HYPOGLYCEMIC EFFECTS OF SOME KENYAN PLANTS USED
TRADITIONALLY IN THE MANAGEMENT OF DIABETES MELLITUS IN
GACHOKA DIVISION, MBEERE DISTRICT
Njagi Joan Murugi
A thesis submitted in partial fulfillment of the requirement for the award of the
degree of Master of Science (Biotechnology) of Kenyatta University.
U IV .. T 18 AR
June 2006
Njagi, Joan Murugi
Hypoglycemic effects
of some Kenyan plants
Declaration
This thesis is my original work and has not been presented for a degree or any
other award in any other university or any other institution.
Joan Murugi Njagi (Bsc, Hons)
Signatur~~.t: Date ...L1.J..~.~.f.o.G
We confirm that the candidate carried out the work reported in this thesis under
our supervision.
Dr. J.J.N. Ngeranwa, PhD
Department of Biochemistry and Biotechnology,
~:::eumversl ilatef1!cJ6f~
Prof. W.M. Njue, PhD
Department of Chemistry,
Kenyatta University.
Signature . ,,/l?b ~. Date /~ .
Prof. E.N.M. Njagi, hD
Department of Biochemistry and Biotechnology,
Kenyatta University.
Signature;:;: ~ Date..141.ClbIIJk .
Prof P.K. Gathumbi, PhD
"Department of Veterinary Pathology, Microbiology and Parasitology,
College of Agriculture and Veterinary Sciences (CAVS),
University of Nairobi. ,I 1
Signature~ S=uu DateuJr/cl.jOb
II
Dedication
I dedicate this work to my parents Samuel Karugu and Margaret Karugu whose
love and support have made me what I am today. To my brothers Jim Murithii and
Martin Mugendi and sister Diana Nyakio who have always been here for me.
111
Acknowledgement
This work could not have been possible without the following persons and institutions
whom I sincerely acknowledge: -
I recognize, with appreciation the great support and tremendous encouragement
from my supervisors Dr. Joseph IN Ngeranwa, Prof Wilson N Njue, Prof Eliud N.M
Njagi and Prof Peter .K. Gathumbi who ensured that all the work went smoothly and
were always available for consultations.
I thank the Catholic German Organization K.A.A.D for their partial scholarship
that enabled me to finish my studies.
I sincerely thank the Chairpersons of Departments of Chemistry and Biological
sciences at Kenyatta University who provided me with the necessary laboratory
materials. I also thank: Messrs Salim Mwatsahu and Maina the Chief technicians of
chemistry Department, who assisted with the trace element analysis by AAS technique.
Mr Solomon Bureti , a technician of Department of Biochemistry and Biotechnology
department, who assisted in breeding and taking care of the laboratory mice.
The director of National Museums of Kenya. Mr .Kisangau and Mr. Wayaya of
National Museums for their unconditional help they provided during freeze drying of the
I'
plant extracts. Dr. Michael Otieno of School of Health Sciences for his involvement and
encouragement through out the project. I thank the director of the Institute of Nuclear
Science University of Nairobi Mr D.Maina together with his staff for their enormous
support in carrying out EXDRF trace element analysis. Mrs. Rose Gitari and Mr. Jackson
Gachoka of Kabete campus for photographing the different histological sections. Mr.
Mugeki for providing folklore literature during collection of the plant samples. I also
IV
thank Mr. Kibiti and Mrs. Eunice parents to Cromwell and Mathew and my parents Mr.
and Mrs. Karugu for the financial, moral and technical support they gave me during this
study.
My fellow student colleagues Dickson, Asman and George for their
encouragement and support. My friends and roommates Mercy, Ruth and Stella who
gracefully put up with my long working hours. Lastly I sincerely thank Mathew Piero
Ngugi and Cromwell M. Kibiti, my colleagues for their moral support from the start to
the end of this work. I finally thank the almighty GOD for keeping me alive and healthy
to do this research.
v
Abstract
Diabetes mellitus is a chronic disorder caused by inherited and/or acquired
deficiencyin production of insulin by the pancreas or by the ineffectiveness of the insulin
produced by the target cells. Diabetes mellitus is characterized by high levels of glucose
in blood, which in turn damages many of the body systems particularly the blood vessels
and nerves. There are two forms of diabetes mellitus: type I diabetes mellitus and type II
diabetes mellitus. Most conventional therapies for the management of type 2 diabetes
include oral hypoglycemic drugs, exercise, diet and physical intervention therapies such
as Acupuncture. Insulin is used in the management of type 1 diabetes mellitus. Insulin
and oral hypoglycemic drugs are expensive and have numerous side effects. Through
ages different communities have used medicinal herbs for diabetes mellitus management.
Today herbal remedies are gaining popularity because the efficacy of conventional
medicine is on the wane. This study was designed to bioscreen 7 aqueous medicinalplant
extracts traditionally used to manage diabetes mellitus and assess their safety.
