Professional Documents
Culture Documents
1.0 Introduction
Diabetes mellitus (DM) is one of the most common problems challenging the public
health in the 21st century (Bailey, 2002). It is characterized by persistent hyperglycemia
and disturbances in the metabolism of fuel compounds as a result of absolute or relative
deficiency in insulin secretion or/and insulin action (Saidu et al., 2010). It occurs when
pancreas cannot produce insulin (type 1 DM) or cannot produce enough of insulin or the
body cannot effectively use the produced insulin for effective uptake of glucose to the
target cells (type 2 DM) (Hajera, et al., 2011). Nowadays, human are suffering not only
on the disease itself but also from the diabetes-related complications. These
complications are significantly important because of their effects on the functions of
several organs in the body (Indah, 2011). The estimated worldwide prevalence of
diabetes among adults in 2010 was 285 million adults (6.4%) and this value is predicted
to rise to around 439 million adults (7.7%) by 2030 where about 90 to 95% of them are of
type 2 DM. (Shaw et al., 2010). Also, the large increases are predicted to occur mostly in
developing countries and in adults especially between 45 and 64 years of age (king et al.,
1998). These predictions indicate a growing burden of diabetes, particularly in
developing countries. In 2011, the prevalence of diabetes mellitus is about 2.7% in
Nigeria (WHO, 2011).
The pathogenesis of both type I and type II diabetes mellitus are different, but
hyperglycemia and its associated complications are common in both conditions. The
immediate metabolic effect of DM is hyperglycemia, which then directly or indirectly
triggers many other biochemical irregularities which can lead to serious microvascular
and macrovascular complications (retinopathy, neuropathy, nephropathy etc.), affecting
the heart and blood vessels, eyes, kidneys, nerves and teeth (Ladan et al., 2007). High
blood pressure, high blood glucose level, elevated cholesterol, and other risk factors in
DM contribute in increasing the risk of cardiovascular complications like atherosclerosis,
which is the primary cause of heart disease and stroke. (Huynh et al., 2008 and Sabitha,
2011). Diabetes like many other metabolic diseases has no known cure but is currently
managed by the use of agents that exhibit hypoglycaemic effects (Saidu et al., 2010).
Epidemiological studies and clinical trials strongly supported the notion that
hyperglycemia is the principal cause of the complications. Effective blood glucose
control is the key for preventing or reversing diabetes complication and improving
quality life in patient with diabetes. Thus, sustained reduction in hyperglycemia will
decrease the risk of developing microvascular and macrovascular complications (Jerums
et al., 2003). Insulin and oral hypoglycaemic agents (sulphonylureas, biguanides and
thiazolidinedione), insulin sensitizer, insulin secretagogues and α- glucosidase inhibitors
are the main agents for the treatment of diabetics and are effective in controlling
hyperglycaemia (Evans, 1999). However, the practical applicability (administration) of
these therapeutic agents’ remains restricted owing to their limited action, secondary
failure rates and accompanying side effects (Kim et al., 2006). Diabetes is also managed
or controlled by non pharmacologic methods, such as diet and exercise. This triggers the
search for new drugs from all possible sources, including traditional medicines (Palsamy
et al., 2008). Throughout the world, many traditional treatments for diabetes exist. This
includes the use of plants such as Albizzia chevalieri, Bitter mellon, Alocasia indica,
Morus indica etc. (Grover et al., 2002, and Saidu et al., 2010). However, few have
received scientific validation and the World Health Organisation has recommended that
plants use for the treatments of diabetes warrant further evaluation (WHO, 1985).
Despite the great strides made in the understanding and management of diabetes, the
disease and disease related complications remain unabated. Phytochemicals identified
from traditional medicinal plants are presenting an exciting opportunity for the
development of new types of therapeutics (Zimmet et al., 1999). It is a known fact that
nutrition and health care are interrelated. Thus, many plants are consumed as food and for
health benefits (Pieroni et al., 2005). The nutraceutical value and the antioxidant activity
of semi-cultivated or neglected vegetables are regarded worldwide as an important area
of the nutritional and phytotherapic research (Eastwood, 1992).
Abelmoschus esculentus, ‘AE’ (Okra or lady’s finger) is a flowering plant in the mallow
family. It is one of the most important vegetables widely cultivated throughout the
tropical and temperate region in the world for its tender fruits and young leaves (Sabitha
et al., 2011). This plant is popular with its mucilaginous properties and has been
acclaimed to have various health benefits which include anti-diabetic properties (Indha,
2011). The fruit of the plant is rich in nutrients like protein, Niacin, Riboflavin,
Phosphorus, Zinc, Cupper, Potassium, vitamins A, B6, C and K, Thiamine, Folate,
Magnesium, Calcium, and Manganese. Okra is rich in phenolic compounds with
important biological properties like quarcetin and flavonol derivatives, catechin
oligomers and hydroxycinnamic derivatives. Okra is also known for being high in
antioxidants activity (Adelakun et al., 2008).
Okra is a powerhouse of valuable nutrients, nearly half of which is soluble fiber in the
form of gums and pectins which help to lower serum cholesterol, reducing the risk of
heart diseases. The other fraction of Okra is insoluble fiber, which helps to keep the
intestinal tract healthy. Okra has several potential health beneficial effects on some of the
important human diseases like cardiovascular disease, type II diabetes, digestive diseases,
ulcer and some cancers. Overall, Okra is an important vegetable crop with a diverse array
of nutritional quality and potential health benefits (Adelakun et al., 2008). Okra mucilage
has medicinal applications when used as plasma replacement or blood volume expander
(Habtamu et al., 2014).
Epidemiological studies show that the incidence of diabetes mellitus will double by 2030,
affecting mostly the developing countries like Nigeria where adequate treatment is often
very expensive (Sarah, 2004 and WHO, 2011). In addition to various health problem
posed by diabetes, it also has severe economic implication in both developed and
developing countries. For example, United States estimated that diabetic related
conditions, leads to health costs of about hundreds billion of dollars yearly (Reaven,
1998). This is frightening as such an economic burden cannot be borne by developing
countries like Nigeria. Management of diabetes mellitus by insulin therapy has several
draw backs such as insulin resistance (Piedrola, 2001). Also, anorexia nervosa, brain
atrophy, and fatty liver are encountered in chronic treatment with insulin (Tobias, 2001).
