ASSESSMENT OF EFFECTS OF ETHANOLIC EXTRACT OF COCO NUCIFERA

(COCONUT) HUSK ON MARKERS OF LIPID PEROXIDATION IN

STREPTOZOTOCIN INDUCED TYPE 2 DIABETES IN FEMALE WISTAR RATS.

HAMZAT TEMITOPE REBECCA

183169

A PROJECT

SUBMITTED TO
DEPARTMENT OF BIOCHEMISTRY,
FACULTY OF BASIC MEDICAL SCIENCES,
LADOKE AKINTOLA UNIVERSITY OF TECHNOLOGY
(LAUTECH), OGBOMOSHO, OYO STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

BACHELOR OF TECHNOLOGY (B.TECH) DEGREE IN BIOCHEMISTRY.

FEBRUARY, 2024.

i
 CERTIFICATION
This is to certify that this project was carried out by HAMZAT TEMITOPE REBECCA under
my supervision at the department of Biochemistry, Faculty of Basic Medical Sciences, Ladoke
Akintola University of Technology, Ogbomosho, Nigeria.

PROF. ADEKUNLE A.S

PROJECT SUPERVISOR DATE

PROF. AFOLABI O.K

HEAD OF DEPARTMENT DATE

EXTERNAL SUPERVISOR DATE

ii
 DEDICATION
This work is dedicated to The Almighty God. He is The Author and Finisher of my faith. To Him
all Majesty and Glory belong.

iii
 ACKNOWLEDGEMENT
My in-depth gratitude goes to God, my maker and giver of all things who showed me His
mercies and saw me through the course of my programme.
I also appreciate my supervisor, Prof. Adekunle A.S for his patience and continued efforts
towards the success of this research work. Your guidance, counsels and corrections made this
work a great deal of success. May God reward you bountifully sir.
To my mother, Mrs Kolawole Adenike Temilade who has been supportive and caring. You will
live long to eat the fruits of your labor in Jesus name. I specially appreciate my sisters, this
success would not have been recorded without you. Thank you for your financial, moral and
spiritual supports, you have always been there, I love you, God bless you
My project colleagues, Setemi, Fathia, Felix, Tina, Moyo, Mary, Taiwo, Hezekiah, Ebenezer,
Pelumi, Ayomidipupo, Fola, Deborah, Dammy. It’s been interesting to work with you all. I hope
to see you at the top.

iv
TABLE OF CONTENTS
CONTENT PAGES
Title page
Certification ii
Dedication iii
Acknowledgement iv
Table of contents v
List of tables
List of figures
Abstract
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
1.2 LITERATURE REVIEW
1.2.1 DIABETES MELLITUS
1.2.2DEFINITION, COMPLICATIONS AND DIAGNOSIS OF TYPE 2 DIABETES
MELLITUS
1.2.2.1 STREPTOZOTOCIN
1.3 INTRODUCTION TO THE RELATIONSHIP BETWEEN TYPE 2 DIABETES AND
COCOS NUCIFERA (COCONUT) HUSK
1.3.1 RELATIONSHIP BETWEEN PHYTOCHEMICALS AND DIABETES
1.3. 2 RELATIONSHIP BETWEEN ETHANOLIC EXTRACT OF COCOS NUCIFERA
HUSK ON LIPID PEROXIDATION MARKERS IN TYPE 2 DIABETES
1.4.0 LIPID PEROXIDATION AND TYPE 2 DIABETES
1.4.1 EECNH AND ANTIOXIDANT ACTIVITY
1.4.2 EECNH AND LIPID PEROXIDATION MARKERS IN STZ- INDUCED T2D
CHAPTER 2
2.0 MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 APPARATUS/ EQUIPMENTS
2.1.2 CHEMICALS

v
2.1.3 EXPERIMENTAL ANIMALS
2.1.4 COMPOSITION OF FEED
2.2.0 METHODOLOGY
2.2.1 GROUPING AND IDENTIFICATION
2.2.2 INDUCTION OF DIABETES THROUGH STREPTOZOTOCIN
2.2.3 PREPARATION OF STREPTOZOTOCIN
2.2.4 PREPARATION OF COCONUT HUSK
2.2.5 ADMINISTERATION OF COCONUT HUSK EXTRACT
2.2.6 SACRIFICING THE EXPERIMENTAL ANIMALS
2.2.7 PREPARATION OF TISSUE HOMOGENATE
2. 3 BIOCHEMICAL ANALYSES
2.3.1 DETERMINATION OF MALONDIALDEHYDE CONCENTRATION
2.3.2 DETERMINATION OF REDUCED GUTATHIONE (GSH) CONCENTRATION
2.3.3ASSAY MIXTURE FOR CALIBRATION OF REDUCED GLUTATHIONE STANDARD
CURVE
CHAPTER THREE
3.0 RESULTS
3.1 HEPATIC TISSUE STATUS OF EXPERIMENTAL RATS
CHAPTER 4
4.0 DISCUSSION AND CONCLUSION
4.1 DISCUSSION
4.2 CONCLUSION
REFERENCES

vi
 LIST OF TABLES
TABLE 1: TABLE 1 SHOWS USES OF THE PARTS OF COCONUT
TABLE 2: INITIAL WEIGHT OF RATS
TABLE 3: COMPOSITION OF FEED
TABLE 4: GROUPING AND IDENTIFICATION
TABLE 3.1: TABLE SHOWING THE MDA AND GSH CONCENTRATIONS

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 LIST OF FIGURES
FIGURE 1: DIAGRAM OF A, COCOS NUCÍFERA L
FIGURE 2: STRUCTURES OF THE MAIN PHYTOCONSTITUENTS ISOLATED
FROM COCOS NUCIFERA (L.)
FIGURE 3: SCHEMATIC DIAGRAM OF COCONUT FRUIT
FIGURE 4: SCHEMATIC DIAGRAM OF COCNUT HUSK (COIR)
FIGURE 5: STRUCTURE OF LIGNIN
FIGURE 6: STRUCTURE OF FLAVOINDS
FIGURE 7: STRUCTURE OF FLAVOINDS
FIGURE 8: STRUCTURE OF CAROTENOIDS
FIGURE 9: STRUCTURE OF ALKALOIDS

viii
 ABSTRACT
This study was designed to investigate the assessment of effects of etanolic extract of cocos
nucifera (coconut) husk on markers of lipid peroxidation in streptozotocin induced type 2
diabetes. Female Wister rats were used as experimental animals. Each rat in the treated group
was administered a dose of 125mg/kg body weight of cocos nucifera husk extract.
Administration was done orally using oral cannula once per day for twenty one days. It was
observed that there’s significant increase in MDA concentration in the diabetic status of rat in
brain compared to control while there’s a decrease in MDA level in the other organ compared to
control.in the treated group, there’s a significant increase in the level of MDA concentration in
brain and kidney but a decrease in heart and liver when compared to control. There’s an increase
in the level of MDA concentration in the liver, kidney and brain but a decrease in heart when
compared to diabetic group.
The diabetic status showed that there’s an increase in the level of GSH concentration in kidney in
the treated group when compared to diabetic status and control, while the other organs showed a
significant decrease when compared to control. In the treated group, there’s a marginal increase
in the level of GSH concentration of all organs when compared to diabetic group. The
observation showed that the administration of cocos nucifera husk may alter the MDA and GSH
concentrations in streptozotocin induced type 2 diabetic Wister rats.

ix
 CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Coconut husks are that parts of coconut that is generally regarded as waste materials of the fruits.
Numerous studies have been conducted and documented that coconut husks are high source of
fiber and other minerals. For thousands of years, medicinal plants have been used as traditional
treatments for numerous human diseases (Jose, et al) in many parts of the world. The natural
products derived from medicinal plants have proven to be an abundant source of biologically
active compounds, making them effective source for alternative medicines. Numerous traditional
medicinal plants have been evaluated for their potential application in the prevention or
treatment of oral diseases particularly those of microbial origin. A number of studies have
investigated the activity of plant extracts and products against specific oral pathogens. The
coconut (Cocos nucifera Linn.) belongs to the Arecaceae family, is native to the coastal areas of
Southeast Asia and is a major crop of the Dakshina Kannada region. Cocos nucifera (L.)
(Arecaceae) is commonly called the “coconut tree” and is the most naturally widespread fruit
plant on Earth. Throughout history, humans have used medicinal plants therapeutically, and
minerals, plants, and animals have traditionally been the main sources of drugs. The constituents
of C. nucifera have some biological effects, such as antihelminthic, anti-inflammatory,
antinociceptive, antioxidant, antifungal, antimicrobial, and antitumor activities. In addition, other
properties such as antihypertensive, anti-inflammatory, antimicrobial, antioxidant,
cardioprotective, antiseizure, cytotoxicity, hepatoprotective, vasodilation, nephroprotective, and
anti-osteoporosis effects were also reported. Because each part of C. nucifera has different
constituents, the pharmacological effects of the plant vary according to the part of the plant
evaluated. Cocos nucifera (L.) is an important member of the family Arecaceae (palm family)
popularly known as coconut, coco, coco-da-bahia, or coconut-of-the-beach (Aragão WM., et al
2002). The plant is originally from Southeast Asia (Malaysia, Indonesia, and the Philippines) and
the islands between the Indian and Pacific Oceans. From that region, the fruit of the coconut
palm is believed to have been brought to India and then to East Africa. After the discovery of the
Cape of Good Hope, this plant was introduced into West Africa and, from there, dispersed to the
American continent and to other tropical regions of the globe (Purseglove JW., et al 2015).

1
The plant is an arborescent monocotyledonous tree of around 25 m in height (giant coconut) with
a dense canopy

Fig 1: A, Cocos nucífera L. (personal collection, agronomist Severo Cortez Lima).

