides for public

health is limited, continued use of DDT was permitted until “locally safe, effective, and

affordable alternatives are available for a sustainable transition from DDT”. A WHO position

statement in 2006 (WHO, 2007) reasserted the public health value of DDT when used for IRS.

Like pyrethroids, DDT has been popular because of its rapid knock-down effect, relative

longevity (6–12 months when used for IRS) and low cost. Despite chemical structural

differences, DDT and pyrethroids have similar modes of action (Brown, 2006).

1.11.3 Organophosphates

Organophosphates comprise a vast range of chemicals, but those recommended for use for IRS

vector control are fenitrothion, malathion and pirimiphos-methyl (WHO, 2009). The insecticides

in this class are highly effective but do not induce an excito-repellency response from the vector,

and in their current formulations have shorter residual activity (2–3 months when used for IRS)

than pyrethroids and DDT (WHO, 1970). In addition, the organophosphates currently used for

malaria control are significantly more expensive than other insecticides. Organophosphates act

on the mosquito vector by inhibiting cholinesterase, preventing breakdown of the

neurotransmitter acetylcholine, resulting in neuromuscular overstimulation and death of the

vector (Brown, 2006).

1.11.4 Carbamates

Carbamates are used for IRS vector control in the form of bendiocarb (WHO, 2009). Like

organophosphates, this compound is highly effective and induces little or no excito-repellency

response from the vector. It has short residual activity (2–6 months when used for IRS) and is

more expensive than pyrethroids and DDT. The mode of action of carbamates is similar to that

of organophosphates (Brown, 2006).

1.12Mechanism of resistance

Insects acquire resistance to insecticides through various means. Resistant insects often exhibit

more than one of these resistance mechanisms at the same time. Resistance mechanisms can be

grouped into four categories (WHO, 2012).

1.12.1 Target-site resistance This occurs when the site of action of an insecticide (typically within the nervous system) is

modified in resistant strains, such that the insecticide no longer binds effectively and the insect is

therefore unaffected, or less affected, by the insecticide. Resistance mutations, known as knock-

down resistance (kdr) mutations, can affect acetylcholinesterase, which is the molecular target of

organophosphates and carbamates, or voltage-gated sodium channels (for pyrethroids and DDT)

(Insecticide Resistance Action Committee, 2011; President’s Malaria Initiative, 2007). Similarly,

cyclodiene (dieldrin) resistance is conferred by single nucleotide changes within the same codon

of a gene for a γ-aminobutyric acid (GABA) receptor. At least five point mutations in the

acetylcholinesterase insecticide-binding site have been identified that singly or in concert causes

varying degrees of reduced sensitivity to OPs and carbamate insecticides. (Sukhoruchenko et al.,

2008)

1.12.2 Metabolic resistance

Metabolic Resistance is related to the enzyme systems that all insects possess to detoxify foreign

materials. It occurs when increased or modified activities of an enzyme system prevent the

insecticide from reaching its intended site of action. The three main enzyme systems are:

esterases, mono-oxygenases and glutathione S-transferases. While metabolic resistance is

important for all four insecticide classes, different enzymes affect different classes (Insecticide

Resistance Action Committee, 2011; President’s Malaria Initiative, 2007). Metabolic resistance

is the most common mechanism and often presents the greatest challenge. Insects use their

internal enzyme systems to break down insecticides. Resistant strains may possess higher levels

or more efficient forms of these enzymes. In addition to being more efficient, these enzyme

systems also may have a broad spectrum of activity (i.e., they can degrade many different

insecticides) (IRAC, 2005).

1.12.3 Behavioural resistance

This is any modification in insect behavior that helps it to avoid the lethal effects of insecticides.

Several publications have suggested the existence of behavioural resistance and described

changes in vectors’ feeding or resting behaviour to minimize contact with insecticides. Studies in

New Guinea and the Solomon Islands showed that Anopheles farauti vectors stopped biting later

in the night (23:00–03:00) after the introduction of indoor DDT spraying and instead bit only in

the earlier part of the evening, before humans were protected by sleeping in a sprayed room

(Mouchet, 2008). In most cases, however, there are insufficient data to assess whether

behavioural avoidance traits are genetic or adaptive; genetic traits could have major implications

for the types of vector control interventions needed. All behavioural traits, however, may not be

negative, as they could lead mosquitoes to feed on non-human animals. It is also possible to

initially mistake the decline of a vector species as behavioural resistance. This mechanism of

resistance has been reported for several classes of insecticides, including organochlorines,

organophosphates, carbamates and pyrethroids (IRAC, 2005).

1.12.4 Cuticular/Penetration resistance

It is a reduced uptake of insecticide due to modifications in the insect cuticle that prevent or slow

the absorption or penetration of insecticides. Examples of reduced penetration mechanisms are

extremely limited and only one study has suggested correlation between cuticle thickness and

pyrethroid resistance in An. funestus (Wood et al., 2010). Microarray experiments have identified

two genes that encode cuticular proteins that are upregulated in pyrethroid-resistant strains of

Anopheles mosquitoes. Experience with other insects suggests that if cuticular resistance

emerges in mosquitoes it could have a significant impact when combined with other resistance

mechanisms. Behavioural and cuticular resistance mechanisms are rarer than the other

mechanisms and are perceived by most experts to be a lesser threat than chemical resistance.

1.13Insecticide Resistance Detection Techniques There are several phenogenetic methods available to diagnose resistance in populations of pest

species which enable the assessment of how shifts in composition and structure of a population

caused by pesticides, may affect its development geographically and over time. Among these,

easy-to-use toxicological methods have gained the most recognition worldwide. They enable the

determination of levels of population susceptibility to pesticides used, in relation to the ratio of

resistant and susceptible genotypes (Sakine, 2012). Resistance can be determined by using

conventional standard bioassay methods published by International Resistance Action

Committee (IRAC) and biochemical, immunological and molecular methods.

1.13.1 Conventional detection methods

The standard method of detection is to take sample of insects from the field and rear them

through to the next generations. Larvae or adults are tested for resistance by assessing their

mortality after exposure to a range of doses of an insecticide. For susceptible and field

populations, LD50 or LC50 values were calculated by using probit analysis.

The results are compared with those from standard susceptible populations. These methods

include some differences for the different pest species. These methods are published by

Insecticide Resistance Action Committee (IRAC, 2011). The other traditional method of

detecting insecticide resistance is to expose individual insects to a diagnostic single dose for a set

time period in a chamber impregnated with the insecticide or on a filter paper impregnated with

the insecticide. These tests only give an indication of the presence and frequency of resistance

and limited information can be gained as to the resistance mechanism (Sakine, 2012). Evolution

of resistance is most often based on one or a few genes with major effect. Before a susceptible

population is exposed to an insecticide, resistance genes are usually rare because they typically

reduce fitness in the absence of the insecticide. When an insecticide is used repeatedly, strong

selection for resistance overcomes the normally relatively minor fitness costs associated with

resistance when the population is not exposed to insecticide (Sakine, 2012).

1.13.2 Immunological detection methods

This method is available only for specific elevated esterases in collaboration with laboratories

that have access to the antiserum. There are no monoclonal antibodies, as yet, available for this

purpose. An antiserum has been prepared against E4 carboxylesterase in the aphid Myzus

persicae. An affinity purified IgG fraction from this antiserum has been used in a simple

immunoassay to discriminate between the three common resistant variants of M. persicae found

in the UK field populations (Devonshire et al., 1996)

1.13.3 Biochemical detection of insecticide resistance Biochemical assays/techniques may be used to establish the mechanism involved in resistance.

