evaluating the neuroprotective effects of fermented

EVALUATING THE NEUROPROTECTIVE EFFECTS OF

FERMENTED ROOIBOS HERBAL TEA IN WISTAR RATS

EXPOSED TO BISPHENOL-A DURING GESTATION AND

LACTATION

By

BUSHRA KHALIFA GAMOUDI

Student Number 3607196

A thesis submitted in fulfilment of the requirements for the degree of

Magister Scientiae (MSc)

Department of Medical Biosciences

Faculty of Natural Sciences

Supervisor

Prof. Okobi Ekpo

http://etd.uwc.ac.za/

ii

DECLARATION

I declare that the thesis titled: “Evaluating the neuroprotective effects of

fermented Rooibos Herbal Tea in Wistar rats exposed to Bisphenol-A during

gestation and lactation” is my work and is hereby submitted for the Master of

Science degree in Medical Biosciences at the University of the Western Cape,

South Africa. This work has not been submitted for any degree or examination in

any other university, and all sources used or quoted have been duly indicated and

acknowledged by complete references

Full name: Bushra Khalifa Gamoudi

Signed.......................................


iii

DEDICATION

To my dearest family for their love, support, encouragement, and making my

dreams a reality.

The Prophet Muhammad (Peace Be upon Him) said, regarding knowledge:

“Acquire knowledge and impart it to the people...One who treads a path in search

of knowledge has his path to Paradise made easy by God” (Sunan Tirmidhi,

Hadith 107; Riyadh us-Saleheen, 245).


iv

ACKNOWLEDGEMENTS

First of all, I would like to acknowledge my Allah for giving me the faith, strength,

good health, wisdom and perseverance, without you nothing is possible, and I

would not have completed my Masters!

This research would not have been completed without the generous help and

support received from different people. I want to express my sincere gratitude for

the valued assistance I received throughout my MSc programme from the

following people:

A special thanks to my husband who stood by me through the rough times

and my children whose presence always brings joy to my life.

My supervisor Prof. Okobi Ekpo for the knowledge gained,

encouragement, for his confidence in me, his patience, support and

motivation throughout the duration and completion of this research. I

acknowledge him for proofreading my thesis. THANK YOU!

I would also like to thank my parents and sisters for their love, care,

prayers, and support during my period of study.

Staff at the histology laboratory of the University of Stellenbosch, for their

help and the technical support.

To my Lab mates and colleagues at the Medical Bioscience department of

UWC, I say thank you for your kind and the friendly working relationship;

I enjoyed my times with you.


v

Lastly, but by no means the least, big thanks to the Libyan government for

funding the project and my studies at the University of the Western Cape.


vi

TABLE OF CONTENTS

DECLARATION ............................................................................................... ii

DEDICATION .................................................................................................. iii

ACKNOWLEDGEMENTS ............................................................................. iv

TABLE OF CONTENTS ....................................................................................... vi

LIST OF FIGURES ............................................................................................... xi

LIST OF TABLES ............................................................................................... xiii

LIST OF ABBREVIATIONS .............................................................................. xiv

ABSTRACT ......................................................................................................... xvi

CHAPTER 1 ........................................................................................................... 1

INTRODUCTION ............................................................................................. 1

1.1. RESEARCH HYPOTHESIS .................................................................... 5

1.2. RESEARCH AIM AND OBJECTIVES................................................... 5

1.2.1. AIM ........................................................................................................ 5

1.2.2. OBJECTIVES ........................................................................................ 5

CHAPTER 2 ........................................................................................................... 7

LITERATURE REVIEW ................................................................................. 7

2.1. THE CENTRAL NERVOUS SYSTEM (CNS) ....................................... 7

2.1.1. THE CEREBRUM ................................................................................. 8

2.1.1.1. CYTOARCHITECTURE OF THE CEREBRAL CORTEX ............. 9

2.1.1.2. FUNCTION OF THE CEREBRAL CORTEX ................................ 10

2.1.1.3. THE HIPPOCAMPUS ...................................................................... 11


vii

2.1.2. THE CEREBELLUM .......................................................................... 13

2.1.2.1. CYTOARCHITECTURE OF THE CEREBELLAR CORTEX ...... 15

2.2. THE ENDOCRINE SYSTEM ................................................................ 16

2.2.1. ENDOCRINE DISRUPTOR COMPOUNDS (EDCs) ........................ 17

2.2.1.1. BISPHENOL A (BPA) ..................................................................... 21

2.2.1.1.1. PHYSICAL AND CHEMICAL PROPERTIES OF BPA ............. 22

2.2.1.2. RELEASE AND HUMAN EXPOSURE TO BPA .......................... 23

2.2.1.3. METABOLISM OF BPA ................................................................. 24

2.2.1.4. ACTIVITY OF BPA ......................................................................... 26

2.3. ROOIBOS (Aspalathus linearis) ............................................................ 28

2.3.1. CHEMICAL COMPOSITION OF ROOIBOS ................................... 29

2.3.1.1. POLYPHENOLS .............................................................................. 30

2.3.2. BIOLOGICAL PROPERTIES OF ROOIBOS TEA ........................... 32

2.3.4.1. NEUROPROTECTIVE EFFECT OF ROOIBOS ............................ 33

CHAPTER 3 ......................................................................................................... 36

MATERIALS AND METHODS .................................................................... 36

3.1. ETHICAL CONSIDERATION .............................................................. 36

3.2. MATERIALS.......................................................................................... 36

3.3. ANIMALS .............................................................................................. 37

3.4. TREATMENT PROTOCOL .................................................................. 38

3.5. PREPARATION OF REAGENTS ......................................................... 39

3.5.1. ROOIBOS HERBAL TEA PREPARATION ..................................... 39

3.5.2. BISPHENOL A (BPA) AND NORMAL SALINE ............................. 40


viii

3.5.3. PREPARATION OF 4% FORMALDEHYDE SOLUTION .............. 40

3.5.4. PHOSPHATE BUFFERED SALINE (PBS) ....................................... 40

3.6. MOTOR FUNCTION TESTS (NEUROBEHAVIORAL TESTS) ........ 41

3.6.1. OPEN FIELD TEST (OFT) ................................................................. 41

3.6.2. NOVEL OBJECT RECOGNITION (NOR) TEST ............................. 43

3.7. ANIMAL SACRIFICE ........................................................................... 44

3.8. HISTOLOGICAL PREPARATION OF BRAIN SAMPLES ................ 44

3.8.1. AUTOMATED TISSUE PROCESSING ............................................ 44

3.8.2. EMBEDDING ..................................................................................... 45

3.8.3. SECTIONING ..................................................................................... 45

3.8.4. STAINING........................................................................................... 45

3.8.4.1. HAEMATOXYLIN AND EOSIN.................................................... 45

3.8.4.2. CRESYL VIOLET (CV) / NISSL STAINING ................................ 46

3.8.4.3. IMMUNOHISTOCHEMICAL (IHC) STAINING .......................... 47

3.9. NEUROCHEMICAL ASSAYS ............................................................. 47

3.9.1. HOMOGENIZATION OF TISSUES .................................................. 48

3.9.1.1. SUPEROXIDE DISMUTASE (SOD) .............................................. 48

3.9.1.2. CATALASE...................................................................................... 48

3.10. STATISTICAL ANALYSIS ................................................................ 49

CHAPTER 4 ......................................................................................................... 50

RESULTS ......................................................................................................... 50

4.1. INTRODUCTION .................................................................................. 50

4.2. EFFECT OF BPA AND FERMENTED ROOIBOS HERBAL TEA ON

BODY MASS ................................................................................................ 51


ix

4.3. NEUROBEHAVIOURAL TESTS ......................................................... 52

4.3.1. THE OPEN FIELD TEST ................................................................... 52

4.3.1.1. THE FREQUENCY OF REARING EPISODES ............................. 52

4.3.1.2. TOTAL FREEZING TIME .............................................................. 53

4.3.1.3. TOTAL SELF-GROOMING TIME ................................................. 54

4.3.2. NOVEL OBJECT RECOGNITION (NOR) TEST ............................. 55

4.4. EFFECT OF BPA AND FERMENTED ROOIBOS HERBAL TEA ON

THE ACTIVITY OF ANTIOXIDANT ENZYMES ..................................... 57

4.4.1. SUPEROXIDE DISMUTASE ............................................................. 57

4.4.2. CATALASE......................................................................................... 59

4.5. HISTOLOGICAL STUDIES .................................................................. 60

4.5.1. LENGTH OF HIPPOCAMPAL CA1 REGION IN EXPERIMENTAL

RATS ............................................................................................................. 61

4.5.2. NUMBER OF PURKINJE CELLS IN PURKINJE CELL LAYER

(PCL) IN EXPERIMENTAL RATS ............................................................. 62

4.6. IMMUNOHISTOCHEMISTRY (IHC) .................................................. 64

4.6.1. GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) .......................... 64

CHAPTER 5 ......................................................................................................... 66

DISCUSSION ................................................................................................... 66

5.1. INTRODUCTION .................................................................................. 66

5.2. ROOIBOS TEA PREVENTED BPA-INDUCED GAIN IN BODY

MASS............................................................................................................. 67

5.3. ROOIBOS TEA MITIGATED BPA-INDUCED

NEUROBEHAVIOURAL DEFICITS .......................................................... 68

5.4. ROOIBOS TEA REGULATED THE ACTIVITY OF ANTIOXIDANT

ENZYMES..................................................................................................... 72


x

5.5. ROOIBOS TEA ATTENUATED NEURODEGENERATION IN RATS

EXPOSED TO BPA ...................................................................................... 73

5.6. CONCLUSION .................................................................................................. 77

REFERENCES ...................................................................................................... 79


xi

LIST OF FIGURES

Figure 2.1: The brain structure 7

Figure 2.2: The layers of the cerebral cortex 8

Figure 2.3: The lobes of the cerebral cortex 9

Figure 2.4: The structure of the Hippocampus 10

Figure 2.5: The layers of the hippocampus proper 11

Figure 2.6: The structure of the cerebellum A) Sagittal and B) Dorsal sections 12

Figure 2.7: The structure of the cerebellar cortex 13

Figure 2.8: Synthesis of Bisphenol A 19

Figure 2.9: Metabolism of BPA 22

Figure 2.10: Rooibos tea; (A) Unfermented (B) Fermented 25

Figure 2.11: Classification of polyphenols 27

Figure 2.12: Chemical structure of Aspalathin 28

Figure 3.1: Flow diagram showing the experimental design for the study 34

Figure 3.2: Open Field Test Apparatus 38

Figure 4.1: Average body mass of experimental rats on PND 42 47

Figure 4.2: Number of rearing episodes of experimental rats on PND 42 49

Figure 4.3: Total freezing time of experimental rats on PND 42 50

Figure 4.4: Total self-grooming time of experimental rats on PND 42 51

Figure 4.5: Recognition index of experimental rats on PND 42 52

Figure 4.6: Effects of different treatments on SOD enzyme activity in the

cerebral homogenate of experimental rats on PND 42 53


xii

Figure 4.7: Effects of different treatments on SOD enzyme activity in the

cerebellar homogenate of experimental rats on PND 42 54

Figure 4.8: Effects of different treatments on CAT enzyme activity in the

cerebral homogenate of experimental rats on PND 42 55

Figure 4.9: Effects of different treatments on CAT enzyme activity in the

cerebellar homogenate of experimental rats on PND 42 55

Figure 4.10: Diagram of the hippocampus showing the landmarks used for

determining hippocampal length in the experimental rats. 57

Figure 4.11: CA1 hippocampal length in experimental rats on PND42 57

Figure 4.12: Number of Purkinje cells in experimental rats on PND42 58

Figure 4.13: Number of GFAP positive cells in experimental rats on PND42 60


xiii

LIST OF TABLES

Table 2.1: Examples of EDCs and their endocrine disruptor effects 16

Table 2.2: Physical properties of BPA 19

Table 2.3: Quantification of the major flavonoids in aqueous rooibos extract 26

Table 3.1: Chemicals used in this study 32

Table 3.2: Equipment used in this study 33

Table 3.3: Tissue Processing Protocol 40

Table 3.4: IHC staining procedure 43


xiv

LIST OF ABBREVIATIONS

•OH

Hydroxyl radical

ANOVA Analysis of variance

ATP Automated tissue processor

BPA Bisphenol A

CA Cornu ammonis

CAT Catalase

CNS Central nervous system

CV Cresyl Violet

DDT Dichlorodiphenyltrichloroethane

DG Dentate gyrus

EDCs Endocrine Disruptor compounds

FRHT Fermented rooibos herbal tea

GFAP Glial fibrillary acidic protein

H&E Haematoxylin and eosin

H2O2 Hydrogen peroxide

HCB Hexachlorobenzene

IHC Immunohistochemistry

NOR Novel Object Recognition

NS Normal saline

NTP National Toxicology Program

O−2 Superoxide

OFT Open Field Test

PAHs Polycyclic aromatic hydrocarbons


xv

PBS Phosphate buffered saline

PCBs Polychlorinated biphenyls

PCDF Polychlorinated dibenzofurans

PCL Purkinje cell layer

PND Post-natal day

PNS Peripheral nervous system

RI Recognition index

ROS Reactive oxygen species

SOD Superoxide dismutase

UGT UDP-glucuronosyl transferase


xvi

ABSTRACT

Exposure to endocrine-disrupting chemicals as bisphenol A (BPA) during

gestation and early postnatal life is known to disrupt normal developmental

processes and alter the body’s endocrine system leading to deleterious effects in

the developing central nervous system (CNS). BPA is an industrial synthetic

chemical commonly used in the production of a range of polymers and consumer

products, despite concerns about its safety. There is therefore the need to protect

the developing CNS from potential damage through the administration of

neuroprotective agents. Most medicinal plants are reported to possess significant

protective potential against tissue damage through different mechanisms that

prevent cell death, oxidative stress, inflammation, immunodeficiency, etc. In this

study, the protective effects of fermented rooibos (Aspalathus linearis) tea against

the deleterious effects of BPA were investigated. Rooibos is a herbal beverage

indigenous to South Africa with widely acclaimed health benefits often linked to

the bioactivity of its polyphenolic compounds, especially aspalathin. The anti-

allergic, cardiovascular, antioxidant and neuroprotective effects of this herb have

been previously reported hence, the present study aims to investigate if regular

consumption of rooibos tea during pregnancy and lactation could protect the

developing brain from the deleterious effects of BPA in a Wistar rat model. A

total of 40 three-month old adult female pregnant dams, with an average weight of

250g, were divided into four groups (n=10). Group 1 control rats received 9%

normal saline ad libitum; group 2 rats received 400µg/kg/day BPA only; group 3

rats received 20% fermented rooibos tea as well as 400µg/kg/day BPA, while

group 4 rats received ad libitum 20% fermented rooibos tea only. Offspring rats

were housed in the same cages as the dams and only separated after weaning on

postnatal day (PND) 21. Neurobehavioural assessment using the open field test

was done on postnatal day (PND) 42 after which the final body masses were taken

before the rats were decapitated under deep anaesthesia, and the desired CNS

parts carefully dissected out and processed for histological, biochemical and

immunohistochemical studies. The results obtained showed that there was


xvii

significant impairment of neurobehavioural activity, decreased cerebral and

cerebellar antioxidant enzyme activity, reduced hippocampal CA1 length,

significant loss of cerebellar Purkinje cells and significant astrocyte activation

demonstrated by increased glial fibrillary acidic protein (GFAP) activity in

experimental rats exposed to BPA only. However, co-administration of rooibos

tea significantly attenuated the BPA-induced distortions. Taken together, these

findings suggest that rooibos could be a potent neuroprotective agent against

BPA-induced structural, functional and biochemical alterations in the developing

CNS.

