CHAPTER ONE

INTRODUCTION

1.1 Background of Study

Pesticides are a family of organic chemicals widely used in agriculture for the
elimination and destruction of rodents, weeds, pests, bacteria, fungi, etc (Jayaraj, 2016).
Members of this family include the herbicides, rodenticides, fungicides, bactericides and
insecticides (NPIC, 2020). Over 98% of sprayed insecticides and 95% herbicides reach a
destination other than their target species, including non-target species, air, water and
soil (Erhunmwunse, 2014). With a global increase in pesticide production and
application, a reported adverse effect has been on the alteration of blood parameters,
renal dysfunction, hepatocellular damage, nervous breakdown etc due to the toxic
components of these pesticides.

Glyphosate, which goes by the trademark RoundUp, is the most commonly used
herbicide as at date, forming the active compound of several pesticide formulations such as
RoundUp, Glycel. Wipeout, Forceup and Delsate. It is a broad-spectrum herbicide used for
the control of weeds in agricultural and non-agricultural areas. Glyphosate[N-(phosphono-
methyl)glycine] inhibits 5-enolpyruvyl shikimate-3-phosphate synthase, a key enzyme in
the synthesis of aromatic amino acids in plants, fungi, and bacteria (Pereira et al., 2008)

It is used on agricultural and non agricultural landscapes to eliminate weeds in

sugarcane, coffee, citrus, wheat and barley plantations among many others (Folarin et al.,
2017), in glyphosate resistant GM crops such as Bt Cotton, Bt Soybean and Bt Corn (Gill,
2017) and as a dessicant in oil seed rape and cereal grains (Monsanto, 2010).

However, glyphosate is not used independently but with adjuvants in different

formulations which help to increase its perforation into plant cells, conversely increasing
its toxicity in the process. (Monsanto, 2010). The surfactant used in a common
glyphosate product (Roundup®) is more acutely toxic than glyphosate itself, but the
synergy of the two is more toxic (Santillo et al., 1989).
 Due to its widespread use on food crops, glyphosate residues have been found in
food and as an environmental pollutant, with the herbicide detected in soil, water and non
target species (Bai, 2016). This has led to growing concerns over the exposure to humans
and animals that could result in adverse effects (Folarin et al., 2017). Exposure could
present as occupational exposure, para-occupational exposure, through residues in food
and environmental pollution (Bertrand, 2019).

While glyphosate is regarded by the EPA as least toxic for animals (Bai, 2016),
RoundUp has shown adverse effects in short term and long term studies, with effects on
reproduction, hepatotoxicity, carcinogenicity, and kidney and liver damage (Casuvoglu,
2011). The herbicide formulations have also been shown to cause the death of human
embryonic, placental and umbilical cells in vitro, even at low concentrations. Glyphosate
passes through human placental cells leading to endocrine disorders and increasing
antioxidant levels in the mother and fetus in rats. Side effects of glyphosate poisoning
include dizziness, body tremors, diarrhoea, epigastric pain, gastrointestinal corrosive
effects, dysphagia and hypotension (Jashni et al., 2013).

With the discovery of the adverse implications of glyphosate use and toxicity of the
formulations, it has become important to search for a means to ameliorate the toxic
effects, especially considering the indiscriminate use of pesticides in Nigeria and the lack
of proper protective equipment during application and handling of pesticides (Bertrand,
2019).

Traditional medicine practice has led to the advent of several known drugs commonly
used today in the treatment of diseases. One of such medicinal plants used in local
medicine is Costus Afer Ker Gawl (C. afer), commonly known as monkey sugarcane,
bush cane or ginger lily. It is known as “Okpete” or “Okpoo” in Igboland, “Kakizawa” in
Hausa, “tete-egun” in Yoruba and “Mbritem” in Efik parts of Nigeria, where it serves
various nutritional and medicinal purposes (Chibueze et al., 2012).

C. afer is a tall perennial plant native to Africa and Asia and has been implicated in the
treatment of malaria, arthritis, hypertension, diabetes mellitus, cough, sore throat,
jaundice, fever, inflammation, hepatic disorder and many other diseases (Boison et al.,
2019). This medicinal benefits can be attributed to the presence of plant bioactives such
as flavonoids, phenols, saponins, tannins and alkaloids (Chibueze et al., 2012).
Studies have shown C. afer to be useful in the treatment of Cyclosporin-a induced
nephrotoxicity (Ezejiofor et al., 2016), on lead induced nephrotoxicity (Ezejiofor,
Orisakwe, 2018), on radiation induced haematological and histopathological damage
(Akomolafe & Chetty, 2021) and on heavy metal induced haematotoxicity (Anyanwu et
al., 2020). This study will elucidate its possible protective effects on glyphosate induced
haematotoxicity and nephrotoxicity.

1.2 Statement of the Problem

The prevalent use of glyphosate has led to an array of potentially harmful effects which
if not treated, could result in organ deterioration, irreversible liver damage, kidney
disease, cancer among many others. This study is therefore designed to verify the
protective effect of Costus Afer leaves’ juice on toxicity induced by glyphosate.