The root of the coconut system is fasciculated. The stem is an unbranched type, and at its apex, a
tuft of leaves protects a single apical bud. The pinnate leaves are feather-shaped, having a
petiole, rachis and leaflets. Under favorable environmental conditions, the giant adult coconut
emits 12–14 inflorescence spikes per year, while the adult dwarf coconut can emit 18 spikes in
the same period. The axillary inflorescence has globular clusters of female flowers. The plant is
monoecious (male and female reproductive organs on the same plant) (Passos EEM., et al 2000).
The coconut fruit comprises an outer epicarp, a mesocarp, and an inner endocarp. The epicarp,
which is the outer skin of the fruit, and the mesocarp, which is heavy, fibrous, and tanned when
dry, have many industrial uses. The endocarp is the hard dark core. Inside is a solid white
albumen of varied thickness, depending on the age of the fruit, and with an oily pulp consistency

and a liquid albumen called coconut water that is thick, sweet, and slightly acidic .
The present review highlights the traditional uses of C. nucifera, phytochemical compounds
isolated from different parts of the plant, and the biological activity and toxicological studies to
date.
Traditional uses

2
All parts of the fruit of the coconut tree can be used. Both the green coconut water and solid
albumen ripe fruits are used industrially and in home cooking in many ways (Rosa M de F.m ., et
al 2001). Additionally, several parts of the fruit and plant have been used by people in different
countries for the treatment of various pathological conditions (Table 1)

Table 1 shows uses of the parts of coconut

Currently, appreciation of natural coconut water is growing. Industry is using the husk fiber from
the pith as raw material for carpets, car seat stuffing, and in agricultural as fertilizers. The hard
core is used to make handcrafts. The stalk and leaves of the coconut tree are useful in
construction, and sugar, vinegar, and alcohol can be extracted from the inflorescence (Fontenele
RES ., et al 2005).
In Brazil, extract from the husk fiber of C. nucifera is used to treat diarrhea (ostes JBF ., et al
2002). In Papua New Guinea, the leaves and roots of young plants are chewed as treatment for
diarrhea and stomachaches . In Fiji, coconut oil is used to prevent hair loss and coconut water is
used to treat renal disease . In Ghana, people use coconut milk to treat diarrhea . In Guatemala,
the husk fiber extract is used as an antipyretic, to reduce renal inflammation, and as a topic
ointment for dermatitis, abscesses, and injuries. In Haiti, a decoction of the dry pericarp is used
for oral treatment of amenorrhea, and the oil is applied as an ointment to burns ; an aqueous
3
extract from the husk fiber is also used for oral asthma treatment. In India, infusions made with
the coconut inflorescence are used for the oral treatment of menstrual cycle disorders (Nagata
JM., et al 2011). In Indonesia, the oil is used as a wound ointment, the coconut milk is used as
an oral contraceptive, and fever and diarrhea are treated with the root extract. In Jamaica, the
husk fiber extract is used to treat diabetes (Calzada F ., et al 2007). In Mozambique, the fruit is
consumed by men as an aphrodisiac. Peruvians use the aqueous extract of the fresh coconut fiber
orally for asthma, as a diuretic, and for gonorrhea. In Trinidad, bark extract is used orally for
amenorrhea and dysmenorrhea, and bark tea is used to treat venereal diseases. In Mexico,
coconut is used to treat various disorders associated with urogenital tract infection
by Trichomonas vaginalis . A decoction of the white flesh of the fruit is used in rural Malaysia to
treat fever and malaria. In Kenya, the fruit is used to relieve skin rash caused by HIV infection
(Nagata JM., et al 2011).
Phytochemistry
Phytochemical studies of the coconut fiber (mesocarp) ethanolic extract revealed that the
presence of phenols, tannins, leucoanthocyanidins, flavonoids, triterpenes, steroids, and
alkaloids, while a butanol extract recovered triterpenes, saponins, and condensed tannins (Costa
CT ., et al 2007). Notably, compounds like flavonoids having antioxidant action are widely
distributed in edible vegetables, fruits, and many herbs (Chao J., et al 2009). Condensed tannins
are reported to possess antihelminthic activity by binding to proteins present in the cuticle, oral
cavity, esophagus, and cloaca of nematodes, thus intensifying the physical and chemical damage
in helminth (Hoste H ., et al 2006).
The lyophilized extract and fractions, as well as ethyl acetate extracts, from the C. nucifera fiber
are rich in polyphenols, compounds such as catechins, epicatechins, tannins, and flavonoids.
The constituents of the liquid albumen were identified as vitamin B, nicotinic acid (B3, 0.64
µg/mL), pantothenic acid (B5, 0.52 µg/mL), biotin (0.02 µg/mL), riboflavin (B2, <0.01 ng/mL),
folic acid (0.003 µg/mL), with trace quantities of vitamins B1, B6, and C, pyridoxine, thiamine,
folic acid, amino acids, L-arginine, plant hormones (auxin, 1,3-diphenylurea, cytokinin),
enzymes (acid phosphatase, catalase, dehydrogenase, diastase, peroxidase, RNA polymerases),
and growth-promoting factors ( Solangih A., et al 2011). Furthermore, oil extracted from the
solid albumen is primarily lauric acid and alpha tocopherol (Arlee R., et al 2013). Root phenolic
compounds were identified as flavonoids and saponins (Pal D., et al 2011). Other compounds

4
identified in leaf epicuticular wax were lupeol methylether, skimmiwallin, [3b-methoxy-25-
ethyl-9,19-cyclolanost-24(241)-ene], and isoskimmiwallin [3b-methoxy-24-ethyl-9,19-
cyclolanost-25(251)-ene] (Figure 2)

Fig 2: Structures of the main phytoconstituents isolated from Cocos nucifera (L.)
1.2 LITERATURE REVIEW
1.2.1 DIABETES MELLITUS
Diabetes Mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia. It
may be due to impaired insulin secretion, resistance to peripheral actions of insulin, or both. It is
one of the oldest diseases known to man (Diabetes mellitus history, 2011). It was first reported in
Egyptian manuscript about 3000 years ago. In 1936, the distinction between type 1 and type 2
was made clearly. Type 2 was first described as a component of metabolic syndrome in 1988
(Patlak M. ,et al,. 2002). Type 2 diabetes mellitus is the most common form of diabetes mellitus
characterized by hyperglycemia, insulin resistance and relative insulin deficiency. Diabetes
mellitus is a metabolic disorder resulting from a defect in insulin secretion, insulin action or
both. The condition itself introduces a need for patient’s lifestyle adjustment to the disease and a
number of everyday therapeutic and diagnostic restrictions. The main indication of diabetes
mellitus is a hyperglycemia in blood which is due to inappropriate pancreatic insulin secretion or
low insulin-directed fostering of glucose by target cells. It is silent killer disease and affects

5
millions of people in the world. It is estimated that in 2010 there was globally 285 million people
suffering from this disease. This number is estimated to increase to 430 million in the absence of
better control or cure. Different types of diabetes mellitus, type 1, type 2, gestational diabetes
and other types of diabetes mellitus are compared in terms of diagnostic criteria, etiology and
genetics. As the disease progresses tissue or vascular damage ensures leading to severe diabetic
complications such as retinopathy, neuropathy, nephropathy, cardiovascular complications and
ulceration. Currently available pharmacotherapy for the treatment of diabetes mellitus includes
insulin and hypoglycemic agents. These drugs act by increasing the secretion of insulin form
pancreas or reducing plasma glucose concentrations by increasing glucose uptake and decreasing
gluconeogenesis. They are specially depression, anxiety disorders, eating disorders and cognitive
disorders including dementia. Various herbal drugs have been also proved effective due to their
beneficial contents in treatment of diabetes Chronic hyperglycemia in synergy with the other
metabolic aberrations in patients with diabetes mellitus can cause damage to various organ
systems, leading to the development of disabling and life-threatening health complications, most
prominent of which are micro-vascular {retinopathy, nephropathy and neuropathy}and macro-
vascular complications leading to an increased risk of cardiovascular diseases. People living with
type 2 diabetes mellitus are more vulnerable to forms of both short and long term complications,
which can lead to their premature death. This tendency of mortality is seen in patients with type
2 diabetes mellitus because of the commonness of this type of diabetes mellitus, its onset and late
recognition, especially in poor resource-poor developing countries like Africa (Saeedi,et al,.
2019). Diabetes mellitus (DM) is a metabolic disorder where in human body does not produce or
properly uses insulin, a hormone that is required to convert sugar, starches and other food into
energy. Absence or reduced insulin in turn leads to persistent abnormally high blood sugar and
glucose in tolerance. It is probably an oldest disease known to man. It is also referred as black-
death from the 14th century (Deepti B., et al 2017). In people with diabetes, blood sugar levels
remain high. This may be due to insulin is not being produced at all, is not made at sufficient
levels, or is not as effective as it should be. The most common forms of diabetes are type 1
diabetes (5%), which is an autoimmune disorder, and type 2 diabetes (95%), which is associated
with obesity. Gestational diabetes is a form of diabetes that occurs in pregnancy, and other forms
of diabetes are very rare and are caused by single gene mutation.
Types of diabetes

6
1. Type 1 diabetes
2. Type 2 diabetes
3. Gestational diabetes
Other types of diabetes
1. Diabetes LADA
2. Diabetes MODY
3. Double diabetes
4. Brittle diabetes
5. Diabetes insipidus
6. Neonatal diabetes mellitus
7. Mixed pathologies in T1DM with obesity and insulin resistance
Type 1 Diabetes – It is a chronic autoimmune disease associated with selective destruction of
insulin producing pancreatic β-cells (Ozougwu J.C., et al 2013). When there is transplantation of
pancreas from twin donors to chronic diabetic twin recipients in the absence of immune
suppression is complicated due to elevated heterogenecity of pancreatic lesions of β-cells which
are rapidly annihilated, and then there is development of massive insulitis by using infiltrating T
lymphocytes which measures an amnestic autoimmune reaction (Deepti B., et al 2017). Type 1
diabetes mellitus accounts for 5% to 10% of diabetes mellitus and is characterized by
autoimmune destruction of insulin producing beta cells in the islet of the pancreas (Sosale,et
al,.2014). As a result, there is an absolute deficiency of insulin. Combinations of genetic
susceptivity and environmental factors such as viral infection, toxins, or some dietary factors
have been implicated as triggers for autoimmunity. T1DM is most commonly seen in children
and adolescents though it can develop at any age.
Type 1 diabetes is often referred to as insulin- dependent (IDDM) or juvenile-onset diabetes.
Symptoms – Frequent urination, thirst, weight loss, extreme fatigue, acetone breath, nausea and
vomiting, blurred vision and itchiness in the genital area.

