Final Project

EVALUATION OF THE TOXICITY OF Alternanthera

brasiliana (L.) O. KUNTZE AND CYPERMETHRIN-

TREATED Amaranthus cruentus FED TO WISTAR RATS

BY

OLAWALE OREOLUWA, SHIRO

B. Agric. (Olabisi Onabanjo University)

MATRIC NUMBER 166698

A PROJECT THESIS IN THE DEPARTMENT OF CROP PROTECTION AND

ENVIRONMENTAL BIOLOGY SUBMITTED TO THE FACULTY OF AGRICULTURE

AND FORESTRY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF THE MASTER OF SCIENCE DEGREE (M. SC) IN ENVIRONMENTAL

BIOLOGY

JUNE, 2013

1

ABSTRACT

Synthetic insecticides are designed to breakdown more slowly than the naturally occurring ones thereby

accounting for their persistence, residual activities and chronic effects to man, plants and the

environment. Botanicals on the other have been heralded for their ability to biodegrade easily and

become harmless compounds in the environment. In this study, the toxicity of Alternanthera brasiliana

and Cypermethrin-treated Amaranthus cruentus was studied on the histopathology of Wistar rats (Rattus

norvegicus). The aim was to investigate the effects of both the botanical and the synthetic when sprayed

on vegetables and consumed within 1 - 3 Days-After-Treatment (DAT). Amaranthus cruentus seeds

were sown by drilling. The treatments were of different concentration levels of 100, 75, 50 and 25% A.

brasiliana extract, Cypermethrin (1ml/100mls) and control (no insecticide). The six (6) treatments were

replicated four times and laid out in a randomized complete block design (RCBD). Different

concentration levels of the treatments were applied on A. cruentus at 3 and 5 Weeks After Sowing

(WAS). Forty four (44) rats were used for the animal studies. They were acclimatized for seven (7) days

and fed on Standard Ration Feed (SRF). Thereafter, they were divided into eleven (11) groups (four

rats/group), out of which ten (10) group were fed on treated A. cruentus while the remaining group

(control) was fed on SRF, all for thirty (30) days. Toxicity effects of the botanical and synthetic

insecticide-treated A. cruentus on rats were assessed on 1 and 3 DAT basis. Data collected during this

study were the growth parameters (plant height, number of leaves, stem girth, leaf area and fresh

weight/yield) of A. cruentus. The histopathology of the livers and kidneys of the rats were then

examined and data analyzed using descriptive statistics and ANOVA at P = 0.05. Results showed no

significant differences in plant height, stem girth, number of leaves and leaf area. The 100% A.

brasiliana-extract treatment had the highest value for plant height while 75% A. brasiliana-extract had

lowest value. Stem girth showed no significant difference at the both one and three day-after treatment

but at 3 DAT, the highest value was for 75% A. brasiliana-extract treatment while the lowest was for

50% A. brasiliana-extract treatment. The 100% A. brasiliana-extract compared very well with the

control and Cypermethrin-treated plot in almost all plant parameters. For the histopathological

examinations, section of the tissues examined displayed congestion at the cortical region, portal and

periportal cellular infiltration by mononuclear cells, necrosis and vacuolization with neuronal

degeneration, shrinkage of glomeruli, necrosis and disruption of renal tubules. This study has shown that

A. brasiliana would not cause similar environmental risks as many of the widely used synthetic

insecticides and thus vegetables treated using its extract are safe for consumption.2

Keywords: Toxicity, Histopathology, Cypermethrin, Botanicals, Alternanthera brasiliana

Word Count: 310

CERTIFICATION

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I certify that Olawale Oreoluwa Shiro of the Department of Crop Protection and Environmental

Biology, University of Ibadan, Ibadan, Nigeria carried out this work under my supervision.

------------------------------ ------------------------------------------

DATE SUPERVISOR

Dr. Olajumoke Oke Fayinminnu,

Environmental Biologist/Toxicologist

Department of Crop Protection and Environmental

Biology,

University of Ibadan, Ibadan.

ACKOWLEDGEMENTS

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I am very much indebted to my supervisor, Dr. Olajumoke Oke Fayinminnu who carefully guided me

through this work. She was able to nurture the research potentials, qualities and ability to do

independent work in me. She was never tired of my shortcomings and was always there to listen to all

my concerns. My gratitude goes to all the members of the Toxicology unit, Crop Protection and

Environmental Biology, University of Ibadan for their support and cooperation during the course of my

project; Dr. Olubunmi Fadina, (Head of Unit). I also appreciate Mr. David Omobusuyi for his assistance

and for allowing me to tap into his wealth of knowledge during the course of this work. Special thanks

to Dr. ‘Tayo Adewunmi of the Faculty of Veterinary Medicine for allowing me use his animal house for

this research and for his availability and willingness to assist at odd hours. A big thank you to my

colleagues who were helpful at different points during this research; Steven Okafor, Ogunseye Israel,

Adewunmi ‘Yinka, Onoja Clement, Opaleye Abiodun, Olubakinde ‘Seun, Falana Modupe, Ajifolukun

‘Desola, Gbemibade Temitayo, Oluyoye Idowu, Ekanade ‘Tosin, Owoeye ‘Femi and Dr. Leonard

Akpheokhai. To the HOD (Dr. R.O. Awodoyin) and other members of staff I say thank you. I am

grateful to my parents Pastor and Mrs. L.O. Shiro for their prayers, contributions and invaluable support.

They also paid the price to give me a sound education. I am greatly indebted to Shiro Oluwatosin

Olufunke, my wife and confidant, for her constant prayers, encouragement, love, kindness and care. You

are a gift sent from God and I pray that God in His infinite mercies will protect, guide you, and make

our dreams and aspirations a reality. Amen. To my son, Shiro Iretomiwa Nathaniel Olamilekan (Mini

me), my jewel of inestimable value, I say “Daddy misses you and I am coming Home”!!! I also thank

my sisters, Akinade Tolulope and Ajayi Abolanle. Thank you all. Finally, I give glory to God Almighty

for making the completion of this programme a reality. Unto the King of kings, I give all glory, Honour

and adoration forever and ever. Amen.

DEDICATION

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This project is dedicated to God almighty for seeing me through, from the beginning to the end of this

programme, I thank Him for His absolute faithfulness. To my wife who was home alone for most of the

duration of this programme, thank you for the love and understanding Dearie.