When a population is well characterised some of the biochemical assays can be used to measure

changes in resistance gene frequencies in field populations under different selection pressure

(Sakine, 2012).

1.13.4 Detection of monooxygenase (cytocrome P450) based insecticide resistance The levels of oxidase activity in individual pests are relatively low and no reliable microtitre

plate or dot-blot assay has been developed to measure p450 activity in single insects. The p450s

are also a complex family of enzymes, and it appears that different cytocromes p450s produce

resistance to different insecticides (Sakine, 2012).

CHAPTER TWO

2.0 METHODOLOGY 2.1 Collection of Plant Materials

Plant samples (leaves and fruits) of Picralima nitida were collected from Umuagwu Akabor in

Oguta Local Government Area of Imo State State, Nigeria. The plant was identified by A.O.

Ozioko, a professional plant taxonomist at the Herbarium section of the International Center for

Ethno-medicine and Drug Development.

2.2 Sample Preparation and Extraction

The leaves of the test plant were rinsed with water to remove dirt. They were then spread out on

a clean surface and allowed enough time to air-dry under shade at room temperature (28 ± 2°C).

The seeds and pulps were extracted from the fruit; dried leaves, fresh seeds and pulp were then

pulverized using an electric blender.

Two kilogram of each pulverized plant samples (leave, seed and pulp of P. nitida), were

subjected to extraction using soxhlet extractor (Electrothermal heating mantle, model Ms-9506

with pyrex soxhlet column, condenser and round bottom flask). The samples were packed into

the soxhlet column to 2/3 its volume. The column was then inserted into the flask and filled with

the solvents. The column was filled until the solvents began to siphon. The soxhlet was then

placed on the heating mantle, and heat adjusted to 40oc. The solvents were allowed to reflux

repeatedly, until refluxing solvent was clear and free from extracts. 2.5L of 95% methanol and

3.0L distilled water were used respectively. The extracted content was then subjected to rotary

evaporator (Bibby Sterlin Ltd, England, RE. 200) until solvents were completely evaporated to

get the solidified crude extracts. The crude extracts thus obtained was stored in sterilized amber

coloured bottles and maintained at 4oC in a refrigerator.

The percentage yields of the extracts were determined as:

% Yield = Weight of dry extract × 100

Weight of sample extracted 1

5000mg each, of the methanolic and aqueous extracts were dissolved in 5ml of

dimethylsulphoxide (DMSO). The extracts were then diluted in 1000ml of distilled water to

obtain a stock solution of 5.00mg/ml. From the stock, graded concentrations of 4.00mg/ml,

3.00mg/ml, 2.00mg/ml, 1.00mg/ml and 0.50mg/ml were then obtained. Using the method

described by WHO (1970).

2.3 Phytochemical Screening

Crude methanolic and aqueous extracts of the leave, seed and pulp of P. nitida were screened for

their phytochemical components using the methods described by Harborne (1984) and Evans,

(2002).

2.3.1 Test for alkaloids

About 20ml of 5% sulphuric acid in 50% ethanol was added to about 2g of each of the sample.

This was then heated on a boiling water bath for 10 minutes, cooled and filtered. The filtrate was

transferred into four test tubes, each containing 2ml of the filtrate and used for the following

tests.

a. Few drops of Dragendoff’s reagent (a solution of bismuth iodide in potassium iodide)

were added to first portion of the filtrate, and homogenized. A brick red precipitate

indicated the presence of alkaloids.

b. About two drops of Wagner’s reagents (a solution of iodine in potassium iodide) were

added to the second portion and swirled for few seconds. A brownish-red precipitate

indicated the presence of alkaloids.

c. About two drop’s of Meyer’s reagent (a solution of mercury iodide in potassium iodide)

were added to the third portion and homogenized for few seconds. A creamy, dirty white

precipitate indicated the presence of alkaloids.

d. Two drops of picric acid (1%) solution was added to the fourth portion and homogenized

for 30 seconds. A reddish precipitate indicated the presence of alkaloids.

2.3.2 Test for saponins

About 20ml of distilled water was added to 0.5g of each of the sample in a 100cm3 beaker and

boiled gently on a hot water bath for 20 minutes. The mixture was filtered hot and allowed to

cool. The filtrate was used for the following test.

a. Frothing test: A volume of 20ml distilled water was added to 5ml of the filtrate in a test

tube, and shaken vigorously. A stable froth (foam) upon standing for about 30 seconds

indicates the presence of saponin.

b. Emulsion test: Two drops of olive oil was added to 5ml of the filtrate in the test tube

above. Formation of emulsion indicates the presence of saponin.

2.3.3 Test for phenols/tannins

About 100g of each of the sample was extracted in 10ml of distilled water. The solution was

heated in a boiling water bath for 3 minutes and filtered. Then 2ml aliquots of the filtrates were

placed in test tube and the following tests were performed.

a. Few drops of 10% neutral aqueous FeCl3 was added to the aliquot of the diluted solution.

Development of green to blue-black precipitates indicates the presence of tannins.

b. A volume of 1ml of 10% lead acetate solution was added to a portion of the filtrate in a

test tube, and homogenized. A coloured precipitate indicates the presence of phenols.

2.3.4 Test for anthraquinones

Approximately 5g of each of the sample was boiled with 10ml aqueous sulphuric acid and

filtered while hot. The filtrate was shaken with 5ml of benzene. The benzene layer was then

separated and 10% ammonia solution was added to half of its volume. A pink, red or violet

colouration in the ammonia phase (lower layer) indicated the presence of anthraquinone.

2.3.5 Test for glycosides

About 300mg of each of the samples was dissolved in 10ml of distilled water and the resulting

solution was filtered. A 5ml of equi-volume mixture of Fehling’s solution 1 and 11 was added to

a 2ml aliquot of the aqueous solution obtained above. The mixture was then homogenized and

heated in a water bath for not less than 5 minutes. Brick red precipitates indicates the presence of

free reducing sugars.

2.3.6 Test for flavonoids About 10ml of ethyl acetate was added to about 0.2g of each of the sample and heated on a water

bath at 40oC for 3 minutes. The mixture was cooled, filtered and used for the following test.

• Ammonium Test: About 4ml of the filtrate was shaken with 1ml of dilute ammonia

solution. Layers were formed and allowed to separate. An intense yellow colour in the

ammoniacal layers indicates the presence of flavonoids.

• To the yellow coloured solution, 3 drops of concentrated sulphuric acid was added.

Disappearance of the yellow colour indicates the presence of flavonoids.

• 1% Ammonium Chloride Solution Test: To 4ml of the filtrate, 1ml of 1% ammonium

chloride solution was added. A yellow colour indicates the presence of flavonoids.

2.3.7 Test for steroids and terpenoids About 5ml of 96% ethanol was added to 1.0g of each of the samples and refluxed for 2 minutes

and filtered. The filtrate was concentrated to 2.5ml on a water-bath and 5ml hot water added. The

mixture was allowed to stand for 1 hr. Waxy matter observed was filtered off. The filtrate was

extracted with 2.5ml chloroform. The layers observed were separated using separating funnel.

A volume of 1ml of concentrated H2SO4 was carefully added to 0.5ml chloroform extract and

shaken to form lower layers. Reddish Brown interface shows the presence of steroids.

Another 0.5ml of the chloroform extract was evapourated to dryness on a water bath at 40oC.

This was heated further with 3ml concentrated H2SO4 for 10 minutes on a water bath at 40oC.

Grey colouration indicated the presence of terpenoids.