Type 2 Diabetes – Type 2 diabetes mellitus is also known as adult-onset diabetes. The
progressive insulin secretary defect on the background of insulin resistance. People with this type
of diabetes frequently are resistant to the action of insulin (Singh N ., et al 2016). Globally, it
affects 5-7% of the world’s population. The disease is usually controlled through dietary therapy,

7
exercise and hypoglycemic agents (Bastaki S., et al 2005). This is the most common form of
diabetes mellitus and is highly associated with a family history of diabetes, older age, obesity
and lack of exercise (Baynest H.W., et al 2015). Type 1 diabetes mellitus accounts for 5% to
10% of diabetes mellitus and is characterized by autoimmune destruction of insulin producing
beta cells in the islet of the pancreas (Sosale,et al,.2014). As a result, there is an absolute
deficiency of insulin. Combinations of genetic susceptivity and environmental factors such as
viral infection, toxins, or some dietary factors have been implicated as triggers for autoimmunity.
T1DM is most commonly seen in children and adolescents though it can develop at any age.
Type 2 diabetes mellitus {T2DM} accounts for around 90% of all cases of diabetes. In T2DM,
the response to insulin is diminished, and this is defined as insulin resistance. During this state,
insulin is ineffective and is initially countered by an increase in insulin production to maintain
glucose homeostasis, but over time, insulin production decreases, resulting in T2DM. T2DM is
most commonly seen in persons older than 45 years. Still, it is increasingly, seen in children,
adolescents, and younger adults due to rising levels of obesity physical, inactivity and energy
dense diets.
Gestational Diabetes- Pregnant women often develop diabetes. During pregnancy large
quantities of hormones are produced, these hormones may reduce insulin action in the mother’s
body,causing insulin resistance. Women that develop diabetes mellitus during pregnancy and
women with undiagnosed asymptomatic type 2 diabetes mellitus that is discovered during
pregnancy are classified with gestational diabetes mellitus (Baynest H.W., et al 2015).. Clinical
importance of GDM lies in the fact that it is associated with significant maternal and fetal
morbidity .
Hyperglycemia, which is first detected during pregnancy, is classified as gestational diabetes
mellitus {GDM}, also known as hyperglycemia in pregnancy. Although it can happen at any
time during pregnancy. GDM generally affects pregnant women during the second and third
trimesters. According to American Diabetes Association {ADA} GDM complicates 7% of all
pregnancies. GDM can be complicated by hypertension, preeclampsia, and hydramnios and may
also lead to increased operative interventions. The fetus can have increased weight and
size{macrosomia} or congenital anomalies. Gestational diabetes mellitus may reflect a
predisposition to type 2 diabetes under the metabolic conditions of pregnancy or it may represent
the extreme manifestation of metabolic alterations that normally occur in pregnancy (Butte,

8
2000). Women with gestational diabetes have decreased insulin sensitivity in comparison with
control groups. Gestational diabetes induces a state of dyslipidemia consistent with insulin
resistance. During pregnancy, women with gestational diabetes do have high serum
triacylglycerol concentrations but lower LDL-cholesterol concentrations than do healthy
pregnant women. During pregnancy, gestational diabetes is associated with a number of
complications for child. Because insulin does not cross the placenta, the fetus is exposed to the
maternal hyperglycemia. The fetal pancreas is capable of responding to this hyperglycemia
(Scollan- Kolippoulos et al., 2006). The fetus becomes hyperinsulinemic, which in turn promotes
growth and subsequent macrosomia (Perkins et al., 2007).
Despite numerous studies, the pathogenesis of GDM remains unclear, and the results obtained so
far indicate a complex mechanism of interaction of many genetic, metabolic and environmental
factors (Plows et al., 2018). The basic methods of treating GDM include an appropriate diet and
increased physical activity, and when these are inadequate, pharmacotherapy, usually insulin
therapy, is used. In developing countries, such as Brazil, oral hypoglycemic agents are also used,
mainly metformin and glibenclamide (glyburide) (Oliveira et al., 2022).

Other types of diabetes

Diabetes LADA- Latent Autoimmune Diabetes of the adults is autoimmune diabetes defined by
adult-onset, presence of diabetes associated autoantibodies, and no insulin treatment requirement
for a period after diagnosis (Laugesen E., et al 2015). It is becoming evident that a proportion of
adults may have a slowly evolving kind of Type 1 diabetes, which is characterized by the
presence of autoantibodies. Some people diagnosed with type 2 diabetes soon find themselves
dependent on insulin; these people may have a slowly progressive form of type 1 diabetes or
LADA.
Diabetes MODY- Maturity onset diabetes of the young is an autosomal dominantly inherited
type of diabetes that results from heterozygous mutations in various transcription factors acting
in the development and maturation of pancreatic β-cells. Characteristics features of MODY are
autosomal inheritance, early onset of diabetes, no signs related to the autoimmune process or
insulin resistance, and preservation of endogenous insulin secretion (Anik A ., et al 2015).

9
Double diabetes- Double diabetes is characterized by the occurrence of hyperglycemia in
children and young adolescents with the combination of markers typical of both type 1 and type
2 diabetes.
Brittle diabetes- Type 1 diabetes is an intrinsically unstable condition. A small group of patients
with type 1 diabetes, mainly young women, suffer chronically by poor metabolic control,
characterized by a severe instability of glycemia values with frequent and unpredictable
hypoglycemic or diabetic ketoacidosis episodes which cannot be attributed to patients or
clinicians’ errors. The quality of life of these patients is dramatically compromised in particular
because of the frequency of acute events, hospital recoveries and precocious appearance of
chronic complications. This clinical condition has been defined as brittle diabetes.
Diabetes Insipidus –Diabetes insipidus is a disease in which large volumes of dilute urine are
excreted due to vasopressin deficiency, AVP resistance or excessive water intake. Polyuria is
characterized by a urine volume in excess of 21/m2/24 h or approximately 150ml/kg/24h at birth,
100-110ml/kg/24h until the age of 2 years and 40-50 ml/kg/24h in the older child and adult
(Dilorgi N., et al 2012).
Neonatal diabetes mellitus –
• It occurs in first six months of life.
• Single gene defect
• Do not produce enough insulin
• Do not gain weight as quickly as expected
• High plasma glucose- mistake for T1DM
Symptoms and causes of Diabetes-
Symptoms of diabetes include-
• Increased thirst and urination Increased hunger
• Fatigue
• Blurred vision
• Numbness or tingling in the feet or hands
• Sores that do not heal
• Unexplained weight loss

10
• Presence of ketones in the urine (ketones are a byproduct of the breakdown of muscle and fat
that happens when there’s not enough available insulin). Frequent infections, such as gums or
skin infections and vaginal infections.
• Male sexual dysfunction.
Causes of diabetes include-

• Obesity
• Excess glucocorticoids
• Excess growth hormone
• Polycystic ovary diseases
• Mutation of insulin receptor
• Lipodystrophy
Causes of type 1 diabetes-
1. A genetic susceptibility to developing type 1 diabetes
2. Certain viruses (e.g. German measles or mumps)
3. Environmental factors
Causes of type 2 diabetes- Type 2 diabetes develops when the body becomes resistant to insulin
or when the pancreas stops producing enough insulin.
Diagnostic tests for diabetes mellitus
Three blood tests are available to diagnose prediabetes and diabetes
1. Casual plasma (blood) glucose/ random plasma test
2. Fasting plasma glucose (FPG)
3. Oral glucose tolerance test
Casual plasma (blood) glucose/ random plasma test- The simplest test and doesn’t require
fasting before taking the test (Laugesen E., et al 2015). The criteria for a diagnosis of diabetes
with this test is the presence of diabetes symptoms and a blood glucose level of 200mg/dl or
higher (Harikumar K., et al 2015)
Fasting plasma glucose (FPG) - A fasting plasma glucose level of 7.0mmol/L correlates most
closely with a 2-hour plasma glucose value of ≥11.1mmol/L in a 75g oral glucose tolerance test
(OGIT) and each predicts the development of retinopathy (Goldenberg R., et al 2013). There

11
should be 8hrs fasting before taking this. Blood glucose more than 126mg/dl on two or more
tests conducted on different days confirms a diabetes diagnosis (Baynest H.W., et al 2015).
Oral glucose tolerance test – 2hr blood glucose level of 200mg/dl or higher, prediabetes is
diagnosed if the 2hr blood glucose level is 140-199mg/dl (Harikumar K., et al 2015).
Hemoglobin A1C (HbA1c) - A1C can be measured at any time of day and is more convenient
than FPG or 2hPG in a 75g OGIT. A1C also avoids the problem of day to day variability of
glucose values as it reflects the average plasma glucose (PG) over the previous 2-3 months
(Goldenberg R., et al 2013). The specificity of HbA1c ≥6.5% is high enough to justify a
diagnosis of diabetes and the sensitivity of HbA1c ≤5.7% is high enough to justify exclusion of a
diagnosis of diabetes.
Diagnostic test for gestational diabetes-
O’Sullivan test – This test is used to detect gestational diabetes. A 50g load of glucose is given
to a fasting patient. Blood is drawn at one hour. Gestational diabetes is suggested by plasma
levels above 1500mg/L (Ngugi M.P., et al 2012)
Treatment for diabetes-
Treatments on diabetes depend on the individual person and the type of diabetes.
Treatment of patients with type 1 diabetes- The patients with type 1 diabetes has lost the
ability to produce insulin and is therefore dependent upon extremely administered insulin
without which they would die.
Insulin therapy –The use of insulin requires daily management of those factors that affect the
insulin dose. Rapid- acting insulin may be given before, during, or immediately after a meal.
1. Conventional therapy- 2 daily injections of mixed insulin. mixed insulin contains rapid-or-
short acting and intermediate acting taken before breakfast and the evening meal.
2. Conventional therapy with a split night-time dose- 1 injection of mixed insulin (rapid-or-short-
acting and intermediate-acting) before breakfast, 1 injection of rapid-or-short–acting insulin
before the evening meal and 1 injection of intermediate-acting insulin before the bedtime snack.
3. Multiple daily injections (MDI) of rapid-or short – acting insulin before every meal with
intermediate –or long-acting insulin once or twice a day.
4. Intensive therapy with a continuous subcutaneous insulin infusion (CSII or insulin pump) - A
bolus dose of insulin is given before meals and snacks based on the amount of carbohydrate
eaten and the measured level of blood glucose.