1.3 Justification of Study

This study is to analyse the viability of C. afer as treatment of glyphosate induced

toxicity. Due to the known toxicity of glyphosate, it is justifiable to investigate the means
of ameliorating the toxicity effects associated with glyphosate exposure. Although
several studies have been conducted on the medicinal properties of C. afer, none has
been done on its effect on glyphosate induced toxicity, hence the need for this study.

1.4 Aim And Objectives of Study

Aim

The aim of this study is to evaluate the ameliorative effects of oral administration of
fresh leaves extract of C. afer on glyphosate induced nephrotoxicity and haematotoxicity
in female wistar rats.

Objectives

The specific objectives of this study are;

 To evaluate the effects of glyphosate on haematological and renal function

indices in rats.
  To evaluate the protective effects of fresh stem juice of C. afer on toxicity
effects of glyphosate in rats.

1.5 Scope of Study

This study is limited to the assessment of the possible protective effects of C. afer crude
extract in glyphosate induced toxicity, using the following indices in a rat model;

1. Renal function (using creatinine, urea, uric acid, sodium, chloride and potassium ion
concentrations).
2. haematological indices (using red blood cell count, white blood cell count,
haemoglobin, packed cell volume and platelet count).
 CHAPTER TWO
LITERATURE REVIEW

2.1 Introduction to Glyphosate

Glyphosate, the active ingredient in herbicides such as RoundUp, is a wide spectrum

non-selective herbicide used for food and non food crops. (Sultana, 2014). It is an
organophosphorus compound [N-(phosphonomethyl)glycine] and is the most used
herbicide worldwide since its commercialization in 1971 (Bai, 2016).

Glyphosate is anionic at physiological pH levels. It is active as a salt with various cations

(e.g. the sodium or isopropylamine salts). As a herbicide, glyphosate is rapidly absorbed
into the plant mainly through plant surfaces. Plants treated with glyphosate slowly die
over a period of days or weeks, and because the chaemical is transported throughout the
plant, no part survives.

2.2 Mechanism of Action of Glyphosate

Glyphosate acts by inhibiting the enzyme 5-enolpyruvoyl shikimate-3- phosphate

synthetase (EPSPS), an enzyme of the shikimate pathway which catalyzes the reaction of
shikimate-3-phosphate and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-
phosphate, which is subsequently dephosphorylated to chorismate, an essential precursor
in plants for the aromatic amino acids, phenylalanine, tyrosine, and tryptophan
(Cavusoglu, 2011). These aromatic amino acids are essential for protein synthesis and
play an important role in the production of secondary metabolites. It is currently
understood that inhibition of EPSPS deregulates the shikimate pathway, resulting in
uncontrolled carbon flow, mostly going into shikimate. This depletes pools of
compounds needed for carbon fixation, causing a general disruption of the organisms
metabolism (Bai, 2016).
FIG. 1 SHIKIMATE PATHWAY AND SITE OF ACTION OF GLYPHOSATE
(Products of the pathway and regulatory feedback inhibition (dotted arrow) are shown)

Source : Duke and Powles (2008)

The shikimate pathway only occurs in higher plants and micoorganisms, hence
glyphosate is categorised as least toxic by the EPA compared to other pesticides (Bai,
2016). However, recent studies have reported adverse effects due to exposure to
glyphosate in animals and humans including hepatotoxicity (Djaber, 2020),
nephrotoxicity (Mesnage et al., 2015), reproductive toxicity (Folarin et al., 2017),
oxidative stress (Cavusoglu et al., 2011) and haematotoxicity (Sultana, 2015).

2.3 Metabolism And Excretion Of Glyphosate

Glyphosate is converted to the major microbial metabolite amino methylphosphonic acid

(AMPA) by C–N bond cleavage. The results of oral administration of glyphosate in rats,
rabbits and goats indicate that absorption from the gastrointestinal tract is incomplete and
amounts to up to 30% of the dose. The most relevant routes of excretion following oral
administration of glyphosate are feces (70–80%) and urine (20–30%). In rats, after a
single oral administration of glyphosate, almost all radioactivity was detected in urine
and feces, and the radiolabeled detected chemical was present as the unchanged parent
compound. Elimination through exhaled air was very low. The AMPA was the only
metabolite detected, accounting for only 0.2–0.3% of the applied dose of glyphosate.
Glyphosate concentrations in urine showed that most part of the administered dose was
excreted as unchanged parent compound upon glyphosate and Roundup exposure, with
an increasing pattern of glyphosate excreted in urine in relation to the duration of
treatment. The adjuvants and the other substances present in Roundup did not seem to
exert a major effect on the absorption and excretion of glyphosate (Panzacchi et al.,
2018). Glyphosate is largely not metabolized in the human body and thus the parent
compound can be measured in urine. Extrapolations from animal toxicological studies
(oral ingestion of glyphosate in rats) suggested an elimination half-life of 33h for
glyphosate for humans (IARC, 2016) while another animal study suggests a first phase
half life of 6h (Williams et al., 2000). However, animal toxicological tests do not always
translate to the human metabolism (Connoly et al., 2019).