2.4 Raising of An. gambiae larvae

The 4th instar Larvae of A. gambiae used in this investigation were raised through the assistance

of Mr. Lucky Uwakwe of Arbo-viral Research Laboratory, Enugu and Dr. Goddy Ngwu of the

University of Nigeria, Nsukka respectively. In the laboratory, the larvae were transferred to

enamel larval trays until adult emergence. After emergence, the mosquitoes were identified by a

professional Parasitologist, Dr Goddy Ngwu of the Department of Zoology, University of

Nigeria, Nsukka. Cyclic generations of A. gambiae were maintained in a 29 cm x 21.5 cm x 56.5

cm cages with potted plants. Mean room temperature of (27± 20C) and a relative humidity of 70-

80 percent were maintained in the insectary. The adult mosquitoes were fed on ten per cent

glucose solution. For continuous maintenance of mosquito colony, the adult female mosquitoes

were blood fed with laboratory reared albino mice. Ovitraps were placed inside the cages for egg

laying. The eggs laid were then transferred to enamel larval trays maintained in the larval rearing

chamber. The larvae were fed with larval food (Quaker oat and yeast in the ratio 3:1). 3rd and 4th

instar larvae were then picked for larvicidal bioassay.

Figure 2.1: The Fourth Instar Anopheles Larva following incubation

2.5 Larvicidal Bioassay

Larvicidal bioassay of individual plant extracts was tested against 4th instar larvae of A.

gambiae. The tests were conducted in 100ml glass beakers, in accordance with (WHO, 2005)

protocol with slight modification. Three replicates and a control were run simultaneously during

each trial. For control, 5ml of 20% DMSO in 995ml of distilled water was used. Twenty healthy

larvae were introduced into each glass beaker and mortality was observed at 24, 48 and 72 hrs

after treatment with extract concentrations of 5.00, 4.00, 3.00, 2.00, 1.00, and 0.5mg/ml. The

treatments were maintained at room temperature. Larvicidal activity of each extract was

determined, by counting the number of dead larvae on daily basis (24hrs interval). The moribund

and dead larvae in the three replicates were combined and expressed as percentage mortality for

each concentration. Dead larvae were recorded when they failed to move after probing with a

needle. Moribund larvae were those unable to rise to the surface within reasonable period of

time. The percentage mortality was calculated and analysis of data was carried out by employing

probit analysis.

% Mortality = Number of Dead Larvae × 100

Number of Larvae Introduced 1

2.5.1 Correction for control mortality

The Corrected percentage mortality was used were a proportion of the insects in the control

batches died during the experiment.

To correct for this the Abbott formula was used (Abbott, 1925).

% Po – % Pc

P = × 100

100 – % Pc

Where P is the corrected mortality, Po is the observed mortality and Pc is the control

Mortality, all expressed in percentages.

2.6 Determination of LC50 and LC95

The 24, 48 and 72h lethal concentration values (LC50 and LC95) will be determined by probit

analysis as described by Finney (1971). SPSS version 16 will be employed in the analysis.

CHAPTER THREE

3.0 RESULTS

3.1 Larvicidal Effect of Plant Extracts on Anopheline Larva

The larvicidal activity of Methanolic and Aqueous extracts of the Leaf, Seed and Pulp of P.

nitida was evaluated on 4th instar larvae of the malaria vector Anopheline mosquito. All the

extracts tested exhibited larvicidal potential against the test organism. All the extracts proved to

be toxic to the test organism, though there was a remarkable difference in the concentrations and

timing of their activity.

3.1.1 Aqueous Leaf Extract

The lethality pattern of the aqueous leaf extracts of P. nitida on the Anopheline larvae. From the

figure, it was evident that at extract concentration of 5.00 mg/ml and 24 h exposure time, the test

organism showed 50% mortality. This 50% mortality was also obtained after 48 h of test at a

concentration of 0.5 mg/ml. However 100% mortality was achieved by extract concentration of

4.00 mg/ml at 72 h of test (Figure 3.1).

3.1.2 Methanolic Leaf Extract

The lethality pattern of the Methanolic leaf extract of P. nitida on Anopheline larvae at

concentrations ranging from 0.5 mg/ml to 5.00 mg/ml revealed that the methanolic leaf extract

could not kill 50% of the test organism at test times of 24 and 48 h. However at 72 h, 95%

mortality was exhibited at concentrations of 3.00 mg /ml to 5.00 mg/ml (Figure 3.2).

Figure 3.1: Effect of Concentrations of Aqueous Leaf Extract of P. nitida on An. gambiae

0

20

40

60

80

100

120

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity

Concentration (mg/ml)

24 hrs

48 hrs

72 hrs

Figure 3.2: Effect of Concentrations of Methanolic Leaf Extract of P. nitida on An. gambiae

3.1.3 Aqueous Seed Extract

The lethality pattern of the aqueous seed extract of P. nitida on Anopheline larvae showed that

though the aqueous seed extract exhibited a certain degree of larvicidal effect on the test

0

20

40

60

80

100

120

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity


24 hrs

48 hrs

72 hrs

organism, its effect was minimal as all concentration of the extracts failed to kill 50% of the

larvae even at the highest time of exposure (72 h) (Figure 3.3).

3.1.4 Methanolic Seed Extract

The lethality pattern of methanolic seed extract of P. nitida on Anopheline larvae revealed that

the methanolic seed extract of P. nitida had an excellent larvicidal potential against the test

organism. 100% lethality was observed at concentrations of 3.00 mg/ml, 1.00 mg/ml and 0.5

mg/ml at 24 h, 48 h and 72 h respectively (Figure 3.4).

Figure 3.3: Effect of Concentrations of Aqueous Seed Extract of P. nitida on An. gambiae

0

5

10

15

20

25

30

35

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity


24 hrs

48 hrs

72 hrs

Figure 3.4: Effect of Concentrations of Methanolic Seed Extract of P. nitida on An. gambiae

3.1.5 Aqueous Pulp Extract

0

20

40

60

80

100

120

0.0 0.5 1.0 2.0 3.0 4.0 5.0

% M

orta

lity


24 hrs

48 hrs

72 hrs

The lethality pattern of the aqueous pulp extract of P. nitida on Anopheline Larva revealed a

moderate larvicidal activity against the Anopheline larva. At concentrations of 5.00 mg/ml, 4.00

mg/ml and 3.00 mg/ml percentage mortalities of 53, 50 and 58 respectively were observed over

exposure times of 24 h, 48 h and 72 h respectively (Figure 3.5).

3.1.6 Methanolic Pulp Extract

The lethality pattern of methanolic pulp extract of P. nitida on Anopheline Larvae also showed

moderate effect on the test organism. At all the concentrations (0.5 mg/ml to 5.0 mg/ml) used in

this research, the extract failed to achieve 50% mortality at the various times of exposure. The

highest concentration (5.00 mg/ml), at the highest exposure time (72 h) only showed 47%

mortality of the test organism (Figure 3.6).

Figure 3.5: Effect of Concentrations of Aqueous Pulp Extract of P. nitida on An. gambiae

0

10

20

30

40

50

60

70

80

0.000 0.500 1.000 2.000 3.000 4.000 5.000

%

Mor

talit

y


24 hrs

48 hrs

72 hrs

Figure 3.6: Effect of Concentrations of Methanolic Pulp Extract of P. nitida on An. gambiae

3.1.7 72 h Effect of the Extracts

0

5

10

15

20

25

30

35

40

45

50

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity


24 hrs

48 hrs

72 hrs

The 72 h lethality pattern of the various extracts on the Anopheline larvae is shown in Figure 3.7.