Keywords: Endocrine disrupting chemicals, Bisphenol A, rooibos, antioxidants,

neurodegeneration, neuroprotection, neurobehavioural activity.


1

CHAPTER 1

INTRODUCTION

Many chemical substances have been identified as Endocrine Disruptor

Compounds (EDCs) and humans become exposed to them either at the workplace,

through food or environment sources (air, water and soil). EDCs have been widely

reported to directly or indirectly affect the normal functioning of the endocrine,

nervous and reproductive systems (Colborn et al., 1993; Clotfelter, et al., 2004;

Kajta and Wójtowicz, 2013). Bisphenol A (BPA) is one of the most recognised

EDCs and is a widespread estrogenic chemical (Krishnan et al., 1993, Eladak et

al., 2015) and more than 8 billion pounds (4 billion kilograms) of BPA are

produced for the manufacturing of polycarbonate plastic food and beverage

containers per year (Rubin, 2011). BPA is also used in the resin lining of metal

cans, dental sealants, and is employed as an additive in a wide array of other

products (Vandenberg et al., 2007).

BPA has been reported to be capable of crossing the placental barrier during

pregnancy and is present in different human fluids including foetal and maternal

serum, as well as in full-term amniotic fluid (Ikezuki et al., 2002, Yamada et al.,

2002). In one study, BPA was identified in developing organs of mice including

the CNS (Mita et al., 2012). Other studies have also reported the effects of BPA

on the developing and adult brain (MacLusky et al., 2005, Zsarnovszky et al.,

2005, Rubin and Soto, 2009, Negri-Cesi, 2015, Gupta et al., 2018). The

hippocampus and cerebellum are some of the CNS structures which have been


2

identified as susceptible targets of BPA-induced distortions. Hippocampal

apoptosis and neurodegeneration (Tiwari, et al., 2015; Xu et al., 2010a) as well as

alteration of early cerebellar morphology (Mathisen, et al., 2013) have been

reported in developing animals. Both these structures were the focus of the

present study.

The cerebellum is a component of the CNS situated at the posterior aspect of the

brainstem, inferior to the occipital and temporal lobes of the cerebral cortex. It is

mainly involved in maintaining balance, posture and the coordination of voluntary

movements through a stabilising control system which receives early warning

signals from each motor impulse (Fonnum and Lock, 2000). It is also involved in

motor, learning and cognitive functions (albeit via poorly understood

mechanisms). Although motor commands are mostly initiated in other CNS

regions, the cerebellum modifies them according to rate, range, force and

sequence of contractions by allowing more adaptive and accurate movements

(Fonnum and Lock, 2000).

The cerebellum belongs to a distributed sensorimotor coordination network that

includes the cerebral cortex, basal motor nuclei, thalamus and the reticular

formation (Doya, 1999). Therefore, any dysfunction of the cerebellum could

disrupt the entire sensorimotor coordination network and manifest as locomotor

dysfunctions. The cerebellum accounts for approximately 10% of the brain’s

volume and contains over 50% of the total number of neurons in the brain

(Herculano-Houzel, 2009). The remarkable diversity in its neuronal population

together with its unique post-natal development makes it an appropriate neural


3

structure for studying the effects of different substances on cellular dynamics

during pre- and post-natal development. Whereas, granule and Purkinje cells are

the most important neurons in the cerebellum, Purkinje cells are very important

because they are the largest neurons in the brain and the sole output of the

cerebellum, thus implying that any distortions in their structure and function could

affect overall cerebellar function (Fonnum and Lock, 2000).

Neurodevelopmental deficits potentially related to cerebellar injury may be arise

from the effects of environmental substances leading to such impaired motor

functions as hypotonia, fine motor incoordination, ataxia and impaired motor

sequencing (Powls et al., 1995, Goyen et al., 1998). Cerebellar injury has also

been implicated in cognitive, social and behavioural dysfunction among older

patients (Berquin et al., 1998, Levisohn et al., 2000) and may contribute to the

long-term cognitive, language, and behavioural dysfunction seen among 25-50%

formerly pre-term infants (Limperopoulos et al., 2007, Messerschmidt et al.,

2008). Only a few studies have focused on the vulnerability of the cerebellum to

toxic agents (Bist and Bhatt, 2009, Sanders et al., 2009), mainly focused on the

effects of lead, polychlorinated biphenyl and mercury on the nervous system (Yun

and Hoyer, 2000, Mervis et al., 2002, Verstraeten et al., 2008).

The functions of the hippocampus in memory acquisition and storage as well as

spatial learning have been adequately described (Sutherland and Rudy, 1989).

Hippocampal CA1 neurons are the most sensitive and susceptible to selective

degeneration in response to such EDCs as BPA, ischemia and other toxic

substances (Kirino, 1982, Pulsinelli et al., 1982, Elsworth et al., 2013, Kimura et


4

al., 2016). In addition, axons of CA1 have been considered the main output of the

hippocampus (Duvernoy, 2013) which informs the choice of this region for

investigation in the current study.

The use of herbal products for the treatment of diseases is as old as humanity, and

certain plants have been reported to contain active compounds which could

protect the brain against such EDCs as BPA. Herbal products are considered to be

natural, green, pure, and with minimal side effects (Prashant et al., 2014), often

representing a source of new compounds with potent antioxidant activity to

address oxidative stress (Auddy et al., 2003). Rooibos (Aspalathus linearis) a

herbal beverage indigenous to South Africa, is one such plant with high

antioxidant activity (McKay and Blumberg, 2007, Joubert et al., 2008). Rooibos

naturally grows in the Western Cape Fynbos region of Clanwilliam and is

naturally caffeine-free, with a low tannin content (Erickson, 2003). The highly

acclaimed antioxidant properties of rooibos could be due to its unique polyphenol

content, aspalathin, known to be capable of delaying, inhibiting, or preventing

oxidation through scavenging of free radicals to diminish oxidative stress (McKay

and Blumberg, 2007, Joubert et al., 2008).

It has been reported that long-term consumption of rooibos could protect rat

brains from age-related changes, possibly due to its ability to prevent the

accumulation of lipid peroxides in the brain (Inanami et al., 1995). Also, rooibos

herbal tea has been shown to decrease steroidal hormone levels, indicating the

possibility that it could protect against the effects of BPA (Schloms et al., 2012).

This study was therefore designed to investigate the potential neuroprotective


5

activity of rooibos herbal tea through the amelioration of the effects of the

endocrine disruptor BPA in an in vivo model.

1.1. RESEARCH HYPOTHESIS

This study hypothesizes that regular consumption of fermented rooibos herbal tea

could protect the developing brain against the deleterious effects of BPA. It is

believed that the polyphenolic compounds in rooibos tea are responsible for its

biological and pharmacological activities.

1.2. RESEARCH AIM AND OBJECTIVES

1.2.1. AIM

This study aims to evaluate the potential neuroprotective effects of fermented

rooibos herbal tea on the development of some CNS regions in rats exposed to

BPA during pregnancy and lactation.

1.2.2. OBJECTIVES

The following objectives were set for this study:

To determine the effects of maternal exposure to rooibos herbal tea and BPA

on the body masses of offspring rats.

To determine the effects of maternal exposure to rooibos and BPA on

neurobehavioural activity in offspring rats.


antioxidant enzymatic activity in cerebri and cerebella of offspring rats.


hippocampal and cerebellar morphology in offspring rats.


6

To determine the effects of rooibos and BPA exposure on the activation of

astrocytes in offspring rats.


7

CHAPTER 2

LITERATURE REVIEW

2.1. THE CENTRAL NERVOUS SYSTEM (CNS)

The nervous system consists of two major parts or subdivisions, the central

nervous system (CNS) and the peripheral nervous system (PNS) (Walsh and

Marshall, 1957). The CNS is the control centre for the entire nervous system and

is comprised of the brain and spinal cord. The brain is further subdivided into

three major sections, cerebrum, brainstem and cerebellum (Figure 2.1). The

cerebrum is the largest part of the human brain and is responsible for the

processing of speech, learning, emotions, muscular contractions as well as the

interpretation of sensory data related to hearing, vision and touch (Abhang et al.,

2016). The brainstem consists of the midbrain, pons and medulla oblongata, and

connects the cerebrum and cerebellum to the spinal cord. The cerebellum lies

posterior to the cerebrum, has important functions in motor control and is

responsible for the maintenance of balance and equilibrium (Morton and Bastian,

2004).


8

Source: https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0024735/

Figure 2.2: The brain structure

2.1.1. THE CEREBRUM

The cerebrum is the largest subdivision of the brain, located superiorly and

anteriorly in relation to the brainstem. It consists of a pair of cerebral hemispheres

(right and left), separated by the midline longitudinal cerebral fissure (the falx

cerebri) (Patestas and Gartner 2016). The cerebral cortex forms the outer covering

of the cerebral hemispheres and consists of a central core of white matter and a

thin outer covering of grey matter. It is estimated to comprise approximately 85%

volume of the adult human brain (Stephan et al., 1981, Rilling and Insel, 1999),

and each hemisphere is further subdivided into four lobes: the frontal, parietal,

temporal, and occipital lobes. The cerebral cortex is few millimetres thick and is


https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0024735/

9

highly convoluted to permit a large surface area of the brain to fit inside skulls

(Hilgetag and Barbas, 2009).

2.1.1.1. CYTOARCHITECTURE OF THE CEREBRAL CORTEX

The cerebral cortex is organized into six layers, and each of these layers has

different roles.

Source: https://medicine.academic.ru/135149/layers_of_cerebral_cortex

Figure 2.2: The layers of the cerebral cortex

The layers of the cerebral cortex, from superficial to deep, are:

Molecular layer (Plexiform) contains scattered horizontal cells and the apical

dendrites of pyramidal neurons of the cerebral cortex

The external granular layer contains small neuronal cell bodies.


https://medicine.academic.ru/135149/layers_of_cerebral_cortex

10

The external pyramidal layer is composed of small and medium pyramidal

cells.

The internal granular layer is made up of densely packed small round

pyramidal cells.

Internal pyramidal layer (Ganglionic) contains larger pyramidal cells and

scattered non-pyramidal cells.

Fusiform or Multiform layer comprises of variably shaped cells.

2.1.1.2. FUNCTION OF THE CEREBRAL CORTEX

Different regions of the cerebral cortex are associated with certain functions. For

instance, the frontal lobe is associated with conscious thought, memory,

personality and control of voluntary muscle movements (Chayer and Freedman,

2001).

Source:https://www.wpclipart.com/medical/anatomy/brain/brain_3/four_lobes_of_the_cerebral_c

ortex.png.html

Figure 2.3: The lobes of the cerebral cortex


https://www.wpclipart.com/medical/anatomy/brain/brain_3/four_lobes_of_the_cerebral_cortex.png.html


11

The parietal lobe gives individuals perspective and to help them understand space,

touch, and volume; the occipital lobe processes visual information while temporal

lobe is involved in auditory reception, helps to understand speech and is involved

in retrieving visual as well as verbal memories (Aversi-Ferreira et al., 2010,

Kiernan, 2012). The process of speech involves two different areas of the

cerebrum, namely, the Broca’s area which is responsible for the coordination of

the muscles for speaking and translation of thought into speech (Fadiga et al.,

2009), and the Wernicke’s area which is involved in the ability to read,

understand, and speak written word (Ardila et al., 2016).

2.1.1.3. THE HIPPOCAMPUS

The hippocampus is a small neuronal curved structure located on the medial

aspect of the temporal lobe of each cerebral cortex and forms an important part of

the limbic system. The hippocampus plays an important role in long-term memory

formation and spatial navigation. The hippocampus consists of two interlocking

U-shaped grey matter structures, the hippocampus proper (cornu ammonis, CA;

Figure 2.4) which is differentiated into four regions, CA1-4, and the dentate gyrus

separated from each other by hippocampal sulcus (Lorente de Nó, 1934;

Duvernoy, 2005).