The limited data currently available on glyphosate pharmacokinetics in vertebrates are

insufficient to predict transport and fate of glyphosate in different mammalian tissues,
organs and fluids in the body, and to determine whether or where bioaccumulation
occurs, although animal metabolism studies indicate kidney and liver as target tissues
(Panzacchi et al., 2018).

2.4 Toxicity of Glyphosate

Glyphosate has been shown to be toxic to both unicellular and multicellular organisms in
aquatic and terrestial habitats (Gill et al., 2017). Effects include the reduction in the rate
of photosynthesis is Euglena, reduction in radial growth of the mycorrhizal fungal
species in unicellular organisms. Its toxicological effects in multicellular organisms have
also been shown in both vertebatates and invertebrates. Effects have been observed in
annelids (earthworms), arthropods (crustaceans and insects), mollusks, echinoderms,
fish, reptiles, amphibians and birds. Toxicological effects like genotoxicity, cytotoxicity,
reproductive toxicity, hormonal disruption, chromosomal aberrations and DNA damage
have also been observed in higher vertebrates like humans.

It is classified by the EPA as slightly toxic based on an LD50 of 5000mg/kgbw for rats
(FAO, 2016) . However, recent studies by the International Agency for Research on
Cancer (IARC) present glyphosate as a possible carcinogen to humans (Group 2A)
(Benbrook, 2019). Effects of glyphosate poisoning as seen in a study of 246 intoxication
case studies include nausea and/or vomiting, abdominal pain, diarrhoea, sore throat,
gastrointestinal mucosal damage, renal and hepatic dysfunction, metabolic acidosis,
pulmonary edema, shock and death (Lee et al., 2000). Leukocytosis (68.0%), low
bicarbonate (48.1%), acidosis (35.8%), hepatic dysfunction (33.6%), hypercapnea
(30.9%), hypoxemia (28.4%), and renal insufficiency (17.1%) were the most common
laboratory abnormalities. Studies have shown glyphosate to induce renal failure,
hepatocellular damage, reproductive effects, oxidative stress and genetic damage
(Cavusoglu, 2011).

However, mechanisms of toxicity of glyphosate formulations are complicated.

Glyphosate is used as five different salts but commercial formulations of it contain
surfactants, which vary in nature and concentration. As a result, human poisoning is not
caused by the active ingredient alone but by the herbicide’s complex and variable
mixtures. It is thus difficult to separate the toxicity of glyphosate from that of the
formulation as a whole or to determine the contribution of surfactants to overall toxicity
(Jasper et al., 2012).

2.5 General Description of Costus Afer

C. afer which belongs to the family Zingiberaceae is a monocot and a relatively tall,
herbaceous, unbranched tropical plant with creeping rhizome. It is commonly found in
moist or shady forest of West and Tropical Africa (Arhoghro, 2014), including Senegal,
Nigeria, South Africa, Guinea, Ghana, and Cameroon and most regions in tropical
Africa, particularly in higher rainfall areas (Umoh, 2019). C. afer is a perennial semi-
woody herb with leafy canes that bear terminal inflorescences of white and yellow
flowers. It is grown locally for medicinal purposes and is used to treat diabetes,
inflammation, rheumatism, arthritis, cough, hepatic disorder, miscarriages, epileptic
attack and haemorrhoids and the plant parts commonly used include the leaves, stem and
rhizome.

Taxonomy of Costus Afer

Domain Eukaryota
Kingdom Plantae
Phylum Spermatophyta
Subphylum Angiosperma
Class Monocotyledonae
Order Zingiberales
Family Zingiberaceae
Genus Costus
Species Costus Afer

2.5.1 Composition of Costus Afer And Medicinal Uses

The medicinal uses of C. afer has been largely attributed to its high phytochemical
composition. Proximate analysis has detected levels of plant bioactives such as tannins,
alkaloids, cardiac glycosides, phenols, flavonoids, saponins and anthraquinones (Boison,
2019). Flavonoids and phenols were abundant in aqueous extract. These are potent
water-soluble antioxidants which prevent oxidative cell damage; it also has antiseptics,
anticancer, anti-inflammatory effects, and mild anti-hypertensive properties. Alkaloids
are known to have an antimicrobial, antifungal, and anti-inflammatory effect, and it also
acts as an anti-hypertensive. In a previous study, C. afer leaf and stem extracts were
proven to be able to reverse histopathological damage of pancreatic β-cells in alloxan-
induced diabetes mellitus, making the plant a potent anti-diabetic (Ezejiofor, 2015).
Fresh stem juice extract of C. afer has also been proven to have ameliorative and
protective potentials on nephrotoxicity of aspirin in albino Wistar rats (Umoh, 2019).
Other findings indicate the potential hepatoprotective, nephroprotective, neuroprotective
and reproprotective effects of the plant extract on heavy metals induced toxicity
(Anyanwu et al., 2021). C. afer leaves’ juice has been shown to contain antioxidants with
nephroprotective potential on nitrocellulose thinner nephrotoxicity in rats (Uboh et al.,
2014) and on haematological parameters, C. afer has also been shown to exhibit
protective effects against heavy metal induced toxicity, thereby preventing
cardiovascular diseases (Anyanwu et al., 2020).