Figure 3.7 revealed that of the six extracts used in this research, only four (Aqueous leaf,

Methanolic leaf, Methanolic seed and Aqueous pulp) exhibited 50% mortality at 72 h, whereas

the aqueous seed and methanolic pulp extracts could not achieve a 50% mortality. Aqueous leaf

and Methanolic seed extracts however showed a100% mortality of the larvae.


The 48 h lethality pattern of the extracts in the test organism, Anopheline larvae is shown

in Figure 3.8. At 48 h test time, three of the extracts (methanolic seed, aqueous leaf and aqueous

pulp) showed 50% mortality. However, only one (methanolic seed) gave 100% mortality at a

concentration of 1.00 mg/ml.


The 24 h lethality pattern of P. nitida extracts on the test organism- Anopheline larvae is shown

in Figure 3.9. The Methanolic seed, aqueous leaf and aqueous pulp extracts were able to elicit

50% mortality of the test organism. However only the Methanolic seed extract could elicit 100%

mortality at a high concentration of 3.00 mg/ml.

Figure 3.7: Comparative Activity of the Extracts on An. gambiae at 72 hours

0

20

40

60

80

100

120

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity


Aqueous Leaf Methanolic Leaf Aqueous SeedMethanolic Seed Aqueous Pulp Methanolic Pulp


0

20

40

60

80

100

120

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity


Aqueous Leaf Methanolic Leaf Aqueous SeedMethanolic Seed Aqueous Pulp Methanolic Pulp


3.2 Relative Median Potency

0

20

40

60

80

100

120

0.000 0.500 1.000 2.000 3.000 4.000 5.000

% M

orta

lity


Aqueous Leaf Methanolic Leaf Aqueous SeedMethanolic Seed Methanolic Pulp Aqueous Pulp

The toxicities of two or more insecticides are compared on the basis of potency or the reciprocal

of an equitoxic dose or concentration. The potency of the extracts was compared using probit

analysis, and results obtained were as follows.

Table 3.1 shows the relative median potency of the aqueous extracts (Aqueous leaf (AL),

Aqueous seed (AS) and Aqueous pulp (AP)) at 24 h trial. A comparism of the different extracts

shows the relative median potency of the extracts as; AL/AS = 0.18, AP/AL = 0.40, AL/AS =

5.48, AP/AS = 2.20, AS/AP = 0.46 and AL/AP = 2.50.

Table 3.2 shows the relative median potency of the 48 h trial of the aqueous extracts. The table

reveals the relative potencies as: AS/AL = 0.05, AP/AL = 0.10, AL/AS = 18.90, AP/AS = 1.85,

AS/AP = 0.54 and AL/AP =10.24.

Table 3.1: Relative Median Potency Estimates for 24 Hrs Aqueous Extracts

Key: Aqueous Leaf (AL), Aqueous Seed (AS), Aqueous Pulp (AP)

Extracts

Relative Median Potency at 95% C.L Relative Median Potency at 95% C.L with LOG Transform

(i) (j) Potency Lower Bound Upper Bound Log Potency Lower Bound Upper Bound AL AS 0.183 0.049 0.393 -0.739 -1.312 -0.406 AP 0.401 0.214 0.602 -0.397 -0.670 -0.220 AS AL 5.479 2.546 20.501 0.739 0.406 1.312 AP 2.197 1.218 5.519 0.342 0.086 0.742 AP AS 0.455 0.181 0.821 -0.342 -0.742 -0.086 AL 2.495 1.660 4.676 0.397 0.220 0.670


Extracts


(i) (j) Potency Lower Bound Upper Bound Log Potency Lower Bound Upper Bound AL AS 0.053 0.012 0.135 -1.276 -1.925 -0.871 AP 0.098 0.033 0.195 -1.010 -1.487 -0.710 AS AL 18.901 7.429 84.123 1.276 0.871 1.925 AP 1.846 1.232 3.221 0.266 0.090 0.508 AP AS 0.542 0.310 0.812 -0.266 -0.508 -0.090 AL 10.238 5.134 30.690 1.010 0.710 1.487 Key: Aqueous Leaf (AL), Aqueous Seed (AS), Aqueous Pulp (AP)

Table 3.3 shows the 72 h relative median potency of the aqueous extracts. The relative potencies

are shown below: AS/AL = 0.04, AP/AL = 0.11, AL/AS = 22.52, AP/AS = 2.44, AS/AP = 0.41

and AL/AP = 9.21.

Table 3.4 shows the relative median potency of the methanolic extracts at 24 h trial. The results

revealed the relative potencies as: MS/ML = 269.76, MP/ML = 2.40, ML/MS = 0.004, MP/MS

= 0.01, MS/MP = 112.49 and ML/MP = 0.42.


Extracts


(i) (j) Potency Lower Bound Upper Bound Log Potency Lower Bound Upper Bound AL AS 0.044 0.003 0.165 -1.352 -2.471 -0.782 AP 0.109 0.019 0.265 -0.964 -1.714 -0.577 AS AL 22.515 6.052 295.53 1.352 0.782 2.471 AP 2.444 1.439 6.359 0.388 0.158 0.803 AP AS 0.409 0.157 0.695 -0.388 -0.803 -0.158 AL 9.212 3.775 51.780 0.964 0.577 1.714 Key: Aqueous Leaf (AL), Aqueous Seed (AS), Aqueous Pulp (AP)

Table 3.4: Relative Median Potency Estimates for 24 Hrs Methanolic Extracts

Extracts


(i) (j) Potency Lower Bound Upper Bound Log Potency Lower Bound Upper Bound ML MS 269.763 16.390 2.247E7 2.431 1.215 7.352 MP 2.398 1.481 11.673 0.380 0.170 1.067 MS ML 0.004 4.450E-8 0.061 -2.431 -7.352 -1.215 MP 0.009 4.592E-7 0.102 -2.051 -6.338 -0.991 MP MS 112.493 9.787 2177504.764 2.051 0.991 6.338 ML 0.417 0.086 0.675 -0.380 -1.067 -0.170 Key: methanolic Leaf (ML), Methanolic Seed (MS), Methanolic Pulp (MP)

Table 3.5 shows the 48 h relative median potencies of the methanolic extracts. From the result,

the relative potencies of the extracts are: MS/ML = 3.55E3, MP/ML = 0.37, ML/MS = 0.000,

MP/MS = 0.000, MS/ MP = 9.703E3 and ML/MP = 2.73.

Table 3.6 shows the relative median potency of the methanolic extracts at a 72 h trial. The results

reveal the relative potencies of the different extracts to be: MS/ML = 25.30, MP/ML = 0.09,

ML/MS = 0.04, MP/MS = 0.003, MS/MP = 291.29 and ML/MP = 11.51.