Ethnobotanical and pharmacological information on the seven plants was gathered from
the traditional healers. The plants collected from Mbeere district of Eastern province
were Caesalpinia volkensii, Vernonia lasiopus, Carissa edulis, Ficus sycomorus, Kleinia
squarrosa, Azadirachta indica and Helichrysum odoratissimum. All of them showed
appreciable degree of hypoglycemic activity. Analysis of these plants the for presence of
trace elements showed that they contained varying amounts of Magnesium, Iron, Nickel
Copper, Zinc, Strontium Molybdenum, Lead, Manganese, Chromium and Vanadium.
Nickel and Strontium were present in one plant extract, two plant extracts had
Manganese, and four plants extracts had Molybdenum. Chromium and Copper were
present in six plant extracts and all the seven plants contained Iron, Lead and Magnesium.
All plant extracts had undetectable quantities of Vanadium. Of the phytochemicals tested
in the seven plants extracts, one plant had bound antraquinones, two had alkaloids, sterol
and triterpenes; three had saponins, four had flavonoids, and five had flavonols, flavones
chalcones and tannins. Free antraquinones were not present in any plant extract. Trace
elements and phytochemical are associated with both the blood glucose lowering effect
and toxicity. Toxicity of single plant extracts is reduced by the practice of using a
combination of different plants extracts by the traditional healers. The study has
established that the plants under study are effective and safe as antidiabetic medicines.
Vl
Declaration
Dedication
Acknowledgement
Abstract
Table of contents
List of tables
List of figures
List of plates
List of appendices
CHAPTER ONE
1.1
1.2
1.3
I' 1.3.1
1.3.2
1.3.3
1.3.3.1
1.3.3.2
1.3.4
1.3.5
1.3.6
Table of contents
11
III
lV
Vl
Vll
Xlll
XlV
xv
XVl
Introduction and literature review
Introduction
1
1
1
Epidemiology, prevalence and incidence of diabetes mellitus 5
Types of diabetes mellitus 8
Type I diabetes mellitus 8
Type II diabetes mellitus 9
Other forms of diabetes 11
Brittle diabetes 11
Gestational diabetes meliitus(GDM) 12
Causes of diabetes mellitus 13
Symptoms of diabetes mellitus 15
Complications of diabetes mellitus 16
Vll
1.3.6.1 Diabetic retinopathy 16
1.3.6.2 Diabetic ketoacidosis (DKA) 17
1.3.6.3 Angiopathy 18
1.3.6.4 Nephropathy 19
1.3.6.5 Hyperglycemia 19
1.3.6.6 Diabetic neuropathy 20
1.3.6.7 Diabetic Foot Disease 20
1.3.6.8 Metabolic disorders 21
1.3.6.8.1 Hypoglycemia 21
1.3.6.8.2 Arteriosclerosis 22
1.3.6.8.3 Lactic acidosis 22
1.3.6.8.4 Hyperosmotic coma 23
1.4 Diagnosis of Diabetes mellitus 24
1.4.1 Functional tests 24
1.4.1.1 Postprandial plasma glucose 24
1.4.1.2 O'sullivan test 25
1.4.1.3 Oral glucose torelance test 25
I'
1.4.1.4 Intravenous glucose torelance test 26
1.5 Management of diabetes mellitus 26
1.5.1 Conventional therapy 26
1.5.1.1 Insulin 26
1.5.1.2 Oral hypoglycemic drugs 30
1.5.1.2.1 Sulfonylureas 31
Vlll
1.5.1.2.2 Biguanides 31
1.5.1.2.3 a-glucosidase inhibitors 31
1.5.1.2.4 Meglitinides 32
1.5.1.2.5 Thiazolidinediones 32
1.5.1.3 Supplements with glucose-lowering effects 34
1.5.1.3.1 Zinc 35
1.5.1.3.2 Manganese 36
1.5.1.3.3 Chromium 37
1.5.1.3.4 Vanadium 37
1.5.1.3.5 Magnesium 37
1.5.1.3.6 Potassium 38
1.5.1.3.7 Inositol 38
1.5.1.3.8 a-lipoic acid and y-linolenic acid 38
1.5.1.3.9 Carnitine 39
1.5.1.3.10 Fiber 39
1.5.1.3.11 Antioxidants 39
1.5.1.3.12 Biotin 40
I'
1.5.1.4 Exercise 40
1.5.1.5 Acupuncture 41
1.5.1.6 Herbal Management of Diabetes Mellitus 42
1.6 Justification 52
1.7 Hypothesis 53
1.8 Main objective 53
IX
1.8.1 Specific objectives 53
CHAPTER TWO
Materials and methods 55
2.1 Collection of plant materials 55
2.2 Preparation of Extracts 55
2.2.1 Preparation of Extracts for Injection into Mice 56
2.3 Pharmacological Testing 56
2.3.1 Animals 56
2.3.2 Collection of blood samples 57