Table of contents

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Title page i

Certification ii

Dedication iii

Acknowledgement iv

Abstract vi

Table of contents vii

List of plates xii

List of tables xiii

Chapter 1

1.0. Introduction

1.1. Justification of the Study

Chapter 2

2.0. Literature review

Chapter 3

3.0. Experimental Sites

3.1. Source of Experimental Materials

3.2. Preparation of Extracts

3.3. Toxicity of Extracts

3.4. Phytochemical Screening

3.4.1. Test for Anthraquinones

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3.4.2. Test for Terpenoids

3.4.3. Test for Flavonoids

3.4.4. Test for Saponins

3.4.5. Test for Alkaloids

3.4.6. Test for Essential oil

3.5. Field Work

3.6. Calibration of Knapsack Sprayer

3.7. Data collection

3.8. Preparation of Feed

3.9. Toxicological Studies

3.10. Data Analysis

Chapter 4


4.1. Toxicity of Alternanthera brasiliana and Cypermethrin-treated Amaranthus cruentus on Wistar

rats

Chapter 5

5.0. Discussion and Conclusion

References

List of plates

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Plate 1: Photomicrographs of a section of Livers and Kidneys of Control Rats exposed to Standard

Ration Feed containing no additives

Plate 2: Photomicrographs of a section of Livers and Kidneys of Rats exposed to feed containing

Cypermethrin-treated Amaranthus cruentus (1ml/100mls)

Plate 3: Photomicrographs of a section of Livers and Kidneys of Rats exposed to feed containing 100%

A. brasiliana-treated Amaranthus cruentus

Plate 4: Photomicrographs of a section of Livers and Kidneys of Rats exposed to feed containing75%






List of tables

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Table 1: Phytochemicals present in the leaf powder of Alternanthera brasiliana

Table 2: Mean values for A. brasiliana extract and Cypermethrin on the plant height (cm) of A.

cruentus for One (1) and Three (3) Days-After treatment.

Table 3: Mean values for A. brasiliana extract and Cypermethrin on the Stem Girth (cm) of A.


Table 4: Mean values for A. brasiliana extract and Cypermethrin on the number of leaves of A.


Table 5: Mean values for A. brasiliana extract and Cypermethrin on the leaf area (cm2) of A.


Table 6: Mean values for A. brasiliana extract and Cypermethrin on the Fresh Weight of A. cruentus

for One (1) and Three (3) Days-After treatment.

Table 7: Summary of the Histopathology results on Tissues of animals exposed to Alternanthera

brasiliana and Cypermethrin-treated Amaranthus cruentus

CHAPTER ONE

1.0. INTRODUCTION

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Vegetables constitute an important part of the human diet since they contain carbohydrates,

proteins, as well as vitamins, minerals and trace elements (Brenner et al., 2000). They play

indispensable roles in human nutrition, especially as a source of vitamins (A, B, C and E), minerals and

dietary fibre (Aletor and Adeogun, 1995). It is an excellent source of bio-available iron, up to 57 ppm

(Rangarajan and Kelly, 1994), and vitamin A, averaging 250 ppm. It is also high in protein (Segura-

Nieto et al., 1994). There are several types of vegetables in tropical African countries including Nigeria

that contribute greatly to the provision of many minerals and vitamins that are deficient in other classes

of food (Aletor and Adeogun, 1995).

Among these vegetables, Amaranthus cruentus seems to be more popular in Nigeria especially in

the south western part of the country (Mensah et al., 2008). Amaranthus cruentus is an herbaceous plant

which belongs to the family Amaranthaceae and genus Amaranthus. According to Brenner et al. (2000)

the genus comprises of approximately 60 species. The genus Amaranthus, of which A. cruentus is one, is

made up of plants mainly cultivated as vegetables for human consumption and animal feed. Other

notable members of the genus include Amaranthus hybridus, Amaranthus

hypochondriacus and Amaranthus caudatus.

Amaranthus is one of the most important annual leaf vegetables in the tropics. Amaranthus

cruentus has a short growing period of four to six weeks which serves as encouragement to farmers

especially the urban and peri-urban farmers to whom it serves as a source of employment (Makinde et

al., 2007).

In Nigeria, Amaranthus leaves combined with condiments are used to prepare soup (Oke, 1983;

Mepha et al., 2007). The leaves can be cooked like spinach, and the seeds can be germinated into

nutritious sprouts. It is an important crop for subsistence farmers.

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Amaranthus cruentus is a common flowering plant species. It is believed to have originated

from Amaranthus hybridus, with which it shares many morphological features. This species was in use

as a food source in Central America as early as 4000 BC. The plant is usually green in color, but a

purple variant was once grown for use in Inca rituals (Jerome, 2001).

The major problem facing vegetable farmers in Nigeria is the lack of adequate storage facilities

and the menace of pests and diseases. Intensive production of vegetable is therefore often accompanied

with frequent spraying of pesticides to improve their outlook thereby increasing profits (Food and

Nutrition Board, 2002). The scourge of these pests and diseases has continuously limited the production

of vegetables.

In the past, various synthetic insecticides have played a major role in vegetable protection and

have immensely benefited mankind both in terms of yield and quality. The modern use of insecticides

has also substantially improved the economic and social well-being of the inhabitants of developing

world by increased food production and by the effective control of public health vector-borne diseases

(IITA, 2000).

One of such synthetic insecticides is Cypermethrin; a synthetic pyrethroid used as an insecticide

in large scale commercial agricultural applications. It is also used in consumer products for domestic

purposes to control various pests, including moth pests of cotton, fruit and vegetable crops (Akubugwo

et al., 2007). Cypermethrin is used for crack, crevice and spot treatment to control insect pests in stores,

warehouses, industrial buildings, houses and apartments, greenhouses, laboratories, ships, railcars,

buses, trucks and aircrafts. It may also be used in non-food areas in schools, nursing homes, hospitals,

restaurants, hotels and food processing plants (Anonymous, 1989).

However, the public concern over the amounts of insecticides that are being applied to the land

and their possible adverse effect on human and animal health, and on the environment has risen sharply.

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Man has become a victim of his own advances with upsurge of many unexplained ailments. A large

decline in population of many species of birds, mainly fish-eating and bird-eating species has been

ascribed to the exposure to insecticides through food chains (Gilden et al, 2010). An upward bio-

magnification of pesticides, development of resistance, toxic residues in food and health hazards to grain

handlers (Obeng-Ofori et al., 1998) necessitates the search for a more sustainable approach to pest

control and natural crop protection (Fayinminnu, 2010).

Originally, the concept of ‘natural pesticides’ arose early in the development of agriculture.

Plants, as long-lived stationary organisms, must resist attackers over their lifetime, so they produce and

exude constituents of the secondary metabolism (PSMs), playing an important role in their defence

mechanisms (Isman, 2008). An interesting way of confirming this for bio-rational pesticides is screening

naturally occurring compounds in plants (Isman, 2006; 2008). Indeed, the Lithica poem (c. 400 B.C.)

states ‘All the pests that out of earth arise, the earth itself the antidote supplies’ (Ibn et al., 1781). In

recent years, there have been efforts internationally at developing new sources of pesticides from the

vast store of naturally occurring substances in plants. However, these natural substances are safe,

biodegradable and environmentally friendly (Olaifa et al., 1997; Fayinminnu et al., 2013). Such

alternatives include the use of botanicals derived from very cheap and renewable sources or at no cost,

especially the tropical plants (Ewete and Alamu, 1990) which are readily available (Fayinminnu, 2010).

These chemical-defensive compounds, often called allelochemicals that ward off attack by

potential herbivores (plant-feeding insects and mites). They may be directly harmful to herbivores or

modify (that is, slow down) their development, thus increasing their susceptibility to natural enemies

such as parasitic wasps (parasitoids) and/or predators (Illinois Pesticide Review, 2004). Humans have

made use of these naturally derived compounds for many years, and a number of botanical insecticides

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have been formulated for use by professionals and homeowners. Botanicals are processed in one of three

ways:

• Preparations of the crude plant material, ground into a dust or powder,

• Extracts from plant resins, formulated into liquid concentrations,

• Isolation of the pure chemicals obtained from plants by extraction or distillation.