These observations have necessitated the analysis of the possible protective effects of C.
afer on glyphosate induced nephrotoxicity and haematotoxicity.

2.6 The Kidney

The kidneys are a pair of reddish-brown bean-shaped organs located on either side of the
spine below the ribs. The functional unit of the kidney is the nephron, and it is composed
of the Bowman’s capsule, the proximal convoluted tubule, loop of Henle, distal
convoluted tubule and collecting tubules.The major functions of the kidneys are to
excrete metabolic waste products as well as to maintain water, pH, electrolyte balance,
production of calcitriol and erythropoietin, detoxification, and excretion of toxic
metabolites and drugs (Vasudevan, 2017). A distortion of kidney function would result in
either decreased excretion of waste products and hence their accumulation in the body or
loss of some vital nutrient from the body. Based on the level of these excretory products
and nutrients in the urine as well as in blood we can make an accurate calculation to
determine the efficiency of the kidney to undertake its various functions.

2.6.1 Nephrotoxicity and Kidney Function Tests

Nephrotoxicity occurs when kidney-specific detoxification and excretion are impaired

due to the damage or destruction of kidney function by exogenous or endogenous
toxicants. (Kim & Moon, 2012). Nephrotoxicity can be diagnosed through a simple
blood test. Basic tests for evaluation of nephrotoxicity resulting in kidney disease
includes the measurement of urea, uric acid, serum creatinine, and concentration of
electrolytes (K+, Cl-, HCO3 and Na+). However, these conventional measurements do not
determine the severity of nephrotoxic induced renal damage (Al-Naimi et al., 2019).

2.6.2 Urea
Urea is the end product of protein metabolism produced in the liver by the urea cycle and
excreted via the kidneys.Normal serum urea value is 20 – 40 mg/dL. The rise in the level
of serum urea is generally seen as a marker of renal dysfunction specially glomerular
dysfunction. Serum urea is increased in all forms of kidney diseases. In acute
glomerulonephritis values may be as high as 300 mg/dL (Vasudevan, 2017). In early
stages of nephrosis, serum urea may be normal, but in late stages serum urea increases
along with decreasing renal function. In malignant hypertension and in chronic
pyelonephritis, the values may reach very high levels. Level of serum urea may also be
decreased in case of hepatic injury (Vasudevan, 2017).

Urea is usually measured calorimetrically by Berthelot’s end point method and is read in
visible range using a calorimeter.

2.6.3 Creatinine

Creatinine is an excretory compound formed as a breakdown product of muscle

metabolism. It is formed from the spontaneous non-enzymmatic conversion of creatine
phosphate to creatinine and is produced at a fairly constant rate, dependent on total
muscle mass. It is therefore excreted via the kidneys as it is freely filtered at the
glomerulus and is also to a very small extent secreted into the tubules. (Vasudevan,
2017). Therefore. any problem with glomerular filtrations has a significant effect on the
excretion of creatinine resulting in a much substantial rise in serum creatinine level.
Normal serum creatinine level is 0.6 to 1.5 mg/dl. Creatinine level more than 1.5 mg/dL
indicates impairment of renal function. Serum creatinine is a better indicator of renal
function and more specifically glomerular function than urea (NIOS, 2012). Creatinine is
most commonly measured in laboratories calorimetrically by Jaffe’s method.

2.6.4 Electrolytes

The purpose of the kidney is not just water balance and excretion but also to maintain the
electrolyte balance of our body. Kidneys actively reabsorb or excrete electrolytes to
maintain the electrolyte balance of the body. Owing to their small size almost all
electrolytes are filtered at the glomerulus. After filtration most of the electrolytes are
absorbed back at the tubular level but any problem at the tubular level will result in non
absorption and excessive loss of electrolytes in urine (NIOS, 2012).

Serum electrolytes that are measured for this purpose are:

Serum Sodium levels (Na+) : 135 to 145 mmols/liter

Serum Potassium level (K+) : 3.5 to 5 mmols/liter

Serum Chloride level (Cl- ) : 95 to 105 mmols/liter

2.6.5 Uric acid

Uric acid is the final oxidation product of purine metabolism and is excreted by the
kidneys. Elevated serum uric acid levels are seen in patients with reduced glomerular
filtration rate, and it has been proposed that uric acid plays a role in the pathophysiology
of chronic kidney disease and acute kidney injury (Giordano, 2015). Normal uric acid
level falls in the range of 4.0 - 8.5 mg/dl in males and 2.7- 7.3mg/dl in females.

2.7 Blood

Blood is a fluid connective tissue composed of the liquid plasma and the blood cells.
Whole blood contains a mixture of about 55 percent plasma and 45 percent blood cells.