For oral hypoglycemic drugs, Sulphonylureas and Biguanide are being used effectively in
controlling hyperglycaemia (Evans, 1999) However; it has been reported that their use
could exert some side effects such as hepatotoxicity, abdominal pain, flatulence,
diarrhoea and hypoglycaemia (Fujisawa et al., 2005). Drug resistance to these medicines
has also been reported after prolonged period of treatment of diabetes with such drugs
(King et al., 1998; Shaw et al., 2010). Even with these therapeutics, diabetes remains an
exceedingly difficult disease to control. No single agent has so far been unequivocally
accepted to be ‘the drug’ for the management of diabetes.
In view of the above, there is urgent need to develop new medications or strategies to
counter the huge increase in prevalence and incidence of diabetes mellitus, high cost of
treatment and the side effects associated with the current medications. Okra fruit are
readily available and affordable in Nigerian communities and it is a good source of
various vitamins and minerals in addition to its high dietary fiber content (Habtamu et al.,
2014). Hence, exploration of potential antidiabetic property of such plant’s fruit for
possible development of Okra based antidiabetic nutraceutical formulation is critical for
future management of diabetes mellitus.
The aim of the present study is to evaluate the antidiabetic effect of different parts
(Whole Okra, Okra Peel and Okra Seed) of Ex-maradi Okra fruit variety (Abelmoschus
esculentus) and to explore their possible antidiabetic mechanism of action against
Alloxan-induced diabetic rats for possible development of Okra-based antidiabetic
nutraceutical formulation that is safe, effective and affordable for the management of
diabetes mellitus.
1.3: The Specific Objectives of this Study are:
Literature Review
Diabetes received its name in the first century B.C from a Greek Physician, Aretaeus of
Cappadocia, after the word diabainein which means “to siphon”, suggesting “the melting
down of flesh and limbs into urine”. This was related to the patients passing excessive
amount of urine. In 1552 B.C, Hesy-Ra, an Egyptian physician was the pioneer who
documented frequent urination with a sweet taste as a symptom of a mysterious disease
that also causes emanciation. Around this time, ancient healers noted that ants seemed to
be attracted to the urine of people who had this disease and use this as a diagnostic test
for what they refer to as “sweet urine disease” (Turnbridge and Home, 1991). Cullen in
1750 added the word “mellitus” (honey-sweet) to the term diabetes.
Right from the origin of the discovery of diabetes to the dramatic breakthrough in its
treatment, many brilliant minds played a part in the fascinating history of diabetes.
Thomas Willis (1675) acknowledged that the sweetness of the urine might be secondary
to the sweetness of the blood. This discovery was revolutionary in cementing the
understanding between the discovery and treatment of diabetes. However, it took about
one and a half century to demonstrate that the sweetness is due to sugar and that the sugar
is glucose. Further discoveries in the understanding of diabetes were the work of Paul
Langerhans in 1869; where he described the islets of the pancrease. Also in 1889; Mering
and Minkowski demonstrated the role of pancrease in diabetes by performing
pancreactomy on dogs. Despite these advances, scientists were unable to provide an
explanation on the function of islets (Murray et al., 2000). Further investigation led
Eugene Opie to suggest link between diabetes and islets cells. Islets cells are clusters of
cells that make insulin in the pancrease (Turnbridge and Home, 1991). As physician
learned more about diabetes, they began to understand how it could be managed. The
realisation that diabetes could be manged by diet and exercise was a steping stone during
the Franco- Prussian War of the early 1870s; where Apollinaire Bouchardat, who was a
French physician, noted that the conditions of his diabetic patients were improving. He
observed that the improvement could be due to war related food rationing. He therefore
developed individualised diets for management of diabetes (Mayne, 1997). The
understanding of the role of insulin was made possible by the findings of Banting and
colleagues in their studies on the effect of pancreatic extracts on laboratory animals
(Banting et al., 1922). After the successful isolation of insulin by Banting and Best with
the collaboration of Jhon Macleod and James in 1922; clinical application of insulin in
the treatment of diabetes started (Banting et al., 1922). The first sulfonylurea
(antidiabetic agent) was identified in 1942 (Guzman and Sower, 1999).
• A 2-hour plasma glucose > 200mg/ dl (11.1 mmol/l) during an oral glucose
tolerance test (OGTT)
Diabetes mellitus has been classified in to two main types namely; insulin-dependent
diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM) (WHO
1999; Satyanarayana, 2012)
Non- Insulin Dependent Diabetes Mellitus (NIDDM) also called type II diabetes;
sometimes it is also referred to as adult onset diabetes mellitus. NIDDM is the most
common type of diabetes usually found in adults from 35 years and above accounting for
about 90 to 95 % of the diabetic population in both developed and developing countries
(Shaw et al., 2010). Majority of Africans have type II diabetes mellitus. As the name
implies, type II diabetes mellitus result from the body’s ineffective use of insulin and it is
largely associated with increasing trends towards overweight and obesity, unhealthy
diets, physical inactivity, and socioeconomic disadvantage (ADA, 2008). Symptoms may
be similar to those of type I diabetes, but are often less marked; this fact makes the
disease diagnosed several years after onset, when complications have already arisen
(WHO, 2013).
The etiology of type I diabetes is still unknown, but the genetic factor is central (ADA,
2004). The ß -cell destruction may be caused by drugs, viruses or autoimmunity due to
certain genetic variation, the ß -cells are recognized as non-self and they are destroyed by
immune mediated injury. Usually, the symptoms of diabetes appear when probably about
80-90% of the β -cells have been destroyed. The pancreas ultimately fails to secrete
insulin in response to glucose ingestion (WHO 2013).
The causative factors of NIDDM includes: genetic, life style and environmental factors.