12
Treatment of patients with type 2 diabetes- In this treatment depends on a number of factors.
1. Body weight
2. Current eating habits
3. Current level of physical activity
4. Severity of symptoms
5. Blood glucose levels
6. Time period of diabetes
Treatments include diet, exercise, medication and insulin therapy
Self-care- It includes Physical exercise, quitting smoking, weight loss, nutritional counseling,
diabetic diet and dietary fiber.
Medications- Anti-diabetic medication, blood thinners, statin and insulin
Preventative- influenza vaccine and pneumococcal vaccine
Pharmacological treatments for diabetes
1. Oral anti-diabetic drugs –blood glucose levels are mainly determined by absorption of glucose
from gut, uptake of glucose by peripheral tissues, hepatic glucose output, and the insulin
secretion from the pancreas.
• Sulfonylureas – sulfonylureas were the first widely used oral hypoglycemic medications
(Goldenberg R., et al 2013). The sulphonylureas bind to specific sulfonylurea receptors on
pancreatic βcells and increase insulin secretion. They are preferably given 15 to 30 minutes
before meals.
• Meglitinide analogues- Meglitinide analogues (repaglinide and netaglinide) are non-
sulfonylurea insulin secretagogues. They are benzoic acid derivatives, which act on separate non-
sulphonylurea receptor binding sites on β-cell and enhance insulin secretion.
• Biguanides- Biguanides reduce hepatic glucose output and increase uptake of glucose by the
periphery, including skeletal muscle. Motorman has become the most commonly used agent for
type 2 diabetes in children and teenagers e.g. Metformin, Phenformin, Buformin (Goldenberg R.,
et al 2013). Metformin is the preferred biguanides.
• Alpha-glucosidase inhibitors- Alpha-glucosidase inhibitors such as acarbose acts by
competitively inhibiting alpha-glucosidase, the enzyme in the small intestine brush border, which
breakdown oligosaccharides and disaccharides into mono- saccharides. The starting dosage is

13
25-50mg once daily, which is increased to 50mg two to three times in a day. It must be ingested
with the first bite of food.
• Thiazolidinediones (Glitazone) - These agents act by improving insulin sensitivity in adipose
tissue and skeletal muscle. It also inhibits hepatic glucose output. Pioglitazone has partial PPAR-
alpha agonist activity. The dosage of rosiglitazone is 2-8mg on one to two divided doses while
that of pioglitazone is 15 to 45mg once a day. The onset action of these drugs start from 2-
4weeks of therapy and the maximum effect is observed after 8-12 week
Diabetes Mellitus is broadly classified into three types by etiology and clinical presentation, type
1 diabetes, type 2 diabetes, and gestational diabetes. Some other less common types of diabetes
include monogenic diabetes and secondary diabetes.
1.2.2 DEFINITION, COMPLICATIONS AND DIAGNOSIS OF TYPE 2 DIABETES
MELLITUS
Type 2 diabetes mellitus {DM} is a chronic metabolic disorder in which prevalence has been
increasing steadily all over the world. Type 2 diabetes was first described as a component of
metabolic syndrome in 1988. (Patlak, et al., 2002.) It is known as insulin dependent and
characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. It results from
interaction between genetic, environmental and behavioral risk. It is primarily due to life style
factors and genetics. A number of lifestyle factors are known important to the development of
type 2 diabetes mellitus. These are physical activity, sedentary lifestyle, cigarette smoking and
generous consumption of alcohol.
COMPLICATIONS
Obesity has been found to contribute to approximately 55% of cases of type 2 diabetes mellitus.
The increased rate of childhood obesity between the 1960s and 2000s is believed to have led to
the increase in type 2 DM in children and adolescents. (Barlow, et al., 2007) Environmental
toxins may contribute to the recent increases in the rate of type 2 DM. There are many medical
conditions which can potentially give rise to, or exacerbate type 2 DM. These include obesity,
hypertension, elevated cholesterol (combined hyperlipidemia), and with the condition often
termed metabolic syndrome (it is also known as Syndrome X, Reaven's syndrome). (Alberti, et
al., 2005). Obesity has become a real concern worldwide due to its increasing prevalence and
associated cluster of diseases that reduce life quality and expectancy. Abnormal deposition of fat
in the adipose tissue due to chronic over nutrition or reduced physical activity or hereditary

14
reasons is called as obesity, (Svestak M, et al. 2016). Obesity increases the risk of type 2
diabetes, cardiovascular disease, cancer, and premature death. The global epidemic of obesity
and T2DM is deterioration According to updated World Health Organization (WHO) reports;
worldwide obesity has almost doubled since 1990 (Obesity and overweight 2013). More than 1.1
billion people are estimated to be overweight of which around 320 million are calculated to be
obese. More than 2.5 million deaths each year are attributed to higher BMI (body mass Index), a
figure that is expected to double by 2030. Incidence rate of obesity is about 300 million adults
worldwide (Kopelman PG 2000). T2DM is a heterogeneous disorder most commonly
characterized by insulin resistance, a state of reduced insulin-mediated glucose uptake, in the
presence of incapacity of the pancreatic beta cells to produce and provide sufficient insulin to
meet the required needs (Lazar MA (2005). A history of 2-3 years of T2DM does not produce
irreversible damages to the beta cells, but as long as the energy overload persists through years,
irreversible impairment of the beta cells occurs, and insulin is required in order to control plasma
glucose (Taylor R 2013). Updated WHO reports mention that 347 million people worldwide
have Diabetes Mellitus (DM) (Wani AI, et al. 2008), of which T2DM comprises the vast
majority (90%). T2DM is closely associated with excessive body fat and physical inactivity. It is
predicted that there will be a growing burden of DM, and that the world prevalence of DM
amongst adults aged 20-79 years will increase to 439 million by 2030 (Zimmet PZ ., et al 2010).
It has been reported that 86% of adults with T2DM are overweight or obese; 52% have obesity
and 8.1% have morbid obesity (Wilding JP, et al. 2006). Obesity and diabetes are closely related
to each other as about 80% diabetics are obese. Obesity is a common finding in T2DM. There is
impaired insulin sensitivity of peripheral tissues such as muscle and fat cells to the action of
insulin in obese individuals (insulin resistance). Weight reduction in such obese patients
produces improvement in the diabetic state (Lin X ., et al 2000). Obesity increases the risk of
T2DM, cardiovascular disease, cancer, and premature death (Aucott LS ,.2008). Pharmacological
factor involved in obesity and diabetes includes lipoprotein lipase, having a central role in the
metabolism of both triglyceride rich particles and High-Density Lipoproteins (HDL).
Lipoprotein lipase is determinant of serum triglyceride and HDL concentrations (Lutsey PL ., et
al 2010). It is a well-known fact that if you are overweight or obese, you are at greater risk of
developing type 2 diabetes, particularly if you have excess weight around your tummy
(abdomen). Abdominal fat causes fat cells to releases ‘pro-inflammatory’ chemicals, which can

15
make the body less sensitive to the insulin it produces by disrupting the function of insulin
responsive cells and their ability to respond to insulin. This is known as insulin resistance - a
major trigger for type 2 diabetes. Having excess abdominal fat (i.e. a large waistline) is known as
central or abdominal obesity, a particularly high-risk form of obesity (Hart CL., et al 2012).
Obesity is also thought to trigger changes to the body's metabolism. These changes cause fat
tissue (adipose tissue) to release fat molecules into the blood, which can affect insulin responsive
cells and lead to reduced insulin sensitivity. Another theory put forward by scientists into how
obesity could lead T2DM is that obesity causes prediabetes, a metabolic condition that almost
always develops into type 2 diabetes. The links between obesity and type 2 diabetes are firmly
established without the intervention of a healthy diet and appropriate exercise; obesity can lead
to type 2 diabetes over a relatively short period of time. The good news is that by reducing your
body weight, by even a small amount, can help improve your body’s insulin sensitivity and
lower your risk of developing cardiovascular and metabolic conditions such as type 2 diabetes,
heart disease and types of cancer. According to the NHS, a 5% reduction in body weight
followed up by regular moderate intensity exercise could reduce your type 2 diabetes risk by
more than 50% (Brettfeld C., et al 2012),. The influence of obesity on type 2 diabetes risk is
determined not only by the degree of obesity but also by where fat accumulates. Increased upper
body fat including visceral adiposity, as reflected in increased abdominal girth or waist-to hip
ratio, is associated with the metabolic syndrome, type 2 diabetes, and cardiovascular disease
(Brettfeld C., et al 2012), although underlying mechanisms remain uncertain. Whether
subcutaneous fat lacks the pathological effects of visceral fat or is simply a more neutral storage
location, for example, requires further study. Beyond differences in body fat distribution,
emerging evidence suggests that different subtypes of adipose tissue may be functionally distinct
and affect glucose homeostasis differentially. Adult humans have limited and variable numbers
of brown fat cells, which play a role in thermo genesis and potentially influence energy
expenditure and obesity susceptibility. Improved understanding of the function of different fat
cell types and depots and their roles in metabolic homeostasis is a priority for investigation into
the pathogenesis and complications of obesity. Likewise, adipose tissue is composed of
heterogeneous cell types. Immune cells within adipose tissue also likely contribute to systemic
metabolic processes. As the study of adipose biology progresses, it will be important to consider
whether additional subtypes of adipocytes or other cell types can be identified to refine our

16
understanding of obesity complications and generate novel approaches to prevention. At least
three distinct mechanisms have been proposed to link obesity to insulin resistance and predispose
to T2DM:
1. Increased production of adipokines /cytokines, including tumor necrosis factor-a, resistin, and
retinol binding protein, that contribute to insulin resistance as well as reduced levels of
adiponectin .
2. ectopic fat deposition, particularly in liver and perhaps also in skeletal muscle, and
dysmetabolic sequelae (Bennett B, et al. 2011).
3. mitochondrial dysfunction, evident by decreased mitochondrial mass and/or function
(Bournat JC,. et al 2010) Mitochondrial dysfunction could be one of many important underlying
defects linking obesity to diabetes, both by decreasing insulin sensitivity and by compromising
b-cell function.
Type 2 DM is characterized by insulin insensitivity as a result of insulin resistance, declining
insulin production, and eventual pancreatic beta-cell failure. This leads to a decrease in glucose
transport into the liver, muscle cells, and fat cells. There is an increase in the breakdown of fat
with hyperglycemia. The involvement of impaired alpha-cell function has recently been
recognized in the pathophysiology of type 2 DM.( Fujioka, et al., 2007). The most prevalent
form of diabetes is type 2, as an estimated 90% of diabetes patients are diagnosed with this form,
and majority of the remaining 10% of patients have type 1 diabetes (T1D), although there are
other rare types. T2D is largely caused by impaired insulin production and secretion by
pancreatic beta-cells, as well as peripheral tissue insulin resistance. Given that ~90% of patients
are obese or overweight at T2D diagnosis, the aetiology of T2D is largely thought to be linked to
diets involving excessive nutrient consumption combined with insufficient energy
expenditure. There are a range of effective treatments that reduce hyperglycaemia in T2D
patients, which mediate their effects by improving insulin secretion or decreasing peripheral
tissue insulin resistance. Despite this, post-diagnosis complications, especially long-term
complications, are prevalent globally. As a result, diabetes remains a leading cause of blindness,
end-stage renal disease, lower limb amputation and cardiovascular disease.
T2D and obesity have such an interdependent relationship that the term “diabesity” has been
coined. In recent decades, the number of people with T2D has more than doubled, and the
increased global burden of T2D is thought to be largely due to an increase in obesity. Obesity has