Plasma

Plasma is a pale yellow fluid constituting about 55% of total blood volume. It contains
91-92% of water and 8-9% of solids which are the organic and inorganic substances such
as plasma proteins, electrolytes, hormones and nutrients. Other constituents of plasma
include enzymes, gases and non-protein nitrogenous substances. The main function of
the plasma is to transport blood cells throughout the body along with nutrients, waste
products, antibodies, clotting proteins, chemical messengers such as hormones, and
proteins that help maintain the body's fluid balance (Mohammad, 2021).
Formed elements

This includes the red blood cells, white blood cells and leucocytes.

Functions of blood

The functions of blood include:

1. Nutritive function

Nutritive substances like glucose, amino acids, lipids and vitamins derived from digested
food are absorbed from gastrointestinal tract and carried by blood to different parts of the
body for growth and production of energy.

2. Excretory function

Waste products formed in the tissues during various metabolic activities are removed by
blood which carried to the excretory organs like kidney, skin, liver, etc. for excretion.

3. Respiratory function

Transport of respiratory gases (O2 and CO2) is done by the blood. Arterial blood is
scarlet red because it contains more oxygen and venous blood is purple red because of
more carbon dioxide.

4. Transport of hormones and enzymes

Hormones which are secreted by ductless (endocrine) glands are released

directly into the blood.

5. Regulation of water and acid-base balance

Water content of the blood is freely interchangeable with interstitial fluid. This helps in
the regulation of water content of the body. Also, plasma proteins and haemoglobin act
as buffers and help in the regulation of acid-base balance.

6. Defensive function
The defensive function of the blood is attributed to the presence of white blood cells.
Neutrophils and monocytes engulf the bacteria by phagocytosis. Lymphocytes are
involved in development of immunity. Eosinophils are responsible for detoxification,
disintegration and removal of foreign proteins.

2.7.1 Haematotoxicity and significance of some haematological indices in clinical

studies

Haematological components serve as an important index for the assessment of body

function (Esikere, 2021) and are a useful tool for early detection of cellular change in the
blood. This could be indicative of damage to the liver, bone marrow and other organs
(Esikere, 2021). Haematotoxicity is the study of blood and blood forming tissues as a
target organ for drugs, chemicals in the environment or workplace, and factors such as
stress, exercise and ionizing radiation (Bloom, 1993). along with organs such as the liver
and kidney, blood is also ranked as a target organ for xenobiotic induced toxicity, as
blood cells are exposed to any agent absorbed or injected into the bloodstream.
Xenobiotics can induce haematologic disorders affecting white blood cells, red blood
cells, platelets and the coagulation system. These syndromes include aplastic anaemia,
agranulocytosis, haemolytic anaemia, megaloblastic anaemia and thrombocytopenia
(Balasubramanian, 2019).

Routine parameters used in the study of haematotoxicity into red blood cell count, white
blood cell count, haemoglobin concentration, haematocrit (PCV), differential leucocyte
count and thrombocyte count. Other special tests for haematotoxicity include reticulocyte
count, platelet aggregation, coagulation factor assay, electron microscopy e.t.c. These
clinical examinations give an overview on the extent of toxicological damage induced in
the blood of an organism.

2.7.2 Red blood cells (RBC) /Erythrocytes

These are the most abundant blood cells with a characteristic red colour due to the
presence of the oxygen carrying pigment haemoglobin. Mature red blood cells are non
nucleated with a biconcave disc which allows easy passage through tiny capillaries. They
play a vital role in transport of respiratory gases (oxygen and carbon dioxide) due to the
presence of haemoglobin as a major constituent of red blood cell and have an average
lifespan of 120 days (Muhammad, 2021).

There are approximately 4 million to 5.5 million red blood cells in each cubic millimeter
of blood, 5 million/cu mm in adult male and 4.5 million/cu mm in adult female.
Abnormal decrease in RBC count is called anemia and an increase is known as
polycythaemia.

2.7.3 White blood cells (WBC)/Leucocytes

White blood cells protect the body from infection and they are the cells that make up
the majority of the immune system. They are much fewer in number than red blood cells,
accounting for about 1 percent of blood volume.They are divided into Granulocytes
(having visible granules or grains inside the cells) and Agranulocytes (free of visible
grains under the microscope).
There are five main types of WBCs.: Neutrophils (granulocytes), Eosinophils
(granulocytes), Basophils (granulocytes), Lymphocytes (non-granulocytes) and
Monocytes (non-granulocytes). Leukopenia is a low white blood cell count that can be
caused by damage to the bone marrow from agents such as medication, radiation, or
chaemotherapy. Leukocytosis is a high white blood cell count that can be caused by a
number of conditions, including various types of infections, inflammatory disease in the
body (Muhammad, 2021).

2.7.4 Platelets / thrombocytes

Unlike red and white blood cells, platelets are not actual cells but rather small
fragments of cells. Platelets help the blood clotting process (or coagulation) by gathering
at the site of an injury, sticking to the lining of the injured blood vessel, and forming a
platform on which blood coagulation can occur. This results in the formation of a fibrin
clot, which covers the wound and prevents blood from leaking out. Fibrin also forms the
initial scaffolding upon which new tissue forms, thus promoting healing.
A higher than normal number of platelets can cause unnecessary clotting, which can lead
to strokes and heart attacks; conversely, lower than normal counts can lead to extensive
bleeding(Muhammad, 2021).