Be that as it may, there is a common and general misconception that natural or botanical

insecticides are always safer than synthetically derived insecticides since they are natural. However, a

closer look at a number of registered botanicals shows that they are toxic to fish, beneficial insects,

mites and mammals. Though extracted from plants, “natural” does not necessarily imply “safe” or “non-

toxic.”

Ames et al., (1990a) estimated that of all dietary pesticides that we eat, 99.9% of the chemicals

that humans ingest are naturally occurring. It was also postulated that the amounts of synthetic pesticide

residues in plant foods are low in comparison to the amount of natural pesticides produced by plants

themselves (Ames et al., 1990a, b; Gold et al., 1997a). Bottom line: Natural compounds derived from

plants may not be inherently less toxic to humans than synthetically derived compounds.

Justification of the Study

The health implication of vegetables sprayed with synthetic insecticides by farmers and

consumed within a short period of time (at most 3 days after treatment) is of great concern to all sundry.

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Many farmers capitalize on consumers’ crave for ‘better quality’ vegetable, which unfortunately is based

mainly on the looks. Consumers consider undamaged, dark green and big leaves as characteristics of

good quality leafy vegetables (Bogusz et al., 2006). Many are ignorant of the fact that the external

morphology of vegetables cannot and does not guarantee safety from contamination. To this end, the

farmers come to market with freshly treated vegetables (few hours to a couple of days) and since all a

consumer sees is the good-looking, green, lush vegetable which appeals to the eyes, he/she purchases

and in the end consumes insecticides in doses that are hazardous to the human body. The campaign for

the use of botanicals informed the need to screen for the naturally occurring compounds in

Alternanthera brasiliana for bio-rational pesticides. It is also needed to examine their toxicity when

vegetables treated with their extracts are consumed within a short period of time.

The objectives of this study, therefore, are

• To identify the phytochemicals present in Alternanthera brasiliana.

• To compare the effects of Alternanthera brasiliana and Cypermethrin on the growth and yield of

Amaranthus cruentus.

• To assess the toxicity of Alternanthera brasiliana and Cypermethrin-treated Amaranthus

cruentus on the histology of Wistar rats.

CHAPTER TWO

1.0. LITERATURE REVIEW

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Plants, as long-lived stationary organisms, must resist attackers over their lifetime, so they

produce and exude constituents of the secondary metabolism (PSMs) which plays an important role in

their defence mechanisms (Isman, 2008). The phytochemicals’ research has its roots in allelochemistry,

involving the complex chemical mediated interactions between a plant and other organisms in its

environment (Chitwood, 2002). These were used in plant protection from the end of 19th century till the

beginning of the Second World War. Many of them are environmentally friendly, pose less risk to

humans and animals, have a selective mode of action, avoid the emergence of resistant races of pest

species, and as a result they can be safely used in Integrated Pest Management (IPM) (Isman, 2006).

The development of botanicals used as pesticides resulted from two parallel methods (Ntalli et al.,

2010):

• The observation of the traditional uses of plants and extracts for cattle and crop protection, followed

by checking the efficiency of these practices and identification of the active molecules. The activity

of nicotine extracted from tobacco (Nicotiana tabacum) and rotenone from Fabaceae Lonchocarpus

nicou and Derris elliptica fall in this category;

• The systematic screening of botanical families followed by biological tests in order to discover the

active molecules. Ryanodine, an alkaloid extracted from Ryania sp., and marketed in the United

States in 1945, is the result of such prospecting, carried out with collaboration between Rutgers

University and Merck in the early 1940s.

In addition, they may have proven suitable as choice products for organic food production. It is

mandatory though to attribute the efficacy of botanicals to specific identified constituent compound(s) in

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order to delineate the mechanisms of bioactivity, biologically and biochemically. They are to fully

exploit the therapeutic potential of extracts (Akhtar and Mahmood, 1994).

Generally, they are designed to breakdown more slowly and persistence can last for as long as

three (3) months, their consumption have been linked to the disruption of the endocrine system known to

adversely affect reproduction and sexual development (Gold et al., 1997) and being xenoestrogens. They

can also increase the amount of estrogens in the body which may lead to breast cancer (Gilden et al.,

2010).

However, current regulatory policy to reduce human cancer risks is based on the idea that

chemicals that induce tumors are potential human carcinogens (Gold et al., 1997a, b, c, 1998, 1999).

The enormous background of human exposures to natural chemicals has not been systematically

examined. This has led to an imbalance in both data and perception about possible carcinogenic hazards

to humans from chemical exposures. The regulatory process does not take into account:

• That natural chemicals make up the vast bulk of chemicals to which humans are exposed,

• That the toxicology of synthetic and natural toxins is not fundamentally different,

• That about half of the chemicals tested, whether natural or synthetic, are carcinogens when tested

using current experimental protocols,

• That testing for carcinogenicity at near-toxic doses in rodents does not provide enough information

to predict the excess number of human cancers that might occur at low-dose exposures,

• That testing at the maximum tolerated dose (MTD) frequently can cause chronic cell killing and

consequent cell replacement (a risk factor for cancer that can be limited to high doses) and that

ignoring this effect in risk assessment can greatly exaggerate risks.

1.1. THE SYNTHETICS

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Synthetic insecticides have been found to increase the yield of agricultural products over tenfold;

but then, the effects of pesticide misuse around the world have been known to include costly

environmental pollution and disruption of the balance of nature (IITA, 2000). The indiscriminate use of

chemicals in the control of pests has led to problems such as pest resistance, pollution of the

environment; leave toxic residues in agricultural produce, adversely affect non-target organisms and

health hazards to the users, or (Hussain et al, 1984). Pesticides work by interfering with an essential

biological mechanism in the pests, but because all living organisms share many biological mechanisms,

pesticides are never specific to just one species. Synthetic Insecticides are derivatives of naturally

occurring ones. They are strongly lipophilic and rapidly penetrate many insects and paralyze their

nervous system (Fuglie, 1998). Various formulations of these pesticides are often combined with other

chemicals (synergists) to increase their potency and persistence in the environment.

Synthetic insecticides applied to vegetables have been reported to cause variable changes in

brain on consumption (Ecobichon et al., 1994) which have been related to hypoxia, hypoglycemia,

and/or damage to cell ion homeostasis. Necrosis of hepatic cells, with pyknotic nuclei and dilatation of

sinusoids with highly disrupted hepatic laminae in rats has also been reported (Biernacki et al., 1995).

Cigankova et al (1993) also reported that synthetics have great impact on the loss of various stages of

spermatogenesis when he observed degeneration and depletion of spermatocytes and spermatids in

supermethrin-exposed adult pheasants.

Their dermal contact in facial area may cause a subjective sensation of tingling or numbness

(Sandhu and Brar, 2000). Slight to severe skin irritation, decreased food consumption, body weight and

absolute and relative gonad weights have been observed in rabbits treated with Cypermethrin

(Handerson and Parkinson, 1981). Besides generalized toxic effects of Cypermethrin, decreased number

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of implantation sites, number of viable fetuses and weight gain of fetuses in rabbits treated with

Cypermethrin have been reported (Elbetieha et al., 2001).