12

Source: https://www.slideshare.net/amandahessborzacchini/hippocampus-13053807

Figure 2.4: The structure of the Hippocampus

The CA is divided into three primary layers, each defined by a particular feature

of the large pyramidal cells or their afferents (Figure 2.5). The polymorph layer is

found between the alveus and the pyramidal layer, and contains cell bodies of the

basket cells and the basal dendrites of the pyramidal cells, as well as afferents

from the septum; the cell bodies of the pyramids dominate the pyramidal layer

(stratum pyramidale), and the molecular layer contains pyramidal dendrites

(Bayer, 1985).

Source: https://www.slideshare.net/drpraveenktripathi/limbic-system-brain

Figure 2.5: The layers of the hippocampus proper


https://www.slideshare.net/amandahessborzacchini/hippocampus-13053807

https://www.slideshare.net/drpraveenktripathi/limbic-system-brain

13

2.1.2. THE CEREBELLUM

The cerebellum is located conspicuously in the hindbrain with pons and medulla

oblongata. Although it is only 10% of total brain mass, the cerebellum has about

75% as much surface area as either of the much larger cerebral hemispheres,

containing approximately 70-101 billion neurons in the brain (Snider, 1958,

Lange, 1975, Andersen et al., 1992). The cerebellum has a relatively longer

developmental period and is one of the first structures in the brain to differentiate

and one of the last to mature (Liu et al., 2011). It is derived from the rhombic lips

and thickenings along the margins of the embryonic hindbrain. By the second

trimester of pregnancy, the fissures of the cerebellar cortex have appeared (Liu et

al., 2011).

The cerebellum consists of two paired lateral extensions known as hemispheres

united by the midline part called the vermis (Figure 2.6). Typically, the

cerebellum is divided into the anterior, posterior and flocculonodular lobes as

defined by the major fissures.


14

Source: http://what-when-how.com/neuroscience/the-cerebellum-motor-systems-part-1/

Figure 2.6: The structure of the cerebellum A) Sagittal and B) Dorsal sections

The cerebellum consists of an outer layer of grey matter called the cortex and an

inner core of white matter known as medulla, containing four paired deep nuclei

namely, dentate nucleus, globose nucleus, emboliform nucleus, and fastigial

nucleus (Snell, 2010). All cerebellar input goes to the cortex, and all output comes

from the deep nuclei. The cerebellum is connected to the brainstem by three pairs

of stalks called cerebellar peduncles. Whereas the inferior peduncles connect the

cerebellum to the medulla oblongata, the middle peduncles connect the

cerebellum to the pons and the superior peduncles connect the cerebellum to the

midbrain (Snell, 2010).


http://what-when-how.com/neuroscience/the-cerebellum-motor-systems-part-1/

15

2.1.2.1. CYTOARCHITECTURE OF THE CEREBELLAR CORTEX

The cerebellar cortex is made up of three layers: the molecular layer, the granule

cell layer and the Purkinje cell layer in the middle (Figure 2.7). The input to the

cerebellum comes via the mossy and climbing fibres. The molecular layer consists

of two main types of neurons: stellate cells and basket cells (Apps and Garwicz,

2005). The Purkinje layer is formed by a single row of large Purkinje cells and

their axons which provide the only efferent pathway to the deep cerebellar nuclei,

thus constituting the sole output of all motor coordination in the cerebellar cortex

(Apps and Garwicz, 2005). The granular layer is densely populated by small

granule cells with dark-staining nuclei and scanty cytoplasm (Apps and Garwicz,

2005).

Source: http://vanat.cvm.umn.edu/neurHistAtls/pages/cns9.html

Figure 2.7: The structure of the cerebellar cortex.

Early studies considered the cerebellum a motor structure because cerebellar

damage mostly leads to impairments in motor function. Although the cerebellum

does not initiate movement, it contributes to coordination, precision, and accurate


http://vanat.cvm.umn.edu/neurHistAtls/pages/cns9.html

16

timing of movements (Penhune et al., 1998, Salman, 2002). The cerebellum

receives input from the sensory system of the spinal cord and other parts of the

brain, and integrate these inputs to fine-tune motor activity (Fine et al., 2002).

Damage to the cerebellum may occur as a result of stroke, head injury, cancer,

cerebral palsy, viral infection, or neurodegenerative diseases and may

subsequently cause individuals to experience tremors, difficulties in maintaining

balance, lack of muscle tone, speech difficulties, lack of control over eye

movement, difficulty in standing upright, and inability to perform accurate

movements (Fine et al., 2002). In addition, cerebellar neurons may become

damaged resulting in the loss of muscle control or coordination of movement in a

condition known as ataxia.

Some studies have shown that ataxia may also be triggered by the exposure to

toxins such as alcohol, drugs, or heavy metals (Manto, 2012, Barsottini et al.,

2014).

2.2. THE ENDOCRINE SYSTEM

The physiological activity of the endocrine system is closely linked to that of the

nervous system, with both systems coordinating the activity of the other (Brück,

1983). However, the differentiating aspect of the endocrine system is that its

effect is deployed through hormones secreted by glands located throughout the

body, conveyed in the bloodstream to particular areas in the body to regulate an

array of activities such as digestion, growth, metabolism and blood pressure

(Hiller-Sturmhöfel and Bartke, 1998).


17

The hypothalamus is the brain structure that connects the nervous and the

endocrine systems (Cleghorn, 1955). It contains a small group of nuclei found at

the base of the forebrain and is responsible for the control of behaviour and such

rudimentary needs as sleep, hunger, thirst and sex as well as emotional and stress

responses (Zha and Xu, 2015). The hypothalamus also regulates the secretion of

hormones from the pituitary glands and other endocrine glands in the body

(Charlton, 2008).

2.2.1. ENDOCRINE DISRUPTOR COMPOUNDS (EDCs)

The United States Environmental Protection Agency (EPA) defines EDCs as “an

exogenous agent that interferes with synthesis, secretion, transport, metabolism,

binding action, or elimination of natural blood-borne hormones that are present

in the body and are responsible for homeostasis, reproduction, and developmental

process” (Diamanti-Kandarakis et al., 2009). Reports show that EDCs alter and

modify natural endocrine function, thus emerging as a major public health

challenge (Diamanti-Kandarakis et al., 2009, Schug et al., 2011, Zoeller et al.,

2014). These deleterious effects are primarily due to their potentially disruptive

activity on physiological processes, particularly through direct interaction with

steroid hormone receptors which are expressed abundantly in the hypothalamus

and other brain areas involved in the regulation of neuroendocrine functions

(Thomas and Doughty, 2004, Tokumoto et al., 2007).

Humans can take up EDCs or its precursors through consumption of contaminated

food, drinking water, inhalation and direct dermal contact with contaminated

products (Caballero-Gallardo et al., 2016). Studies have linked EDCs to adverse


18

biological effects in animals, giving rise to concerns that its exposure might cause

serious medical effects (Colborn et al., 1993). Examples of some common EDCs

and their effects are summarized in Table 2.1.


19

Table 2.3: Examples of EDCs and their endocrine disruptor effects

Name Uses Effects References

Endrin Insecticide and

pesticide

Competitively binds to androgen receptors; Toxic to fish and the

CNS

(Coble et al., 1967, Bhattacharya

and Mukherjee, 1975, Lemaire et

al., 2004)

Polychlorinated dibenzofurans (PCDF)

Insulators and

lubricants

Toxic to infants and is associated with memory and attention

deficits, decreased verbal abilities, and adverse behavioural as

well as emotional effects in early childhood.

(Henretig, 2009, Kodavanti et al.,

2017)

Heptachlor Insecticide and

pesticide

Toxic to the liver and the central nervous system as well as

reproductive, hematopoietic, immune, and renal systems.

(Fendick et al., 1990)

Hexachlorobenzene (HCB) Pesticide In animals, it induces neurological symptoms such as paralysis,

tremors and convulsions. In humans, it causes damage to the

liver, thyroid, nervous system, bones, kidneys, blood, and

immune and endocrine systems

(Addae et al., 2013)

Mirex Insecticide Human carcinogen and high toxicity to fish and other aquatic

animals

(Sanders et al., 1981, Spinelli et

al., 2007)

Dichlorodiphenyltrichloroethane

(DDT)

Pesticide Carcinogenic, early pregnancy loss, toxic to the reproductive

and nervous systems.

(Longnecker et al., 1997, Salleh et

al., 2015)

Aldrin Pesticide Carcinogenic (de Jong et al., 1997, Hooker et al.,

2014)

Chlordane Insecticide Carcinogenic, toxic to the reproductive systems (Fry, 1995, Persson and

Magnusson, 2015, Xiong, 2017)


20

The adverse effect of EDCs can be seen during critical developmental stages such

as intrauterine, perinatal and in puberty, during which neuroendocrine systems are

modulated by steroid and other hormones (Frye et al., 2012). EDCs cross the

blood-placental and blood-brain barriers to interfere with development and

function, thus affecting pregnant mothers and children, both categories of which

have been considered the most susceptible to the harmful effects of exposure to

EDCs (Perera and Herbstman, 2011, Schug et al., 2015). Various reports indicate

that foetuses and offspring animals are exposed to EDCs through the placenta and

breast milk respectively, with such resultant effects as learning difficulties,

infertility and increased vulnerability to cancer not becoming evident until much

later in life (Poongothai et al., 2007, Li et al., 2013b). The major adverse health

effects arising from exposure to EDCs in humans include a myriad of

reproductive problems such as reduced count, motility and quality of sperms,

decreased female fertility, longer time to conception, higher miscarriage rates

(Singleton and Khan, 2003, Crain et al., 2008, Campion et al., 2012), increased

occurrence of testicular, prostate and breast cancer (Manibusan and Touart, 2017,

Balabanič and Klemenčič, 2018) and premature puberty in females (Zama et al.,

2016).

Some nervous system disorders may have their origins in endocrine disruptions,

particularly if the hippocampus and hypothalamus are targeted resulting in such

conditions as schizophrenia, bipolar disorders and cognitive dysfunctions (Brown

Jr, 2008; Meeker, 2012). A typical EDC that has been implicated in organ

function disruption and several other nervous system pathologies is 2, 2-bis (4-

hydroxyphenyl) propane, commonly known as BPA (Brown Jr, 2008).


21

2.2.1.1. BISPHENOL A (BPA)

BPA is an industrial synthetic chemical commonly used in the production of a

range of polymers such as polycarbonate plastics and epoxy resins, as well as in

the production of several indoor applications and consumer products such as

thermal printer paper, in the lining of cans presently used for food and beverages.

It is also used for electronic equipment, water pipes, dental sealants, sports

equipment, medical devices, tableware, among others (Geens et al., 2011, Ribeiro

et al., 2017)

BPA is reported to be one of the highest chemicals produced worldwide, and there

has been an exponential increase in its use during the last 30 years, thus making

its potential for food and environment contamination higher (vom Saal and Myers,

2008, Gao et al., 2015). Human exposure to BPA is recognized as a widespread

occurrence, especially in developed countries, with reports indicating that

approximately 100 tonnes of BPA are released into the atmosphere each year, and

analyses of human samples showed the presence of BPA in most tested subjects

(Geens et al., 2012). The consumption of BPA-contaminated food is anticipated to

contribute to the overall BPA environmental exposure by more than 90% for all

age groups and its presence in the air, waste and drinking water, as well as in dust

particles has been reported (Vandenberg et al., 2007, Rudel et al., 2011, vom Saal

and Welshons, 2014).

Structurally, BPA is a diphenyl compound composed of two hydroxyl groups in

the ‘‘para’’ position connected by a methyl bridge, with two methyl functional

groups attached to the bridge making it structurally similar to hormones such as


22

synthetic oestrogen, diethylstilbestrol (Kang et al., 2006). BPA is synthesized by

the condensation of acetone with two equivalents of phenol, and the reaction is

catalyzed by strong acids like hydrochloric acid (Figure 2.8) or a sulfonated

polystyrene resin (Uglea and Negulescu, 1991).

Figure 2.8: Synthesis of Bisphenol A

Prenatal BPA exposure is associated with neural, behavioural and bipolar

disorders in neonates, infants and children (Brown Jr, 2008). Reports indicate that

BPA induces hyperinsulinemia relating to type 2 diabetes mellitus and obesity at

very low doses and also triggers a high risk of miscarriage in women (Sugiura-

Ogasawara et al., 2005, Alonso-Magdalena et al., 2010).

2.2.1.1.1. PHYSICAL AND CHEMICAL PROPERTIES OF BPA

The physical and chemical properties of BPA are listed below.

Table 2.4: Physical properties of BPA.

Properties

Boiling point 220 oC (4 mmHg)

Melting point 158-159 oC

Molecular formula (CH3)2C(C6H4OH)2

Molar Mass 228.29 g·mol−1

Water solubility 21.5 oC

Vapour Pressure 25 oC

Density 1.20 g/cm³

BPA Acetone

Phenol Phenol


https://en.wikipedia.org/wiki/Catalysis

https://en.wikipedia.org/wiki/Hydrochloric_acid

https://en.wikipedia.org/wiki/Sodium_polystyrene_sulfonate

https://en.wikipedia.org/wiki/Sodium_polystyrene_sulfonate

23

2.2.1.2. RELEASE AND HUMAN EXPOSURE TO BPA

BPA has received considerable attention in recent years due to widespread

sources for human exposure through such routes as the environment (soil, air, and

aquatic) and contaminated foods. As a result of the high quantities of BPA

produced and an increase in the number of products based on epoxy resins and

polycarbonate plastics, there is widespread environmental contamination and

well-documented human exposure to BPA (Le et al., 2008). Although BPA is a

colourless solid soluble in organic solvents and poorly soluble in water, studies

have shown that BPA leaches from baby bottles, tin cans, reusable plastic water

bottles, and polycarbonate plastic containers into water, beverages, drink solutions

and food liquors, mostly due to the exposure of these BPA-containing plastics to

high temperatures (Grumetto et al., 2008, Le et al., 2008, De Coensel et al., 2009).