17
become a global pandemic over recent decades (Zhou X, et al., 2016). Weight loss is associated
with an improved prognosis for overweight T2D patients and obese individuals. Better
glycaemic control has been reported in T2D patients who have lost weight, and excess body
weight is associated with the risk of cardiometabolic complications, which are major causes of
morbidity and mortality in T2D and obese individuals. Bariatric surgery has proven to be an
effective treatment for diabesity, but it is expensive and there are numerous post-surgery
complications: for example, vomiting and dumping syndrome, iron and B12 deficiency, and
secondary hyperparathyroidism. GLP-1 analogues (eg liraglutide and exenatide) are used to treat
T2D as they promote insulin secretion and induce weight loss.( D’Alessio, et al.,2011). Since
the GLP-1 receptor (GLP-1R) agonists are effective in treating diabesity, they could be
pharmacological alternatives to bariatric surgery but without the post-surgery complications.
Obesity is caused by excessive energy intake and subsequent storage coupled with insufficient
energy expenditure resulting in weight gain. It has become a huge healthcare concern over the
last few decades, especially in developed countries. However, in some obese individuals,
excessive dietary intake may have a genetic aetiology, such as leptin deficiency. Obese
individuals have a BMI of ≥30 kg/m2, and individuals with a BMI of ≥25 and <30 are classified
as overweight. Obesity and overweight incidences have more than doubled since 1980, giving
rise to >2.1 billion individuals with a BMI of >25 globally, as stated by the WHO. The WHO
estimated that there were ~600 million obese individuals in 2014, and this number will continue
to rise in the future. Obesity is associated with an increased risk for T2D, hypertension,
dyslipidaemia, cardiovascular diseases, musculoskeletal disorders (such as osteoarthritis), certain
types of cancer, and premature mortality. It is well established that there is a generally directly
proportional relationship between BMI and both fasting and postprandial insulin levels. A
similar relationship also exists between BMI and the degree of insulin resistance. The
hyperinsulinemia associated with rising BMI is necessary to overcome insulin resistance and
maintain normoglycemia. Not all obese individuals develop insulin resistance though; one study
reported that 19, 34 and 60% of individuals with a BMI of <30, ≥30–35 and >35, respectively,
were insulin resistant. (Ferrannini, et al., 1997).
DIAGNOSIS: Type 2 diabetes is usually diagnosed using the glycated hemoglobin (A1C) test.
This blood test indicates your average blood sugar level for the past two to three months. Results
are interpreted as follows:

18
  Below 5.7% is normal.
 5.7% to 6.4% is diagnosed as prediabetes.
 6.5% or higher on two separate tests indicates diabetes.
If the A1C test isn't available, or if you have certain conditions that interfere with an A1C test,
your health care provider may use the following tests to diagnose diabetes:
Random blood sugar test. Blood sugar values are expressed in milligrams of sugar per deciliter
(mg/dL) or millimoles of sugar per liter (mmol/L) of blood. Regardless of when you last ate, a
level of 200 mg/dL (11.1 mmol/L) or higher suggests diabetes, especially if you also have
symptoms of diabetes, such as frequent urination and extreme thirst.
Fasting blood sugar test. A blood sample is taken after you haven't eaten overnight. Results are
interpreted as follows:
 Less than 100 mg/dL (5.6 mmol/L) is considered healthy.
 100 to 125 mg/dL (5.6 to 6.9 mmol/L) is diagnosed as prediabetes.
 126 mg/dL (7 mmol/L) or higher on two separate tests is diagnosed as diabetes.
Oral glucose tolerance test. This test is less commonly used than the others, except during
pregnancy. You'll need to not eat for a certain amount of time and then drink a sugary liquid at
your health care provider's office. Blood sugar levels then are tested periodically for two hours.
Results are interpreted as follows:
 Less than 140 mg/dL (7.8 mmol/L) after two hours is considered healthy.
 140 to 199 mg/dL (7.8 mmol/L and 11.0 mmol/L) is diagnosed as prediabetes.
 200 mg/dL (11.1 mmol/L) or higher after two hours suggests diabetes.
TREATMENT
Management of type 2 diabetes includes:
 Healthy eating.
 Regular exercise
Factors affecting type 2 diabetes
Type 2 diabetes is the main focus in this review and it is a complex condition that can be
influenced by a combination of genetic, lifestyle and environmental factors. They are;
(1) Genetic factors: There is evidence to suggest that certain genetic variations can
increase the risk of developing type 2 diabetes. Having a family history of the
condition can also predispose individuals to an increased risk.

19
 (2) Obesity and Body Weight: Being overweight or obese is a significant risk factor for
type 2 diabetes. Excess body weight, especially around the abdomen can lead to
insulin resistance, where the body`s cells become less responsive to the effects of
insulin.
(3) Sedentary Lifestyle: Lack of physical activity and a sedentary lifestyle contribute to
the development of type 2 diabetes. Regular exercise helps improve insulin sensitivity
and promotes better blood sugar control.
(4) Unhealthy Diet: A diet high in processed foods, sugary beverages, unhealthy fats, and
low in fruits, vegetables, and whole grains is associated with an increased risk of type
2 diabetes. Consuming excessive calories and refined carbohydrates can contribute to
weight gain and insulin resistance.
(5) Age and Ethnicity: Type 2 diabetes is more common in older adults, although it is
increasingly being diagnosed in younger individuals.
(6) Gestational Diabetes: Women who have had gestational diabetes during pregnancy
have a higher risk of developing type 2 diabetes later in life.
(7) Other medical condtions: Certain medical conditions, such as polycystic ovary
syndrome (PCOS), hypertension, and metabolic syndrome, increase the risk of
developing type 2 diabetes.
1.2.2.1 STREPTOZOTOCIN
Streptozotocin (STZ) is an antibiotic that causes pancreatic islet β-cell destruction and is widely
discussed experimentally to produce a model of type 1 diabetes mellitus (T1DM) and also in
type 2 diabetes. In this study the model albino rats were induced with streptozotozin after 13
weeks of feeding with high fat diet which allows for easy access with the introduction of diabetes
in their bodies. T2D is a complex metabolic disorder essentially characterized by alterations in
lipid metabolism, insulin resistance and pancreatic β-cell dysfunction (Reed, et al,.2017).
Obesity is the most common risk factor for the development of T2D (Reed, et al,.2017); this may
lead to elevated serum triglycerides, hypertension, and insulin resistance. Owing to the
complexity of this disease, preclinical animal models that accurately reflect the pathogenesis of
the human disease are essential (Reed, et al,.2017).. Most animal models that are used for
induction of diabetes make use of toxic chemicals, which target the pancreatic β-cells; for
example, alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyuracil) (ALX) which was first used in

20
1943 generates free radicals that lead to fragmentation of the β-cells; however, ALX can cause
kidney toxicity because it has a very narrow effective dose range (Asrafuzzaman et al,. 2017),
thus it is seldom used. The most common chemical used to induce diabetes is streptozotocin
(STZ) which was first used in 1963; it can be used for induction of both type-1 and T2D
(Asrafuzzaman et al,. 2017). High-doses of STZ severely impair insulin secretion, a feature that
is similar to type-1 diabetes; moreover, this dose of STZ may also lead to ketone body formation
and spillage into the urine, a characteristic of uncontrolled type-1 diabetes (Rivellese et al,.
2013). On the other hand, low-doses of STZ causes mild impairment in insulin secretion that is
more closely resembled in the later stages of T2D. However, the low dose STZ model does not
address the insulin resistance axis typically seen in T2D. Of note, some previous studies have
indicated that animals that were overfed a high-fat diet (HFD) developed insulin resistance.
Therefore, a model which incorporates a HFD to induce peripheral insulin resistance, followed
by low dose STZ to target the pancreatic β-cells would closely mimic not only the phenotype but
also the pathogenesis of human T2D . Feeding a HFD leads to development of hyperinsulinemia,
obesity, and insulin resistance but not frank hyperglycemia or diabetes( Medinsky et al,.2014);
therefore, to induce diabetes it would be necessary to administer low dose STZ.
Mechanism of STZ action
Streptozotocin is an antibiotic derived from Streptomyces achromogenes and is essentially a
nitrosourea analogue. Its nitrosourea moiety causes β-cell damage, while it's deoxyglucose
moiety is responsible for transporting the native molecule across the cell membranes. Since STZ
uses glucose transporter 2 (GLUT2) for transportation into the pancreatic β-cells, other organs
which also express this transporter such as the kidney, liver, and intestine.

1.3 INTRODUCTION TO THE RELATIONSHIP BETWEEN TYPE 2 DIABETES AND

COCOS NUCIFERA (COCONUT) HUSK

Coconut fiber is the most well-known fibrous waste coconuts cultivation. Coconut husk is
composed of 30% fiber and 70% pith, with lignin and phenolic content (panyakaew and fotos ,.
2011). Due to the high lignin content, coconut fiber is very elastic, durable, and resistant to

21
rotting. (Danso,.2011). Coconut husk, also known as coconut coir, contains various organic
compounds, including phytochemicals.

Figure 3: Schematic diagram of coconut fruit

Figure 4 : Schematic diagram of cocnut husk (coir)

Phytochemicals are chemically naturally occurring compounds found in plants that have
potential health benefits and often possess antioxidant, anti-inflammatory, and anti- microbial
properties. The specific phytochemicals present in coconut husk may vary, They are;

22
(1) Lignin: Lignin is a complex polymer present in the cell walls of plants, including coconut
husk. It has antioxidant and antimicrobial properties.

Fig 6: structure of lignin

(2) Tannins: Tannins are polyphenolic compounds found in many plants, including coconut
husk. They have antioxidant properties and can contribute to the astringent taste and color
of certain foods. tannins are a heterogeneous group of high molecular weight
polyphenolic compounds with the capacity to form reversible and irreversible complexes
with proteins (mainly), polysaccharides (cellulose, hemicellulose, pectin, etc.), alkaloids,
nucleic acids and minerals, etc (Mangan, et al,.1988). On the basis of their structural
characteristics it is therefore possible to divide the tannins into four major groups:
Gallotannins, ellagitannins, complex tannins, and condensed tannins (Serrano,et, al
2009).
(1) Gallotannins are all those tannins in which galloyl units or their meta-depsidic
derivatives are bound to diverse polyol-, catechin-, or triterpenoid units.
(2) Ellagitannins are those tannins in which at least two galloyl units are C–C coupled to
each other, and do not contain a glycosidically linked catechin unit.
(3) Complex tannins are tannins in which a catechin unit is bound glycosidically to a
gallotannin or an ellagitannin unit.