NIDDM more commonly occurs in obese individuals. Overeating coupled with
underactivity leading to obesity is associated with the development of NIDDM (WHO
2013). Obesity acts as a diabetogenic factor in genetically predisposed individuals by
increasing the resistance to the action of insulin. This is due to a decrease in insulin
receptors on the insulin responsive (target) cells (Satyanarayana, 2012). The patients of
NIDDM may have either normal or even increased insulin levels. lt is suggested that
over-eating causes increased insulin or reduction but decreased synthesis of insulin
receptors. This is based on the fact that weight reduction by diet control alone is often
sufficient to correct NIDDM (WHO, 2013).
2.7: Glycated Hemoglobin.
People with diabetes have an increased risk of developing a number of serious health
problems (Nathan et al., 2005). Long-term complications of diabetes develop gradually.
Consistent high blood glucose levels can lead to serious diseases that eventually affect
the heart and blood vessels, eyes, kidneys, nerves and teeth (cardiovascular disease,
blindness, kidney failure, and lower limb amputation). In addition, people with diabetes
also have a higher risk of developing infections. In general, diabetes complications may
be disabling or even life-threatening (DCCTRG, 1995; Ahmed et al., 2008).
Complications of diabetes mellitus could be broadly divided into two classes, viz: acute
and chronic complication. Acute complications include ketoacidosis while chronic
complications include atherosclerosis, hematological abnormalities, kidney, eye,
neuropathic diseases and foot alceration (WHO 1994).
III. Hypoglycemia
V. Respiratory Infections
The immune response is impaired in individuals with diabetes mellitus (Ahmed et al.,
2008). Cellular studies have shown that hyperglycemia both reduces the function of
immune cells and increases inflammation. The vascular effects of diabetes also tend to
alter lung function, all of which leads to an increase in susceptibility to respiratory
infections such as pneumonia and influenza among individuals with diabetes. Several
studies also show diabetes associated with a worse disease course and slower recovery
from respiratory infections (Ahmed et al., 2008)
VI. Periodontal Disease
Diabetes associated with periodontal disease (gum disease) may make diabetes more
difficult to treat. Gum disease is frequently related to bacterial infection by organisms
such as Porphyromonas gingivalis and Actinobacillus Actinomycete (Lakschevitz et al.,
2011) A number of trials have found improved blood sugar levels in type II diabetics who
have undergone periodontal treatment (Mombelli, 2012).
I. Diabetic Cardiomyopathy
Damage to the heart muscle, leading to impaired relaxation and filling of the heart with
blood (diastolic dysfunction) and eventually heart failure; this condition can occur
independent of damage done to the blood vessels over time from high levels of blood
glucose. (Kobayashi et al., 2014).
Abnormal and decreased sensation, usually in a 'glove and stocking' distribution starting
with the feet but potentially in other nerves, later often fingers and hands; when
combined with damaged blood vessels this can lead to diabetic foot. Other forms of
diabetic neuropathy may present as mononeuritis or autonomic neuropathy.
Approximately one half of people with diabetes have some form of peripheral neuropathy
(PN), either polydiabetic or monodiabetic neuropathy (Dyck, 1993). People with diabetes
also frequently have autonomic neuropathy, including cardiovascular autonomic
dysfunction, which is manifested as abnormal heart rate and vascular control (Vinik,
2003). Physical therapists commonly encounter diabetes- associated PN in the evaluation
and treatment of balance and movement disorders because these disorders frequently
affect lower-extremity sensation and can cause lower-extremity pain in people with
diabetes. Loss of lower-extremity sensation coupled with impaired peripheral vascular
function can contribute to lower-extremity (commonly foot) ulceration (Boulton, 1997).
V. Diabetic Retinopathy
Diabetic retinopathy (DR) is a microvascular complication that can affect the peripheral
retina, the macula, or both. It is characterized by growth of friable and poor-quality new
blood vessels in the retina as well as macular edema (swelling of the macula), which can
lead to severe vision loss or blindness (Nathan et al., 2005). Retinal damage (from
microangiopathy) is a leading cause of visual disability and blindness in people with
diabetes. Also, is the most common cause of blindness among non-elderly adults in the
US (Nathan et al., 2005). The severity of DR ranges from non proliferative and pre
proliferative to more severely proliferative DR, in which the abnormal growth of new
vessels occurs (Harding, 2003). Total or partial vision loss can occur through a vitreous
hemorrhage or retinal detachment, and central vision loss can occur through retinal vessel
leakage and subsequent macular edema (Sheetz, 2002).
There are different classes of anti-diabetic drugs, and their selection for use depends on
the nature of the diabetes, age and situation of the person, as well as other factors. With
the exceptions of insulin injection, most of the antidiabetic drugs are administered orally,
thus called oral hypoglycemic agents (drugs).
A. Insulin
Type I diabetes mellitus is a disease caused by the absence of insulin. Therefore, Insulin
injection must be used in management of Type I diabetes mellitus. Insulin is usually
administered subcutaneously, either by injections or by an insulin pump. In acute-care
settings, insulin may also be carefully administered intravenously. In general, insulin
could be grouped in to three classes based on the rate at which they are being metabolized
by the body.
(a) Rapid acting insulins e.g. Regular insulin (Humulin R, Novolin R) Insulin lispro
(Humalog) Insulin aspart (Novolog) Insulin glulisine (Apidra) Prompt insulin zinc
(Semilente, Slightly slower acting).
(b) Intermediate acting insulins e.g. Isophane insulin, neutral protamine Hagedorn (NPH)
(Humulin N, Novolin N) Insulin zinc (Lente)
(c) Long acting insulins e.g. extended insulin Zinc insulin (Ultralente) Insulin glargine
(Lantus) Insulin detemir (Levemir) (Fuhlendorff et al., 1998).
It is a known fact that herbal medicines are used for primary health care by about 80%
the world’s population particularly in the developing countries where resources are
meager and medical facilities are limited; many plants are used for diabetic medication
because of their cultural acceptability, safety, efficacy, potency, inexpensiveness/
affordable, and lesser side effects (Pullaiah and Chandrasekhar, 2000). Ethnobotanical
information indicates that more than 800 plants species are used as traditional remedies
for the treatment of diabetes mellitus (Pushparaj et al., 2000). Throughout the world,
numbers of traditional herbal medicinal plants that have good potentials for use in the
treatment of diabetes have been studied and about 200 pure compounds fractionated from
such plants have showed hypoglycemic activity (Grover et al., 2002). However, there is
insufficient evidence to determine their effectiveness because many of such plants have
not been scientifically scrutinzed (WHO, 2007). The WHO Expert Committee
recommended the importance of investigating hypoglycemic agents from plant origin
which were used in traditional medicine for the treatment of diabetes mellitus. Also, they
recommended the development of herbal medicine in this concern. This suggestion was
made because; there could be a better source for the treatment of this debilitating
metabolic disease than the currently available synthetic drugs (WHO, 2000). Recently,
much attention has been focused on natural hypoglycemic agents to delay the onset or
progression of diabetic complications (Rahman and Ismail, 2009; and Peng et al., 2011).