For instance, boiling to sterilize infant feeding bottles or addition of very hot

water or beverages to drinking bottles was reported to increase the rate of BPA

leaching by up to 55-fold (Le et al., 2008). Also, the discolouration, scratching of

polycarbonate, abnormal increase in pH and prolonged storage of water, drinks or

food in BPA-containing products increases the chances of leaching (Munguia-

Lopez et al., 2005, Biedermann-Brem et al., 2008).

In most industrialized countries, BPA detection in human amniotic fluid, serum,

breast milk and urine using various evaluation techniques has been reported

(Vandenberg et al., 2007) leading to the observation that BPA concentration in

human serum ranges from 0.2–1.6 ng/mL or 0.88–7.0 nM (Sajiki et al., 1999,

Takeuchi and Tsutsumi, 2002). In the United States, BPA was found to be present

in 95% of urine samples of 394 adults with a median concentration of 1.28 μg/L


24

(Calafat et al., 2004) and according to the U.S. Environmental Protection Agency,

a reference dose of BPA is 50 mg kg/day was set while the European Union set a

no-observed-adverse-effect level (NOAEL) of 5 mg kg/day (Moriyama et al.,

2002, Gao et al., 2015).

2.2.1.3. METABOLISM OF BPA

Following oral intake of BPA in humans and rodents, reports show that BPA is

absorbed from the gastrointestinal tract and is glucuronidated by UDP-

glucuronosyltransferase (UGT, Figure 2.9) as a first-pass metabolism in the liver

to its main metabolite, BPA-glucuronide (Pottenger et al., 2000, Pritchett et al.,

2002, Völkel et al., 2002). The glucuronidation of BPA is a detoxification reaction

by UGT to detoxify BPA, its metabolite BPA-glucuronide is quickly excreted in

the urine due to its half-life of less than 6 hours. Bisphenol A-sulphate on the

other hand is reported to be a minor urinary BPA metabolite in humans (Ye et al.,

2005, 2006).

Following the oral intake of BPA in humans, the first-pass metabolism in the liver

is reported to be a super-effective process that results in a tremendously reduced

systemic availability (Pottenger et al., 2000, Upmeier et al., 2000). Also, BPA-

glucuronide and BPA-sulphate do not hinder hormonal control of reproduction,

thus indicating that these reactions are strictly detoxification pathways (Snyder et

al., 2000, Shimizu et al., 2002, Willhite et al., 2008). In rats, BPA is also

predominantly glucuronidated, with sulphation representing a minor pathway

(Pottenger et al., 2000), however the BPA-glucuronide formed is excreted from

the liver via the bile into the gastrointestinal tract, cleaved back to BPA and


25

reabsorbed into the blood. Thus it undergoes enterohepatic recirculation resulting

in a significantly slower elimination of BPA including its conjugate in rodents

when compared with humans (Vandenberg, 2010).

Source: (Aschberger et al., 2010)

Figure 2.9: Metabolism of BPA

Several studies have suggested that the absorption and distribution of BPA in

maternal organs and foetuses are extremely rapid and that BPA easily crosses the

placenta after oral administration to pregnant rats (Miyakoda et al., 1999,

Takahashi and Oishi, 2000). Currently, there is an increase in research on the

adverse effects of foetal BPA exposure due to the ease of movement across the

placenta and the detection of low concentrations of endogenous UGT in foetal

tissues (Nishikawa et al., 2010, Angle et al., 2013). BPA exposure alters

secondary sexual characteristics and triggers neuronal, behavioural, and immune

disorders in foetuses and young children (Gies and Soto, 2013, Pouzaud et al.,

2018). In the human foetal liver, studies have shown that UGT is present at a

concentration five-times lower than in the adult liver and the UGT detoxification


26

activities are not detected in foetal rat liver, thus suggesting that foetuses may be

more susceptible to the deleterious effects of BPA as most of it could be

accumulating in the tissues with potential consequencies (Matsumoto et al., 2002,

Jalal et al., 2017).

2.2.1.4. ACTIVITY OF BPA

BPA possesses estrogenic properties and acts as an agonist for oestrogen receptors

(Matsushima et al., 2010, Shanle and Xu, 2010b). For instance, the E-SCREEN,

often regarded as the most sensitive assay for oestrogenicity based on its ability to

differentiate between partial and full agonists, has confirmed the estrogenic

properties of BPA (Matsushima et al., 2010). Prepubescent CD-1 mice treated

with BPA demonstrated estrogenic responses such as increased expression of the

oestrogen-inducible protein lactoferrin, improved uterine wet weight and luminal

epithelial height (Markey et al., 2001). In another study, ovariectomized rats

exposed to BPA treatment experienced triggered proliferation of the uterine and

vaginal epithelial cells and induced estrogenic responses in the mammary and

pituitary glands (Colerangle and Roy, 1997, Steinmetz et al., 1998).

There is evidence in literature which indicates that exposure to BPA during early

development may increase breast cancer risk (Murray et al., 2007). This assertion

is supported by studies showing that BPA activates genes involved in the growth

of mammary glands and promotes breast cancers in rats after exposure to known

carcinogens at a dose that would not cause cancer in BPA-untreated mice

(Betancourt et al., 2010, Wadia et al., 2013). In treated male rats, a reduction in

sperm count, testosterone and luteinizing hormone (LH) as well as in the weights


27

of the testes, epididymis, seminal vesicle and prostate gland were reported

(Sakaue et al., 2001, Akingbemi et al., 2004, Nakamura et al., 2010).

A report by the National Institutes of Health in Norway indicates that BPA has

adverse effects on foetal and infant brain development and behaviour. In

agreement with the previous report, the U.S. National Toxicology Program (NTP)

expressed concerns on the effects of BPA on the prostate gland, brain and

behaviour in foetuses, infants, and children (Abdallah, 2016). In rodents and non-

human primates, it has been reported that BPA affects the brain even at relatively

low exposure levels (Leranth et al., 2008, Nakagami et al., 2009). In-vivo studies

demonstrate that perinatal or neonatal BPA exposure modifies brain sexual

differentiation (Patisaul et al., 2006, Rubin et al., 2006), and initiates aggression,

anxiety, cognitive deficits, and learning-memory impairment (Miyagawa et al.,

2007, Tian et al., 2010, Xu et al., 2010b). BPA also induces changes in the

differentiation of ectodermal tissues, including neural tissues in cynomolgus

monkeys (Yamamoto et al., 2007), and triggers apoptosis in the central neurons of

tadpoles leading to head, vertebral and abdominal developmental deficits (Oka et

al., 2003).

Whereas BPA exposure in male rats during lactation is linked to hyperactivity and

the loss of dopaminergic neurons (Ishido et al., 2007), exposure during gestation

and lactation led to significant changes in the Glutamate/Aspartate (G/A) ratio in

the hippocampus of the cerebrum in Sprague-Dawley rats, thus suggesting

neuronal and glial developmental alterations (Kunz et al., 2011).


28

2.3. ROOIBOS (Aspalathus linearis)

Rooibos tea is prepared from a leguminous shrub, Aspalathus linearis which is

native to the Cederberg Mountains of the Western Cape region of South Africa

(Joubert et al., 2004) and is commonly used in the fermented (red; Figure 2.10) or

unfermented (green) form (Joubert et al., 2004). Fermentation is the enzymatic

oxidation of polyphenols and other components in the leaves of the plant, thus

converting the green grassy-smelling and malty tasting tea to the sweet-smelling

and fruity red tea (Krafczyk and Glomb, 2008).

(A) (B)

Source: http://www.carmientea.co.za/tea-variants/rooibos/ & http://amavida.com/learn/

Figure 2.10: Rooibos tea; (A) Unfermented (B) Fermented

Rooibos tea has become a popular and reputable herbal beverage due to its

characteristic taste, absence of alkaloids and low tannin content (Cheney and

Scholtz, 1963, Jaganyi and Wheeler, 2003). In 1968, Annetjie Theron discovered

that an infusion of rooibos alleviated prolonged restlessness, vomiting and

stomach cramps when administered to her colicky baby, thus leading to its

classification as a healthy beverage. This led to a broader consumer base, and


http://www.carmientea.co.za/tea-variants/rooibos/

http://amavida.com/learn/

29

several babies have since been reared with rooibos either added to their milk or

given as a weak brew (Joubert et al., 2008).

Other pharmacological reports suggest that rooibos relieves ingestion, heartburn

and nausea, improves appetite, decreases nervous tension, enhances sound sleep

and has anti-allergic effects (Morton, 1983, Van Wyk et al., 1997). Rooibos

formulations have also been reported to be capable of relieving such

dermatological complications as eczema, rashes and acne, leading to the

production of toiletries and cosmetic products, now sold through many

supermarkets and beauty shops (Joubert et al., 2008).

2.3.1. CHEMICAL COMPOSITION OF ROOIBOS

The leaf tannin content of fermented rooibos is between 3.2% to 4.4% (Reynecke

et al., 1949, Blommaert and Steenkamp, 1978) and traces of the alkaloid,

sparteine, flavonoids (aspalathin and aspalalinin) have been detected in this herbal

tea (Rabe et al., 1994, Shimamura et al., 2006). Other compounds detected in

rooibos include nothofagin, flavones (orientin, iso-orientin, vitexin, isovitexin,

chrysoeriol, luteolin and luteolin-7-O-glucoside), flavanones (dihydro-orientin,

dihydro-iso-orientin and hemiphlorin), flavonols (quercetin and its glycosides,

quercetin-3-robinobioside, hyperoside, isoquercitrin and rutin), phenolic acids,

lignans, esculetin, orientin, iso-orientin, and trace quantities of the aglycones,

luteolin, quercetin and chrysoeriol (Joubert et al., 2008).


30

Table 2.3: Quantification of the major flavonoids in aqueous rooibos extract (mg/500 ml)

brewed with 500 ml of boiled water and 10 g of dried green rooibos tea leaves.

Phenolic compounds mg/500 ml µmol

Aspalathin 287 ± 0.1 636 ± 20

Nothofagin 34.4 ± 0.0 79 ± 3.1

Isoorientin 26 ± 0.0 58 ± 2.6

Orientin 17 ± 0.0 38 ± 1.8

Rutin 7.9 ± 0.0 13 ± 0.3

Hyperoside 0.8 ± 0.0 1.7 ± 0.1

Isoquercitrin 2.9 ± 0.0 6.3 ± 0.4

Vitexin 3.1 ± 0.0 7.1 ± 0.2

Isovitexin 2.8 ± 0.0 6.6 ± 0.3

Luteolin-O-galactoside 0.9 ± 0.0 1.9 ± 0.2

TOTAL 383 ± 0.1 848 ± 29

Source: (Breiter et al., 2011)

2.3.1.1. POLYPHENOLS

The potential health benefits and bioactivity of rooibos have been linked to its

naturally-occurring polyphenolic content (Joubert and Ferreira, 1996, McKay and

Blumberg, 2007, Joubert and de Beer, 2011). Polyphenols have received great

reviews in the past decade due to their abundance in diets as well as their

antioxidative action, regulation of several protein functions and the prevention of

such oxidative stress-related diseases as cancer and neurodegenerative disorders

(Joubert and Ferreira, 1996, Canda, 2012). Polyphenols scavenge for free radicals

and neutralize reactive oxygen species (ROS) by altering free radical chain

reactions as well as control the activities of enzymes and chelating metal ions that

partake in the formation of free radicals (Perron and Brumaghim, 2009, Fraga et

al., 2010).


31

Polyphenols are subdivided into flavonoids and phenolic acids (Figure 2.11), and

may also be further classified as monomeric molecules (containing one unit) or

polymeric molecules (containing more than one unit) (Erickson, 2003).

Source: Modified from (Mentor, 2014)

Figure 2.11: Classification of polyphenols

Regardless of its considerable reduction during fermentation, aspalathin (Figure

2.12) is the main flavonoid of unfermented rooibos and is one of the major

components of the water extract of fermented rooibos (Joubert, 1996, Bramati et

al., 2002).

Figure 2.12: Chemical structure of Aspalathin


32

2.3.2. BIOLOGICAL PROPERTIES OF ROOIBOS TEA

As a result of its high polyphenolic contents, rooibos contains antioxidants that

scavenge free radicals and thus prevent cellular oxidative damage (Joubert et al.,

2008). The antidiabetic potential of the rooibos tea extract was confirmed using a

glucose-lowering test of its flavonoids in streptozotocin-treated rats (Muller et al.,

2012). In this regard, aspalathin has been reported to have a beneficial effect on

glucose homoeostasis through the stimulation of glucose uptake in muscle tissues

and increased insulin secretion from pancreatic β-cell, thus preventing type II

diabetes in mice (Kawano et al., 2009).

The aqueous extract of rooibos tea extract also exhibited bronchodilatory,

antispasmodic and blood pressure lowering activities in rabbits, guinea-pigs and

rats through the activation of KATP with the selective bronchodilatory effect, thus

validating its therapeutic potential in the treatment of congestive respiratory

diseases (Khan and Gilani, 2006). In different studies, treatment of rats with

rooibos tea improved sperm concentration, motility and viability (Opuwari and

Monsees, 2014) while consumption of the fermented tea considerably improved

the lipid profiles and redox status in adults predisposed to the development of

cardiovascular diseases (Marnewick et al., 2011).

The natural antioxidant and hepatoprotective activity of rooibos tea extract were

demonstrated via its inhibition of increased concentrations of triacylglycerols,

cholesterol, malondialdehyde, aminotransferases, bilirubin and alkaline

phosphatase in rats treated with carbon tetrachloride (Bosek and Nakano, 2003).