Exposures to lindane have shown long-term effects on myometrial functions that are necessary

for parturition, inhibiting spontaneous phasic contractions in late gestation rat uterus and gap junction

intercellular communication in myometrial cell cultures (Rita, 2003). Intrauterine growth retardation has

also been associated with elevated maternal blood concentrations of lindane (Siddiqui et al., 2003).

Additionally, in preliminary studies we have found that simultaneous exposure of uterine tissues to

lindane reduces the force and oscillatory activity of spontaneously contracting rat uterine strips (Goel et

al., 1998).

Again, Dichlorodiphenyltrichloroethane (DDT) has the potential to disrupt the endocrine system

of humans (Colburn et al., 1996). There is also evidence that DDT causes teratogenic effects in test

animals (Mellanby, 1992).

1.2. THE BOTANICALS

In a bid to increase environmental awareness, The European Union through the Commission of

the European Communities in 2006 launched a Thematic Strategy on the Sustainable Use of Pesticides;

it decided to minimize the hazards and risks to health and the environment caused by the use of plant

protection products. This framework directive was accepted by the European Parliament accepted in

2009.

The directive states that “when pesticides are used, appropriate risk management measures

should be established and low-risk pesticides as well as biological control measures should be

considered in the first place“. Biological control comprises various technologies of which one option is

the use of botanical products. On the other hand, there is a quest to explore the ability of the nature and

the abundant resources for chemicals available for plant defence and suitable in pest management for

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crop protection. Many kinds of plant species and technologies have been used in the production of

botanical pesticides.

Essential oils from sunflower (Asteracaeae) have been used as chemical defence against

insecticides, acaricides, avoiding bacterial or fungi phyto-pathogen colonization, attracting natural

enemies of herbivores (Bakali et al., 2008; Yadav, et al., 2008; Karamanoli et al., 2005; Iacobellis et al.,

2005; Flamini, 2003; Karamanoli, 2002).

Neem (Azadiracta indica) is a mixture of more than 100 limonoid compounds, including

azadirachtin, salannin, and nimbin and their analogues provoking repellence, feeding deterrence and

insect growth inhibition (Schmutterer, 1990). They are known to possess insecticidal and antifungal

properties (Akhtar et al., 2008; Carpinella, et al., 2003).

The use of neem extract (Azadirachta indica) for the production of a wide range of commercial

formulations exhibiting good efficacy against more than 400 insect species (Akhtar et al., 2008; Lee et

al., 1991), mites (Flamini, 2003) and nematodes is a norm (Akhtar, 2000; Oka et al., 2007). Obeng-

Ofori et al. (2003) evaluated the seed extracts of the neem tree, Azadirachtha indica (A. Juss) on okra

pests; and found that the extracts reduced the damage done to the leaves, flowers and fruits of the crop.

At 5 ml/L and 6 ml/L rates of application, neem oil extracts were most effective in preventing the

development of Euphestia cautella larvae in stored grains (Eziah et al., 2011)

Kuriyama et al., (2005) evaluated that extracts of Quassia amara, Cassia camara and Picrasma

exelca acts as non-competitive antagonists of the ionotropic GABARs to stabilize the closed

conformation of the channel, resulting in the inhibition of the action of GABA in nematodes. They have

also been known to act against insects, nematodes and weeds (Koul, 2008; Powell et al., 1998; Leskinen

et al., 1984; Chitwod, 2002; Lin et al., 1995).

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Plant Secondary Metabolites from the soapbark tree, Quillaja saponaria, possesses significant

antifeedant, fungicidal and nematicidal properties (Chitwood, 2002; Koul, 2008; Duke et al., 2003;

D'Addabbo et al., 2006; 2010; Ribera et al., 2008; Martin and Magunacelaya, 2005). An extract of the

plant Macleaya cordata is known to exhibit fungicidal properties (Newman et al., 1999).

The efficacy of mixtures of piper retrofratctum (Piperaceae) and Annona squamosa

(Annonaceae) extracts, Aglaia odorata (Meliaceae) and Annona squamosa extracts were evaluated at

0.05% and 0.1% against deltamethrin at 0.04% and Bacillus thuringiensis at 0.15%. The mixtures were

found to decrease the population of Crocidolomia pavonana and Plutella xylostella and it did not affect

the insect pests’ natural enemies: Diadegma semiclausum and Eroborus argentiopilosus (Dandang and

Djoko, 2011).

In single assays, the seed extract of Annona squamosa exhibited high insecticidal activity against

Crocidolomia pavonana larva with the LC50 being 0.208% (Basana and Prijono, 1994). The Aglaia

odorata extract was noted to be effective against several agricultural insect pests including P. xylostella

and C. pavonana larvae. Treatment of the ethanol twig extract of A. odorata caused 100% mortality to

Spodoptera litura (Koul et al., 1997).

Both Jatropha curcas and Annona muricata seed crude extracts have been shown to act as

contact and stomach poisons against Sitophilus zeamais on rice grain. By dipping method, the weevil

mortality were 90% and 70% respectively at concentration 20% (v/v), whilst by surface protectant

method, the weevil mortality was 100% at 0.4% (v/w) concentration for both crude extracts (Asmanizar

et al., 2012).

Ntalli et al (2010b; 2010c) tested the paralytic activity of extracts obtained with hydro-

distillation from 15 botanical species on root knot nematodes and its activity against Melodogyne

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incognita. It was found to decrease in the order O. vulgare, O. dictamnus, M. pulegium, M. officinalis,

F. vulgare, P. anisum, E. meliodora and P. terebinthus.

Natural products from common weeds such as Ageratum conyzoides (L.), crops like tomato,

Solanum lycopersicum, cashew, Anacardium occidentale (L.) and some ornamental plants such as the

morning glory, Ipomea carnea (Jacq.) used in various forms and concentrations have been found

effective in controlling insect pests at different stages of their development and activities (Dale, 1996).

Chinaberry demonstrated bio-fumigant properties when incorporated as pulverized fruits in Melodogyne

incognita infested soil to be tested for its effect on nematode life cycle (EC50=0.34 % w/w) (Ntalli et al.,

2010a).

In a choice bioassay, a laboratory experiment involving birch tar oil on aphids (Myzus persicae)

eggplants effectively killed (95 %) when sprayed once (1% v/v aq. solution) (Tiilikkala and Segerstedt

2009). When painted on fences and pots, it most efficiently prevented the molluscs Arianta arbustorum

and Arion lusitanicus (Lindqvist et al., 2010) from crossing the barriers to reach the food behind the

fence or in the pots. The repellence of birch tar oil was also noticed with the egg laying psyllids (Trioza

apicalis).

Velmurugan et al., (2009) showed that wood vinegar made from bamboo and broad-leaved trees

are effective against sap-staining fungi. The antifungal efficiency of which is reported to be strongly

dependent on their phenolic compound content (Baimark et al., 2009). The variability of botanical

products is a well-known phenomenon and carefully considered e.g. in the production of botanical

medicine (Shane-McWhorter 2001).