2.7.5 Packed cell volume

Packed cell volume (PCV), also known as haematocrit value, is the volume of red
blood cells expressed in percentages (Muhammad, 2021). It is the portion of whole blood
volume occupied by the blood cells after centrifugation of whole blood filled in a micro-
haematocrit capillary. PCV reference ranges in adults ranges from 40-52% in males and
37-46% in females (Kaslow, 2011) and haematocrit value is one of the precise methods
of determining the degree of anaemia or polycythaemia (Esikere, 2021).

2.7.6 Haemoglobin

Haemoglobin is a tetrameric conjugated protein consisting of a heme group and a

globin portion. It is a characteristic red pigment of red blood cells and functions in the
transport of the respiratory gases, oxygen and carbon (iv) oxide (Muhammad, 2021). A
nomal haemoglobin concentration ranges from 13.2-16.2 g/dl in males and 12.0-15.2 g/dl
in females . Haemoglobin deficiency decreases the oxygen carrying capacity of the
blood, which could result in anemia (Esikere, 2021).
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Materials

3.1.1 Chemicals and Reagents

All the chemicals used in this study were of analytical grade and were utilized according
to prescriptions in the manufacturer’s manual. They include chloroform, normal saline,
Glyphosate (Force up ® ,containing 360g Glyphosate in the form of isopropylamine salt)
and distilled water.

3.1.2 Equipment and Glassware

Spectrophotometer (SP-300, Optima, Japan), Sysmex automated haematology analyzer

(BC-2600 KX-2IN, Sysmex Corporation, Kobe, Japan), refrigerator, table centrifuge,
electronic weighing balance, dissecting set, test tubes, test tube racks, micropipette,
pipette tips, sterile syringes,plain sample bottles, EDTA sample bottles, plastic sieves,
chess cloth, mortar and pestle, beakers, hand gloves,cotton wool and nose masks.

3.1.3 Glyphosate

Glyphosate (Force up ®, containing 360g Glyphosate in the form of isopropylamine salt)

was obtained from a chemical store in Watt market, Calabar, Cross River State.

3.1.4 Experimental animals

Fifteen female albino rats weighing 70-110g were purchased from the University of
Nsukka, and transported to the animal house, College of Medical Sciences, University of
Calabar, Calabar.
3.2 Methods

3.2.1 Collection of plant sample

Fresh mature C. Afer leaves were harvested from the bushes around Staff Quarters,
University of Calabar, Cross River State.

3.2.2 Preparation of C. Afer leaves’ juice

The fresh leaves were washed to remove sand particles, shredded into pieces and
pounded with a mortar and pestle to obtain the leaf juice. The fresh juice was then
separated into a clean plastic container using a chess cloth. Leaf juice was refrigerated
and a fresh sample was prepared every two days.

3.2.3 Animal handling and groupings

The rats used in this study were kept in wooden cages at room temperature (25 ± 2 oC).
The animals were weighed and acclimatized for two weeks prior to the commencement
of exposure. They were fed with Vital growers feed from Grand cereals Ltd., and water
ad libitum. The animals were divided into three groups of five rats each; Group I (control
group), Group II and III (experimental groups). All experimental and surgical procedures
were done in accordance with the University of Calabar guidelines on animal care.
Table 1: Experimental design

Group Number of animals

1 5 Control group

2 5 Pesticide only group

3 5 Pesticide and treatment (C. Afer )

group
3.2.4 Mode of Exposure

The experimental animals were subjected to oral exposure. 1.5ml of Glyphosate

(containing 1ml Glyphosate and 0.5ml water) was administered to Group II and Group
III daily using a locally constructed beaded syringe needle as an oral gavage.

3.2.5 Administration of C. afer leaves’ juice

Daily administration of leaf juice was done one hour after glyphosate was administered.
This was carried out for Group III animals only using 1.3ml of C. afer leaves’ juice

3.2.6 Collection and handling of blood samples for analysis

At the end of the experimental period, the rats were fasted overnight for 12 hours and
anaesthesized using chloroform. Blood samples were collected from the heart by cardiac
puncture using 5ml syringes. Blood was collected into EDTA sample bottles for
heamatological analysis and plain red sample bottles for biochemical analysis.

The clotted blood in the plain sample bottles were centrifuged for 15 minutes at 2000rpm
and the supernatant (serum) was collected with a micropipette into another plain red
sample bottle for laboratory analysis.

3.3 Estimation of haematological indices

The haematological parameters assayed in this study were evaluated using an automated
haematology analyzer. Haematological parameters including red blood cells (RBC)
count, white blood cells (WBC) count, platelets (PLT) count, haemoglobin (HGB) count
and packed cell volume (PCV) count were determined using the Sysmex Automated
Hematological Analyzer (BC-2600 KX-2IN).