Fermented rooibos tea was reported to prevent obesity through the inhibition of


33

adipogenesis and adipocyte metabolism in 3T3-L1 adipocytes, thus highlighting

its potential in preventing obesity (Sanderson et al., 2014). In a different study,

rooibos increased the activity of cytosolic glutathione S-transferase alpha,

microsomal UGT and glutathione, in addition to the modulation of phase II drug

metabolizing enzymes and oxidative status in the liver of treated rats, thus

suggesting that it is important in protecting against the adverse effects related to

mutagenesis and oxidative damage (Marnewick et al., 2003). Similar

antimutagenic properties of fermented and unfermented rooibos tea in preventing

the transformation of a mutagenic compound into a mutagen was investigated

with the Salmonella typhimurium mutagenicity assay, and rooibos showed potent

antimutagenic activity against 2-acetylaminofluorene and aflatoxin B1-induced

mutagenesis (Van der Merwe et al., 2006).

2.3.4.1. NEUROPROTECTIVE EFFECTS OF ROOIBOS

The brain is one of the most sensitive targets of oxidative stress (Garbarino et al.,

2015). It accounts for approximately 2% of total body mass but consumes 15–

20% of the energy produced in the human body, and this increase in mass-specific

metabolic rate is ascribed to the high amount of omega-three polyunsaturated fatty

acids in brain tissue which are vulnerable to peroxidation (Hulbert et al., 2007). In

the brain tissue, there are high amounts of redox-active iron and copper, thereby

increasing its susceptibility to oxidative damage. Also, the brain is mostly unable

to replace impaired cells since it is composed generally of terminally

differentiated glia and neurons (Garbarino et al., 2015).


34

When there is excessive production of ROS, endogenous defence mechanisms

against ROS may not be sufficient to suppress ROS-associated oxidative damage,

consequently, exogenous dietary antioxidants are needed to supplement and

protect neurons against oxidative damage (Liu et al., 2018). Several natural

beverages such as rooibos are reported to be good sources of antioxidants due to

its polyphenolic content and have been subsequently utilized against a variety of

oxidative stress-induced neurodegenerative disorders such as Alzheimer’s and

Parkinson’s disease (Hong et al., 2014). In one study, rooibos tea showed efficient

protection against immobilization-induced oxidative stress in rats through the

reduction of stress-related metabolites (5-HIAA and FFA), prevention of lipid

peroxidation, restoration of stress-induced protein degradation, regulation of

glutathione metabolism and modulation of such antioxidant enzymes as

superoxide dismutase and catalase (Hong et al., 2014). The administration of

rooibos was observed to suppress the age-related accumulation of lipid peroxides

in several regions of rat brains (Inanami et al., 1995). Also, prolonged

consumption of fermented rooibos tea was found to offer neuroprotection against

ischemic brain injury through the attenuation of brain oedema and neuronal

apoptosis as well as improving neurobehavioural outcomes in rats given rooibos

tea compared to the untreated rats (Akinrinmade et al., 2017).

Although there is evidence in literature on the protective activity of rooibos

against some oxidative stress-inducing agents, its neuroprotective activity on the

development and function of the developing CNS is not clearly known. In the

present study, the neuroprotective effects of rooibos on the developing Wistar rat

hippocampus and cerebellum exposed to BPA during gestation and lactation was


35

investigated. Results from this study are expected to provide the first research

evidence on the benefits of rooibos tea as a remedy for ameliorating potential

BPA toxicity in the developing CNS using Wistar rats exposed to BPA.


36

CHAPTER 3

MATERIALS AND METHODS

3.1. ETHICAL CONSIDERATION

Ethical clearance for this study was provided by the Faculty of Natural Science

Research Ethics Committee of the University of the Western Cape, Cape Town,

South Africa. Ethical and project registration numbers were assigned to the

research project before commencement (Ethics Registration No: 2013-0410).

3.2. MATERIALS

The materials and equipment used in this study are shown in Table 3.1 and 3.2.

Table 3.4: Chemicals used in this study

Chemicals Supplier

Bisphenol A (BPA) Sigma-Aldrich (USA)

Ethanol Sigma-Aldrich (USA)

Sodium Chloride (NaCl) Merck ( SA)

Formaldehyde Sigma-Aldrich (USA)

Sodium hydroxide (NaOH) Merck ( SA)

Di-sodium hydrogen phosphate (anhydrous) (Na2HPO4) Merck (Germany)

Sodium di-hydrogen orthophosphate (Anhydrous)

(NaH2PO4)

Associated chemical enterprises

(SA)

Potassium chloride (KCL) Merck ( SA)

Sodium pentobarbitone Norpham Medical (SA)

DPX Kimix (SA)

Paraffin wax Merck (Germany)

Haematoxylin and eosin (H&E) stain Kimix (SA)

Cresyl Violet (CV) stain Kimix (SA)

Perchloric acid (PCA) Sigma-Aldrich (USA)


37

Table 3.5: Equipment used in this study

Product Supplier

Automatic tissue processor Duplex processor, Shandon Elliott (UK)

Autostainer machine Leica AutoStainer XL, (Germany)

Weighing Balance Adam, Keynes (USA) and Radwag Wagi (Poland)

Centrifuge Eppendorf centrifuge (Germany)

Embedding system Leica, EG1160 (Germany)

Liquid chromatographic system Waters Acquity (USA)

Microtome Leica, RM 2125 RT (Germany)

Water bath Leica SMM (Germany)

3.3. ANIMALS

Forty (40) adult female Wistar rats approximately three months old, with an

average weight of 250g were used in this study. Animals were procured from the

University of Stellenbosch animal facility, Cape Town, South Africa and

maintained under standard laboratory conditions at the University of the Western

Cape animal house. Daily body masses of rats were measured using a weighing

balance, and progressive weight changes relative to the initial weights were noted.

Mating was implemented by keeping one male rat with one female rat (1:1) in a

cage for seven days (Figure 3.1).

During mating, pregnancy and post-partum periods, animals were allowed free

access to standard rat chow and tap water. Successful mating was confirmed by

the presence of a vaginal plug or spermatozoa in the vaginal smear the following

morning (Ramírez-López et al., 2016). Pregnant dams were randomly assigned to

four treatment groups of ten dams each (n = 10; total = 40).


38

Figure 3.3: Flow diagram showing the experimental design for the study (10 rats per

group; G1=control, G2= BPA only, G3= rooibos + BPA and G4= rooibos tea only

3.4. TREATMENT PROTOCOL

Throughout pregnancy, dams were randomly assigned to four different groups of

ten rats each (n=10) and housed in separate cages as summarized below:

G1 received 9 % normal saline ad libitum (NS)

G2 received 400µg/ kg/day BPA (BPA)

G3 received 20 % fermented rooibos tea and 400µg/ kg/day BPA (RB)

G4 received 20 % fermented rooibos tea ad libitum (RT)

After parturition, the pups remained with mothers in the respective cages

maintained at temperature 21–24ºC under a 12 hours’ light and 12 hours’ darkness

cycle. The treatment was stopped after delivery on PND 21 (weaning day) and the

pups were kept in separate cages and were allowed food and tap water ad libitum.

Female Rat

G1: NS G2: BPA G3: RB G4: RT

Male Rat


39

The day of birth was considered as PND 0 and pups were sacrificed at the end of

the experiment on postnatal day (PND) 42. It is important to note that the

treatment was only applied to the pregnant dams in the groups and not the

offspring. The offspring rats were thus exposed to treatment only during

pregnancy (via the placenta) and lactation. A total of 12 pups per group were

sacrificed on each of the assigned days.

3.5. PREPARATION OF REAGENTS

3.5.1. ROOIBOS HERBAL TEA PREPARATION

The fermented rooibos herbal tea used in this study was a gift from Rooibos Ltd

(Clanwilliam, South Africa) to the research laboratory of Prof. Thomas Moonses

at the Department of Medical Biosciences, University of the Western Cape, South

Africa. A concentration of 0.02g/ml of fermented rooibos herbal tea reported to be

the routine amount used by rooibos consumers for tea-making purposes

(Marnewick et al., 2003, Pantsi et al., 2011) was used throughout this study.

Briefly, 1000ml freshly boiled tap water was added to 20g of fermented rooibos

leaves and stems for 5 minutes followed by filtration with cheesecloth and

Whatman’s filter papers (no 4 and 1 respectively). The aqueous extract was then

allowed to cool off to room temperature before administration to rats ad libitum.

Fresh tea was prepared daily while intake of rooibos tea and water was measured

throughout the experimentation period by subtracting the volume of the remaining

fluid from the initial volume. No major fluid leakage from water bottles was

observed, and the average consumption rate per cage was 40ml/day.


40

3.5.2. BISPHENOL A (BPA) AND NORMAL SALINE

BPA was dissolved in 1% absolute ethanol, diluted in water, and administered

orally as the vehicle at a concentration of 400µg/ kg/day. This concentration was

selected based on the findings from a previous study (Ahmed and Eid, 2015). An

oral route of BPA administration to the pregnant and lactating female was chosen

throughout this study to mimic the most likely route of exposure of the compound

in humans and wildlife. Glass water bottles were used in this study to ensure that

related compounds did not leach from plastic water bottles to possibly confound

the results of this study. The preparation of 0.9% normal saline was done by

adding 9g of Sodium Chloride (NaCl) to a litre (1000ml) of distilled water.

3.5.3. PREPARATION OF 4% FORMALDEHYDE SOLUTION

The formaldehyde solution was needed to preserve the specimens for histological

and immunohistochemical studies. Briefly, a litre of 4% formaldehyde was

prepared by adding 40 g of paraformaldehyde powder into 700 ml of distilled

water at 60 °C. The paraformaldehyde solution was stirred on a burner, and two

drops of 2 M sodium hydroxide (NaOH) solution were added until the

paraformaldehyde solution was clear. The volume of solution was made up to 1 L

with distilled water. The solution was prepared in small amounts as needed to

ensure that only fresh solutions were used each time.

3.5.4. PHOSPHATE BUFFERED SALINE (PBS)

PBS was prepared by adding 1.44 g anhydrous di-sodium hydrogen phosphate

(Na2HPO4), 0.24 g anhydrous sodium di-hydrogen orthophosphate (NaH2PO4), 8

g of sodium chloride (NaCl) and 0.2 g of potassium chloride (KCL). The


41

chemicals were dissolved in 800 ml until complete dissolution. The pH of the

solution was confirmed to be 7.4 by a pH meter and was thereafter made up to

1000 L.

3.6. MOTOR FUNCTION TESTS (NEUROBEHAVIORAL TESTS)

Neurobehavioral studies are commonly used to evaluate behavioural changes

induced by exposure to treatments of varying types, concentrations and dosages

(Seale et al., 2012). Common neurobehavioral parameters include locomotor

activity, cognitive function (e.g. memory deficit, etc.), gait, depression, anxiety,

equilibrium, etc. In this study, only the open field test (OFT) and the novel object

recognition (NOR) tests were done due to some constraints.

3.6.1. OPEN FIELD TEST (OFT)

The OFT is a commonly used neurobehavioral assessment tool that provides

simultaneous measurement of locomotion and anxiety in laboratory animals

(Kendigelen et al., 2012). On PND 42, the OFT was performed in the OFT

apparatus (Figure 3.2), a square plexiglass box (72×72 cm with 36cm walls) with

a video camera (Samsung HMX-F90, South Korea) positioned above the

apparatus to record each trial for subsequent analysis.

The open-field arena was divided into 16 equal squares, via a 4 × 4 grid, for ease

of data analysis. Animals were tested singly, each transported from the housing

room to the testing room and allowed to acclimatize in the OFT apparatus. Pre-

testing was done for two days to prepare the animals. On the third day, each rat

was placed in the centre zone of the OFT arena and observed for 5 minutes. This

was repeated twice after which the rat was returned into its home cage and the


42

OFT box cleaned with 70% ethanol before testing the next rat. Video recording of

all tests was done using the overhead camera, and recordings were analysed using

the Smart video tracking software version 3.0, from Panlab Harvard Apparatus

(Massachusetts, USA) to measure the locomotor activity of each rat by extracting

the total distance travelled in the OFT arena. As a measure of anxiety, the total

distance travelled in the 12 squares near the walls was compared with the distance

travelled in the four squares at the centre of the arena. These assays were

performed my the researcher as well as two blind” observers, and the results were

averaged.

Source: http://www.neuralconnections.net/2011/10/stress-receptors-and-responses.html

Figure 3.4: Open Field Test Apparatus.

The activities evaluated include the frequency of rearing episodes (the number of

times the rat stood on its hind limbs with its forelimbs against the wall of the


http://www.neuralconnections.net/2011/10/stress-receptors-and-responses.html

43

testing cage), total freezing time and total self-grooming activity (the duration that

a rat spends licking or scratching itself while remaining stationary).

3.6.2. NOVEL OBJECT RECOGNITION (NOR) TEST

The NOR test is widely used to evaluate cognition, particularly recognition

memory, in rodent models of CNS disorders. This test measures the ability of rats

to recognize a novel object in an otherwise familiar environment, thus reflecting

the use of learning and recognition memory (Ennaceur and Delacour, 1988,

Ennaceur, 2010). The apparatus for the NOR test included a square plastic/glass

box (40x40x25 cm), with a digital camera (Samsung HMX-F90, South Korea)

mounted directly above it. The object test arena was divided into four equal

squares via a 2 × 2 grid, and rats were tested singly.

The NOR test was conducted with two different kinds of objects which were

different in shape, colour, and texture. The task procedure consisted of three

phases: habituation, familiarization, and test phase. During habituation, the rats

were allowed to explore an empty arena. Twenty-four hours after habituation

(familiarization), the rats were exposed to the familiar arena with two identical

objects placed at an equal distance. The next day (test phase), the rats were

allowed to explore the arena in the presence of the familiar object (old object) and

a new object to test recognition memory. Recognition memory was evaluated

using a recognition index (RI) for each animal calculated using the formula: RI =

TN/ (TN + TF) where TN and TF represent the time an animal spends with the

novel object (TN) versus a familiar (old) one (TF) respectively. The testing box

was cleaned with 70% ethanol before testing the next rat.


