ABSTRACT
The extracts of the root barks of Detarium microcarpum Guill&Perr and
Sclerocarya birrea (A.Rich) Hochst. have found traditional use in Nigeria and
other African countries for treatment of dysentery, skin diseases and as antifeedant
against stored product pests.
These claims have been verified through antifeedant, cytotoxicty and
microbial tests. Phytochemical analyses carried out on the extracts of both plants
(using solvents of varying polarities) showed the presence of reducing sugars,
alkaloids, saponin glycosides, sterols, resins and balsams, flavonoids and tannins.
All the extracts (except for the petroleum spirit extract) from the two plants were
active against Bacillus subtilis, Escherichia coli, Cornybacterium pyogenes,
Staphyloccocus aureus and Salmonella typhi.
The methanol and ethyl acetate extracts from both plants were active in the
shrimp lethality bioassay (Cytotoxicity Test). The methanol extracts of the plants
had good antifeedant activity. Purification of the methanol fraction of the S. birrea
extract was carried out to determine the components of the plant. The petroleum
spirit extract tested positive for aldehyde and this was oxidized to the acid
derivative in order to facilitate its isolation and purification. Infra Red and Nuclear
Magnetic Resonance spectroscopic analyses were carried out to determine the
possible structures of compounds obtained
TABLE OF CONTENTS
Title Page………………………….…………………………………….. ii
Declaration………………………….………………………….………… iii
Certification………………………….…………………………………… iv
Dedication………………………….…………………………………….. v
Acknowledgement. ………………………….…………………………… vi
Abstract………………………….……………………………………….. ix
Table of Contents………………………….……………………………… x
List of Tables………………………….…………………………………. xv
List of Figures………………………….…………………………….…. xvi
CHAPTER ONE
1.0 Introduction………………………….…………………………………. 1
1.1 Pesticides………………………….…………………………………….. 1
1.1.1. Natural Pesticides From Plants………………………….……………… 2
1.2. Antifeedants………………………….…………………………………… 3
1.2.1. Azadirachtin………………………….…………………………………… 4
1.2.2. Lantadenes………………………….…………………………………….. 5
1.2.3. Quassin………………………….……………………………………….. 6
1.2.4 Diterpenes. ………………………….…………………………………… 7
1.2.5 Quinolizidine Alkaloids………………………….………………….…… 9
1.2.6. Harrisonin………………………….……………………………………… 10
1.2.7. Warburganal………………………….………………………….………… 10
1.2.8. Guineensine………………………….…………………………………….. 11
1.2.9. (-) Polygodial………………………….………………………………….. 11
1.2.10 Annonacin………………………….…………………………………….. 12
1.2.11 Sesquiterpenes………………………….…………………………………. 13
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1.3. Qualities of a Good Antifeedant …………………………………………. 14
1.4. Project Objectives………………………….……………………………… 14
1.5. Justification for Research………………………….……………………… 15
CHAPTER TWO
2.0. Literature Review………………………….…………………………… 17
2.1 Detarium microcarpum. Guill&Perr. (Caesalpinaceae)……………………… 17
2.1.1. Brief Description of Genus Features of the Family…………………… 17
2.1.2. Description of General Features of the General – Detarium ……………. 18
2.1.3. The Plant – Detarium microcarpum (Guill&Perr). ……………………… 18
2.1.4. Vernacular names………………………….……………………………… 18
2.1.5. Location………………………….………………………………………. 18
2.1.6. General Features………………………….……………………………… 18
2.1.7. General Uses………………………….…………………………………. 19
2.1.8. Medicinal and other Related Uses………………………………………… 19
2.1.9. Other Genus in the Caesalpinaceae Family………………………………. 21
2.2 Sclerocarya birrea. (A.Rich).Hochst. (Anacardiaceae)………………………….. 22
2.2.1. Brief Description of General Features of the Family…………………… 22
2.2.2. Description of the General Features of the Genus-Sclerocarya ……….. 23