23
 (4) Condensed tannins are all oligomeric and polymeric proanthocyanidins formed by
linkage of C-4 of one catechin with C-8 or C-6 of the next monomeric catechin. ff

Fig 6: structure of tannins

(3) Flavonoids: Flavonoids are a diverse group of plant compounds with antioxidant and
anti-inflammatory properties. They are commonly found in fruits, vegetables, and plant-
derived products, including coconut husk. Flavonoids are polyphenolic compounds that
are ubiquitous in nature. More than 4,000 flavonoids have been recognized, many of
which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks
(Harborne, et al,.1999). The flavonoids appear to have played a major role in successful
medical treatments of ancient times, and their use has persisted up to now. Flavonoids are
ubiquitous among vascular plants and occur as aglycones, glucosides and methylated
derivatives. More than 4000 flavonoids have been described so far within the parts of
plants normally consumed by humans and approximately 650 flavones and 1030
flavanols are known (Pridham, et al,.2000). Small amount of aglycones (i.e., flavonoids
without attached sugar) are frequently present and occasionally represent a considerably
important proportion of the total flavonoid compounds in the plant.

24
Fig 7: structure of flavoinds
(4) Phenolic acids: Coconut husk contains phenolic acids, such as ferulic acid and caffeic
acid, which are known for their antioxidant and anti-inflammatory activities.

(5) Carotenoids: Carotenoids are pigments responsible for the yellow, orange, and red colors
in fruits and vegetables. While they are predominantly found in the flesh of coconut,
traces of carotenoids may also be present in the husk.
It's important to note that the concentration and specific composition of phytochemicals
in coconut husk can vary depending on factors such as the variety of coconut, maturity of
the husk, and processing methods. The potential health benefits of these compounds are
an area of ongoing research, and their specific effects on human health are still being
studied.

Fig 8: structure of carotenoids

(6) ALKALOIDS
It is a class of naturally occurring organic nitrogen -containing bases. Alkaloids have diverse and
important physiological effects on humans and other animals. Well-known alkaloids
include morphine, strychnine, quinine, ephedrine, and nicotine (Mueller,et al,.1999).
Alkaloids are found primarily in plants and are especially common in certain families of
flowering plants. The medicinal properties of alkaloids are quite diverse. Morphine is a powerful
narcotic used for the relief of pain, though its addictive properties limit its usefulness. Codeine,
the methyl ether derivative of morphine found in the opium poppy, is an excellent analgesic that

25
is relatively non-addictive. Certain alkaloids act as cardiac or respiratory stimulants. Quinidine,
which is obtained from plants of the genus Cinchona, is used to treat arrhythmias, or irregular
rhythms of the heartbeat. Many alkaloids affect respiration, but in a complicated manner such
that severe respiratory depression may follow stimulation. The drug lobeline (from Lobelia
inflata) is safer in this respect and is therefore clinically useful. Ergonovine (from the
fungus Claviceps purpurea) and ephedrine (from Ephedra species) act as blood-vessel
constrictors. Ergonovine is used to reduce uterine hemorrhage after childbirth, and ephedrine is
used to relieve the discomfort of common colds, sinusitis, hay fever, and bronchial asthma.Many
alkaloids possess local anesthetic properties, though clinically they are seldom used for this
purpose. Cocaine (from Erythroxylum coca) is a very potent local
anesthetic. Quinine (from Cinchona species) is a powerful antimalarial agent that was formerly
the drug of choice for treating that disease, though it has been largely replaced by less toxic and
more effective synthetic drugs. The alkaloid tubocurarine is the active ingredient in the South
American arrow poison, curare (obtained from Chondrodendron tomentosum), and is used as a
muscle relaxant in surgery. Two alkaloids, vincristine and vinblastine (from Catharanthus
roseus, formerly Vinca rosea), are widely used as chemotherapeutic agents in the treatment of
many types of cancer. Nicotine obtained from the tobacco plant (Nicotiana tabacum) is the
principal alkaloid and chief addictive ingredient of the tobacco smoked in cigarettes, cigars, and
pipes. Some alkaloids are illicit drugs and poisons. These include the hallucinogenic
drugs mescaline (from Lophophora species) and psilocybin (from Psilocybe mexicana).
Synthetic derivatives of the alkaloids morphine and lysergic acid (from Claviceps purpurea)
produce heroin and LSD, respectively. The alkaloid coniine is the active component of the
poison hemlock (Conium maculatum). Strychnine (from Strychnos species) is another powerful
poison.
Special methods have been developed for isolating commercially useful alkaloids. In mcases,
plant tissue is processed to obtain aqueous solutions of the alkaloids. The alkaloids are then
recovered from the solution by a process called extraction, which involves dissolving some
components of the mixture with reagents. Different alkaloids must then be separated and purified
from the mixture. High-performance thin-layer chromatography (HPTLC) and related techniques
may be used for the efficient quantitative analysis of alkaloids. Alkaloids in crystalline form may
be obtained using certain solvents.

26
Fig 9: structure of alkaloids

1.4 RELATIONSHIP BETWEEN PHYTOCHEMICALS AND DIABETES

An increased awareness of a healthy lifestyle linked to food preference and consumption
has been highlighted in the global market. Currently, food manufacturers are trying to utilize
natural sources because consumers prefer natural over synthetic antioxidants. Evidence suggests
that agricultural products rich in phenolic compounds, including coconut (Cocos nucifera) fruit,
provide significantly positive antioxidant effects, (Adekola, K.A., et al 2017).
Phenolic compounds described in coconut fruit are catechins and phenolic acids, such as
protocatechuic, chlorogenic, and vanillic acid (Onyechi, O., et al 2010). The level and
composition of the phenolic compounds in coconut fruits may differ among the varieties (Kibria,
A.A., et al 2018). However, frequently consumed young coconut fruit provides considerable
antioxidant effects. The green coconut water mitigates the oxidative stress in hypoglycemia and
hypertensive rat model (Perera, C., et al 2014). Additionally, the maturation level is also
essential in characterizing the antioxidant compounds in the fruit (Solangi, A.H., et al 2011).

27
Henceforth, the aforementioned variables affecting the antioxidant activities need to be studied
to optimize the use of coconut fruit.
Indonesia is one of the largest coconut producers, contributing up to 27% of world
production of coconuts. Within the country, the fruit is typically processed by food industries
into oil, copra, virgin coconut oil (VCO), coconut milk, and desiccated coconut. These coconut-
derived products require a specific type of fruit as the raw material, mainly based on the maturity
levels: young (6-month-old) and mature (12- month-old) fruit. Young coconut flesh is most
suited as an ingredient for beverage products. In contrast, mature fruit is frequently processed
into several products, such as coconut milk, dried shredded coconut flesh, and VCO. Due to the
coconut processing, some by-products were generated, including the meso- and endocarp of the
fruits.
Furthermore, in keeping with the zero waste strategy, the utilization of by-products
generated from food and agricultural industries is recently favorable due to the availability of
advanced extraction technologies (Kalina, S, et al 2018). Earlier reports have disclosed a
considerable amount of phenolic compounds and the antioxidant effects contained in extracts
from tomato pomace, grape peel, coffee spent, and other agro-industrial by-products. As many
parts of the coconut have proven to contain phenolic compounds providing antioxidant activities
(Chemat, F., et al 2020), it is reasonable to suppose that the extract of coconut processing by-
products may deliver similar benefits.
The coconut mesocarp is the fibrous mid-part contributing 85% of the whole fruit, whilst
the endocarp is the hardest part accounting for 10% of the coconut fruit. Some previous studies
have reported that both meso- and endocarp contain a considerable amount of antioxidant
compounds. Young coconut mesocarp has been reported to provide radical scavenging activities
(DPPH) ranging from 5.72 (Muritala, H.F, et al 2018) to 0.032 mg mL−1, whereas the endocarp
exerts 10.89 µg mL−1 in a cell line (Elsbaey, M, et al 2019). To earn these advantages, effective
extraction of the antioxidant compounds from coconut by-products is therefore essential.
The relationship between the phytochemicals present in coconut husk and diabetes is not
extensively studied, and there is limited scientific evidence specifically examining the effects of
coconut husk phytochemicals on diabetes. However, some general points can be discussed:
1. Antioxidant Properties: Many phytochemicals found in plants, including coconut husk,
have antioxidant properties. Antioxidants help protect cells from damage caused by unstable

28
 molecules called free radicals. Oxidative stress, which occurs when there is an imbalance
between free radicals and antioxidants in the body, is believed to contribute to the
development and progression of diabetes. Antioxidant-rich foods and compounds are often
associated with potential benefits in managing diabetes by reducing oxidative stress.
Ethanolic extract of coconut husk contains various antioxidants compounds like phenolics,
flavonoids and tannins which can directly scavenge free radicals and prevent them from
damaging lipids.(Rajakumar,k., et al 2009).EECNH may stimulate the activity of endogenous
antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx), further bolstering cellular defenses against oxidative stress. (Vijaya
kumar,M., et al 2004).
2. Anti-inflammatory Effects: Some phytochemicals in plants, including coconut husk, exhibit
anti-inflammatory properties. Chronic low-grade inflammation is associated with insulin
resistance and the progression of type 2 diabetes. Therefore, consuming foods or compounds
with anti-inflammatory properties may have a positive impact on diabetes management
(Naskar et al,. 2011).
3. Dietary Fiber: While not a phytochemical; coconut husk is rich in dietary fiber. High-fiber
diets have been associated with improved blood sugar control and reduced risk of type 2
diabetes. Fiber slows down the digestion and absorption of carbohydrates, which can help
regulate blood sugar levels and improve insulin sensitivity (Dua K, et al,. 2013).
4. Reducing hyperglycemia: Chronic hyperglycemia in T2DM contributes to oxidative stress.
EECNH has shown hypoglycemic effects in some studies, potentially reducing the indirect
contribution of high blood sugar to lipid peroxidation. (Rajakumar, K.., et al 2009).
5. Improving insulin sensitivity : EECNH may improve insulin sensitivity, leading to better
glucose uptake and utilization by cells, thereby indirectly reducing the generation of free radicals
associated with hyperglycemia.(Sreelatha, S., et al 2010).