44

3.7. ANIMAL SACRIFICE

On PND 42, the final body masses of the animals were taken before injection of

150 mg/bw (i.p) sodium pentobarbital and subsequent decapitation under deep

anaesthesia. The neural specimens of interest were carefully dissected out and

either fixed in 4% paraformaldehyde solution (for histology and

immunohistochemical studies) or stored in cold phosphate buffered saline (PBS)

(mainly for biochemical studies).

3.8. HISTOLOGICAL PREPARATION OF BRAIN SAMPLES

3.8.1. AUTOMATED TISSUE PROCESSING

After two days in 4% paraformaldehyde solution, brain specimens were put in

appropriately labelled cassettes before processing in a Leica-2125 automated

tissue processor, ATP (Leica, Germany) for 7 hours in preparation for sectioning,

staining and microscopic analysis. The programme selected for the specimens

consisted of 4 hours of dehydration (through different grades of ethanol to remove

water), 1 hour 30 minutes of xylene clearing (to remove ethanol) and 1 hour 30

minutes of infiltration with molten paraffin wax (to displace xylene, Table 3.3).

Table 3.6: Tissue Processing Protocol

Step Solution Time (min) Temperature (ºC)

1 10% Formalin 30 Room Temperature

2 70% Ethanol 30 Room Temperature



5 99.9% Ethanol 30 Room Temperature



8 Xylene 30 Room Temperature

9 Xylene 30 Room Temperature

10 Paraffin 60 60

11 Paraffin 60 60

12 Paraffin 60 60


45

3.8.2. EMBEDDING

The cassettes of CNS specimens were removed from the last change of paraffin

wax in the ATP (Leica, Germany), and samples were manually removed from

cassettes. After that, samples were carefully placed in steel moulds containing

liquid paraffin wax in the desired orientation, and more liquid wax was added

until the samples were completely covered by paraffin wax. The moulds were

then transferred to the refrigerating plate of an embedding machine to allow the

wax to solidify. Tissue blocks were later removed from the moulds and

refrigerated until sectioning.

3.8.3. SECTIONING

Tissue blocks were removed from refrigeration and allowed for two hours before

trimming to appropriate dimensions followed by sectioning at 5 microns using a

Leica TP-1020 microtome (Leica, Germany). Each section was placed in warm

water to allow for spreading before mounting on a labelled glass slide.

3.8.4. STAINING

Standard haematoxylin and eosin (H&E), cresyl violet as well as

immunohistochemical (GFAP) staining were done for morphometric studies.

3.8.4.1. HAEMATOXYLIN AND EOSIN

H & E staining was done with an autostainer machine (Leica AutoStainer XL) at

the Histology Laboratory of the University of Stellenbosch, South Africa using a

standardized protocol. Briefly, the glass slides were placed on a staining rack,

deparaffinized in two changes of xylene for 5 minutes, rehydrated by immersion


46

in two changes of 99% ethanol for 2 minutes followed by 96% and 70% ethanol

for two minutes each. Thereafter the slides were rinsed in running tap water for at

least two minutes before staining in haematoxylin for 8 minutes. The slides were

then placed in running water at room temperature for 5 minutes, differentiated in

acid alcohol for 2-3 seconds before being rinsed in running water. Slides were

counterstained in eosin for 4 minutes, and thereafter rinsed in running tap water

for at least one minute followed by dehydration in three different baths of alcohol

(70%, 96%, and 99%) for 30 seconds, one minute and 30 seconds respectively and

finally cleared in xylene for one minute. Coverslips were then mounted on the

slides using DPX mounting medium and left to dry for 48hours (Cardiff et al.,

2014).

3.8.4.2. CRESYL VIOLET (CV) / NISSL STAINING

The CV staining technique is commonly used to study cellular cytoarchitecture in

neural tissues because it presents clearly delineated neuronal perikaryons in

comparison to the modest rendition of shape and size of cell bodies offered by

staining with haematoxylin and eosin (Akinrinmade et al., 2017). Briefly, sections

were deparaffinized in xylene twice for 10 minutes each and then hydrated in

100% ethanol twice for 5 minutes each, then in 95% ethanol for 3 minutes and

again in 70% ethanol for 3 minutes. Hydrated sections were then rinsed in tap

water and distilled water, stained in 0.1% Cresyl violet for 5 minutes and

thereafter rinsed quickly in distilled water. Sections were further differentiated in

95% ethanol for 2 minutes and then dehydrated in 100% ethanol for 10 minutes,

cleared in xylene for 10 minutes before coverslip mounting with DPX and drying

for 48 hours.


47

3.8.4.3. IMMUNOHISTOCHEMICAL (IHC) STAINING

All immunohistochemical (IHC) staining procedures were done using the Leica

Bond Autostainer and the Bond Polymer at the Histology Laboratory of the

University of Stellenbosch, South Africa using a standardized protocol. The tissue

sections were then manually rehydrated in ethanol grades, cleared in xylene,

mounted with DPX solution and then left to dry for 48 hours. (Table 3.4).

Table 3.4: IHC staining procedure

Step Type Incubation

Time

Temperature Dispense Type

1 Peroxide Block 5 min Ambient selected vol.

2-4 Bond Wash Solution 0 min each Ambient selected vol.

5 Primary Antibody 15 min Ambient selected vol.


9 Post Primary 8 min Ambient selected vol.


13 Polymer 8 min Ambient selected vol.


16-17 Deionized Water 0 min each Ambient selected vol.

18 Mixed DAB Refine 10 min Ambient selected vol.

19-21 Deionized Water 0 min each Ambient selected vol.

22 Haematoxylin 5min Ambient selected vol.

23-25 Deionized Water 0 min Ambient selected vol.

Rehydration and clearing

Step Solution Duration

1 70% alcohol 5 dips

2-3 96% alcohol 5 dips each

4-5 99% alcohol 5 dips each

6-7 Xylene Dip for 1 min each

Mounting for observation was done using DPX and slide cover slips.

3.9. NEUROCHEMICAL ASSAYS

Neurochemical assays are often done to determine the quantities of biological

molecules and compounds of interest, ranging from neurotransmitters,

neuromodulators, reactive oxygen species, neurosteroids, ions, neurohormones,

neuro enzymes, immunogens, ingested drugs, neurotoxins etc. The neurochemical


48

assays done in this study include superoxide dismutase (SOD) and catalase (CAT)

mainly to evaluate cerebral and cerebellar antioxidant enzyme activity.

3.9.1. HOMOGENIZATION OF TISSUES

Brain tissues were homogenized in 10 times (w/v) 0.1M PBS (pH 7.4) in a Teflon

glass homogenizer (IKA Laboratories, Germany) for two periods of 10 seconds

each. The homogenate obtained was then centrifuged at 15,000 rpm in a

microcentrifuge at 4°C for 10 minutes and the supernatant collected and

transferred into newly marked Eppendorf tubes for relevant neurochemical assays.

3.9.1.1. SUPEROXIDE DISMUTASE (SOD)

SOD activity assays measure the amount of enzyme that inhibited the oxidation of

pyrogallol, expressed as units per milligram of protein. In this study, the

supernatants were collected on ice and the SOD colorimetric activity kit (Life

Technologies, USA) was used to measure SOD activity according to the

manufacturer’s instructions. Briefly, 10μL of standards and samples, 50μL of 1X

substrate and 25µL of 1X xanthine oxidase were added to each well of a 96 well

half area plate and then incubated for 20 minutes at room temperature.

Absorbance was read using a Polarstar Omega plate reader (BMG Labtech, USA)

at 450nm and a curve-fitting software was used to generate the standard curve

using a four parameter algorithm.

3.9.1.2. CATALASE

The catalase colorimetric activity kit (Life Technologies, USA) was used to

measure the activity of catalase in the samples. Catalase is a highly conserved


49

enzyme expressed in all mammalian tissues and catalyzes the hydrolysis of

hydrogen peroxide (H2O2) into H2O and O2, to prevent the potential harmful

effects of excessive levels of H2O2 (Nagata et al., 1999, Agati et al., 2012). The

supernatants were collected on ice and experiments were performed according to

manufacturer’s instructions. Briefly, 25μL of standards, 25μL of the sample and

25μL of hydrogen peroxide reagent were added into each well and incubated for

30 minutes at room temperature. Thereafter, 25 µL of the substrate and 25μL of

1X HRP solution were added to each well and further incubated for 15 minutes at

room temperature. Absorbance was read using a Polarstar Omega plate reader at

560nm, and a curve-fitting software was used to generate the standard curve using

a four parameter algorithm.

3.10. STATISTICAL ANALYSIS

The GraphPad Prism Software V7 was used for all statistical analyses

(www.graphpad.com/scientific-software/prism/) and data obtained was expressed

as mean ± standard error of mean (SEM). A one-way analysis of variance

(ANOVA) followed by Tukey's multiple comparison post-hoc test was performed

to determine statistical significance (p<0.05).


50

CHAPTER 4

RESULTS

4.1. INTRODUCTION

In this study, the potential neuroprotective effects of fermented rooibos herbal tea

on the development of the cerebrum and cerebellum was evaluated in rats exposed

to BPA during pregnancy and lactation. Two groups of pregnant Wistar rat dams

(RB and RT) were administered fermented rooibos herbal tea ad libitum during

pregnancy and lactation while the third group of pregnant Wistar rat dams (BPA)

were administered with BPA only. The fourth group of dams (NS) was

administered normal saline ad libitum and offspring from this group served as

controls.

Daily consumption of rooibos tea and water was measured throughout the

experimentation period by deducting the volume of the remaining fluid from the

original volume; the average intake per cage was 40ml/day throughout the study

period. No major fluid leakage from the water bottles was detected. Rats were

allowed ad libitum access to tap water, normal saline and rooibos tea depending

on the treatment groups. Offspring rats from each group (n = 12) were subjected

to neurobehavioural assessment on PND 42, weighed and sacrificed for further

neurochemical, histological and immunohistochemical evaluation.

The control group (NS) results were used as non-pathological reference for all

other groups and a one-way ANOVA multiple comparisons test and Turkey’s


51

multiple comparisons post-hoc test (GraphPad Prism version 7.0) were used to

compare differences between respective treatment groups for all parameters.

Findings from this study will provide information on the ability of natural

products to protect the developing brain against the deleterious effects of EDCs.

4.2. EFFECTS OF BPA AND FERMENTED ROOIBOS HERBAL TEA ON

BODY MASS

Previous studies have indicated that perinatal BPA exposure leads to body mass

increases in offspring rats (Rubin et al., 2001, Miyawaki et al., 2007). The effects

of rooibos tea and BPA treatments on changes in body mass in offspring Wistar

rats was investigated as part of the present study. Findings show that by PND 42,

BPA treatment led to a significant increase (p<0.0001, Figure 4.1) in body masses

when compared to the controls. The consumption of rooibos tea appeared to have

prevented the BPA-induced body mass increment as corroborated by a significant

reduction (p<0.0001) in the body masses of offspring rats in the RB group when

compared to the BPA group. There was no significant difference (p>0.05) in the

body masses of offspring rats in the RT group when compared to the control

group.


52

NS

BP

A

RB

RT

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

Av

era

ge

bo

dy

we

igh

t (g

)

NS: Normal saline, BPA: Bisphenol A, RB: Rooibos tea + Bisphenol A, RT: Rooibos tea.

Bars represent the mean + SEM (n=12). P<0.0001 vs NS; фP<0.0001 vs BPA

Figure 4.2: Average body mass of experimental rats on PND 42

4.3. NEUROBEHAVIOURAL TESTS

4.3.1. THE OPEN FIELD TEST

The open field test is one of the most commonly used measures of animal

neurobehavioral deficits. In the present study, video recordings were analysed to

evaluate the frequency of rearing episodes, total freezing time and total self-

grooming time on PND 42. Three different observers captured the data and results

were averaged to obtain the final values which were analysed statistically for

comparisons between the respective groups.

4.3.1.1. THE FREQUENCY OF REARING EPISODES

Rearing episodes indicate the frequency at which the animal stands on hind limbs

or leans against the walls of the open field apparatus with the forelimb paws

(Davies et al., 2012). It is considered one of the measures of anxiety in rodents


53

and has been reported to reveal exploratory behaviour triggered by new stimuli

(Van Abeelen, 1977, Alves et al., 2012). Findings show that BPA treatment led to

a significant reduction (p=0.0005, Figure 4.2) in rearing episodes when compared

to the control, however, in the RB group of rats, there was a significant increase

(p<0.0001) in rearing episodes when compared to BPA group of rats. Also, there

was no significant difference (p>0.05) between the RT group of rats and the NS

control group.

NS

BP

A

RB

RT

0

1 0

2 0

3 0

4 0

Re

ari

ng

ep

iso

de

s


Bars represent the mean + SEM (n=6). P<0.001 vs NS; фP<0.0001 vs BPA

Figure 4.2: Number of rearing episodes of experimental rats on PND 42

4.3.1.2. TOTAL FREEZING TIME

Freezing (complete absence of body movements) is commonly used as an

indicator of anxiety and high-stress conditions (Walsh and Cummins, 1976, Díaz-

Morán et al., 2014). Total freezing time (seconds) was determined by scoring with

a stopwatch and reviewing video recordings of each rat during the test period.

Findings show that BPA treatment led to a significant increase (p<0.0001, Figure


54

4.3) in total freezing time when compared to control. Conversely, rooibos

treatment significantly reduced (p=0.0343) the total freezing time in the RB group

of rats when compared to the BPA group of rats and there was significant increase

(p>0.0044) in total freezing time in the RT group of rats when compared to the

NS control group.