2.2.3. The Plant – Sclerocarya birrea (A.Rich) Hochst……………………… 23
2.2.4. Vernacular Names………………………….……………………………. 23
2.2.5. Location………………………….……………………………………….. 23
2.2.6. General Features…………………………..……………………………… 23
2.2.7. General Uses………………………….…………………………………… 24
2.2.8. Medicinal and other Related Uses ……………………………………….. 25
2.2.9. Other Genus in the Anacardiaceae Family………………………………. 27
2.3. Micro-organisms and their Pathogenic Activities ……………………… 28
2.3.1. Staphylococcus aureus ……….………………………………………….. 28
2.3.2. Salmonella typhi .. ………………………….……………………………. 29
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2.3.3 Bacillus subtilis ….………………………….…………………………… 29
2.3.4 Cornybacterium pyogenes ….………………………….………..……… 29
2.3.5. Escherichia coli ……………..…………………………………………… 30
CHAPTER THREE
3.0. Experimental ……………………………………………………………. 31
3.1. Apparatus and Reagents………………………………………………….. 31
3.1.1. Apparatus…………………………………………………………………. 31
3.1.2. Reagents………………………………………………………………….. 32
3.2. Plant Samples Collection and Preparation……………………………….. 34
3.3. Extraction………………………………………………………………… 34
3.3.1. Bulk Extraction………………………………………………………….. 35
3.4. Preliminary Phytochemical Screening Tests……………………….……. 36
3.4.1. Tests for Carbohydrates……………………………………………….….. 36
3.4.1.1 Molisch’s Test………………………………………………………….… 36
3.4.1.2 Fehling’s Test………………………………………………………….…. 36
3.4.1.3 Standard Test for Combined Reducing Sugar …………………………… 36
3.4.2. Tests for Glycosides………………………….………………………….. 37
3.4.2.1 Hydrolysis of Glycosides – by Dilute Mineral Acid……………………… 37
3.4.2.2 Test for Cyanogenetic Glycosides……………………………………….. 37
3.4.2.3 Test for Saponin Glycosides………………………….………….……… 37
3.4.2.4 Test for Cardiac Glycosides …………………………………….….…… 38
3.4.3 Test for Sterols: Salkowski Reaction …………………………………… 39
3.4.4 Test for Flavonoids: Shinoda Test …………………………………..….. 39
3.4.5 Test for Alkaloids …………………………………………………..…… 40
3.4.5.1 Preparation of Extracts…………………………………………………… 40
3.4.5.2 Mayer’s Reagent Test ……………………………………………..……. 40
3.4.5.3 Dragendorff’s Reagent Test ……………………………………………… 40
3.4.5.4 Wagner’s Reagent Test …………………………………………………… 41
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3.4.6 Tests for Resin and Balsams …………………………………………….. 41
3.4.6.1 Test For Resin …………………………………………………………… 41
3.4.6.2 Tests for Balsam ……………………………………………………… 41
3.4.7. Test for Tannins ………………………………………………………….. 41
3.5. Antibacterial Screening Test Using the Agar-Diffusion method …………. 42
3.5.1. Isolation of Cultures……………………………………………………….. 42
3.5.2. Preparation and Storage of Media…………………………………………. 43
3.6 Cytotoxicity Test…………………………………………………………… 44
3.6.1. Materials…………………………………………………………………… 44
3.6.2 Procedure………………………………………………………………….. 45
3.6.3. Determination of LC50 ……………………………………………………. 46
3.7 Antifeedant Tests …………………………………………………………. 46
3.7.1. Preparation of Extracts …………………………………………………… 46
3.7.2. Insect Culture……………………………………………………………… 46
3.7.3 Procedure………………………………………………………………….. 46
3.8. Functional Group Test of the Sclerocarya birrea Pet. spirit extract
[SBPe]……………………………………………………………………… 48
3.8.1. Oxidation of the Aldehyde Functional Group………………………………………. 48
3.9. Purification and Characterization of the Sclerocarya birrea methanol
extract. [SBMe]. …………………………………………………………… 49
3.9.1 Acid Hydrolysis of SBMe Extract ………………………………………… 49
3.9.1.1 Test for Reducing Sugar ………………………………………………….. 50
3.9.1.2 Extraction of SBMe Hydrolysate …………………………………………. 50
3.9.2 Functional Group Test ……………………………………………… 50
3.9.2.1 Alkaloid Test …………………………………………………………….. 50
3.9.2.2 Test for Cardiac Glycosides: Salkowski Test ……………………………. 50
3.9.2.3 Test for Phenols…………………………………………………………… 51