2.9.2 OBESITY
Obesity is a medical condition in which excess body fat has accumulated to
the extent that it may have a negative effect on health. People are generally
considered obese when their body mass index (BMI), a measurement obtained by
dividing a person's weight by the square of the person's height, is over 30 kg/m2,
with the range 25–30 kg/m2 defined as overweight. Obesity increases the
likelihood of various diseases and conditions, particularly cardiovascular diseases,
type 2 diabetes, obstructive sleep apnea, certain types of cancer, osteoarthritis and
depression, (Kushner, 2007).
Obesity is increasing at alarming rates in industrialized countries. Two
changes in the environment seem to have directly contributed to this increase. The
first is a reduction in the energy expenditure required for daily living, as a result of
technological improvements, and the second is an increase in energy intake
resulting from the increasing availability of palatable, low cost, high fat, energy
dense food.
2.9.3 Causes of obesity
The balance between calorie intake and energy expenditure determines a
person's weight. If a person eats more calories than he or she burns
(metabolizes), the person gains weight (the body will store the excess energy
as fat). If a person eats fewer calories than he or she metabolizes, he or she
will lose weight. Therefore, the most common causes of obesity are
overeating and physical inactivity. Ultimately, body weight is the result of
genetics, metabolism, environment, behavior, and culture.
Genetics; A person is more likely to develop obesity if one or both parents
are obese. Genetics also affect hormones involved in fat regulation. For
example, one genetic cause of obesity is leptin deficiency. Leptin is a
hormone produced in fat cells and in the placenta. Leptin controls weight by
signaling the brain to eat less when body fat stores are too high. If, for some
reason, the body cannot produce enough leptin or leptin cannot signal the
brain to eat less, this control is lost, and obesity occurs. The role of leptin
replacement as a treatment for obesity is under exploration.
Over eating; Overeating leads to weight gain, especially if the diet is high in
fat. Foods high in fat or sugar (for example, fast food, fried food, and
sweets) have high energy density (foods that have a lot of calories in a small
amount of food). Epidemiologic studies have shown that diets high in fat
contribute to weight gain.
Frequency of eating; the relationship between frequency of eating (how
often you eat) and weight is somewhat controversial. There are many reports
of overweight people eating less often than people with normal weight.
Scientists have observed that people who eat small meals four or five times
daily, have lower cholesterol levels and lower and/or more stable blood
sugar levels than people who eat less frequently (two or three large meals
daily). One possible explanation is that small frequent meals produce stable
insulin levels, whereas large meals cause large spikes of insulin after meals.
Physical inactivity; sedentary people burn fewer calories than people who
are active. The National Health and Nutrition Examination Survey
(NHANES) showed strong correlations between physical inactivity and
weight gain in both sexes.
Medications; Medications associated with weight gain include certain
antidepressants (medications used in treating depression), anticonvulsants
(medications used in controlling seizures such as carbamazepine, some
diabetes medications (medications used in lowering blood sugar such as
insulin, sulfonylureas, and thiazolidinediones), certain hormones such as
oral contraceptives, and most corticosteroids such as prednisone.
Psychological factors; for some people, emotions influence eating habits.
Many people eat excessively in response to emotions such as boredom,
sadness, stress, or anger. While most overweight people have no more
psychological disturbances than normal weight people, about 30% of the
people who seek treatment for serious weight problems have difficulties
with binge eating.
Social issues: There is a link between social issues and obesity. Lack of
money to purchase healthy foods or lack of safe places to walk or exercise
can increase the risk of obesity.
Diseases such as hypothyroidism, insulin resistance, polycystic ovary
syndrome, and Cushing's syndrome are also contributors to obesity. Some
diseases, such as Prader-Willi syndrome, can lead to obesity.
One medication, orlistat, is current widely available and approved for long
term use. Weight loss however is modest with an average of 2.9 kg (6.4 lb) at 1 to
4 years and there is little information on how these drugs affect longer-term
complications of obesity. Its use is associated with high rates of gastrointestinal
side effects, (Rucker D, et al., 2007).
B. A triglyceride is an ester derived from glycerol and three fatty acids (from
tri- and glyceride), (Nomenclature of Lipids, 2007).Triglycerides are the
main constituents of body fat in humans and other animals, as well as
vegetable fat, (Nelson, et al., 2000).They are also present in the blood to
enable the bidirectional transference of adipose fat and blood glucose from
the liver, and are a major component of human skin oils, (Lampe, et al.,
1983). There are many different types of triglyceride, with the main division
between saturated and unsaturated types. Saturated fats are "saturated" with
hydrogen — all available places where hydrogen atoms could be bonded to
carbon atoms are occupied. These have a higher melting point and are more
likely to be solid at room temperature. Unsaturated fats have double bonds
between some of the carbon atoms, reducing the number of places where
hydrogen atoms can bond to carbon atoms. These have a lower melting point
and are more likely to be liquid at room temperature, (Lampe, et al., 1983).
C. High-density lipoproteins (HDL) are one of the five major groups of
lipoproteins, (LDL and HDL: Bad and Good Cholesterol, 2017).
Lipoproteins are complex particles composed of multiple proteins which
transport all fat molecules (lipids) around the body within the water outside
cells. They are typically composed of 80-100 proteins per particle (organized
by one, two or three ApoA; more as the particles enlarge picking up and
carrying more fat molecules) and transporting up to hundreds of fat
molecules per particle. Increasing concentrations of HDL particles are
strongly associated with decreasing accumulation of atherosclerosis within
the walls of arteries. This is important because atherosclerosis eventually
results in sudden plaque ruptures, cardiovascular disease, stroke and other
vascular diseases. HDL particles are sometimes referred to as "good
cholesterol" because they can transport fat molecules out of artery walls,
reduce macrophage accumulation, and thus help prevent or even regress
atherosclerosis, but studies have shown that HDL-lacking mice still have the
ability to transport cholesterol to bile, suggesting that there are alternative
mechanisms for cholesterol removal, (Betteridge, et al., 2008).