3.3.1 Biochemical Assay

The renal function parameters assayed in this study include; serum creatinine,serum urea,
serum uric acid, serum potassium level, serum chloride level and serum sodium level.
3.3.2 Estimation of serum creatinine

The concentration of serum creatinine was assayed based on the reaction of creatinine
with picric acid in an alkaline medium to form a red complex. The red complex which is
proportional to the concentration of creatinine in the sample was measured
spectrophotometrically.

Preparation and Stability of Working Reagents

Mix one volume of creatinine dye reagent (R1) (2 × 50mls) with one volume of creatinine
base reagent (R2) (2 × 50mls). The working reagent is stable for 30 days at room
temperature.

Creatinine reacts with picric acid to produce a red colored complex, creatinine alkaline
picrate. The change in absorbance is proportional to the creatinine concentration.

The working reagent was stored at room temperature of 28oC.

The standard solution and the working reagents were pipetted into respective tubes.

Laboratory Procedure

Pipette 1ml of R1 (Butter), 10ml of standard and 10ml of sample. This was mixed and the
optical density was determined 60 seconds after the addition of standard.

The creatinine concentration is gotten as follows;

Creatinine concentration (mg/dl) = (T2 - T1) of sample ×2

(T2 - T1) of sample

3.3.3 Estimation of serum urea

3.3.4 Estimation of serum uric acid

3.3.5 Estimation of Serum Potassium

Principle

At an alkaline pH, potassium ions and turbidimetric tetraphenyl borate (TPB) form a
turbid emulsion, the increase of which can be measured quantitatively in a photometer at
578nm. The increase in absorbance (A) is directly proportional to the concentration of
potassium in the sample.

Procedure

Pipette 1ml of sodium hydroxide 0.05mmol/L, TPB - Na 240mmol/L, 20ml of standard

and 20ml of sample. Mix and incubate for 3 minutes at 37 0C and for 5 minutes at 250C.
Afterwards, mix thoroughly and read absorbance of sample A and standard A against
blank.

Calculation

Serum potassium (mmol/L) = A sample ×5

A standard

3.3.6 Estimation of serum sodium

Serum sodium was estimated using the magnesium uranyl acetate method described in
DIALAB diagnostic kits.

Principle

The level of sodium in the serum sample was estimated using magnesium-uranyl acetate
reaction method on modified Maruna and Tinder’s method. In this method, sodium and
potassium are precipitated together by magnesium uranyl acetate as uranyl magnesium
sodium acetyl salt. Excess of uranyl salts reacts with potassium ferrocyanide to produce a
brownish color. The intensity of the color is inversely proportional to the sodium
concentration in the sample specimen and is measured photometrically at 530nm.
Uranyl ions + Magnesium ions + sodium ions Uranyl magnesium acetyl salt

Free uranyl ions + K4Fe (CN)6 Brown colored complex

Procedure

Pipette 1ml of sodium (precipitating reagent), 10ul of sodium standard and 10ul of
serum. Shake vigorously at room temperature for an interval of 5 minutes. Afterwards,
centrifuge at 3000rpm to get a clear supernatant fluid. Immediately after centrifugation,
transfer the supernatant fluid for test and standard.

3.3.7 Estimation of Serum Chloride

The level of serum chloride was determined using mercuric thiocyanate reaction method
described by Teco Diagnostic Kit. The earlier method for the determination of chloride
involved in the precipitation of an insoluble salt. Chloride can be determined by the
titration of the chloride with standard mercuric nittrate solution using phenylcarbazone as
the indicator. This method requires protein precipitation.

Principle

Hg (SCN)2 + 2CL- HgCl2 + 2SCN-

SCN + Fe 3+ 4Fe (SCN)3 red complex

Chloride ions from a soluble non ionized compound with mercuric ions. It displaces
thiocyanate ions from non-ionized mercuric thiocyanate. The thiocyanate ions when
dissociated from the complex, reacts with ferric ions to from a colored comlex that
absorbs light at 480nm. The intensity of the color produced is directly proportional to the
concentration of chloride.

3.4 Statistical analysis

All data were expressed as mean ± standard deviation error of mean (SEM). The results
were analyzed by one-way analysis of variance (ANOVA), followed by pair wise
comparison between test and control groups using students t-test. Differences between
control and respective test groups were considered significant at p<0.05.
 CHAPTER FOUR

RESULTS

The results obtained from the study, protective effect of C. Afer on glyphosate induced

nephrotoxicicty and haematotoxicity in female wistar rats are presented below.

4.1 Protective effect of C. Afer on glyphosate induced toxicity on serum urea,

creatinine and uric acid concentration in wistar rats.

The results of protective effect of C. Afer on glyphosate induced toxicity on serum urea,

creatinine and uric acid concentration is presented in figure 4.1. The result of glyphosate

treated group showed a significant increase in serum urea concentration, compared with

the control group. The result of experimental group treated with C. Afer after glyphosate

induction showed no significant difference compared with the control group. There was a

significant decrease in serum urea concentration in group treated with C. afer after

glyphosate induction compared with glyphosate treated group.