3.9.3 Chromatographic Separations …………………………………………….. 51
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3.9.3.1 Preparation of Plates ……………………………………………………… 52
3.9.3.2 Purification of the SBMe Hydrolysate by Column Charomatography……. 53
CHAPTER FOUR
4.0. Results and Discussion………………………………………………… 54
4.1 Bulk Extraction ……………………………………………………….. 54
4.2 Preliminary Phytochemical Screening …………………………………. 54
4.2.1. Detarium microcarpum ………………………………………………… 54
4.2.2 Sclerocarya birrea …………………………………………………… 60
4.3. Antibacterial Screening Tests …………………………………………. 64
4.3.1 Detarium microcarpum ……………………………………………….. 64
4.3.2. Sclerocarya birrea …………………………………………………….. 66
4.4 Cytotoxicity Test ……………………………………………………… 69
4.5. Antifeedant Tests …………………………….……………………….. 72
4.5.1 Observations …………………………………………………………… 76
4.5.2 Relative Comparison ………………………………………………….. 78
4.6. S. birrea Petroleum spirit Extract [SBPe]……………………………………… 79
4.6.1 Spectroscopic Analysis of the SBPe – oil……………………………….. 79
4.6.2. Spectroscopic Analysis of Acid derivative [SBPeA]……………………….. 79
4.7 Purification of S. birrea Methanol Extract Hydrolysate [SBMeH]………… 82
4.7.1 Functionality Test……………………………………………………… 82
4.7.2. Chromatographic Separation of S. birrea Methanol Extract Hydrolysate…. 83
4.7.3 Spectroscopic Analysis and Physical Constants………………………. 83
CHAPTER FIVE
5.0 Conclusion and Recommendation …………………………………….. 84
5.1 Conclusion……………………………………………………………… 84
5.2 Recommendation………………………………………………………. 88
References……………………………………………………………… 89
Appendix ……………………………………………………………… 95
CHAPTER ONE
1.0. INTRODUCTION
1.1. Pesticides
Pesticides are chemical agents which are intended to prevent, destroy, repel
or otherwise decrease the ability of pests to compete with desired organisms such
as cultivated crops, animals or humans. Under this broad definition are the
insecticides, fungicides, herbicides, molluscicides, nematocides, ovaricides,
repellents and antifeedants, etc. They could be synthetic or natural in origin.
Some of the synthetic pesticides include carbamates, pirimiphose, permethrin,
malathion, decamethrin etc. (Austin, 1984). Many of these compounds are highly
potent towards the pests. However, they possess several disadvantages, some of
which are lack of selectivity, built-up resistance, toxicity to man and accumulation
in the environment [Kroschwitz and Howe-Grant, 1993]. The indiscriminate use
of chemical pesticides has given rise to many well-known and serious problems
including genetic resistance of pest species, toxic residues in stored products,
hazards from handling etc. The problem caused by pesticides and their residues
have increased the need for effective biodegradable pesticides with greater
selectivity.
The use of synthetic pesticides and the apparent health hazard posed by
these pesticides have led to the consideration of biopesticides as a viable
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alternative in the control of pests. Biopesticides employ the natural defences that
plants and trees have to kill insects. They act by blocking the insect receptor- that
is the neurotransmitter that regulates an insect’s movement, behaviour and
metabolism. (Salihu, 1999).
An area which is receiving considerable attention presently is the isolation
and identification of active constituents from plants that have been found to repel
or kill some insects, deter feeding and/or distort their life cycles. After isolation
and characterization, the aim is to either synthesize their templates for large
production or modify the traditional method of processing to make the repellent,
antifeedant, insecticidal properties etc. more refined.