Key: methanolic Leaf (ML), Methanolic Seed (MS), Methanolic Pulp (MP)

Extracts


(i) (j) Potency Lower Bound Upper Bound Log Potency Lower Bound Upper Bound ML MS 3.553E3 71.978 4.007E9 3.551 1.857 9.603 MP 0.366 0.041 0.729 -0.436 -1.383 -0.138 MS ML 0.000 2.496E-10 0.014 -3.551 -9.603 -1.857 MP 0.000 1.209E-11 0.009 -3.987 -10.917 -2.064 MP MS 9.703E3 115.758 8.269E10 3.987 2.064 10.917 ML 2.731 1.373 24.179 0.436 0.138 1.383


Extracts


(i) (j) Potency Lower Bound Upper Bound Log Potency Lower Bound Upper Bound ML MS 25.302 2.977 364.351 1.403 0.474 2.562 MP 0.087 0.025 0.188 -1.061 -1.601 -0.726 MS ML 0.040 0.003 0.336 -1.403 -2.562 -0.474 MP 0.003 0.000 0.040 -2.464 -3.969 -1.394 MP MS 291.293 24.754 9301.309 2.464 1.394 3.969 ML 11.513 5.320 39.899 1.061 0.726 1.601 Key: methanolic Leaf (ML), Methanolic Seed (MS), Methanolic Pulp (MP)

3.3 Median Lethal Concentration

The median lethal concentration or dose is a quantitative expression of tolerance of a particular

species under a given condition or location. It is a definitive biological characteristic and

depends on other physiological and physical characteristics such as age, sex, rearing conditions

and temperature. Usually the abbreviation LC50 is used for the 50% lethal concentration. The

other levels are abbreviated LD90 or LD95 to refer to the 90% and 95% lethal concentrations,

respectively. The higher the LC50 value, the lower the toxicity of the extracts. The larvae in this

research were tested for resistance by assessing their mortality after exposure to a range of

concentrations of the extracts over a range of time. Results obtained are presented below.

Table 3.7 shows the LC50 and LC95 of the aqueous leaf extracts at 24, 48 and 72 h interval. The

table indicates that at 24 h trial, the LC50 and LC95 of the extract were 3.141 mg/ml and 42.154

mg/ml respectively. At 48 h, the LC50 and LC95 were 0.352 mg/ml and 4.730 mg/ml and at 72

hours LC50 and LC95 were found to be 0.164 mg/ml and 2.201 mg/ml.

Table 3.8 shows the 24, 48 and 72 h LC50 and LC95 of the methanolic leaf extract. From the table,

the LC50 and LC95 are; 48.38 mg/ml and 657.79 mg/ml at 24 h, 5.82 mg/ml and 79.09 mg/ml at

48 h and 0.33 mg/ml and 4.52 mg/ml respectively.

Table 3.7: LC50 and LC95 of Aqueous Leaf Extracts at Varying Time Intervals.

Time

Lethality

LC50 and LC95 at 95% C.L Conc.

(mg/ml) Lower Bound

Upper Bound

Log Conc.

Lower Bound

Upper Bound

24 hrs LC50 LC95

3.141 42.154

2.399 22.846

4.286 110.030

0.497 1.625

0.380 1.359

0.632 2.042

48 hrs LC50 LC95

0.352 4.730

0.199 3.246

0.529 7.911

-0.453 0.675

-0.702 0.511

-0.277 0.898

72 hrs LC50 LC95

0.164 2.201

0.077 1.463

0.278 3.562

-0.785 0.343

-1.116 0.165

-0.556 0.552

Table 3.8: LC50 and LC95 of Methanolic Leaf Extracts at Varying Time Intervals

Time

Lethality


(mg/ml) Lower Bound

Upper Bound

Log Conc.

Lower Bound

Upper Bound

24 hrs LC50 LC95

48.383 657.786

23.534 234.509

116.317 4237.819

1.685 2.818

1.372 2.370

2.066 3.627

48 hrs LC50 LC95

5.817 79.089

3.826 39.093

8.530 303.078

0.765 1.898

0.583 1.592

0.931 2.482

72 hrs LC50 LC95

0.333 4.523

0.134 3.209

0.539 8.163

-0.478 0.655

-0.873 0.506

-0.269 0.912

Table 3.9 shows the LC50 and LC95 of the aqueous seed extracts at the varying time intervals.

The table shows the LC50 and LC95 of the extracts to be 17.67 mg/ml and 216.06 mg/ml at 24 h

trial, 13.46 mg/ml and 164.61 mg/ml at 48 h, 10.56 mg/ml and 129.10 mg/ml respectively.

Table 3.10 shows the LC50 and LC95 of the methanolic seed extract at various time intervals; 24

h, 48 h and 72 h. From the table, the 24 h LC50 and LC95 are 0.87 mg/ml and 0.74 mg/ml, at 48 h

the LC50 and LC95 are 0.02 mg/ml and 0.18 mg/ml respectively. At 72 h, the LC50 and LC95 are

0.02 mg/ml and 0.12 mg/ml respectively.

Table 3.9: LC50 and LC95 of Aqueous Seed Extracts at Varying Time Intervals

Time

Lethality


(mg/ml) Lower Bound

Upper Bound

Log Conc.

Lower Bound

Upper Bound

24 hrs LC50 LC95

17.668 216.064

11.320 73.074

41.480 3476.249

1.247 2.335

1.054 1.864

1.618 3.541

48 hrs LC50 LC95

13.461 164.614

9.053 55.593

31.224 2750.668

1.129 2.216

0.957 1.745

1.494 3.439

72 hrs LC50 LC95

10.557 129.102

7.390 45.079

22.497 1995.113

1.024 2.111

0.869 1.654

1.352 3.300

Table 3.10: LC50 and LC95 of Methanolic Seed Extracts at Varying Time Intervals

Time

Lethality


(mg/ml) Lower Bound

Upper Bound

Log Conc.

Lower Bound

Upper Bound

24 hrs LC50 LC95

0.087 0.739

0.000 0.000

0.793 3.864

-1.062 -0.132

-16.378 -7.210

-0.101 0.587

48 hrs LC50 LC95

0.021 0.182

0.000 0.000

0.231 0.951

-1.671 -0.741

-19.039 -9.797

-0.636 -0.022

72 hrs LC50 LC95

0.015 0.124

0.000 0.000

0.255 1.174

-0.835 -0.905

-21.315 -12.123

-0.594 0.070

Table 3.11 shows the 50 and 95% lethal concentrations of the aqueous pulp extract at 24, 48 and

72 h interval. The result showed the LC50 at 24, 48 and 72 h to be 4.98 mg/ml, 4.30 mg/ml and

2.79 mg/ml respectively. The LC95 were shown to be 18.541 mg/ml, 16.01 mg/ml and 10.40

mg/ml respectively.

Table 3.12 shows the LC50 and LC95 of the methanolic pulp extract at 24, 48 and 72 h

respectively. The finding showed the 24 h LC50 and LC95 to be 12.29 mg/ml and 96.99 mg/ml

respectively. At 48 h the LC50 and LC95 was shown to be 9.700 mg/ml and 76.58 mg/ml

respectively. At 72 h, the values for the LC50 and LC95 were 6.49 mg/ml and 51.24 mg/ml

respectively.

Table 3.11: LC50 and LC95 of Aqueous Pulp Extracts at Varying Time Intervals

Time

Lethality


(mg/ml) Lower Bound

Upper Bound

Log Conc.

Lower Bound

Upper Bound

24 hrs LC50 LC95

4.979 18.541

4.334 14.464

5.821 25.489

0.697 1.268

0.637 1.160

0.765 1.406

48 hrs LC50 LC95

4.299 16.008

3.770 12.637

4.970 21.667

0.633 1.204

0.576 1.102

0.696 1.336

72 hrs LC50 LC95

2.792 10.397

2.475 8.489

3.157 13.452

0.446 1.017

0.394 0.929

0.499 1.129

Table 3.12: LC50 and LC95 of Methanolic Pulp Extracts at Varying Time Intervals

Time

Lethality


(mg/ml) Lower Bound

Upper Bound

Log Conc.