1.5 RELATIONSHIP BETWEEN ETHANOLIC EXTRACT OF COCOS NUCIFERA

HUSK ON LIPID PEROXIDATION MARKERS IN TYPE 2 DIABETES
Several studies have investigated the effects of EECNH on markers of lipid peroxidation in
streptozotocin induced T2DM animal models. The potential of natural products in managing

29
chronic diseases (T2D) has gained significant research interest. Coconut husk has emerged as a
promising candidate due to its reported antioxidant and anti-diabetic properties.(vijaykumar,M.,
et al 2010). The detrimental effects of T2D extend beyond hyperglycemia, impacting various
organs and systems. One crucial aspect is oxidative stress, characterized by an imbalance
between free radicals and antioxidant defenses, leading to cellular damage and complications.
Lipid peroxidation, the oxidative degradation of lipids is a prominent marker of oxidative stress
and plays a significant role in T2D pathogenesis. Coconut palm has been used traditionally for
various aliment, including diabetes. This review delves into the effects of ethanolic extract of
cocos nucifera husk on markers of lipid peroxidation in streptozotocin induced T2D models.
Studies demonstrate that EECNH administration significantly reduces MDA, a major marker of
lipid peroxidation in the blood, liver, kidney and brain of T2D rats compared to control
(Jayaprakasam T., et al 2011). Improved histological finding: reveals that reduced cellular
damage and improved tissue architecture compared to diabetic controls, suggest potential
protection against oxidative stress induced damage(Rajendran R., et al 2009).
1.5.1 LIPID PEROXIDATION AND TYPE 2 DIABETES
T2D is characterized by hyperglycemia, leading to increased oxidative stress and free radical
generation. These free radicals attack lipid in cell membrane, triggering a chain reaction known
as lipid peroxidation. This process damages cellular structures and contributes to various T2D
complications, including cardiovascular disease, neuropathy and retinopathy. Elevated levels of
lipid peroxidation markers like malondialdehyde(MDA) and thiobabituric acid reactive
substances (TBARS) are observed in T2D patients.(vijaykumar,M., et al 2010)
1.5.2 ETHANOLIC EXTRACT OF COCOS NUCIFERA HUSK AND ANTIOXIDANT
ACTIVITY
Ethanolic effects of cocos nucifera husk is rich in bioactive compounds like phenolics,
flavonoids and terpenoids, known for their antioxidant properties. These compounds scavenge
free radicals, preventing lipid peroxidation and protecting cellular membranes. (Rajendran, R., et
al 2014)
1.5.3 ETHANOLIC EFFECTS OF COCOS NUCIFERA HUSK AND LIPID
PEROXIDATION MARKERS IN STZ- INDUCED T2D
Several animal studies have investigated the effects of ethanolic effects of cocos nucifera husk
on lipid peroxidation markers in STZ- induced T2D models. Here are some key findings:

30
  Reduced MDA and TBARS levels: Several studies report significant reductions in MDA
and TBARS levels in diabetic rats treated with EECNH compared to untreated controls.
This indicates decreased lipid peroxidation and improved cellular protection.( Das, S., et
al 2012)
 Enhanced antioxidant enzyme activity: EECNH administration has been shown to
increase the activity of antioxidant enzymes like SOD, CAT, GPx in diabetic rats. These
enzymes directly neutralize free radicals, further mitigating oxidative stress.
 Improved tissue histology: Studies have observed improved liver and kidney tissue
morphology in diabetic rats treated with EECNH , suggesting reduced oxidative damage.
Here are some key findings :
 Reduced malondialdehyde (MDA) levels : MDA is a major product of lipid peroxidation
and serves as a common marker. Studies have shown that EECNH administration
significantly reduced MDA levels in liver, pancreas, kidney and brain of diabetic animals
compared to control group.(vijaykumar,M., et al 2010).
 Increased antioxidant enzyme activity: EECNH treatment has been reported to increase
the activity of SOD, CAT and GPx in diabetic animals, suggesting enhanced antioxidant
defense mechanism antioxidant defense mechanism.
 Improved lipid profile: some studies observed positive changes in lipid profiles, including
decreased triglycerides and LDL cholesterol, which might indirectly contribute to
reduced lipid peroxidation.

CHAPTER 2
2.0 MATERIALS AND METHODS
2.1 MATERIALS

31
The materials used in conducting this research project can be categorized as apparatus/
equipments, chemicals and experimental animals.
2.1.1 APPARATUS/ EQUIPMENTS
The apparatus/ equipment used are:
 Sensitive weighing balance
 Centrifuge
 Micro pipette
 Conical flask
 Thermometer
 Refrigerator
 Cuvette
 Beakers
 Test tubes
 Test tube rack
 Sample bottles
 Water bath
 Ph meter
 Needle and syringe
 Oral cannula
 Spectrophotometer
 Measuring cylinder
 Homogenizer
 Mortal and pestle

2.1.2 CHEMICALS
 Phosphate buffer
 Ethanol
 Chloroform
 Dimethyl sulfoxide
 Streptozotocin
 Normal saline

32
  Potassium chloride (KCL)
 Thiobabituric acid (TBA)
 Hydrochloric acid (HCL)
 Elman’s reagent
 Sodium hydroxide (NAOH)
 Phosphate buffer
 Sulphosalicyclic acid
 NaH2PO4.2H2O
 Gluthatione (GSH)
 Trichloroacetic acid (TCA)

2.1.3 EXPERIMENTAL ANIMALS

Fifteen (15) female Albino wistar rats of average weight of 127.8kg was obtained from a
commercial breeding center Okefia , Ogbomosho, Oyo state and housed in a well-ventilated
animal house. They were arranged into their respective cages and given feed and water.

2.1.3 COMPOSITION OF FEED

Table 3
INGREDIENT STANDARD FEED HIGH FAT DIET
FAT 5% 43.7%
Coconut oil (PKC) - 21.2%
Lard oil - 16Kg
Cholesterol - 2.5%
PROTEIN 33% 13.8%
Fish meal 14.5% 4.4%
Soya beans 17.5% 8.5%
Yeast 1% 0.9%
CARBOHYDRATES 60% 40%
Cornstarch 32% 25%
Wheat flour 28% 15%
SALT 1% 1%

33
 NaCl 1% 0.9%
VITAMIN PREMIX 0.5% 0.5%

MINERAL PREMIX 0.5% 0.5%

2.2 STUDY DESIGN

The fifteen animals were grouped into three groups and five rats in each box of A, B and C
amounting to fifteen animals altogether. The animals were acclimatized for two weeks and re-
grouped according to their weight. The first group was labeled the control group and group two
diabetic rats induced with streotozotocin without treatment. The third group was diabetic rats
induced with streptozotocin treated with cocos nucifera husk.

2.21 GROUPING AND IDENTIFICATION

Table 4
GROUPS FEED TYPE COLOUR GROUP TYPE
INDICATION
Group A Standard Feed Green head Control group
Group B High fat diet Green tail Diabetic Untreated
Group D High fat diet Green tail and head Diabetic + extract
125mg/kg

2.2.2 PREPARATION OF STREPTOZOTOCIN

 0.0813g of streptozotocin was measured for 10 rats by using their average weights
 5ml of normal saline was withdrawn into 5ml sample bottle .

34
  The measured streptozotocin was dissolved into 5ml of normal saline and stirred it to
dissolve.
 The streptozotocin solution was withdrawn using the 5ml syringe and each rat was given
0.5ml of the strep solution by inter-peritoneal method.
 The rats were given 5% glucose water for 24hrs to prevent hypoglycemia which was
prepared by measuring 5g of glucose which was dissolved in 100ml of distilled water.

2.2.3 INDUCTION OF DIABETES THROUGH STREPTOZOTOCIN

After two weeks of acclimatization, rats in group B and D were fed with high fat diet for 13
weeks to help induce obesity which is required for the induction of type 2 diabetes in them. The
rats in control group were given normal diet for the same period of time. At 13th week, weights of
250kg was obtained in the rats. The rats were made to fast overnight for 10hrs before being
induced.
2.2.4 PREPARATION OF COCONUT HUSK
Large amount of coconut husk(cocos nucifera) was bought, gotten or harvested and pounded
using mortal and pestle. After pounding to fine substance, the finer substance of coconut husk
which was measured using weighing balance and was then poured into a neat covered bowl. It
was then macerated and soaked in ethanol for three days (72 hrs). On the third day, it was sieved
and the filtrate was poured in a clean bottle. The filtrate was gradually poured into the rotary
evaporator to remove the impurities, the ethanol and get the extract needed. The extract gotten
was air-dried and packed in a closed glassware.

CONCENTRATION OF COCONUT HUSK

Weight of the coconut husk: 150g of coconut husk
Volume of ethanol: 125ml of ethanol
Concentration: 150g/125ml
=1.2g/ml

35
2.2.5 ADMINISTERATION OF COCONUT HUSK EXTRACT
The already diabetic rats whose glucose level to be over 200mlM/L were given different doses of
coconut husk extract. The administration was done by using a standard dosage formula of
125mg/kg body weight with 5% DMSO. It was then administered to each rat using oral cannula.
Subsequent dose for the treated group is ;
GROUP D: 125mg per kg body weight

2.2.6 ANIMALS SACRIFICE

On the 21st day the rats were made to fast overnight for 10hrs and then made unconscious using
chloroform. While unconscious, each rat was opened up and blood was collected directly from
the heart, centrifuged at 4000rpm for 10mins to obtain serum. The following tissues, liver,
kidney, heart, pancreas, brain, small intestine and adipose tissue were excised from the rats. All
traces of blood were cleaned from the tissues using phosphate buffer. The serum and tissues were
kept in plain bottles and placed inside freezer at -4oC until further analysis.
2.2.7 Preparation Of Tissue Homogenate
The organs that were quickly excised were immediately placed in blotting paper to remove
blood. Sample of organs were immediately rinsed in 0.1M phosphate solution to remove the
hemoglobin. The organ sample was homogenized in aqueous phosphate buffer (0.1 molar, PH
7.4) volumes of four times the weight of sample using Teflon homogenizer. The resultant
homogenates were centrifuged at 4000rpm for 10 minutes. The supernatant was gently picked
into sample bottles and stored for use during assay for the biomarkers of oxidative stress.