1.1.1. Natural Pesticides From Plants
To combat insect attacks, plants have developed a number of protective
mechanism such as repellency, feeding deterrency, etc, and a number of natural
products have been identified from these plant extracts. These have found use as
insecticides, antifeedants, repellents etc. although many of them could not be
profitably extracted. However, several of these extracts have provided valuable
contact insecticides which possess the advantage that their uses do not appear to
result in the emergence of resistant insect strain to the same degree as the
application of synthetic insecticides [Cremlyn, 1991]. The major natural products
among these are nicotine, derris, pyrethrum, ryania, azadirachtin to mention just a
few. Crude extracts of some plants producing these natural products have a long
– 18 –
history of use as fish poisons, insecticides, antifeedants and repellants among
many communities.
The bioactive constituent of these plants in most cases do not kill the pests
outright, but exert pesticidal effects through deterrence of feeding or inhibition of
growth. Inhibition of pest growth pattern and production of phytotoxic symptoms
by certain plants is a well established phenomenon of repellents and antifeedants
which when present cause such plants to be significantly protected against insect
and other predational pests. These deterrents negatively affect the biting, feeding
and maintenance of feeding activity by the insects. Toxic action may induce
sickness or weakness, inhibit growth, slow maturation, decrease reproduction and
promote premature death [Bloszyk et al, 1995]. Plants with these natural pesticidal
constituents are termed antifeedants [van Beek, 1993].
1.2. Antifeedants
Antifeedants are substances that are not necessarily food repellants but
cancel out the signal to the appropriate organ in the insect to initiate feeding on the
host [Cremlyn, 1991]. They are substances in plants which when perceived by the
insects or pests reduce or prevent insect feeding. Antifeedants in plant body
constitute defence mechanism against insect attack. They are plant substances or
constituents which interact with taste receptors of herbivores (insects, pests etc) in
such a way that the process of consuming is strongly repressed. In the presence of
the antifeedant substance the insect may starve to death while remaining on the
host plant, possibly because it makes the host plant distasteful to the insect so
– 19 –
feeding is inhibited. The majority of antifeedants do not directly kill the insect.
Antifeedants, unlike insecticides are often selectively toxic towards some insect
species, whereas they are not harmful to others and to mammals. They are
environmentally friendly because they attack only specific pests without affecting
the non-target organisms [Cremlyn, 1991; van Beek, 1993; Paruch, 2000].
Plants with bitter and/or pungent taste have been observed to be typical of
tissues containing alkaloids and other classes of toxic compounds such as the
sesquiterpenoids, the triterpenoids or limonoids e.g. azadirachtin, and these are
known to have some of these antifeedant properties. [Bloszyk et al, 1995]. Some
classes of compounds that have been isolated as antifeedants include:
1.2.1. Azadirachtin
One of the most successful antifeedants is a group of tetranortripenoids
from the neem tree Azadirachta indica [Meliaceae]. The main constituent
azadirachtin [I], a naturally occurring insect repellent and antifeedant has provided
Indian and South American peasant farmers with a cheap or even free insect pest
control technique. It has two main effects on several common insect pests
including caterpillars and locust:
(a) It repels them from eating the leaves or seeds of the plant
(b) It upsets the cycle of moults by which they grow, slows down their
growth and eventually kills them. [Kraus, et al, 1993; Rembold et al,
1987].
– 20 –
Azadirachtin is the most active antifeedant against lepidopterous species known; it
is lethal at a concentration of only 10ppm [Ley, 1990]. The azadirachtin is a
highly complex compound which has not yet been synthesized though its structure
has been elucidated [Kraus et al, 1993; Rembold et al, 1987]. Various products
have been formulated from the neem concentrate, the most popular being
Margosan-O
[R] [Larson, 1986]. Currently, efforts are being directed towards
designing synthetic compounds which can mimic azadirachtin [Ley, 1990].
1.2.2. Lantadenes
Substantial work has been carried out on the utilization of Lantana camara
[Verbaneaceae] for insecticidal activity. Reports have shown that they contain a
wide range of triterpenoids, some of which are hepatotoxic to animals [Barre,
1997]. Some of these are pentacyclic triterpene acids such as Lantadene A and B,
– 21 –
Iceterogenin and dihydrobutandene A [II]. These compounds though with
insecticidal activity have also been implicated in lantana poisoning of ruminants
[Tripathi et al, 1992].