Abelmoschus esculentus, (AE) (Okra or lady’s finger) is a flowering plant in the mallow
family. It is one of the most important and utilized species of vegetables widely known
and cultivated throughout the tropical and temperate regions in the world for its tender
fruits (Naveed et al., 2009). This plant is an economically important vegetable crop
characterized by its mucilaginous properties and has been acclaimed to have various
health benefits which include antidiabetic properties (Ndangui et al., 2010). Okra plant
was previously included in the genus Hibiscus. Later, it was designated to Abelmoschus,
which distinguished it from the genus Hibiscus (Aladele et al., 2008). Okra has so far
been acclaimed to originate from Ethiopia (Sathish and Eswar, 2013; Habtamu et al.,
2014). Then propagated in North Africa, Arabia India and other parts of the world
(Nzikou et al., 2006). Okra is a multipurpose crop due to uses of its various parts ranging
from the fresh leaves, buds, flowers, pods, stems and seeds (Mihretu et al., 2014). Okra
immature fruits (green seed pods), which are consumed as vegetables, can be used in
salads, soups and stews, fresh or dried, fried or boiled (Ndunguru and Rajabu,
2004). It offers mucilaginous consistency after cooking. Often, the extract obtained from
the fruit is added to different recipes like soups, stews and sauces to increase the
consistency.
The fruit of this particular plant is rich in nutrients like protein, niacin, riboflavin,
phosphorus, zinc, copper, potassium, vitamins A, B6, C and K, thiamine, magnesium,
folate, calcium, and manganese. Okra is rich in phenolic compounds with important
biological properties like quarcetin and flavonol derivatives, catechin oligomers and
hydroxycinnamic derivatives (Adelakun et al., 2010). Okra has been called “a perfect
villager’s vegetable” because of its robust nature, dietary fiber, and distinct seed protein
balance of both lysine and tryptophan amino acids (unlike the proteins of cereals
and pulses) (Sanjeet et al., 2010). The amino acid composition of Okra seed protein is
comparable to that of soybean and the protein energy ratio (PER) is higher than that of
soybean (Adetuyi et al., 2011 and 2012) and the amino acid pattern of the protein
renders it an adequate supplement to legume or cereal based diets (Ndangui et al.,
2010). Okra seed is known to be rich in high quality protein especially with regards to
its content of essential amino acids relative to other plant protein sources (Oyelade
et al., 2003). Okra seed is rich in protein and unsaturated fatty acids such as
linoleic acid Hence, it plays a vital role in the human diet (Farinde et al., 2007).
Okra is one of the classical examples of good source of dietary fibers used not only as
food but also for unique therapeutic significance owing to its several health beneficial
effects such as hypoglycemic, hypocholesterolemic, antioxidant, and antimicrobial, anti-
inflammatory, anti-constipation anti ulcer, anti cancer activities and host of other
potentials (Viuda-Martos et al., 2010). Okra is a powerhouse of valuable nutrients, nearly
half of which is soluble fiber in the form of gums and pectin’s which help to lower serum
cholesterol and reducing the risk of heart diseases. The other fraction of Okra is insoluble
fiber, which helps to keep the intestinal tract healthy (Sengkhamparn et al., 2009). These
beneficial effects have been partly attributed to compounds which possess antioxidant
activity in the Okra. The major antioxidants in Okra fruit are vitamins C and E,
carotenoids, and phenolic compounds, especially flavonoids. Nutrient antioxidants may
act together to reduce reactive oxygen species level more effectively than single
dietary antioxidants, because they can function in synergy (Rossetto et al., 2002).
In addition, a mixture containing both water-soluble and lipid-soluble antioxidants
is capable of quenching free radicals in both aqueous and lipid phases (Trombino et
al., 2004). These antioxidants scavenge radicals and inhibit the chain initiation or break
the chain propagation (the second defense line). Vitamin E and carotenoids also
contribute to the first line of defense against oxidative stress, because they quench singlet
oxygen (Krinsky, 2001). Okra mucilage has medicinal applications when used as plasma
replacement or blood volume expander (Habtamu et al., 2014).
Evidences have been gathered that Okra fruit is not toxic. There has not been any report
of Okra fruit toxicity since antiquity (Anupam et al., 2014). In animal studies, Okra failed
to induce any signs of toxicity or mortality in mice and rats that received acute and sub
chronic regimes as there were no significant hematological, hepatic or histopathological
changes in the animals.
Acute oral toxicity of aqueous and methanolic Okra seed extracts has been determined as
per Organization for Economic Cooperation and Development (OECD) guidelines by
Sathish et al., (2014) where a single oral dose (50, 300, and 2000 mg/kg) of the extract
was administered to a group of four (4) mice. The mice were observed individually at
least once during the first 30 minutes, periodically during the first 24 hours, with special
attention given during the first 4 hours and daily thereafter, for a total of 14 days for
abnormal signs, diarrhea, food and water intake. The mice did not show any sign of
toxicity up to 2000 mg/kg. No record of toxicity signs on histopathological studies on all
vital organs in the experimental rats and the hematological parameters as well. Hence, the
LD50 cut- off value might exceed 2000 mg/kg of aqueous and metanolic Okra seed
extract. Similarly, Alqasoumi, (2012) had performed acute toxicity test on Okra aqueous
extract where he administered the extract at various doses, ranging from (500, 1000,
2000, 4000 up to 8000 mg/kg) in five groups of ten (10) mice (five male and five female
mice each). No any behavioral abnormality and clinical signs and symptoms of toxicity
were observed from 30 minutes up to 6 hours on the first day and thereafter, everyday up
to 7 days. No toxicity symptoms were recorded as no lethality was observed up to 8000
mg/kg of the Okra extract in the mice (Al-Rehaily et al., 2002).