2. 3 BIOCHEMICAL ANALYSES
2.3.1 DETERMINATION OF MALONDIALDEHYDE CONCENTRATION
Lipid peroxidation was determined by measuring the thiobabituric acid reactive substances
(TBARS) produced during lipid peroxidation as described by Ohkawa et al (1979).
Principle: This method is based on the reaction between 2-thiobabituric acid (TBA) and
malondialdehyde ; an end product of lipid peroxidation. On heating in acidic pH, the product
MDA-TBA adduct, is pink chromophore which absorbs light maximally at 532nm and is

36
extractables into organic solvents such as butanol. MDA is often used to calibrate this test and
the results are expressed as the amount of free MDA produced.
Preparation of reagents:
30% Trichloroacetic acid (TCA)
30g of TCA was dissolved in 100mls of distilled water and stored at 4ºC.
0.75% Thiobarbituric acid (TBA) in 0.1M HCL
0.075g of TBA was dissolved in 0.075g of TBA (Sigma Chemicals Co, London) was dissolved
in 10ml of 0.1M HCl. Dissolution was aided by shaking in a boiling water bath. It was prepared
fresh.
0.1M Tris- KCl buffer, pH 7.4
1.12g of KCl and 2.36g of Tris base were dissolved separately in distilled water and made up to
100ml . The pH was adjusted to 7.4
Procedure:
Sample Tris buffer TCA TBA
0.4ml 1.6ml 0.5ml 0.5ml
An Aliquot of 0.4ml of sample was mixed with 1.6ml of Tris-KCl buffer (0.1 M, pH 7.4) to
which 0.5ml of 30% TCA was added. Then 0.5ml of 0.75% TBA was added and placed in a
water bath for 45 minutes at 800C. This is then cooled in ice and centrifuged for 15 minutes at
3000 x g. The absorbance of the resulting clear pink solution was measured at 532nm against a
reference blank of distilled water. Lipid peroxidation was calculated with a molar extinction
coefficient of 1.56 x 105M-1cm-1, using the formula below:
MDA (μM/mg protein) = Absorbance x Volume of mixture
E532nm x Volume of Sample

2.3.2 DETERMINATION OF REDUCED GUTATHIONE (GSH) CONCENTRATION

Principle:
The reduced form of glutathione comprises, in most instances, the bulk of cellular non-
protein sulfhydryl groups. This method is therefore based upon the development of a relatively
stable (yellow) colour when 51, 51-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent) is added to
sulfhydryl compounds. DTNB interacts with GSH to form 5-thio-2-nitrobenzoic acid (TNB),

37
which is a chromophoric product. The rate of TNB formation is proportional to the total GSH
concentration and is estimated spectrophotometrically at 412nm. The absorbance of this complex
at 412nm is proportion to the level of reduced glutathione in the test sample (Anderson 1997).

Preparation of reagents:
(1) Reduced Glutathione (GSH) working standard
Forty milligrams of GSH (Sigma, Mol. Wt. 307.3g) was dissolved in 0.1M Phosphate buffer, pH
7.4 and made up to 100ml with the same and then stored at 40C.
(2) 0.1M Phosphate buffer (pH 7.4)
Solution A: 7.1628g of Na2HPO4.12H2O (Mol.Wt. 358.22) was dissolved in 200ml of distilled
water.
Solution B: 1.5603g of NaH2PO4.2H2O (Mol.Wt.358.22) was dissolved in 100ml of distilled
water. Finally 0.1M phosphate buffer was prepared by adding 200ml of (solution A) to 100ml of
(solution B) and the pH adjusted to 7.4 with drops of HCl or NaOH as the case may be.
(3) Ellman’s Reagent [5’,5’-dithiobis-(2-nitrobenzoate) (DTNB)]
This was prepared by dissolving 40mg of Ellman’s reagent in 0.1M phosphate buffer and made
up to 100ml. DTNB is stable for at least 3 weeks in the refrigerator.
(4) Precipitating Solution.
4% sulphosalicyclic acid (C7H6O6S.2H2O, Mol.Wt. 254.22) was prepared by dissolving 4g of
sulphosalicyclic acid in little quantity of distilled water and made up to 100 mark in a standard
volumetric flask. This reagent is stable for approximately 3 weeks at 4oC.

Procedure:
The Test sample (0.1 ml) was diluted with 0.9 ml of distilled water to give 1 in 10
dilutions. Three milliliters of 4% sulphosalicyclic acid solution (precipitating solution) was
added to the diluted test sample to deproteinize it. The mixture was centrifuged at 3,000g for 10
minutes. The supernatant (0.5 ml) was added to 4ml of 0.1 M phosphate buffer followed by
addition of 4.5 ml of Ellman’s Reagent. A blank was prepared with reaction mixture of 4ml of
0.1 M phosphate buffer, 0.5 ml of the diluted precipitating solution (addition of 3ml of
precipitating solution and 2ml of distilled water) and 4.5 ml of Ellman’s Reagent. All readings

38
were taken within 5 minutes at 412nm, as color developed is not stable, followed by addition of
Ellman’s Reagent. Reduced glutathione (GSH) is proportional to the absorbance at 412nm. GSH
concentration was determined by plotting the standard curve for GSH and the equivalent
concentrations for absorbance values were extrapolated from the curve.

2.3.3 ASSAY MIXTURE FOR CALIBRATION OF REDUCED GLUTATHIONE

STANDARD CURVE

GSHStock Phosphate buffer Ellman’s Reagent GSH Absorbance

solution (ml) (PO4) (ml) (ml) Concentration (412nm)
(μg/ml)
0.00 0.48 4.50 8 0.033
0.05 0.45 4.50 20 0.099
0.10 0.40 4.50 40 0.246
0.20 0.30 4.50 40 0.346
0.30 0.20 4.50 80 0.505
0.40 0.10 4.50 100 0.683

CHAPTER THREE

3.0 RESULTS

39
The table below shows the ethanolic effect of cocos nucifera husk extract on streptozotocin
induced type 2 diabetes. In liver, the diabetic status caused a statistically significant decrease in
the concentration of MDA and GSH. At 125mg/kg body weight of ethanolic extract of the husk,
the concentration of both MDA and GSH were significantly reduced (p<0.05) when compared
with the control. In kidney, the diabetic status of the rats caused a significant decrease in the
concentration of MDA and GSH, but in the treated group, there’s an increase in the
concentration of MDA and GSH when compared to control. In heart, the diabetic status of rats
caused a significant decrease in the concentration of GSH and MDA, but in the treated group,
there’s a significant decrease(p<0.05) in the concentration of MDA and GSH when compared to
control. Finally in brain, the diabetic status of the rats showed a significant increase in the
concentration of MDA and decrease in the concentration of GSH. At 125mg/kg body weight of
the husk extract, there’s an increase in concentration of MDA and a decrease (p<0.05) in the
concentration of GSH when compared to control.

40
ORGANS CONTROL DIABETIC GROUP TREATED GROUP

MDA GSH MDA GSH

MDA GSH

LIVER 0.736 ± 25.40 ± 0.251 ± 23.80 ± 0.358 ± 24.40 ±

0.357 1.342 0.105a 0.447 0.182b 0.894

KIDNEY 0.616 ± 28.56 ± 0.499 ± 27.36 ± 0.650 ± 29.20 ±

0.136a 0.555 0.098b 1.016 0.171bc 0.828a

HEART 0.584 ± 31.20 ± 0.435 ± 28.80 ± 0.383 ± 30.80 ±

0.473 1.095 0.209a 1.095a 0.137b 1.095b

BRAIN 0.518 ± 29.60 ± 1.042 ± 29.32 ± 1.322 ± 29.34 ±

0.434 0.548 0.3823a 0.295 0.272b 1.191

Table 3.1 shows the concentration of MDA and GSH in various rat organ in control,
diabetic and treated groups.

41
 CHAPTER FOUR
4.0 DISCUSSION AND CONCLUSION
4.1 DISCUSSION
Oxidative stress has become a focus of interest in most medical disciplines and an area of wide
research work. Increasing evidence shows that oxidative stress is associated with the
pathogenesis of diabetes and recent studies suggest a possible relationship between lipid
peroxidation and diabetes complications (Jalali M et al.,2008). There’s recent evidence that
increased oxidative stress in diabetes mellitus contributes to the development of diabetic
complications. (Singer DE et al.,1992). MDA is a stable end product of lipid peroxidation and its
measurement is considered a reliable marker of oxidative damage. (Baynes JW,1991).
Table 3.1 showed that there’s significant increase in MDA concentration in the diabetic status of
rat in brain which is due to the development of diabetic complications which could lead to an
increase in lipid peroxidation and also a decline in defense system in brain. The findings in
accordance with Mahreen et al (2010). The occurrence of oxidative stress in the brain as shown
by significant elevation of MDA level might be due to the fact that the brain is highly susceptible
to oxidative damage due to its high oxygen consumption, high lipid content and low level of
antioxidants compared to the other organs but MDA level is decreased in the hepatic tissue,
cardiovascular tissue and renal tissues.
GSH is a tri-peptide molecule that serves as a major intracellular antioxidant, protecting cells
from oxidative stress caused by free radicals and helps to detoxify harmful compounds but in
TABLE 3.1, the level of GSH is increased in kidney in the treated group compared to the
diabetic group and control. Studies have shown that cocos nucifera husk has higher phenolic
contents which showed higher radical scavenging activity and also have a major antioxidant
capacity of the husk extract (Oliveira et al ., 2013). As observed in table 3.1, there’s a marginal
increase in the level of GSH concentration in all the organs compared to the diabetic group. We
can say that the effect of cocos nucifera husk is more evident in cardiovascular, brain and hepatic
tissues which indicates its ability to inhibit lipid peroxidation and protect cell membranes but not
in renal tissues as there’s a reduction in the level of GSH in liver, heart and brain compared to
control.

42
4.2 CONCLUSION
Type 2 diabetes is one of the causatives of death for both men and women in the United States
and in many other Western countries. Studies showed that many factors which includes; obesity
and body weight, genetic factor, sedentary lifestyle, unhealthy diet, age and ethnicity, gestational
diabetes and other medical factors. Several studies suggest that cocos nucifera husk extract may
offer protective effects against lipid peroxidation thereby potentially alleviating oxidative stress
associated diabetes. Oxidative stress occurs when there’s an imbalance between the production
of reactive oxygen species and the body’s ability to neutralize them. In diabetes, this imbalance
can lead to nephropathy, cardiovascular diseases, neuropathy and other lipid and protein damage.
By assessing markers of lipid peroxidation, the study is aimed to understand how the ethanolic
extract of cocos nucifera affects oxidation of lipids, a process which can lead to cellular damage.
Overall, this study contributes to our understanding of the potential benefits of natural
compounds such as cocos nucifera husk extract, in combating oxidative stress and addressing the
complex pathophysiology of type 2 diabetes.

43
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