1.2.3. Quassin
Quassia amara and Picrasma excelsa [Simaroubaceae] are plants that
contain bitter-tasting seco-triterpenes such as quassin, neoquassin and 18-
hydroxyquassin which are used as a bitter tonic, a hop imitate, an anthelmintic and
or an insecticide. Quassia extracts prepared from the wood of both trees were
widely used against ectoparasites (lice) and flies, the extract were also used as
spray solution on farms. The constituent, Quassin [III] acts as a contact, stomach
and systemic poison in insects (ectoparasites, lepidopteran larvae, beetles and
flies) and nematodes. Quassin seems to be a selective insecticide with no recorded
– 22 –
toxic effects on humans, though if applied in high concentrations, irritation of
stomach and vomiting can occur. [Stoll, 1986].
1.2.4. Diterpenes
Tetranorditerpenes have been isolated from the leaves of the plant
Detarium microcarpum [Caesalpinaceae]. These had antifeedant activity against
wood termite Reticulitermes speratus. The isolated compound was reported as
ent-18-oxo-3, 13-clerodadien-15-oic acid. [IV] [Lajide et al, 1995].
Clerodane diterpenoids and phytoecoysteroids with potential insect
antifeedants and moulting hormone activities respectively have been isolated from
Ajuga plants. Some clerodanes were active against larvae of Egyptian cotton
– 23 –
leafworm Spodoptera littoralis and it has been observed that the medicinal plant
Ajuga remota [Labiatae = Lamiaceae] leaves were not attacked by African
armyworms. The observation has led to the isolation of three moderately strong
antifeedants named ajugarins I – III [V – VI], [Camps et al, 1993]. The lactone,
ajugarin I has antifeedant activity against several insects including Locusta
migatoria [Ley et al, 1990].
– 24 –
1.2.5 Quinolizidine Alkaloids
Another group of potential antifeedants are the quinolizidine alkaloids.
These are the constituents of many leguminosae especially in the genera Lupinus,
Genista, Cytisus, Baptisia, Thermopsis, Sophora and Ormosia. The quinolizidine
alkaloids were found to be feeding deterrents for a number of oligo- and polyphagous insects including aphids, moths and butterfly larvae, beetles,
grasshoppers, flies, bees and ants, that is, it has a wide spectrum of activity on a
wide range of insect order [Wink, 1993]. Sophora occidentalis [Dapilioaceae] is
reported to have fish poisoning property with nicotine – like action in its root and
seed extract [Dalziel, 1934]. Some of these isolated alkaloids include hypanine
[VII] and spartein [VIII]. [Wink, 1993].
– 25 –
1.2.6. Harrisonin
Harrisonia abyssinica [Simaroubaceae] is a shrub widely used in East
Africa folk remedies as a remedy for bubonic plague, haemorrhoid and snake bite.
Phytochemical analysis of the plant led to the isolation of harrisonin [IX] which is
known to have antifeedant activity against monophagous army worm S. exempta
[Nakanishi, 1980].
1.2.7. Warburganal
A constituent Warburganal [X] isolated from the bark of Warburgia
ugandensis [Canellacea] has been found to exert potent antifeedant effects against
S. exempta and S. littoralis, both insect pests of plants [Kubo et al, 1976].
– 26 –
1.2.8. Guineensine
The powdered dry fruits of Piper guineense [Piperaceae] are used as local
spice and as an insecticide against bruchid [Ivbijaro et al, 1986]. An insecticide
guineensine [XI] has been isolated from the plant [Okogun et al, 1974]. The
effectiveness of the essential oil of P. guineense as seed dressing for cowpea
production has been reported [Olaifa et al, 1986].
1.2.9. (-) Polygodial
This is a less complex antifeedant isolated from the marsh pepper
Polygonium hydropiper [Piperaceae]. Plants treated with low concentrations of
the compound were not colonized by aphids and consequently infection by plantvirus diseases was significantly reduced. Polygodial has been synthesized [Ley,
– 27 –
1985], but unfortunately, the (+) isomer is phytotoxic so the synthetic (racemic)
product requires a tedious resolution before it can be used in crop protection.