Lower Bound

Upper Bound

24 hrs LC50 LC95

12.285 96.993

8.647 41.004

23.991 643.793

1.089 1.987

0.937 1.613

1.380 2.809

48 hrs LC50 LC95

9.700 76.584

7.143 34.136

16.997 452.613

0.987 1.884

0.854 1.533

1.230 2.656

72 hrs LC50 LC95

6.490 51.236

5.057 24.076

10.158 271.486

0.812 1.710

0.704 1.382

1.007 2.434

3.4 Median Lethal Time (LT50)

The median lethal time which is the time taken to kill 50% of the test organism was also

estimated. Result obtained is presented in Figure 3.10. The figure shows the LT50 values of the

extracts at concentrations ranging from 0.5 mg/ml to 5.0 mg/ml. From the figure it can be seen

that the methanolic seed extract has the lowest range of LT50, with 3.6 h LT at concentration 1.0

mg/ml. The aqueous leaf extracts also has moderately low values with 28 hrs as its LT50 at 1.0

mg/ml. The methanolic leaf extract showed the third lowest LT values with an LT50 of 57 h at

concentration of 1.0 mg/ml.

Figure 3.10: Median Lethal Times of Samples.

Key: Methanolic Leaf (ML), Aqueous Leaf (AL), Methanolic Seed (MS)

1.871.76 1.74 1.71 1.68 1.65

1.72

1.44 1.43 1.41 1.36 1.33

0.75

0.560.43

0.30 0.27 0.26

0.00

0.50

1.00

1.50

2.00

2.50

0.50 1.00 2.00 3.00 4.00 5.00

Log

Tim

e (H

rs)


ML AL MS

3.5 Percentage Yield of Samples

Table 3.13 shows the percentage yield of the samples. The table reveals that the leaf showed a

greater yield for both aqueous and methanol. However, there was no significant difference in

yield. The methanolic seed sample showed a relatively higher yield compared to the aqueous

seed extract. The pulp sample showed the least yield for both aqueous and methanol, though the

methanolic yield was remarkably higher than the aqueous yield.

Table 3.13: Percentage Yield of Samples

Samples Methanolic Aqueous

Leaf 21.65 21.37

Seed 8.33 3.85

Pulp 3.93 0.89

3.6 Phytochemical Screening of Samples

The phytochemical screening of the crude methanolic and aqueous extracts of P. nitida revealed

the presence of bioactive substances, which includes: alkaloids, cardiac glycosides, saponins,

tannins, flavonoids, terpenes and steroids (Table: 3.14).

Tabe 3.14: Phytochemical Result of Picralima nitida Samples

Key: + Present - Absent

Samples Alkaloids Cardiac Glycosides

Flavonoid Saponin Tannins Terpenes Steroids

Methanolic Leaf Extract + - + + + + +

Methanolic Seed Extract + + - + + + +

Methanolic Pulp Extract + - - - + - +

Aqueous Leaf Extract + + + + + + -

Aqueous Seed Extract - + + + + + -

Aqueous Pulp Extract - - + + + + -

CHAPTER FOUR

4.0 DISCUSSION AND CONCLUSION

4.1 Discussion

The plant kingdom has proved to be a reliable reservoir of potent phytochemicals which can

serve as suitable, efficient, readily available and eco-friendly alternatives in the fight against

insect pest. In line with the search for compounds with excellent activity against insect pest, this

work evaluates the larvicidal potentials of crude methanolic and aqueous extracts of different

parts (Leaf, Seed and pulp) of the plant Picralima nitida on the malaria vector, Anopheles

gambiae.

It is pertinent to state that no previous work on the larvicidal potential of seed and pulp samples

of this plant has been conducted, though Ubulom et al. (2012) investigated the larvicidal

potentials of crude ethanolic and aqueous extracts of the leaf. The leaf sample used in this work

gave yield value of 21.65 and 21.37% w/w with methanol and aqueous extraction respectively

(Table 3.13), contrary to the 5.26 and 3.52% w/w yield reported for aqueous and ethanolic

extraction of the leaf sample by Ubulom et al. (2012). This difference in yield might have

resulted due to the difference in extraction procedures given that Ubulom et al. (2012) used cold

maceration whereas Soxhlet extraction was used in this research. Other factors such as the age of

the plant might have also contributed (Sukumar et al., 1991). Although, there was no significant

difference (P≤0.05) in the methanolic and aqueous yield of leaf sample, this was not true for the

seed and pulp extract. The seed sample gave a yield of 8.33% w/w with methanol as against

3.85% w/w yield with aqueous extraction (Table 3.13). The pulp sample was next with a yield of

3.93%w/w with methanol and then 0.89%w/w yield with aqueous (Table 3.13). This difference

in yield seen between the samples might be due to differences in concentration of

phytochemicals present within individual samples, nature of the samples, sample grain size and

solubility of phytochemicals (Ghosh et al., 2012). The difference in the results of percentage

yield obtained from this research with reports of previous works might be as a result of

difference in solvents used, bearing in mind that most previous works made use of ethanol and

not methanol. In line with Ghosh et al. (2012), difference in the methods of extraction can also

be a determining factor, since most previous works made use of cold maceration as against the

Soxhlet extraction employed in this research. The developmental stage of plant samples and age

of plant might also be a factor to be considered (Sukumar et al., 1991).

Phytochemical screening of crude aqueous and methanolic extracts of the sample revealed the

presence of alkaloids, cardiac glycosides, saponins, tannins, flavonoids, terpenes and steroids

(Table 3.14). This result corresponds to results of previous works (Iroegbu and Nkere, 2005;

Ubulom et al., 2012; Nwakile and Okore, 2011). The phytochemical compounds detected in the

samples of P. nitida have been reported to exhibit significant biological activity (Lee, 2000;

Wiesman and Chapagain, 2006). Kouitcheu et al. (2008), conducted a phytochemical screening

and toxicological profile of methanolic extract of P. nitida fruit –rind and reported high level of

toxicity which might be attributed to the presence of bioactive phytochemicals. Larvicidal assay

employed in this research revealed that all the extracts used showed activity against An. gambiae

larvae. However, the extracts were not of equal potency. This finding is in line with the report of

Ubulom et al. (2012), whose work reported that the 72 h LC50 of ethanolic and aqueous leaf

extracts were 0.66% and 1.06% w/v respectively.

Variations in the potency of the extracts provokes a thought of doubt, since a closer look at the

phytochemical result reveals the same or similar classes of phytochemicals in almost all the

extracts. It is hence worthy of mention that this might be as a result of variation in the

concentration of individual phytochemical owing to the fact that the phytochemical screening

conducted was only qualitative and not quantitative. It can also be as a result of interactions

between phytochemicals present in the individual extract. Milugo et al. (2013) reported

antagonistic effect of alkaloids and saponins on bioactivity in the quinine tree (Rauvolfia caffra

sond.).

The 72 h LC50 for the leaf extracts used in this research were found to be 0.33mg/ml and

0.16mg/ml for methanol (Table 3.8) and aqueous leaf extracts (Table 3.7) respectively, hence a

variation from that obtained by Ubulom et al. (2012). This observed variation may be as a result

of age of plant parts (young, mature or senescent), solvent used during extraction as well as upon

the available vector species (Sukumar et al., 1991). It might also have resulted from the various

interplays of ecological factors (biotic and abiotic) which might affect the survival rate of the

larvae (Paaijmans, 2008; Mutuku et al., 2006). Changes in the larvicidal efficacy of plant

extracts have also been reported to have occurred due to geographical origin of the plant in

Citrus sp (Bagavan et al., 2009; Sumroiphon et al., 2006; Mgbemena, 2010), Jatropha sp

(Rahuman et al., 2007; Sakthivadivel et al., 2008), and Ocimum sanctum (Mgbemena, 2010;

Anees, 2008; Rahuman and Venkatesan, 2008).