Alternanthera brasiliana (L.) O. kuntze; an important herb found as a perennial herb, native to

tropical and subtropical regions of Australia and South America, is one of such plants being prospected

for its insecticidal properties as propounded by Isman (2006). It belongs to the family Amaranthaceae,

22

genus Alternanthera and species brasiliana. It is prostrate and branchy 7.5- 45.0 cm long, presenting a

circular to polygonal stem in transection, long internodes and swollen nodes, at which opposite leaves

attach. Branches are 7.5- 45.0 cm long, glabrous, the ultimate ones with two lines of hair, nodes often

villous; leaves 2.5-7-5 cm, even longer when growing in watery places, rather fleshy, sometimes

obscurely denticulate; flowers inconspicuous, white, in clusters; seeds 1.25-1.5mm, sub- orbicular.

The inflorescence is cymes, composed of hermaphrodite, actinomorphous and monocyclic

flowers. The leaves are simple, entire, decussate, oval-lanceolate and purple, presenting uniseriate

epidermis, pluricellular non-glandular trichomes coated by papillose cuticle, anomocytic and diacytic

stomata on both surfaces; the mesophyll is dorsiventral, with collateral vascular bundles and druses. The

stem, in secondary growth, has the dermal system similar to the leaf; the angular collenchyma alternates

with the chlorenchyma; it occurs as druses and a cambial variant, consisting of concentrical arcs of

extra-cambia outside the first cambium and aligned vascular bundles in the pith (Duarte and Debur,

2004).

Traditionally, the plant is used as a galactagogue (Induces milk secretion), abortifacient (Causes

abortion) and febrifuge (Alleviates fever). It is also used for indigestion. The leaves are used like

spinach and in soups. It is claimed to be a good fodder which increases milk in cattle. In some parts of

Bihar, the plant is used for hazy vision, night blindness, diarrhea, dysentery and post-natal complaints.

The poultice (Dressing by covering with a therapeutic substance) of the herb is reported to be used for

boils (Anon, 2005). An ether extract of the plant yields an active principle having anti-ulcerative

property. It has very high iron content, and may be used as a salad. The herb is said to possess diuretic

properties and its decoction is taken in gonorrhea (Anon, 2005).

23

The aerial part of A. brasiliana is used in cystitis, throat and general infarction, antibiotic and

antiviral against virus-herpes simplex I. (Coelho de Souza et al., 2004) and antioxidant activities

(Mariani et al., 2008). The plant is known to contain medicinal and insecticidal properties.

However, not much has been done to exploit its insecticidal potentials. Alternanthera brasiliana

seems to be a promising plant as an insecticide with low mammalian toxicity since its extract has shown

lymphocyte proliferation in man (Khare, 2007). Bell et al. (1990) reported that secondary compounds

like alkaloids, terpenoids, phenolic, flavonoids, chromenes and other minor chemicals can affect insects

in several ways and also postulated by Isman (2006).

Wound healing activity of methanolic extract of leaves of Alternanthera brasiliana evaluated by

Chorioallantoic membrane (CAM) model, showed a higher percent contraction of wound at (5% w/w)

and it significantly increased angiogenesis and tensile strength (Barua et al., 2009). Methanolic and

hydro - alcoholic extracts obtained from A. brasiliana in-vitro cultivated plantlets and callus presented

analgesic properties with different in vivo pharmacological models (Silva et al., 2005). Alternanthera

brasiliana is also known to show antimicrobial activities against Staphylococcus aureus,

Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis, Micrococcus luteus, Candida albicans,

and Saccharomyces cerevisiae. (Coelho de Souza et al., 2004).

CHAPTER THREE

3.0. MATERIALS AND METHODS 3.1. Experimental Sites

24

The experiment was carried out in the Teaching and Research Farm Unit, Toxicology Laboratory

of The Department of Crop Protection and Environmental Biology (CPEB), Central Animal House,

College of Medicine and The Experimental Animal unit of The Department of Veterinary Anatomy, all

in the University of Ibadan, Ibadan Nigeria.

3.2. Source of Experimental Materials

The seeds of Amaranthus Cruentus used were collected from the Practical Year Training Plot

Unit, Faculty of Agriculture and Forestry. Leaves of Alternanthera brasiliana were harvested from the

Teaching and Research Farm Unit and experimental animals (Rats) were from the Central Animal

House, College of Medicine, University of Ibadan. The Cypermethrin used was bought from the open

market, in Dugbe Area of Ibadan

3.3. Preparation of Extracts

The extraction procedure was carried out according to the method of Ahn and Chung (2000) with

a modification, one hundred and forty-four grams (144gms) of the plant was used instead of the seventy

two grams (72gms) used in Ahn and Chung (2000) to prepare the extract. Leaves of A. brasiliana were

air-dried for seven (7) days, cut into chips and milled into powder. The milled material was soaked for

24 hours after which the solution was filtered through muslin cloths to remove the debris.

Filtrate obtained was then passed through Whatman No.1 filter paper. The final filtrate of plant

part was considered as the full strength (100%) of the aqueous extracts. Using serial dilution method,

volume of distilled water was added to the full strength filtrate to obtain 75%, 50% and 25% (v/v)

strength. The extracts were stored in refrigerator at 20oC for 24hours prior to use to prevent putrefaction

and degradation of allelochemicals present in them. The extracts were used for the bioassay.

3.4. Toxicity of Extracts

Toxicity of the aqueous extracts of Alternanthera brasiliana was tested by preparing

concentrations corresponding to 100, 75, 50 and 25% and using the recommended dose of Cypermethrin 25

(1ml/100mls) as control, the different concentrations of the plant extract were applied to vegetables on

the field with each treatment replicated four times.


The screening was performed on the powder of Alternanthera brasiliana in the Organic

Laboratory of the Chemistry Department, University of Ibadan. The following compounds were tested

for according to Harbone and Sofola (2007):

(i) Anthraquinones

0.5g of the extract was boiled with 10ml of concentrated tetraoxosulphate (VI) acid, H2SO4 and

filtered while hot. The filtrate was shaken with 5ml of chloroform. The chloroform layer was removed

through pipette into another test tube and 1ml of dilute ammonia was be added. The resulting solution

was observed for color changes.

(ii) Terpenoids (Salkowski test)

To 0.5g each of the extract, 2ml of chloroform was added. 3ml of concentrated H2SO4 was

carefully added to form a layer. A reddish brown coloration of the interface indicates the presence of

terpenoids.

(iii) Flavonoids

5ml of dilute ammonia was added to a portion of an aqueous filtrate of the extracts. 1ml of

concentrated H2SO4 was then added. A yellow coloration that disappears on standing indicates the

presence of flavonoids.

(iv) Saponins

26

5ml of distilled water was added to 0.5g of extract in a test tube. The solution was shaken

vigorously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil

and shaken vigorously after which it was observed for the formation of an emulsion.

(v) Tannins

About 0.5g of the extract was boiled in 10ml of water in a test tube and then filtered. A few

drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration.