Polygodial [XII] has also been reported to have been isolated from the plant
Warburgia salutaris [Canellacea] [Mahlori et al, 1999].
1.2.10. Annonacin
Annona squamosa [Annonaceae] found in tropical America, Asia and
Africa contain strong insecticidal, larvicidal, repellent and antifeedant constituents
with remarkable activity on aphids, grasshoppers, diamond back-moth, red
pumpkin beetle, green bug, etc. [Gaby, 1988; Nayar, 1995].
Chemical investigation of the seeds of A. squamosa led to the isolation of
novel insecticidal acetogenins, neoannonin [XIII] and annonacin [XIV] [Kawazu
et al, 1989]. Alkaloids such as anonaine, ligenamine, anolobine etc and
flavonoids, quercetin-3- rutinoside (rutin) have also been isolated from the leaves
and root of the plant [Wagner et al, 1980; Sectharaman, 1986].
– 28 –
1.2.11. Sesquiterpenes
The sesquiterpenes Xanthorrhool, have been reported present in the
rhizomes of Curcuma xanthorrhiza or Curcuma zedoaria as having insecticidal
activity against the larvae of the vigorous insect pest Spodoptera littoralis when
applied topically via the larval integument [Nugroho et al, 1996]. Sesquiterpenes
have been isolated from Celastus angulatus which have been used as natural
insecticides for a long time [Liu et al, 1995].
1.3. QUALITIES OF A GOOD ANTIFEEDANT
A good natural insecticide or antifeedant should have the following
qualities:
a. Effectivity – such as in insecticidal activity or insect deterrence.
b. Low toxicity in vertebrates and beneficial insects.
c. More than one mode of action to reduce resistance to pest.
d. Low persistence in plants, soil and water.
e. Extraction and formulation should be simple, cheap and easy
f. Plant material should be one that can be easily sourced.
Although not all criteria can be fulfilled in most cases, some of the
examples given above come rather close to these demands. For example,
azadirachtin, from Azadirachta indica (the Neem plant).
1.4. PROJECT OBJECTIVES
– 29 –
It is estimated that 10-35% of all crops are lost either before harvest on the
field or during storage. [Cremlyn, 1991].
This project aims to find a solution to the post-harvest loss which occurs
due to pest destructive activities on stored grains by sourcing for plants which
could be used as antifeedants against agronomic pests.
The plants selected for this work are:
i. Detarium microcarpum. Guill&Perr.
ii. Sclerocarya birrea. (A. Rich) Hochst.
The crude extracts of these plants would undergo the following tests:
i. Phytochemical screening to identify the constituents present.
ii. Antibacterial screening to determine its biological activity against
bacterially induced ailments.
iii. Cytotoxicity tests for the lethality bioassay.
iv. Antifeedant tests to determine its effectiveness or otherwise against insect
pests. A reference standard pesticide for stored products, Pirimiphos
methyl – 2% Dust, will be used for a comparative antifeedant study with
the crude extracts. The rust red flour bettle, Tribolium casteneum Hbst. is
to be used as the test organism.
v. Isolation of constituents of the plants, characterization and structural
elucidation of these constituents using spectroscopic techniques would also
be carried out.
1.5. JUSTIFICATION FOR RESEARCH
– 30 –
One of the developmental priorities of a nation is to grow enough food to
support her population. This idea is often not realized because of damages to food
crops by pests. The development of insecticide – based technique for protecting
crops in small traditional farms in Africa has only been partially successful
because of the high cost of synthetic insecticides and erratic supply due to foreign
exchange constraints. The harmful effects of some of these synthetic pesticides on
non-target organisms especially humans as a result of its persistence calls for great
concern.
The need to sustain and increase our capacity for food production in order
to meet increasing demand without irreversible damage to ecological and other
natural resources cannot be over-emphasized hence the need for this research into
the natural antifeedant constituents of plants. The establishment of the antifeedant
properties of these plants: Detarium microcarpum and Sclerocarya birrea by the
farmers motivated the interest in order to establish a scientific proof of their claim
and the possibility of isolating the active constituent(s) for increased
insecticidal/antifeedant activity against stored product pest.
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