The result showed a strong time - dependent correlation between the concentration of the extracts

and the mortality rate of the larvae. This is evident in carefully examining the effect of time on

the mortality rate. It can be observed that at a longer time of 48 and 72 hours of exposure, more

larval mortality was recorded at same concentration of extracts (Figures 3.1 - 3.9). This finding

corresponds with that of Khanna and Kannabiran (2007); Suwannee et al. (2006). It hence

follows that the lethal concentrations of the samples decrease with increase in time of exposure.

The seed extracts showed a deviation from what was obtainable from the leaf extracts. The

methanolic seed extracts showed a remarkable higher activity than the aqueous seed extract. The

24 h LC50 for the aqueous and methanolic seed extracts were 17.67 mg/ml and 0.09 mg/ml

respectively (Table 3.9 and 3.10), and their 72 h LC50 values were 10.56 mg/ml for aqueous

extract (Table 3.9) and 0.02 mg/ml for the methanolic extract (Table 3.10). The observed low

activity of the aqueous seed extract could be attributed to the inability of the aqueous solution to

extract more of the bioactive compounds (given its high oil content) which were readily available

using the methanol which eluted more phytochemicals including less polar compounds, many of

which possess larvicidal properties. This justifies the assertion made by Ghosh et al. (2012), that

polar solvent will extract polar molecules and non-polar solvents extract non-polar molecules.

The absence of alkaloids in the aqueous seed extract which might have arisen as a result of the

insolubility of alkaloid in water may be of significance given that alkaloids have been reported to

be the most active component of P. nitida (Meyer et al.,2006). Elena et al. (2010) reported that

alkaloids are representative of compounds that interact with ion channels, neurotransmitter

converters such as acetylcholinerase (AChE) and monomine oxidase, indicating their possible

effects on the nervous system. Devonshire et al. (1992) reported that alkaloids are the major sites

of action of both organophosphate, pyrethroids and carbamate insecticides. The presence of

alkaloids in the aqueous leaf extract despite the reported insolubility of alkaloids in water is

subject to debate. This might be as a result of forced solubility arising from the mild heat

associated with the extraction method. However, this would amount to very little of alkaloids in

the aqueous leaf extract indicating that other phytochemicals not alkaloids might have also

exhibited some level of lethality. The aqueous pulp sample was also more potent compared to the

methanolic pulp extract. The 24 and 72 h LC50 values for the pulp extracts were 4.98mg/ml and

2.80 mg/ml for the aqueous pulp extract (Table 3.11) while the methanolic pulp extract values

were 12.29mg/ml and 6.50mg/ml (Table 3.12). This result further reveals that alkaloids might

not be the only bioactive components of the plant, justified by the phytochemistry of the pulp

extracts which reveals that the aqueous pulp extract though lacking the alkaloids which were

reported by Meyer et al., (2006) as the most active components of P. nitida showed a better

activity when compared to the pulp methanolic extract which contained the alkaloids.

Comparatively, the six extracts used in this research indicate that the methanolic seed, aqueous

leaf and methanolic leaf extracts showed significantly high larvicidal activity against the

immature vector of malaria (An. gambiae larvae) at the various times of exposure (Figures 3.7 -

3.9). Other extracts (aqueous seed, aqueous pulp and methanolic pulp), though showed some

level of toxicity on the larvae had relatively high median lethal concentration values when

compared with the results obtained by previous research.

A critical observation of the timely relative potency of the methanolic leaf and methanolic pulp

extracts reveals that at 24 hours, the methanolic pulp extract exhibited more activity than the

methanolic leaf extract, with a relative potency value of 2.40 times that of the methanolic leaf

extract (Table 3.4). However, a reversal in trend was observed at 48 and 72 h with the

methanolic leaf extract proving to be more potent than the methanolic pulp with a relative

potency 2.73 and 11.51 times that of the methanolic pulp extract respectively (Table 3.5 and 3.6).

This observation is also evident in Figures 3.7 to 3.9. This delayed activity of the methanolic leaf

extract as against methanolic pulp extract may be as a result of the molecular weight, structure

and mode of action of the active ingredient which might have affected its solubility and rate of

penetration. Ghosh et al .(2012) reported that mode of action as well as nature of phytochemicals

plays an important role in its toxicity. The overall trend showed a direct proportional relationship

between time of exposure and mortality.

An estimation of the LT50 values also showed that methanolic seed, aqueous leaf and methanolic

leaf extracts of P. nitida required a time range of 3.61 ± 6h, 27.66 ± 4h and 57.49 ± 6hrs to kill

50% of the test organisms at a concentration of 1.0 mg/ml (Figure 3.10).

4.2 Conclusion

Results obtained from this study showed that methanolic and aqueous extracts of parts of the

plant P. ntida may serve as an alternative to synthetic insecticides in the control of the deadly

malaria vector, A. gambiae. Phytochemicals are environmentally friendly, readily available and

inexpensive and hence could serve as a more favourable option in the eradication of mosquitoes

and other insect pests from our environment.

It is important to highlight that this work is a preliminary assay and hence calls for extensive

work to be done especially in phytochemical analysis of the extracts, the application of column

chromatography and thin layer chromatography (TLC) to purify and isolate specific toxic

phytochemical with bioactive potentials requires urgent attention. Determination of the structure

of active ingredients by Infra Red (IR) Spectroscopy, Nuclear Magnetic Resonance (NMR), Gas

Chromatography and Mass Spectroscopy (GCMS) analysis and studies on the effects of active

ingredient on non target organisms and field evaluation of the active principle before its

recommendation in vector control programme and commercial production. Again, it is also

pertinent to state that changes in environmental factors such as turbidity of the water, the pH,

water temperature etc arising from the introduction of extracts might have altered the ecological

balance of the habitat, thereby contributing to the death of the larvae. Hence subsequent research

should standardize these factors to its ambient levels before carrying out the bioassay.

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Appendix 1: Effect of Aqueous Leaf Extracts of P. nitida on A. gambiae at various time intervals

Conc. (mg/ml)

Mean±

SD

24hrsMean

% Mortality

Mean±

SD

48hrsMean

% Mortality

Mean±

SD

72hrsMean

% Mortality

0.0 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

0.5 2.3 ± 0.6 12 10.0 ± 1.7 50 12.0 ± 1.0 60

1.0 7.7 ± 1.2 38 17.0 ± 1.0 85 19.3 ± 1.2 97

2.0 8.0 ± 2.6 40 17.0 ± 1.7 85 19.7 ± 0.6 98

3.0 9.3 ± 1.5 47 18.3 ± 0.6 92 19.7 ± 0.6 98

4.0 9.7 ± 3.2 48 18.7 ± 1.5 93 20.0 ± 0.0 100

5.0 11.0 ± 1.0 55 19.0 ± 1.0 95 20.0 ± 0.0 100

Appendix 2: Effect of Methanolic Leaf Extracts of P. nitida on A. gambiae at various time intervals

Conc. (mg/ml)

Mean±

SD

24hrsMean

% Mortality

Mean±

SD

48hrsMean

% Mortality

Mean±

SD

72hrsMean

% Mortality

0.0 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

0.5 0.0 ± 0.0 0 2.7 ± 0.6 13 8.3 ± 1.5 42

1.0 0.0 ± 0.0 0 4.3 ± 1.2 22 16.7 ± 1.5 83

2.0 0.0 ± 0.0 0 5.0 ± 1.0 25 17.7 ± 1.5 88

3.0 0.3 ± 0.6 2 5.7 ± 1.5 28 19.0 ± 1.0 95

4.0 1.0 ± 1.0 5 7.0 ± 2.0 35 19.3 ± 0.6 97

5.0 3.0 ± 1.0 15 7.7 ± 0.6 38 19.3 ± 0.6 97

Appendix 3: Effect of Aqueous Seed Extracts of P. nitida on A. gambiae at various time intervals