Glyphosate treated group showed significant increase in serum creatinine concentration

compared to control group. Group treated with C. afer after glyphosate induction showed

slight decrease in serum creatinine concentration compared to control group. There was a

significant increase in serum creatinine concentration in glyphosate treated group

compared to the group treated with C. Afer after glyphosate induction.

There is no significant difference in serum uric acid concentration in both glyphosate

treated group and group treated with C. Afer after glyphosate induction compared to the

control group. There was also no significant difference observed in glyphosate treated

group compared to group treated with C. Afer after glyphosate induction.

Figure 4.1: Protective effect of C. Afer on glyphosate induced toxicity on serum urea,

creatinine and uric acid concentration in wistar rats.

 Values are expressed as mean ± SEM, n=5
*: denote significant difference compared to control group (p>0.05)
a: denotes significant difference compared to Glyphosate group (p>0.05)

4.2 Protective effect of C. Afer on glyphosate induced toxicity on serum sodium,

potassium and chloride concentration in wistar rats.

The results of protective effect of C. Afer on glyphosate induced toxicity on serum

sodium, potassium and chloride concentration is presented in figure 4.2. There was a

significant decrease in serum sodium concentration of glyphosate treated group

compared to control group. There was also a significant decrease in sodium

concentration in group treated with C. Afer after glyphosate induction compared to

control group. Group treated with C. Afer after glyphosate induction produces a

significant increase in serum sodium concentration compared to glyphosate treated

group.

There was no significant difference in serum potassium concentration observed in

glyphosate treated group and group treated with C. Afer after glyphosate induction

compared to control group. There was no significant difference in serum potassium

concentration in glyphosate treated group compared to group treated with C. Afer after

glyphosate induction.

There was a significant increase in serum chloride concentration in glyphosate treated

group compared to the control group. There was also a significant increase in serum

chloride concentration in group treated with C. Afer after glyphosate induction compared

to control. There was a significant decrease in serum chloride concentration in group

treated with C. Afer after glyphosate induction compared to glyphosate treated group.
 Figure 4.2 Protective effect of C. Afer on glyphosate induced toxicity on serum

sodium, potassium and chloride concentration in wistar rats.

Values are expressed as mean ± SEM, n=5

*: denote significant difference compared to control group (p>0.05)
a: denotes significant difference compared to Glyphosate group (p>0.05)

4.3 Protective effect of C. Afer on glyphosate induced toxicity on white blood cell

count (WBC), red blood cell count (RBC) and haemoglobin (Hb) concentration in

wistar rats.

The results of protective effect of C. Afer on glyphosate induced toxicity on white blood

cell count (WBC), red blood cell count (RBC) and haemoglobin (Hb) concentration in

wistar rats is presented in figure 4.3. There was a significant increase in white blood cell

count in glyphosate treated group compared to control group. Group treated with C. Afer

after glyphosate induction showed significant increase in white blood cell count
compared to control group. There was a significant increase in white blood cell count in

group treated with C. Afer after glyphosate induction compared to glyphosate treated

group.

There was no significant difference in red blood cell count observed in glyphosate

treated group compared to control group. There was no significant difference of red

blood cell count in group treated with C. Afer after glyphosate induction compared to

control. There was no significant difference in red blood cell count in glyphosate treated

group compared to group treated with C. Afer after glyphosate induction.

There was no significant difference in haemoglobin concentration observed in

glyphosate treated group compared to control group. There was no significant difference

in haemoglobin concentration in group treated with C. Afer after glyphosate induction

compared to control. There was no significant difference in haemoglobin concentration

in glyphosate treated group compared to group treated with C. Afer after glyphosate

induction.
 Figure 4.3 Protective effect of C. Afer on glyphosate induced toxicity on white blood

cell count (WBC), red blood cell count (RBC) and haemoglobin (Hb) concentration in

wistar rats.

Values are expressed as mean ± SEM, n=5

*: denote significant difference compared to control group (p>0.05)
a: denotes significant difference compared to Glyphosate group (p>0.05)

4.4 Protective effect of C. Afer on glyphosate induced toxicity on packed cell volume
(PCV) and platelet count in wistar rats.

The results of protective effect of C. Afer on glyphosate induced toxicity on packed cell

volume (PCV) and platelet count in wistar rats is presented in figure 4.4. There was no

significant difference in packed cell volume in glyphosate treated group and group

treated with C. Afer after glyphosate induction compared to control. There was no

significant difference in packed cell volume in glyphosate treated group compared to

group treated with C. Afer after glyphosate induction.

There was a significant increase in platelet count in glyphosate treated group compared

to control group. There was also a significant increase in platelet count in group treated

with C. Afer after glyphosate induction compared to control.

There was a significant decrease in platelet count in group treated with C. Afer after

glyphosate induction compared to glyphosate treated group.

Figure 4.4 Protective effect of C. Afer on glyphosate induced toxicity on packed cell

volume (PCV) and platelet count in wistar rats.

Values are expressed as mean ± SEM, n=5

*: denote significant difference compared to control group (p>0.05)
a: denotes significant difference compared to Glyphosate group (p>0.05)