CHAPTER THREE
All the chemicals and reagents used for this work are of analytical grade.
Table 1: List of Chemicals and Reagents
Chemicals Manufacturer
3.2 Equipments
Standard laboratory equipments were used for this work.
Table 2: List of Equipments /Materials and Glass wares
Twenty (20) apparently healthy young Wister albino rats of both sexes weighing between
100 – 200 g were used for this study. The rats were kept at animals’ house under normal
environmental conditions and maintained with free access to pelletized growers feed, and
access to water ad libitum. The animals were allowed to acclimatize for two weeks (14
days).
The Okra sample was thoroughly sorted to remove out any unwanted matter. Whole Okra fruit (W) (500
g) was ground to powder using a mortar and pestle. The peels (P) of another 500 g of the Okra fruit was
obtained while the same procedure was repeated to obtain the seed (S) both of which were ground to
powder the powdered samples were sieved with a fine mesh, placed in a labeled sealed container and
stored at normal laboratory conditions until needed. Okra powdered sample was prepared by
The range of diabetogenic dose of alloxan is quite narrow even in the same species of
albino rats. Therefore; even slight overdosing may be generally toxic causing the loss of
many animals (Lenzen et al, 1996). To prevent the toxic side effects, ranges of 80 to 180
mg/kg of alloxan (20 mg interval) was tested and 120 mg/kg was selected as minimum
and safest dose for induction of Alloxan-diabetes in this work. The Alloxan diabetic rat
models were prepared by adopting the method of Kandur and Goyal (2005). All rats,
except the Normal Control Group were intraperitoneally injected with 120 mg/kg body
weight of the prepared alloxan. After 6 hours of alloxan administration, rats in their cages
were then allowed 10 % glucose solution for the next 24 hours in other to prevent
alloxan- induced hypoglycemia. The animals were observed for polydipsia, polyuria,
polyphagia as well as general reduction of body weight. Seventy two hours (three days)
after alloxan administration, the animals were fasted overnight and diabetes was
confirmed from the rats by measuring their fasting blood glucose level with the aid of a
single touch glucometer. Rats that have fasting blood glucose level >7.0 mmol/l
(126mg/dl) were considered diabetic and included in the study (Kandur and Goyal,
2005).
Simple random sampling technique was used in grouping the rats for this study. By
applying this method, rats were randomly selected and divided in to six (6) groups;
Group 1, Normal control (NC): Normal healthy rats fed on WHO recommended rat feed
pellets.
Group 2, Diabetic control (DC): Induced with 120 mg/kg body weight of prepared
Alloxan.
Group 3, Metformin treated group (MC): In Addition to 120 mg/kg body weight of
prepared Alloxan, received 500mg/kg body weight of metformin.
Group 4, Okra peel treated group (OP): In Addition 120 mg/kg body weight of prepared
Alloxan, received 300mg/kg body weight of Okra peel.
Group 5, Okra seed treated group (OS): In Addition to 120 mg/kg body weight of
prepared Alloxan, received 300mg/kg body weight of Okra seed.
Group 6, Whole Okra treated group (WO): In Addition to 120 mg/kg body weight of
prepared Alloxan, received 300mg/kg body weight of whole Okra.
Each group of the rats were housed in a labelled cage as described above, feed with
pelletized growers feed (Vital feed, Jos, Nigeria), and allowed access to water ad libitum
throughout the period of the study.
Okra sample solution (OS) was administered orally every morning by intubation using
intravenous cannula tube at doses of 100, 200 and 300 mg/kg body weight to the
respective rats in their respective groups by single forced oral feeding once per day for a
period of 21 days. The rats in the Metformin group were administered with 500 mg/kg
body weight Metformin.
24 hours after the last treatment, the animals were subjected to 12 hours fasting after
which the animals were anaesthetized by dropping each individual animal in a plastic jar
saturated with chloroform vapour. The animals were then removed from the jar and blood
samples collected from them through cardiac puncture into labelled plastic specimen
sample bottles containing EDTA (disodium ethylinediamine tetraacetate) for hemoglobin
and glycated hemoglobin assay; the remaining blood was collected into plastic centrifuge
tubes without anti-coagulant and were allowed to clot then centrifuged at 4000 g for ten
minutes. The sera obtained were pipette into labeled specimen test tubes for estimation of
serum glucose and lipid profiles
Serum glucose was estimated by glucose oxidase/ peroxidase method using Randox kit
(Trinder, 1969).
Principle
Glucose was determined after enzymatic oxidation in the presence of glucose oxidase.
The hydrogen peroxide formed reacts, under catalysis of peroxidase, with 4-
aminophenazone and phenol to produce a red-violet colored quinoneimine complex that
can be measured spectrophotometrically at 500nm. (Glucose oxidase catalyses the
oxidation of glucose to hydrogen peroxide and glutamic acid). In the presence of
peroxidase, H2O2 is broken down and the oxygen released is reacted with 4-
aminophenazone and phenol to produce a red-violet (pink) colored quinoneimine
complex that can be measured spectrophotometrically at 500nm).
Serum (µl) - 10 -
Standard - - 10
glucose(µl)
10µl of serum, standard (5.5 mmol/L) and distilled water were respectively pipette in to
the test tubes. Each test tube is then followed by 1000 µl of the reagent as shown above.
The tubes were mixed properly, incubated at 37oC for 10 minutes and the absorbance of
standard and tests read against the blank at 500nm using spectrophotometer.
Calculation: The glucose concentration was calculated using the relation:
Absorbance of Test
Serum glucose (mmol/L) = Absorbance of Standard
Serum total cholesterol (TC) was estimated by enzymatic method using Randox kit
(Allain et al., 1974).
Principle
The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator
quinoneimine is formed from hydrogen peroxide and 4 – aminoantipyrine in the presence
of phenol and peroxidase. The absorbance of the dye is measured spectrophotometrically
at 500nm. (Cholesterol ester in serum is hydrolyzed by cholesterol esterase. The Total
cholesterol is then oxidized by cholesterol oxidase to the corresponding ketone. The H 2O2
formed is decomposed by peroxidase in the presence of 4 – aminoantipyrine and phenol
to yield a quinoneimine dye. The absorbance of the dye was measured
spectrophotometrically at 500nm is proportional to the concentration of cholesterol).