NS

BP

A

RB

RT

0

1 0

2 0

3 0

4 0

Imm

ob

ilit

y d

ura

tio

n (

se

c)

*


Bars represent the mean + SEM (n=6). P<0.001 vs NS; фP<0.05 vs BPA; *P<0.005 vs

NS

Figure 4.3: Total freezing time of experimental rats on PND 42

4.3.1.3. TOTAL SELF-GROOMING TIME

Previous reports have demonstrated an increase in the total self-grooming time in

rodent models of anxiety and stress-exposed rats or mice (Kalueff and Tuohimaa,

2005, Kalueff et al., 2007). In this study, the cumulative duration that a rat spent

licking or scratching itself while remaining stationary was used as a measure of

self-grooming. Whereas BPA treatment led to a significant increase (p<0.0001,


55

Figure 4.4) in total self-grooming time when compared to the control, rooibos

treatment significantly reduced (p<0.0001) the total self-grooming time as seen in

the RB group when compared to the BPA group. Also, there was no significant

difference (p>0.05) in total freezing time in the RT group of rats when compared

to the NS control group.

NS

BP

A

RB

RT

0

1 0

2 0

3 0

4 0

5 0

Gro

om

ing

tim

e (

se

c)


Bars represent the mean + SEM (n=6). P<0.001 vs NS; фP<0.05 vs BPA.

Figure 4.4: Total self-grooming time of experimental rats on PND 42

4.3.2. NOVEL OBJECT RECOGNITION (NOR) TEST

The NOR test was used in this study to determine the ability of a rat to recognize

and spend longer time exploring a novel object introduced into its environment. In

this study, rats were exposed to both novel (N) and familiar (old, F) objects for 3

minutes, and the Recognition Index (RI) was calculated from the formula below:


56

𝑅𝐼 = 𝑇𝑁

(𝑇𝑁 + 𝑇𝐹)… … … … … . 𝐹𝑜𝑟𝑚𝑢𝑙𝑎 1,

Where RI = Recognition Index, TN = Time spent with a novel object and TF = time spent

with a familiar object

Findings show that BPA treatment led to a reduction (p=0.6289, Figure 4.5) in RI

when compared to the control, however, in the RB group of rats, there was a

significant increase (p<0.0269) in RI when compared to the BPA group of rats.

Also, there was no significant difference (p>0.05) between the RT group of rats

and the NS control group.

NS

BP

A

RB

RT

0

5

1 0

1 5

2 0

2 5

Re

co

gn

itio

n I

nd

ex


Bars represent the mean + SEM (n=6). фP<0.05 vs BPA.

Figure 4.5: Recognition index of experimental rats on PND 42


57

4.4. EFFECT OF BPA AND FERMENTED ROOIBOS HERBAL TEA ON THE

ACTIVITY OF ANTIOXIDANT ENZYMES

4.4.1. SUPEROXIDE DISMUTASE

Reactive oxygen species (ROS) are generally regarded as cytotoxic agents that

induce oxidative damage by attacking cell membranes and DNA (Ozaydın et al.,

2018). The production of ROS may be triggered by inflammation, radiation,

drugs, aging and such chemical substances as BPA. However, antioxidant

enzymes such as SOD and CAT are known to inhibit the damage caused by ROS.

An increase in ROS generation or a reduction in cellular antioxidant levels results

in a condition known as oxidative stress (Hassan et al., 2012, Abdel-Wahab,

2014). BPA has been reported to cause damage in many organs such as the liver,

kidney and brain by lowering antioxidant activity, thus increasing oxidative stress

(Song et al., 2014, Moghaddam et al., 2015).

Superoxide dismutases (SOD) are enzymes that counteract oxidative damage by

catalyzing the dismutation of superoxide radical (O2-) into either molecular

oxygen O2 or H2O2 which are less toxic molecules (Hollman and Katan, 1999).

Findings from this study show that BPA treatment resulted in a significant

decrease (p<0.005, Figure 4.6-4.7) in cerebral and cerebellar SOD activity;

however, in the RB group of rats, there was a significant increase (p<0.05) in

SOD activity when compared to BPA group of rats.

There was no significant difference (p<0.05) in SOD activity in the RT group of

rats when compared to the NS control group.


58

T re a tm e n t

SO

D a

cti

vit

y (

U/m

g p

ro

tein

)

NS

BP

AR

BR

T

0 .0 0

0 .0 5

0 .1 0

0 .1 5

0 .2 0

0 .2 5



Figure 4.6: Effects of different treatments on SOD enzyme activity in the cerebral

homogenate of experimental rats on PND 42

T re a tm e n t

SO

D a

cti

vit

y (

U/m

g p

ro

tein

)

NS

BP

AR

BR

T

0 .0 0

0 .0 5

0 .1 0

0 .1 5

0 .2 0



Figure 4.7: Effects of different treatments on SOD enzyme activity in the cerebellar



59

4.4.2. CATALASE

CAT is involved in the detoxification of H2O2 through the conversion of two

molecules of H2O2 to two molecules of H2O and one molecule of O2 in tissues

(Nagata et al., 1999, Agati et al., 2012). The rate of H2O2 disintegration is directly

proportional to catalase concentration hence, higher CAT concentrations indicate

highly potent antioxidant effects and vice versa. Following treatment of rats with

BPA, there was a significant reduction in the cerebral and cerebellar catalase

activity (p<0.05, Figure 4.8-4.9). In the RB group, there was a significant increase

(p<0.05) in CAT activity when compared to BPA group. Also, there was no

significant difference (p<0.05) in CAT activity in the RT group of rats when

compared to the NS control group.

T re a tm e n t

CA

T a

cti

vit

y (

U/m

l)

NS

BP

AR

BR

T

0

2

4

6

8



Figure 4.8: Effects of different treatments on CAT enzyme activity in the cerebral



60

T re a tm e n t

CA

T a

cti

vit

y (

U/m

l)

NS

BP

AR

BR

T

0

2

4

6

8



Figure 4.9: Effects of different treatments on CAT enzyme activity in the cerebellar


4.5. HISTOLOGICAL STUDIES

In this study, coronal sections of the cerebrum were obtained and stained to study

the potential neuroprotective effects of fermented rooibos herbal tea on the

development of CNS structures in rats exposed to BPA during pregnancy and

lactation. Coronal sections were cut and stained with H&E and Cresyl violet (CV)

for cell counting and histological analysis of the shapes and sizes of cell bodies in

the (CA1) region of the hippocampus while GFAP immunostaining was used for

analysis of damage responses in astrocytes. Similar analysis was done for the

Purkinje cells and the glial cells in the cerebellum.


61

4.5.1. LENGTH OF HIPPOCAMPAL CA1 REGION IN EXPERIMENTAL

RATS

The H & E staining revealed very clear outlines of the pyramidal cell layer of the

hippocampal region of interest - the CA1 region. The length of this region was

determined by measurement along the dotted red landmark using an appropriate

Image-j tool as shown in Figure 4.10. In order to ensure that the sampling size

was sufficient for this analysis, specimens from six rats were assigned to each

group and three (3) separate staggered serial sections were used per animal, giving

a total of 18 sections per group. The singular CA1 region of each slide was

measured and average values were computed and plotted for each group using the

Graph Pad Prism software version 6.

CA - cornua ammonis (subregions - CA1, CA2 and CA3); DG – Dentate gyrus.

Figure 4.10: Diagram of the hippocampus showing the landmarks used for determining

the hippocampal length in experimental rats.

Findings show that on PND 42, there was a significant reduction (p<0.0001,

Figure 4.11) in the hippocampal CA1 length of rats treated with BPA when


62

compared to the rats in the control group. In the RB group of rats, there was a

significant increase (p=0.0082) in the CA1 length when compared to the BPA

group. Findings also show that there was a significant reduction (p=0.0004) in the

RT group of rats when compared to the control rats.

T re a tm e n t

CA

1 l

en

gth

(

m)

NS

BP

AR

BR

T

0

2 0 0 0

4 0 0 0

6 0 0 0

*


Bars represent the mean + SEM (n=3). P<0.0001 vs NS; фP<0.01 vs BPA; *P<0.0005

vs NS

Figure 4.11: CA1 hippocampal length in experimental rats on PND42

4.5.2. NUMBER OF PURKINJE CELLS IN THE PURKINJE CELL LAYER

(PCL) IN EXPERIMENTAL RATS

A loss or significant reduction in the Purkinje cells in the cerebellum has been

linked to motor disorders (Darmanto et al., 2000, Maloku et al., 2010, Celik et al.,

2018) and in the current study, the number of Purkinje cells significantly

decreased (p=0.0002, Figure 4.12) following treatment with BPA. However, in

rats treated with rooibos tea and BPA (RB), Purkinje cell numbers significantly


63

increased (p<0.0001) when compared to rats treated with BPA only. In the RT

group, there was no significant difference (p=0.9849) in the number of Purkinje

cells when compared to rats in the NS control group.

T re a tm e n t

Nu

mb

er o

f P

urk

inje

ce

lls

NS

BP

AR

BR

T

0

2 0

4 0

6 0

8 0



Figure 4.12: Number of Purkinje cells in experimental rats on PND42


64

4.6. IMMUNOHISTOCHEMISTRY (IHC)

4.6.1. GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP)

GFAP is an essential protein of intermediate glial filaments in astrocytes used for

the assessment of damage responses in astrocytes (Cikriklar et al., 2016). An

increase in the expression of GFAP is a key feature in astrocytic and CNS damage

(Hausmann et al., 2000, Yu et al., 2004). A total of three rats were assigned per

group, and tissue sections were processed for immunostaining to investigate

GFAP. The immunoreactive astrocytes for GFAP were determined in six counting

boxes (440 × 330 µm) per slide along the CA1 region, which surrounded the

polymorphic and molecular layers adjoining the pyramidal layer. The average

values were computed, plotted and compared between the different treatment

groups using the Graph Pad Prism software version 6.

Results showed that there were significantly more (p<0.0001, Figure 4.13) GFAP-

immunoreactive astrocytes in the BPA group of rats when compared to control

while the rooibos (RB) group of rats had significantly fewer (p<0.0001) GFAP-

immunoreactive astrocytes when compared to the BPA only -treated group. Also,

there was no significant difference (p>0.05) between the RT group of rats and the

rats in the NS control group.


65

T re a tm e n t

No

. o

f G

FA

P p

os

itiv

e c

ell

s

NS

BP

AR

BR

T

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0



Figure 4.13: Number of GFAP positive cells in experimental rats on PND42


66

CHAPTER 5

DISCUSSION

5.1. GENERAL INTRODUCTION

Endocrine-disrupting chemicals (EDCs) are a wide range of compounds that

disrupt normal growth and development by halting or modifying the action of

hormones upon ingestion or absorption into the body (Schug et al., 2015).

Examples of EDCs include a broad group of natural and synthetic chemical

substances, such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated

biphenyls (PCBs), pesticides, herbicides, fungicides, BPA and

dichlorodiphenyltrichloroethane (DDT). EDCs are abundant in our environs, diet,

and consumer products, which makes exposure to these toxins very frequent and

regular (Waring and Harris, 2005). Exposure to EDCs during initial

developmental stages is known to interrupt normal developmental processes,

thereby altering the body’s endocrine system to initiate deleterious effects in

various systems in the human body (Schug et al., 2015). For instance, EDCs

initiates neurotoxic effects that disturb the synthesis, transport, and release of such

neurotransmitters as glutamate, dopamine, serotonin, norepinephrine, which play

important roles in regulating behaviour, cognition, learning, and memory

(Dickerson and Gore, 2007, Rasier et al., 2007).

This study was designed to investigate if continuous consumption of fermented

rooibos herbal tea (FRHT) could offer diverse protection against potential damage


67

to the developing CNS of offspring Wistar rats exposed to BPA, following ad

libitum maternal consumption during and after pregnancy as observed on PND 42.

The offspring were considered to have ‘formed’ and ‘developed’ in the presence

of ad libitum rooibos tea consumption including postnatal consumption through

breastfeeding. After the evaluation of neurobehavioural activity, rats were

sacrificed, and cerebral as well as cerebellar samples were examined for changes

in antioxidant enzyme activities and histomorphology. The results obtained from

this study are discussed in the following sections.

5.2. ROOIBOS TEA PREVENTED BPA-INDUCED GAIN IN BODY MASS

In this study, findings revealed an increase in the body mass of experimental rats

treated with BPA only. This is in line with previous reports demonstrating that

perinatal BPA exposure increases body mass when compared to controls (Rubin

et al., 2001, Miyawaki et al., 2007, Ezz et al., 2015). There are confirmed reports

which link BPA to weight gain and obesity (Newbold et al., 2009, Rubin and

Soto, 2009, Shankar et al., 2012). For instance, Li and colleagues reported that

high BPA exposure might promote childhood obesity and might advance global

obesity epidemic in the human population (Li et al., 2013a). In the same regard,

Harley and co-workers revealed that elevated urinary BPA concentrations in

children aged nine were linked to higher odds of obesity, increased waist

circumference, BMI z-score and percentage body fat (Harley et al., 2013). An

appraisal of available literature reveals other reports of body mass gain in

offspring of rodent mothers exposed to BPA during gestation and/or lactation, or

during the early postnatal period (Howdeshell et al., 1999). Also, postnatal

exposure of BPA in male rat pups to investigate the effects of BPA on anxiety


68

revealed that exposed males substantially weighed more than the controls

(Patisaul and Bateman, 2008). Consequently, the increase in body mass gain in

this study highlights the ability of BPA to promote the progression of obesity

which could in turn further aggravate several metabolic, cardiovascular and

neurological disorders associated with BPA exposure.

Several mechanisms have been proposed to explain the association between BPA

exposure and body mass gain including the ability of BPA to enhance

differentiation of adipocytes and promote lipid accumulation in target cells

(Masuno et al., 2002, Wada et al., 2007), as well as increased gene expression of

adipogenic transcription factors (Phrakonkham et al., 2008). Others include the

ability of BPA to inhibit the release of adiponectin from human adipose tissue

explants (Hugo et al., 2008), dysregulate neuronal appetite controlling circuits

through its action on the hypothalamus (Wade and Schneider, 1992, Simerly,

2008), and inhibit the signalling of PPARgamma, which is essential for lipid

homeostasis and differentiation of brown and white adipocytes (Grun and

Blumberg, 2006, Grün and Blumberg, 2007). Findings from this study confirm

that although perinatal BPA exposure is a major challenge with respect to weight

gain and possibly childhood obesity, these effects could be attenuated by rooibos

through its protective activity against BPA.