(vi) Alkaloids

0.5g of extract was diluted to 10ml with acid alcohol, boiled and filtered. To 5ml of the filtrate,

2ml of dilute ammonia was added. 5ml of chloroform was added and shaken gently to extract the

alkaloidal base. The chloroform layer was extracted with 10ml of acetic acid. This was divided into two

portions. Mayer’s reagent was added to one portion and Draggendorff’s reagent to the other. The

formation of a cream (with Mayer’s reagent) or reddish brown precipitate (with Draggendorff’s reagent)

is regarded as positive for the presence of alkaloids.

(vii) Essential oil

The oil was extracted using soxhlet extraction method.

3.6. Calibration of Knapsack Sprayer

The calibration of the capacity of the knapsack sprayer was done according to Akobundu (1987)

in order to evaluate the amount of extract to be used per plot and consequently the gross area.

3.7. Field Work

The gross area was 20m x 30m with individual plot sizes of 2m x 1m and an alley of 1m on

which Amaranthus cruentus seeds were sown by drilling. Each of the five (5) treatments was replicated

four times (4) and laid out in a completely randomized block design (CRBD). The plots were adequately

watered before the seeds were sown and subsequently, watering was done twice daily.

27

The Plants were thinned two weeks after sowing (WAS) and the six (6) different

concentrations/treatment levels were applied and plants evaluated on days-after-treatment basis i.e. one

day after treatment (1DAT) and three days after treatment (3DAT). The plants were sprayed at 3 WAS

and 24hours before harvesting at 5 WAS. The plants were terminated at the end of the fifth week by

harvesting before emergence of the inflorescence. Harvested plants were properly bulked, marked and

air-dried for a period of seven days

3.8. Data Collection

At weekly intervals, the plants were assessed for growth parameters by taking the plant height

from soil surface (using meter rule), stem diameter at 1cm above soil level (using a pair caliper), number

of leaves produced, leaf area and yield.

3.9. Preparation of Feed for Wistar Rats

The drying process was monitored to be gradual with adequate manual turning by raking the

spread plants to facilitate uniform drying and eliminate moulds. Samples were obtained randomly from

the dried plant for the dry weight measurement. The drying process was followed by milling the brittle

leaves and stems (edible parts) after sieving with a 0.5mm sieve to obtain into particle size that could be

mixed with animal feed (1g of additive added to 99g of animal feed).

Standard Ration Feed (SRF) was milled and divided into eleven (11) feed portions. The different

levels of milled A. cruentus were then mixed with the feed portions and each group mixed thoroughly in

the proportion of (1g of A. cruentus to be used for feed formulation added to 99g of animal feed).

3.10. Toxicological Studies on Rats fed Alternanthera brasiliana and Cypermethrin-treated

Amaranthus cruentus

Prior to the arrival of the rats, the rat house and cages were properly cleaned and disinfected.

Cages were properly arranged and fitted with drinkers that could comfortably drop water when imbibed

by rats, and feeders properly fixed to eliminate feed spillage.

28

A total of 44 rats were subjected to acclimatization for a period of 7days during which standard

rat feed and water were given to them in ad libitum. At the expiration of the acclimatization period the

rats were divided into 11groups of 4rats each and housed in stainless box per group for a 30-day

experimental period. Feeders and drinkers were fitted into each box to provide food and water. Each

group of the animals was exposed to each of the 11 feed compositions:

• No additives + Standard ration feed

• 1ml/100ml concentration of 1DAT Cypermethrin- treated vegetable + SRF

• 100% Alternanthera brasiliana 1DAT extract + SRF




• 1ml/100ml concentration of 3DAT Cypermethrin- treated vegetable + SRF





The animal room temperature was maintained at ambient temperature of 270C. Body weights of

the animals were taken on the 0-day of the experiment and this was repeated weekly till the 30 th day of

the experiment to assess the weight gain/loss. Daily routine observation was done to check for mortality

and abnormal clinical manifestations such as salivation and aggressiveness.

After 30 days of treatment with Amaranthus cruentus additives the rats were sacrificed by

cervical dislocation. The abdomens of all rats were dissected immediately to remove livers, kidneys and

29

testes. Blood was taken by ocular puncture, preserved in heparin bottles and refrigerated for further

analysis.

The tissues (Liver and kidneys) were weighed and preserved in 10% formalin for

histopathological examination evaluation. They were then processed and stained with hematoxylin and

eosin stain for histopathology examination. The slides were examined single-blindly by a qualified

pathologist.

3.11. Data analysis

The experimental design used was randomized complete block design (CRBD). All the data were

analyzed using one-way analysis of variance (ANOVA) at P = 0.05 after carrying out appropriate

transformations and means were separated. Differences were considered to be statistically significant at

P = 0.05. Web Agri-Stat Package (WASP) was used for the analysis.

CHAPTER FOUR

4.0. RESULTS4.1. Phytochemical Screening.

Analysis of the powdered leaf extract of Alternanthera brasiliana indicated the presence of

saponins, flavonoids, reducing sugars, glycosides and resins while tannins, phlobatanins, alkaloids,

phenols, anthraquinones and steroids were absent as shown in Table 1.

30

4.1.1. Effects of A. brasiliana extract and Cypermethrin on the plant height (cm) of A. cruentus for

One (1) and Three (3) Days-After Treatment.

The results as in Table 2 showed that at 1 DAT there was no significant difference (P≤0.05)

between the values for the control, Cypermethrin (1ml/100mls), 100 and 25% for plant height.

Treatments 100, 75 and 50% were significantly different (P≤0.05) from each other. The highest mean

value was recorded for the plots treated with 100% extract, while the lowest value was recorded from

the 75% extract treated plots. At 3 DAT there was no significant difference (P=0.05) recorded for plant

height between the control, 50 and 25% treatments but were significantly different at P=0.05 from the

Cypermethrin, 100 and 75% treatments.

4.1.2. Effects of A. brasiliana extract and Cypermethrin on the stem girth of A. cruentus for


Stem girth at 1 DAT as shown in Table 3 recorded no significant difference (P=0.05) between

the control and 25% treatments but with significant differences (P=0.05) when compared with 100, 75

and 50% treatments. At 3 DAT, no significant differences (P=0.05) were observed between the control

and other treatments. Significant differences were observed between the Cypermethrin-treated plots, 100

and 50% treatments while 75 treatment and 25% extract treated plots recorded no significant difference

(P=0.05) from each other.

4.1.3. Effects of A. brasiliana extract and Cypermethrin on the number of leaves of A. cruentus

for One (1) and Three (3) Days-After Treatment.

There was no significant difference (P=0.05) amongst all the means for the treatments at 1 DAT

as shown in Table 4. It also showed that the control plot recorded the lowest value with a significant

difference (P=0.05) from other plots at 3 DAT. It also showed 25% extract treated plots recording the

highest value with no significant difference (P=0.05) from 50 and 100% treated plots. Also these

treated plots recorded no significant difference (P=0.05) from the Cypermethrin and 75% treated plots

31

4.1.4. Effects of A. brasiliana extract and Cypermethrin on the leaf area (cm2) of A. cruentus for


Table 5 showed no significant difference (P=0.05) among all treatments, with the control

recording the highest value for leaf area at 1 DAT. For 3 DAT no significant differences (P=0.05) was

observed amongst all the treatments. Plots treated with 75% extract recorded the highest value for leaf

area. Although not significantly different from 50% and Cypermethrin-treated plots. No significant

difference was also observed from 100 and 25% treated plots. However, the control recorded the lowest

value for leaf area but it was not significantly different (P=0.05) from the 100 and 25% treated plots.