The equations for the reaction are:
Cholesterol ester + H2O Cholesterol esterase
Cholesterol + fatty acids
Cholesterol + O2 Cholesterol oxidase
Cholestene – 3 – one + H2O2
2H2O2 + Phenol + 4 – aminoantipyrine proxidase Quinoneimine + 4H2
Procedure
Three test tubes were set up and labeled blank, test and standard as follow:
Blank Test Standard
Serum (µl) - 10 -
In to test tubes labeled test, standard and blank, 10 µl of serum, standard (200 mg/dl) and
distilled water were respectively pipette in to the test tubes. Each test tube is then
followed by 1000 µl of the reagent as shown above.
The test tubes were mixed, incubated at 37oC for 5 minutes and the absorbance of the
standard and test were read against the blank at 500 nm against the reagent blank.
Calculation
Cholesterol concentration was obtained using the relation:
Absorbance of Test
Serum total cholesterol (mg/dl) = Absorbance of Standard x Conc of Standard
This was done by enzymatic method of Burstein et al., (1970) using Randox Kit
Principle
Low density lipoproteins, very low density lipoproteins and chylomicron fractions are
precipitated quantitatively by the addition of phosphotungastic acid in the presence of
magnesium ions. After centrifugation, the cholesterol concentration in the high density
lipoprotein fraction, which remains in the supernatant, is determined
spectrophotometrically at 500 nm.
Procedure
Into centrifuge tubes, 200 µl of serum and 500 µl of precipitant (0.55 mmol/L
phosphotungastic acid and 25 mmol/l Magnesium Chloride) were added, mixed and
allowed to stand for 10 minutes at room temperature. The tubes were centrifuged for 10
minutes at 4000 rpm. The supernatant was collected and used for the analysis.
Three test tubes were then set up and labelled blank, standard and test, as follows:
Blank Standard Test
Standard - 100 -
supernatant(µl)
The tubes were mixed and incubated for 5 minutes at 37 0 C and the absorbance of the
samples and standard were measured against the reagent blank at 500nm.
Calculation
The HDL-C concentration was obtained from the relation:
Absorbance of Test
Serum HDL-C (mg/dl) = Absorbance of Standard x Conc. of Standard
Serum (µl) - 10 -
Standard triglyceride - - 10
(µl)
The tubes were mixed and incubated at 37 oC for 5 minutes and the absorbance of the
standard and tests were read at 500nm against the blank.
Absorbance of Test
Serum TG (mg/dl) = Absorbance of Standard x Conc. of Standard
The results were expressed as mean ± S.D. The data were analyzed using the
Independent students t-test was used to compare means, and values were
Results
The results of the effect of treatment with Okra on body weigh changes are presented in
Table 1. The result indicated that alloxan injection resulted in drastic decrease in body
weight of the animals compared with that of the normal control. The results of the
repeated treatment with Okra and Metformin for three weeks resulted in increase in body
weight of all the treated rats. During the weekly body weight observation of the treated
and untreated diabetic rats, there was no improvement in the observed body weight
changes in the entire treated groups in comparison with the normal control rats after the
first week of treatment with Okra peel, seed and whole (each in doses of 300 mg/kg)
respectively. Same thing applied to the Metformin control group treated with 500mg/kg
Metformin (table 3). However, the effect of repeated treatment for 14 days resulted in
considerable increases in body weight in all the animals treated with Okra fruit and
Metformin. This progress continued up to the last day (day 21) of the experimental period
where most of the individual animals were observed to regain their initial body weight.
On the other hand, there was progressive decrease in body weight of the diabetic
untreated rats as compared with that of the normal control and the entire treated groups
(table 1).
Table 3: Table 3: Effect of Administration of Different Parts of the Okra Fruit on
Body Weight Changes in rats
GRP Body Weight Body Weight After Alloxan Induction (g) Before Alloxan
Induction (g) Initial day 7th Day 14th Day 21st Day
Key:
Values are expressed as mean ± S.E.M.
Key: NC: Normal Control, DC: Diabetic Control, MC: Metformin Control (500 mg/kg)
body weight of Metformin; OP: Okra peel, OS: Okra seed, OW: Whole Okra (each 300
mg/kg) body weight of Okra Peel, Seed and Whole respectively.
4.2: Effect of Administration of Okra Fruit on Serum Glucose level
The results of the effect of treatment with Okra fruit on serum glucose is presented in
Table 2. Generally, the results indicated significant increase (p<0.05) in the levels of
serum glucose levels in the diabetic untreated group (DC) as compared with the normal
control group (NC).
Results of the effect of treatments with Okra fruit for three weeks resulted in significant
(p<0.05) decrease in serum glucose levels as compared to the diabetic untreated group
(DC). The serum glucose level ranged from (5.84±0.12 to 7.78±0.65 mmol/l) which are
significantly (p<0.05) lower than that of the diabetic untreated group (DC) where the
serum glucose level was found to be (14.50±1.21 mmol/l). Again, the result showed that,
there was no significant (p>0.05) difference in serum glucose level in the normal control
group and the Metformin control group. However, OP treated group showed the highest
hypoglycaemic effect with (5.84±0.12 mmol/l) serum glucose level.
Glu/Grps NC DC MC OP OS OW
Values are expressed as mean ± S.E.M., Mean values having different superscript letter in
the same column are significantly different at (p<0.05).
Key: NC: Normal Control, DC: Diabetic Control, MC: Metformin Control (500 mg/kg)
body weight of Metformin; OP: Okra peel, OS: Okra seed, OW: Whole Okra (each 300
mg/kg) body weight of Okra Peel, Seed and Whole respectively.