Conc.(mg/ml)

Mean±

SD

24hrs Mean

% Mortality

Mean±

SD

48hrs Mean

% Mortality

Mean±

SD

72hrs Mean

% Mortality

0.000 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

0.500 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

1.000 1.3 ± 1.2 7 1.3 ± 1.2 7 1.7 ± 1.5 8

2.000 1.7 ± 1.2 8 2.0 ± 1.0 10 3.3 ± 0.6 17

3.000 1.7 ± 0.6 8 2.7 ± 0.6 13 4.0 ± 1.0 20

4.000 3.3 ± 0.6 17 4.3 ± 1.5 22 4.7 ± 1.2 23

5.000 4.0 ± 2.0 20 5.7 ± 1.5 28 6.3 ± 1.2 32

Appendix 4: Effect of Methanolic Seed Extracts of P. nitida on A. gambiae at various time intervals

Conc. (mg/ml)

Mean±

SD

24hrsMean

% Mortality

Mean±

SD

48hrs Mean

% Mortality

Mean±

SD

72hrs Mean

% Mortality

0.0 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

0.5 18.7 ± 1.5 93 19.7 ± 0.6 98 20.0 ± 0.0 100

1.0 19.3 ± 1.2 97 20.0 ± 0.0 100 20.0 ± 0.0 100

2.0 19.7 ± 0.6 98 20.0 ± 0.0 100 20.0 ± 0.0 100

3.0 20.0 ± 0.0 100 20.0 ± 0.0 100 20.0 ± 0.0 100

4.0 20.0 ± 0.0 100 20.0 ± 0.0 100 20.0 ± 0.0 100

5.0 20.0 ± 0.0 100 20.0 ± 0.0 100 20.0 ± 0.0 100

Appendix 5: Effect of Aqueous Pulp Extracts of P. nitida on A. gambiae at various time intervals

Conc. (mg/ml)

Mean±

SD

24hrsMean

% Mortality

Mean±

SD

48hrs Mean

% Mortality

Mean±

SD

72hrsMean

% Mortality

0.0 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

0.5 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

1.0 0.3 ± 0.6 2 1.3 ± 1.5 7 2.0 ± 1.0 10

2.0 2.7 ± 1.5 13 3.0 ± 2.0 15 8.0 ± 1.7 40

3.0 3.3 ± 0.6 17 3.7 ± 0.6 18 11.7 ± 1.5 58

4.0 9.3 ± 1.5 47 10.0 ± 1.7 50 12.7 ± 0.6 63

5.0 10.7 ± 1.2 53 13.3 ± 1.5 67 14.0 ± 1.0 70

Appendix 6: Effect of Methanolic Pulp Extracts of P. nitida on A. gambiae at various time intervals

Conc. (mg/ml)

Mean±

SD

24hrsMean

% Mortality

Mean±

SD

48hrsMean

% Mortality

Mean±

SD

72hrsMean

% Mortality

0.000 0.0 ± 0.0 0 0.0 ± 0.0 0 0.0 ± 0.0 0

0.500 0.3 ± 0.6 2 0.3 ± 0.6 2 0.3 ± 0.6 2

1.000 0.7 ± 0.6 3 1.7 ± 0.6 8 2.0 ± 1.0 10

2.000 1.0 ± 1.0 5 2.0 ± 1.0 10 3.7 ± 1.2 18

3.000 3.0 ± 1.7 15 3.7 ± 1.5 18 5.0 ± 1.0 25

4.000 4.3 ± 0.6 22 4.3 ± 0.6 22 6.7 ± 0.6 33

5.000 5.0 ± 2.0 25 6.3 ± 1.5 32 9.3 ± 1.5 47

Appendix 8: Probit Estimate for Methanolic Seed Extract

Parameter

Estimates

Appendix 7: Probit Estimates For Methanolic Leaf

Parameter Estimates

Parameter Estimate Std. Error Z Sig.

95% Confidence Interval Lower

Bound Upper Bound

PROBITa Conc 1.451 .251 5.792 .000 .960 1.942

Interceptb 24 hrs -2.445 .425 -5.754 .000 -2.870 -2.020

48 hrs -1.110 .239 -4.638 .000 -1.349 -.871

72 hrs .694 .107 6.503 .000 .587 .800

a. PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm.)

b. Corresponds to the grouping variable Time.



Bound Upper Bound

PROBITa Conc 1.768 .812 2.176 .030 .176 3.360

Interceptb 24 hrs 1.878 .815 2.304 .021 1.063 2.693

48 hrs 2.955 .643 4.595 .000 2.312 3.598

72 hrs 3.245 .892 3.637 .000 2.353 4.137


b. Corresponds to the grouping variable VAR00006.

Appendix 9: Probit Estimate for Aqueous Seed Extract

Parameter Estimates

Parameter Estimate Std.

Error Z Sig.


Bound Upper Bound

PROBITa

Conc 1.513 .349 4.336 .000 .829 2.196

Interceptb

24 hrs -1.887 .344 -5.482 .000 -2.231 -1.542

48 hrs -1.708 .286 -5.978 .000 -1.994 -1.422

72 hrs -1.548 .260 -5.965 .000 -1.808 -1.289


Appendix 10: Probit Estimate for Aqueous Leaf Extract

Parameter Estimates



Bound Upper Bound

PROBITa

conc 1.459 .140 10.418 .000 1.184 1.733

Interceptb 24 hrs -.725 .084 -8.584 .000 -.810 -.641

48 hrs .661 .087 7.556 .000 .573 .748

72 hrs 1.145 .105 10.916 .000 1.040 1.250




Appendix 11: Probit Estimate for Methanolic Pulp Extract

Parameter Estimates

Parameter Estimate Std. Z Sig. 95% Confidence Interval

Appendix 13: Median Lethal Times for Methanolic Leaf, Aqueous Leaf and Methanolic Seed

Error Lower Bound

Upper Bound

PROBITa

Concentration 1.833 .359 5.107 .000 1.130 2.536

Interceptb 24 hrs -1.997 .272 -7.354 .000 -2.268 -1.725

48 hrs -1.809 .261 -6.930 .000 -2.070 -1.548

72 hrs -1.489 .220 -6.761 .000 -1.709 -1.269


b. Corresponds to the grouping variable Treatment.

Appendix 12: Probit Estimate for Aqueous Pulp Extract

Parameter Estimates

Parameter Estimate Std.

Error Z Sig.


Bound Upper Bound

PROBITa

Concentration 2.881 .206 13.982 .000 2.477 3.285

Interceptb

24 hrs -2.009 .135 -14.883 .000 -2.143 -1.874

48 hrs -1.825 .129 -14.111 .000 -1.954 -1.695

72 hrs -1.285 .116 -11.062 .000 -1.401 -1.169


b. Corresponds to the grouping variable Treatment.

conc (mg/ml)

ML AL MS Time Log Time Time Log Time Time Log Time

0.50 73.97 1.87 52.67 1.72 5.60 0.75 1.00 57.49 1.76 27.66 1.44 3.61 0.56 2.00 55.11 1.74 26.88 1.43 2.72 0.43 3.00 51.59 1.71 25.51 1.41 1.97 0.30

4.00 48.20 1.68 22.88 1.36 1.86 0.27 5.00 44.20 1.65 21.19 1.33 1.82 0.26

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LARVICIDAL ACTIVITY OF Picralima nitida - University Of