4.1.5. Effects of A. brasiliana extract and Cypermethrin on the fresh weight (g/plot) of A. cruentus

for One (1) and Three (3) Days-After Treatment.

Results on fresh weight in Table 6 showed no significant difference (P=0.05) among all

treatments at 1 DAT and 3 DAT. For the one day after treatment, the 100% extract treated plots had the

highest value and 25% treatment the lowest but for Table 6b, the highest value was for 75% treatment

while the lowest was for the control.

Table 1: Phytochemicals present in the leaf powder of Alternanthera brasiliana

Compound Powdered Leaf Extract

Saponins +

Flavonoids +

Tannins -

Phlobatanins -

32

Cardiac glycosides -

Alkaloids

Reducing Sugar

-

+

Phenol -

Anthraquinones -

Glycosides +

Resins +

Steroids -

+ indicates presence

– indicates absence

Table 2: Mean values for A. brasiliana extract and Cypermethrin on the plant height (cm) of A.

cruentus 5 WAS.

Treatments (%) 1 DAT 3 DATControl

Cypermethrin (1ml/100mls)

17.310ab ± 1.33

20.412a ± 3.12

17.25b ± 4.79

15.32ac ± 6.79100% Extract 21.530a ± 3.39 21.73a ± 4.0875% Extract 16.233b ± 0.54 21.20a ± 4.0850% Extract 14.915b ± 1.89 16.73c ± 6.2925% Extract 16.735ab ± 0.76 16.98c ± 6.45

LSD (<0.05) 4.801 0.97

33

Means followed by the same alphabet in each column are not significantly different from each other.

Table 3: Mean values for A. brasiliana extract and Cypermethrin on the Stem Girth (cm) of A.

cruentus at 5 WAS.



0.11c ± 0.386

0.11c ± 0.386

0.13c ± 2.246

0.19a ± 2.251100% Extract 0.14a ± 1.935 0.25a ± 0.2575% Extract 0.13ab ± 2.246 0.18b ± 2.24650% Extract 0.13ab ± 0.964 0.23a ± 0.96425% Extract 0.12bc ± 0.704 0.18b ± 2.246

LSD (<0.05) 0.01 0.01

Means followed by the same alphabet in each column are not significantly different from each other

34

Table 4: Mean values for A. brasiliana extract and Cypermethrin on the number of leaves of A.

cruentus at 5 WAS.



11.350a ± 0.386

12.350a ± 0.865

8.00c ± 2.50

10.00a ± 4.08100% Extract 13.150a ± 1.935 11.00a ± 4.79

75% Extract 11.450a ± 2.246 10.00ab ± 4.0850% Extract 12.750a ± 0.964 11.75a ± 4.7925% Extract 14.550a ± 0.704 12.00a ± 7.50

LSD (<0.05) 4.121

NS

0.5

Means followed by the same alphabet in each column are not significantly different from each other.

NS = Not significant

35

Table 5: Mean values for A. brasiliana extract and Cypermethrin on the leaf area (cm2) of A.

cruentus at 5 WAS.



33.075a ± 5.543

36.405a ± 4.128

57.63c ± 4.79

73.68ab ± 5.50100% Extract 46.035a ± 6.696 70.50bc ± 4.7975% Extract 35.285a ± 8.558 76.45a ± 8.6650% Extract 36.405a ± 4.128 72.68ab ± 4.0825% Extract 37.080a ± 9.209 67.85bc ± 7.50

LSD (<0.05) 22.714

NS

4.45

Means followed by the same alphabet in each column are not significantly different from each other.NS = Not significant

Table 6: Mean values for A. brasiliana extract and Cypermethrin on the Fresh Weight of A.

cruentus at 5 WAS.

Treatments (%) 1 DAT 3 DATControl 32.93a ± 12.71 32.93a ± 10.50

36

Cypermethrin (1ml/100mls) 32.96a ± 10.50 42.26a ± 18.49100% Extract 57.49a ± 13.05 46.77a ± 10.3575% Extract 38.95a ± 4.94 48.17a ± 9.1950% Extract 42.26a ± 18.49 41.83a ± 9.1925% Extract 32.93a ± 12.71 38.95a ± 4.94

LSD (<0.05) 35.95

NS

27.32

NS

Means followed by the same alphabet in each column are not significantly different from each otherNS = Not significant

4.2. Toxicity of Alternanthera brasiliana and Cypermethrin-treated Amaranthus cruentus on

Wistar rats.

Results on the histopathological examinations carried out on the experimental rats is summarized

in Table 7. The Standard Ration Feed group (control) showed no visible lesions in two while the

remaining two showed mild portal cellular infiltration by mononuclear cells (Plate 1). There were no

apparent morphological changes in the livers and kidneys of the control rats. However, it produced

sloughing off renal tubular epithelial, but no effect on glomeruli. The control group showed no symptoms of

any gross abnormalities therefore, there were no adverse effects.

37

Observations from the 100% A. brasiliana extract-treated experimental rats (Plate 3) showed

three of the rats examined having no observable lesions except one in which there was a mild periportal

cellular infiltration. The livers of the rats exposed to 75% A. brasiliana extract-treatment (Plate 4)

showed some marked renal cortical congestion even though there were no observable lesions in some

members of the group.

Necrotic changes were observed in the livers of some members of the 50% A. brasiliana extract-

treatment (Plate 5) in addition to hemorrhages and necrosis. There were development of lesions

indicated by mild periportal cellular infiltration by mononuclear cells and necrosis of hepatic cells with

pyknotic nuclei in the livers of members exposed to the 25% A. brasiliana extract-treatment (Plate 6)

In the Kidneys of the experimental rats, there were marked renal cortical congestion in almost all of

the rats exposed to 50 (Plate 5) and 25% (Plate 26) A. brasiliana extract-treatments. Shrinkage of

glomeruli, necrosis, and disruption of renal tubules with severe portal and central venous congestion

were observed in the 75% A. brasiliana extract-treatment (Plate 4). For the control (Plate 1) and 100%

A. brasiliana extract-treatment group (Plate 3), no visible lesions were observed.

Table 7: Summary of the Histopathology results on Tissues of animals exposed to Alternanthera

brasiliana and Cypermethrin-treated Amaranthus cruentus

38

39

Treatment Liver Kidney

Control Rat (Standard ration

feed)1

No visible lesions No visible lesions

Control Rat (Standard ration feed)2

Mild lesions observed Mild portal cellular infiltration


feed)3

No visible lesions Mild renal cortical congestion


feed)4

No visible lesions Mild renal cortical congestion

Cypermethrin-treated

(1ml/100mls) Rat 1

No visible lesions Mild congestion at the cortical

region. There are numerous

sites showing protein casts in

the tubular lumen.Cypermethrin-treated

(1ml/100mls) Rat 2


(1ml/100mls) Rat 3


(1ml/100mls) Rat 4

Mild portal cellular infiltration No visible lesions observed

by mononuclear cells

No visible lesions observed Marked renal cortical

congestion

Mild portal cellular infiltration No visible lesions observedby mononuclear cells