4.3: Effect of Administration of Okra fruit on Serum Lipid Profile and Atherogenic
Index in an Alloxan induced diabetic Albino rat
The results of the effect of treatment with Okra fruit on serum lipid profile and
atherogenic index is presented in Table 3. The result indicated significant increase
(p<0.05) in the levels of serum total cholesterol (TC), triglyceride (TG), very low-density
lipoprotein (VLDL-C), low density lipoprotein (LDL-C) and atherogenic index (AIX) in
the alloxan-induced diabetic untreated group (DC) as compared with that of the normal
control group (NC). In the same vain, there was significant decrease (p<0.05) in HDL-C
in the alloxan-induced diabetic untreated group (DC) as compared with the normal
control group (NC). Results of treatments with Okra after three weeks showed significant
(p<0.05) decrease in the levels of serum TC, TG, VLDL-C, LDL-C and AIX while the
serum HDL-C levels was significantly increase (p<0.05) in all the groups treated with
Okra as compared with the diabetic untreated group (DC).
The serum TC; TG; VLDL-C; LDL-C and AIX levels in OS groups, the levels ranged
from (80.81±2.48 to 90.27±2.44; 80.82±1.44 to 87.83±3.83; 16.16±0.28 to 21.36±0.76;
13.32±1.63 to 25.03±1.62 and 0.51±0.03 to 0.83±0.17 mg/dl) respectively. All of which
are significantly (p<0.05) lower than that of the diabetic untreated group (DC) where the
serum levels of the aforementioned parameters (TC; TG; VLDL-C; LDL-C and AIX)
were found to be (104.59±2.88; 94.10±2.30; 18.82±0.46; 63.09±3.25 and 2.81±0.22
mg/dl) respectively.
Table 3: Effect of Administration of Okra fruit on Serum Lipid Profile and
Atherogenic Index in an Alloxan induced diabetic Albino rat.
Values are expressed as mean ± S.E.M., Mean values having different superscript letter in
the same column are significantly different at (p<0.05).
Key: NC: Normal Control, DC: Diabetic Control, MC: Metformin Control (500 mg/kg)
body weight of Metformin; OP: Okra peel, OS: Okra seed, OW: Whole Okra (each 300
mg/kg) body weight of Okra Peel, Seed and Whole respectively.
DISCUSSION
The current study showed that fasting blood glucose level returns to normal range in
normal control (NC) rats that were re-fed for one week after fasting. But, in diabetic
control (DC) group of rats, the reverse was the case, where the fasting blood glucose
level remains significantly (p>0.05) higher than that of the normal. This is in agreement
with the findings of Renold et al., 1953; Friedmann et al., 1965; Ross, 1967; Anderson,
1974 and Nilesh et al., 2012. The increased blood glucose levels in the diabetic rats as
compared to that of the normal group could be due to the destruction of the pancreatic
cells caused by the alloxan which leads to inadequate insulin secretion and this could
results in decrease synthesis of glycogen and increase in glycogenolysis and
gluconeogenesis (Sumana and Suryawashi, 2001). The current results clearly show that
Okra peel treated group, Okra seed treated group and whole Okra treated group (OP, OS
and WO) at the doses of 300 mg/kg for one week resulted in significant (p>0.05)
decrease in fasting blood glucose level as compared with the diabetic control (DC)
groups. Similar results was also found in Metformin treated group (MC) after fasting for
the 36 hours, re-fed and treated with 500 mg/kg Metformin for one week. Although, all
the treatment with the different parts of the Okra fruit (WO, OP and OS) maintained the
blood glucose level within the normal ranges (Table 1). However, the hypoglycemic
effect was found to be more in the OP treated group followed by OS and the least was
WO group. This showed the significant antidiabetic effect of Okra fruit especially in the
Okra Peel group. This significant hypoglycemic effect might be due the ability of some
constituents of Okra fruits to stimulate insulin secretion
This study also showed significant increase (p<0.05) in the levels of serum total
cholesterol (TC), triglyceride (TG), very low-density lipoprotein (VLDL-C), low density
molc`lipoprotein (LDL-C), and atherogenic index (AIX) in the alloxan-induced diabetic
untreated group (DC) as compared with that of the normal control group (NC) , and
significant decrease in high density lipoprotein as compared with that of the normal
group. Similar results were seen in a study carried out by Pearce et al, (2008), on
the levels of lipid profile in alloxan-induced diabetic patients. They found out that
there are increased levels of total cholesterol, triglycerides and HDL in patients
with overt and subclinical hypothyoidism. Results also showed that treatments with
whole Okra after three weeks showed significant (p<0.05) decrease in the levels of serum
TC, TG, VLDL-C, LDL-C, and AIX while the serum HDL-C levels was significantly
increase (p<0.05) in all the groups treated with whole Okra as compared with the diabetic
untreated group (DC). This is in line with the work carried out by Daher CF et al,
(2006), they discovered significant decrease in total cholesterol, triglyceride, low
density lipoprotein level after daily administration of aqueous extract of Okra fruit.
However, similar research confirmed that okra pods are important vegetable source of
viscous fiber, an important dietary component to lower cholesterol (Kendall and Jenkins,
2004, kumar et al., 2010). The soluble fiber of okra helps to reduce serum cholesterol and
therefore decreases the chance of cardiovascular disease.
The study also recorded a change in body weights of the rats fed with high fats diet,
possibly due to high fats contents from the diet. This study also that treatment with Okra
fruit significantly reduced the body weights of the rats. A study has reported that increase
in body weight of rats after feeding on diets rich in fats was suggested to have induced
obesity. It also confirmed decrease in obesity after treatment with whole Okra (Lai et al.,
2016, Kim et al., 2016).
CHAPTER FIVE
5.2 CONCLUSION
The possible mechanism of antidiabetic effects of Ex-maradi Okra fruit might involve stimulation of
glycogenesis, delay intestinal glucose diffusion, increase glucose adsorption capacity and possible
regeneration of pancreatic islets cells. These mechanisms may significantly contribute to the control of
postprandial hyperglycemia that is important in management of diabetes mellitus.
Based on the findings of this study, it is concluded that the methanol extracts of selected
varieties of Abelmoschus esculentus fruit have components that have the capacity to exert
some biological activity hence, their usefulness in disease intervention. The methanol
extracts of the two varieties of Abelmoschus esculentus fruit both have components that
have the ability to lower hyperlipidemia.
5.3 RECOMMENDATIONS
Isolate bioactive components of the two potent Okra varieties and compare their effects
with available hypolipidemic drugs.
To study the hypolipidemic effect of the active components of the two potent varieties at
molecular level.