5.3. ROOIBOS TEA MITIGATED BPA-INDUCED NEUROBEHAVIOURAL

DEFICITS

In this study, the open field test (OFT) was employed to investigate certain

neurobehavioral tests such as the frequency of rearing episodes, total freezing


69

time, total self-grooming time and the novel object recognition (NOR) test. The

OFT, initially developed in 1934, is a routine test that evaluates emotionality,

exploratory behaviour and general activity in rodents (Hall, 1934). It is one of the

most commonly used measures of behaviour in animal psychology because it

offers a prompt and easy evaluation of precise behaviours that require little or no

training to the test subject and the person administering the test (Walsh and

Cummins, 1976, Prut and Belzung, 2003). Also, its popularity stems from the

psychological and physiological conceptions underlying the tests, which are

largely uncomplicated and easy to understand (Seibenhener and Wooten, 2015).

Although the primary outcome of interest is “movement”, it is sometimes

influenced by other factors such as motor output, exploratory drive, freezing, fear-

related behaviour and sickness (Gould et al., 2009). The OFT is also employed to

assess the toxic effects of compounds in rodents and thus evaluates different

facets of behaviour other than movement/locomotion (Gould et al., 2009).

The frequency of rearing episodes is regarded as a measure of exploratory activity

in the OFT and indicates the number of times rats stood on both hind paws in a

vertically upright position (Erdoğan et al., 2004, Ennaceur, 2014). Reports show

that a reduction in rearing episodes is indicative of increased anxiety (Costall et

al., 1989) and is further demonstrated under experimental conditions, such as

stress (Masood et al., 2003, Fan et al., 2011), drug treatment (van Lier et al.,

2004) and brain stimulation (Racine, 1972, Hannesson et al., 2001). In this study,

the reduction in the frequency of rearing episodes in the group of rats exposed to

BPA only could be indicative of increased anxiety and stress as demonstrated in

previous studies. However, following exposure to rooibos, it was observed that


70

experimental rats exhibited an increase in rearing episodes, thus indicating that

rooibos was protective against BPA-induced reduction in the frequency of rearing,

possibly indicative of induced anxiety or stress.

Freezing activity, an important indicator of anxiety in rodents (Díaz-Morán et al.,

2014), is defined as the duration of the OFT within which the rodent was

stationary. It is conspicuous in conditions where stress levels are noticeably high

and occurs in response to apparent danger, environmental displacement, sudden

changes and fright stimuli (Walsh and Cummins, 1976, Brandao et al., 2008). In

line with earlier studies showing increased freezing activity in stress conditions

(Ranjbar et al., 2017) and in rats with impaired brain function (Saikhedkar et al.,

2014), total freezing time was significantly elevated in this study in the group of

rats exposed to BPA only. Conversely, we observed a significant decline in total

freezing time in the BPA-and-rooibos treated rats, thus confirming the protective

activity of rooibos against BPA in experimental rats.

Total self-grooming time is defined as the duration of time the experimental rats

expended in licking or scratching itself while motionless. It is a very important

behavioural activity in rodents and is involved in biologically essential processes,

such as thermoregulation, social communication and de-arousal (Spruijt et al.,

1988, Spruijt et al., 1992, Leonard et al., 2005). Previous reports indicate that self-

grooming is enhanced by exposure to stress (Jolles et al., 1979, Katz and Roth,

1979) and in rodent models of anxiety such as stress-induction in rats or mice

after exposure to anxiogenic drugs (Kalueff and Tuohimaa, 2005, Kalueff et al.,

2007).


71

In this study, the increase in self-grooming observed in the BPA-only group could

be indicative of increased anxiety and stress. However, following exposure to

rooibos in the BPA-and-rooibos group, it was observed that the rats exhibited a

significant reduction in self-grooming, thus demonstrating that rooibos tea

consumption could have offered protection against BPA-induced anxiety-like

behaviour demonstrated as increase in self-grooming in this study.

The integrity of the medial temporal lobe is essential for memory processing and

this can be evaluated through the recognition index (RI), which is a measure of

the capability of a person or an animal to determine if a previously encountered

item is familiar or not (Squire et al., 2007). Tests that evaluate RI are becoming

gradually valuable tools for research designed to explore the neural basis of

memory (Winters et al., 2004). One such tests is the novel object recognition task

(NOR), studied for the first time by Ennaceur and Delacour to evaluate short-term

memory in rodents (Ennaceur and Delacour, 1988) which highlights the capacity

of the rodent to recall information for a short amount of time (Mathiasen and

DiCamillo, 2010). The NOR can be designed to evaluate working memory,

attention, anxiety, and preference for novelty in rodents (Silvers et al., 2007,

Goulart et al., 2010). It has also been utilized to investigate the effects of several

pharmacological treatments and brain impairment in rodents (Goulart et al.,

2010).

Findings from the present study showed that RI was significantly lowered in

BPA-only treated rats when compared to the NS controls. This is in line with

previous reports which show reduced RI in stress conditions (Nagata et al., 2009,


72

Eagle et al., 2013). Conversely, when compared to rats exposed to BPA only, a

significant increase in RI was observed in the RB group thus suggesting a

neuroprotective role by rooibos tea against possible BPA-induced short-term

memory loss.

5.4. ROOIBOS TEA REGULATED THE ACTIVITY OF ANTIOXIDANT

ENZYMES

Reports show that exposure to BPA during pregnancy can result in a variety of

foetal malformations and growth retardations as well as early embryonic losses

(Bajaj et al., 1993, Friedler, 1996, Khattak et al., 1999), possibly via the

production of excessive ROS known to be toxic to cells (Nicol et al., 2000, Wells

et al., 2005). BPA-induced ROS production has been investigated in many cell

types and at different concentrations (Huc et al., 2012, Babu et al., 2013, Gassman

et al., 2015). For instance, nanomolar BPA concentration ranges were found to

result in short and transient elevations of ROS (Koong and Watson, 2015, Pfeifer

et al., 2015); nevertheless prolonged and increased levels of oxidative stress have

been reported (Huc et al., 2012, Porreca et al., 2016). BPA exposure in

micromolar concentration ranges have been reported to induce higher levels of

oxidative stress (Huc et al., 2012, Babu et al., 2013, Gassman et al., 2015, Leem et

al., 2017), and cytotoxicity via ROS-triggered DNA damage (Ooe et al., 2005,

Huc et al., 2012, Leem et al., 2017). Different in vivo studies have shown an

association between high urinary BPA levels and elevated concentrations of such

oxidative stress biomarkers as lipid peroxidation and malondialdehyde (Watkins

et al., 2015, Zhang et al., 2016), thus supporting the pro-oxidant activity of BPA.


73

Cellular antioxidants mitigate the deleterious effects arising from interactions

between lipid, protein, DNA molecules and ROS (Hassan et al., 2012).

Irrespective of the existence of a potent antioxidant system, an imbalance in the

generation of ROS due to natural or synthetic chemicals may result in some

disorders. The concentration of free radical scavengers and the levels of such

antioxidant enzymes as SOD and CAT regulate the antioxidant capacity of cells;

thus numerous endpoints have been investigated following BPA exposure to

evaluate its effect on antioxidant capacity (Gassman, 2017).

The enzyme SOD is known to defend tissues from oxidative damage by

catalyzing the conversion of O2− to H2O2 (Fridovich, 1997), while catalase is

responsible for the detoxification of the H2O2 generated by SOD through the

conversion of two molecules of H2O2 to form two molecules of H2O and one

molecule of O2 (Nagata et al., 1999, Agati et al., 2012), thus providing protection

against ROS. Findings from the current study showed that whereas BPA induced

marked oxidative damage by inhibiting the activities of the antioxidant enzymes

SOD and CAT, the intake of rooibos tea could attenuate these effects of BPA.

These findings are in agreement with other studies that report a significant

decrease in SOD and CAT levels following BPA treatment (Eid et al., 2015,

Moghaddam et al., 2015, Ozaydın et al., 2018).

5.5. ROOIBOS TEA ATTENUATED NEURODEGENERATION IN RATS

EXPOSED TO BPA

The development of the brain involves a complex interplay between dendritic

growth and the production of neuronal connections to form a neural network

(McAllister et al., 1997, Wong and Ghosh, 2002). Different brain regions are


74

tasked with different functions, and histological damage to these brain regions

hinders their function (Mcallister et al., 2004, Kimura et al., 2016). The

hippocampus is subdivided into two main parts which have been studied

comprehensively for spatial navigation, learning and memory functions; these are

the cornu ammonis (subfields: CA1, CA2, CA3, CA4) and the dentate gyrus

(Bannerman et al., 2014, Moser et al., 2014). Alterations in hippocampal

morphology are linked to changes in cognitive function (Van Praag et al., 2000)

and the hippocampal CA1 (H-CA1) neurons in rodents are reported to be essential

for the processing of hippocampus-dependent memory (Bartsch et al., 2011).

These neurons are also the most sensitive and most susceptible neurons to

selective degeneration in response to such EDCs as BPA, ischemia and other toxic

substances (Kirino, 1982, Pulsinelli et al., 1982, Elsworth et al., 2013, Kimura et

al., 2016).

Previous studies have shown that there was a reduction in the dendritic spines of

neurons in the H-CA1 region following exposure of mice to BPA (Eilam-Stock et

al., 2012, Kimura et al., 2016, Liu et al., 2016). The results obtained from the

present study are in agreement with the above findings as there was a significant

reduction in the average length of the H-CA1 region in rats exposed to BPA-only

when compared to the NS control group. However, a significant increase in the

average H-CA1 length was observed in rats treated with BPA-and-rooibos when

compared to BPA-only rats. These morphological findings indicate a protective

effect of rooibos in the CA1 region of the hippocampus in rats, thus attenuating

BPA-induced impairment of cognition in the rats.


75

In certain human inherited disease conditions, such as ataxia telangiectasia,

paraneoplastic cerebellar degeneration and lysosomal storage diseases, a

significant loss of Purkinje cells has been reported (Gilman et al., 1981, Anderson

et al., 1987) as a noticeable component of the histopathological alterations

detected in cerebellar injury. Some of the clinical indicators of cerebellar

dysfunction frequently manifested in these conditions include ataxia and tremor

(Riedel et al., 1990). Purkinje cell loss is a significant observation in many

diseases but the exact cause is unknown.

In some in vivo studies, the significant reduction in Purkinje cell population is due

to the neurotoxic effect of EDCs on Purkinje cells (Kimura-Kuroda et al., 2007,

Ibhazehiebo et al., 2011) and the reduction in Purkinje cells observed in this study

is similar to findings from these previous studies, possibly indicating that the

deleterious effects of BPA on the cerebellum (Ahmed and Eid, 2015). The number

of Purkinje cells however significantly increased in rats treated with BPA-and-

rooibos tea when compared to the BPA-only rats, thus suggesting a

neuroprotective role for rooibos tea.

Apart from neuronal damage caused by most EDCs, it is plausible to also

investigate possible damage to the support cells of the CNS. Astrocytes (also

called astroglia) are the most abundant support cells in the CNS (Zuchero and

Barres, 2015) and contain GFAP which is an important protein of intermediate

glial filaments in astrocytes commonly used to investigate astrocytic damage

response (Cikriklar et al., 2016). In this study, there was an increase in the

expression of GFAP-positive astrocytes in BPA-exposed rats when compared to


76

controls. Increased GFAP expression is prominent in conditions of CNS injury

and astrocyte damage (Hausmann et al., 2000, Yu et al., 2004, Cai et al., 2006)

previous studies have reported on the various mechanisms of astrocyte activation

possibly associated with BPA exposure. For instance, increased generation of

hydroxyl radicals has been reported in the adult mouse brain following BPA

exposure (Kabuto et al., 2003) and elevated concentrations of lipid peroxidation in

postnatal mouse brains was reported following BPA exposure during pregnancy

and lactation (Kabuto et al., 2004). Consequently, BPA-triggered oxidative stress

and peroxidation could be said to be responsible for the astroglial activation

reported in this study. However, rats exposed to rooibos tea had significantly

reduced GFAP levels when compared to BPA-only treated rats. Taken together,

findings from this study seem to indicate that rooibos tea is protective against the

deleterious effects of BPA in the developing brain.


77

5.6. CONCLUSION

This study provides the first research evidence of the neuroprotective potential of

fermented rooibos herbal tea against BPA exposure on brain development in

Wistar rats. The protective activity observed is possibly due to the natural and

bioactive constituents of this herb, reported to be responsible for the regulation of

cellular antioxidants which inhibit BPA-induced oxidative damage. These

findings suggest that rooibos tea offers protection against BPA toxicity, and may

provide the basis for clinical trials and policy decisions aimed at regulating the

use of products containing such EDCs as BPA.

Limitations of this study include investigation of few immunohistochemical

markers in the brains of experimental rats, few neurobehavioural tests to confirm

BPA-induced impairment of cognitive function, and the evaluation of only few

oxidative stress markers to fully elucidate the impact of oxidative stress on BPA

exposure. These limitations are partly due to financial constraints and the need to

pitch the volume of research data at the MSc level.

Future recommendations

Isolation of the bioactive compounds in rooibos and investigation of their

neuroprotective activity against BPA.

Investigation of additional immunohistochemical, apoptotic, inflammatory and

oxidative stress biomarkers to corroborate findings from this study.


78

Assessment of additional OFT activities and neurobehavioural tests (such as

T-maze test and water maze test) to offer more information on BPA-induced

impairment of locomotion, learning and memory.


79

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