100% A. brasiliana Extract- No visible lesions observed No visible lesions observed

treated Rat 1

100% A. brasiliana Extract Periportal cellular infiltration Marked renal cortical congestion

-treated Rat 2 by mononuclear cells (mild)

100% A. brasiliana Extract- No visible lesions observed Mild necrosis and vacuolization with

treated Rat 3 neuronal degeneration

100% A. brasiliana Extract- No visible lesions observed Moderate congestion of the cortical

treated Rat 4 vessels

75% A. brasiliana Extract- No visible lesions observed Severe portal and central venous

treated Rat 1 congestion

75% A. brasiliana Extract- Marked renal cortical Necrosis of hepatic cells

treated Rat 2 congestion

75% A. brasiliana Extract- Marked renal cortical Shrinkage of glomeruli, necrosis and

treated Rat 3 congestion disruption of renal tubules

75% A. brasiliana Extract- No visible lesions observed Mild portal congestion

treated Rat 4

50% A. brasiliana Extract- Mild portal congestion and Marked renal cortical congestion

treated Rat 1 cellular infiltration

50% A. brasiliana Extract- Congestion and hemorrhage Marked renal cortical congestion

40

treated Rat 2 with thickening of inter-alveolar septa

50% A. brasiliana Extract- No visible lesions observed Marked renal cortical congestion

treated Rat 3

50% A. brasiliana Extract- Hemorrhages, disruption Marked renal cortical congestion

treated Rat 4 in branching structure and

early necrotic changes

25% A. brasiliana Extract- No visible lesions observed Marked renal cortical congestion.

treated Rat 1 There are foci of interstitial cellular

infiltration

25% A. brasiliana Extract- Mild periportal cellular infiltration Marked renal cortical congestion

treated Rat 2 by mononuclear cells

25% A. brasiliana Extract- No visible lesions observed Mild portal cellular infiltration by

treated Rat 3 mononuclear cells

25% A. brasiliana Extract- Mild necrosis of hepatic cells Mild portal cellular infiltration by

treated Rat 4 with pyknotic nuclei mononuclear cells

41

Liver 1 Liver 2 Liver 3 Liver 4

42

Kidney 1 Kidney 2 Kidney 3 Kidney 4

Plate 1: Photomicrographs of a section of Livers and Kidneys of Control Rats exposed to Standard

Ration Feed containing no additives.

Key:

Liver 1 = Standard Ration-fed Rat 1 Kidney 1 = Standard Ration-fed Rat 1




43


44


Plate 2: Photomicrographs of a section of Livers and Kidneys of Rats exposed to feed containing

Cypermethrin-treated Amaranthus cruentus (1ml/100mls)

Key:

Liver 1 = Cypermethrin-treated Rat 1 Kidney 1 = Cypermethrin-treated Rat 1




45


46



A. brasiliana-treated Amaranthus cruentus.

Key:

Liver 1 = 100% A. brasiliana-treated Rat 1 Kidney 1 = 100% A. brasiliana-treated Rat 1




47


48


Plate 4: Photomicrographs of a section of Livers and Kidneys of Rats exposed to feed containing75% A.

brasiliana-treated Amaranthus cruentus.

Key:





49


50




Key:





51


52




Key:





53

CHAPTER FIVE

5.0 DISCUSSION AND CONCLUSION

In this study, for plant parameters, the 100% extract of A. brasiliana compared favourably with

the Cypermethrin treatment than all other treatments applied in the growth and yield parameters

analyzed thereby giving credence to Mariani et al (2008) who advocated that the plant is known to

contain insecticidal properties. The performance of the 100% extract, may be due to the presence of high

concentrations of plant secondary metabolites (Flavonoids and Glycosides) that can affect insects since

there was no dilution.

On the other hand, study of tissues has been and continues to be important in understanding

fundamental molecular mechanisms of toxicity as well as in assessment of risks to humans. Microscopic

observation revealed that the 100% A. brasiliana-treated animal group’s hepatic tissue showed normal

large polygonal cells i.e. the histopathological study of the liver and kidney showed a normal

architecture. This may be due to the many pharmacological activities of the extracts of A. brasiliana like

anti-inflammatory, analgesic, wound healing, antitumor, immunostimulant and antimicrobial activities in

accordance with Kumar et al., (2011). There was no visible lesion which may suggest significant

presence of contaminant(s).

The kidneys also showed some structural changes through the manifestation of lesions at

different levels. The degree of manifestation however was not dose-dependent (did not follow the

concentrations of the extract) on the feed of the different animal groups. Observable lesions at varying

magnitudes were noticed across treatment groups. This is in conformity with the findings of Vainio et

al., (1995) that various natural toxins, which have been present throughout vertebrate evolutionary

history, nevertheless cause cancer in vertebrates. The lesions found in the tissues of the rats in the

control group may be in conformation with the works of Lois et al (2001), who postulated that while 54

research scientists pay so much attention to the toxicity of chemical substances present in foods, they

sometimes overlook the other contents whose actions may either be synergistic or antagonistic.

The extracts of A. brasiliana were found to increase angiogenesis, tensile strength and inhibit

writhing especially in the 100% extract-treated rats and this is also in conformity with Barua et al.,

(2009). Based on this work and those of Akhtar, M. (2000, 2008). Baimark et al., (2009), D’Addabbo et

al (2010) and Asmanizar et al (2012) it seems evident that plant extracts are biodegradable and thus will

not cause similar environmental risks as many of the widely used synthetics.

To this end, it can be concluded that it is safe to consume Amaranthus cruentus vegetables

sprayed with A. brasiliana extract at either One day-after treatment (1DAT) or Three days-after

treatment (3DAT). Due to A. brasiliana’s wound healing, anti-inflammatory, analgesic, antibacterial

coupled with its actions against infarction (Localized necrosis resulting from obstruction of the blood

supply). Again, where the 100% A. brasiliana extract treatment is not used, the 75% A. brasiliana extract

concentration can be used as it’s performance was fairly effective too, just that due to the extracts

increasing biodegradability vis-à-vis the level of dilution, there may be the need to apply more of it with

fewer days in between the days of application (Matsumura et al., 1972).

Amongst the various types of botanicals being prospected for use as insecticides, A. brasiliana is

one of the readily available ones which can be gotten without much problems. This is because the plant

can be seen growing along river banks, edges of farmlands and in most cases, found among weeds

growing in freshly cultivated plots. One other encouragement farmers can is the ease of preparation as

one only needs water for the extraction.

Conclusively, longer exposure periods to A. brasiliana-treated A. cruentus in different

concentrations is further recommended for toxicity tests in experimental animals to ascertain its

55

insecticidal, phytochemical and pharmacological studies. In addition, studies that will examine the

contents of food compositions to ascertain their mode of interaction (Synergistic or Antagonistic) with

A. brasiliana extracts is also recommended.

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56

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and Pseudaletia unipuncta. Phytochem. Rev., 7: 77–88, ISSN: 1568-7767, EISSN: 1572-980X.

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