2.3.3 Blood Glucose Determination 57
2.4 Phytochemical screening 58
2.4.1 Alkaloids 58
2.4.2 Sterols and Terpenoids 58
2.4.3 Saponins 59
2.4.4 Flavonoids 59
2.4.5 Tannins 59
2.4.6 Anthraquinone 59
I' 2.5 Trace Elements Determination 60
2.5.1 Energy Dispersive X-ray Fluorescence (EDXRF) 60
2.5.2 Atomic Absorption Spectrophotometry (AAS) 60
2.5.2.1 Preparation of Standard Solutions 61
2.5.2.2 Preparation of working standards 61
2.5.2.3 20% Hydrochloric Acid (w/w) 61
x
2.5.2.4 Lanthanum Solution 62
2.5.3 Digestion of plant materials 62
2.5.3.1 Total Elemental Content Determination 63
2.6 Preliminary in vivo toxicity assay 64
2.6.1 Histopathological Examination of Tissues 64
2.7 Data analysis 65
CHAPTER THREE
Results 66
3.1 In vivo hypoglycemic activity assay 66
3.1.1 Effect of Ficus sycomorus on Blood Glucose in alloxan-induced diabetic
rmce 66
3.1.2 Effect of Caesalpinia volkensii on blood glucose in alloxan-induced
diabetic mice 69
3.1.3 Effect of Carissa edulis on Blood Glucose in alloxan-induced diabetic
Mice 72
3.1.4 Effect of Kleinia squarrosa on blood glucose m alloxan-induced diabetic
rmce 75
3.1.5 Effect of Helichrysum odoratissimum on blood glucose in alloxan-induced
diabetic mice 78
3. 1.6 Effect of aqueous leaf extracts of Azadirachta indica on blood glucose in
alloxan-induced diabetic mice 81
3.1.7 Effect of aqueous leaves extract of Vernonia lasiopus on blood glucose in
alloxan- induced diabetic mice 84
3.2 Trace metal analysis 87
3.3 Classes of Compounds in the aqueous Plant extracts 90
Xl
4.1
4.2
4.3
3.4 Preliminary in vivo toxicity
CHAPTER FOUR
92
Discussion, Conclusion and Recommendations
Discussion
Conclusion
Recommendations
References
Appendix
95
95
101
102
104
122
XlI
List of tables
Table 1: Effects of Ficus sycomorus extract on blood glucose levels in alloxan induced
diabetic mice 67
Table 2: Effects of Caesalpinia volkensii extract on blood glucose levels in alloxan
induced diabetic mice 70
Table 3: Effects of Carissa edulis extract and insulin on blood glucose levels in alloxan
induced diabetic mice 73
Table 4: Effects of Kleinia squarrosa extract on blood glucose levels in alloxan induced
diabetic mice 76
Table 5: Effects of H. odoratissimum extract on blood glucose levels in alloxan induced
diabetic mice 79
Table 6: Effects of Azadiracta indica extract on blood glucose levels in alloxan induced
diabetic mice 82
Table 7: Effects of Vernonia lasiopus extract on blood glucose levels in alloxan induced
diabetic mice 85
Table 8: Trace metals present in the hypoglycemic plants as analyzed by EDXRF 89
Table 9. Trace metals present in the hypoglycemic plants as analyzed by AAS 90
Table 10: Phytochemistry of the seven medicinal plants 91
Xlll
List of Figures
Figure 1: Percentage reduction in blood glucose by varying doses of Ficus sycomorus in
diabetic mice 68
Figure 2: Percentage reduction in blood glucose by varying doses of Caesalpinia
volkensii in diabetic mice 71
Figure 3: Percentage reduction in blood glucose by varying doses of Carrisa edulis in
diabetic mice 74
Figure 4: Percentage reduction in blood glucose by varying doses of Kleinia squarrosa in
ili~~cmi~ n
Figure 5: Percentage reduction in blood glucose by varying doses of Il.odoratissimum in
diabetic mice 80
Figure 6: Percentage reduction in blood glucose by varying doses of Azandiracta indica
in diabetic mice 83
Figure 7: Percentage reduction in blood glucose by varying doses of Vernonia lasiopus in
diabetic mice 86
XlV
List of Plates
Platel:Histological section of skeletal muscle of a mouse treated with an aqueous stem
bark extract of Ficus sycomorus 93
Plate 2:Histological section of normal spleen of a mouse treated with normal saline 94
Plate3: Histological section of a spleen of a mouse treated with an aqueous stem
bark extract of Helichrysum odoratissimum 94
xv .
List of Appendices
Appendix 1: Instrumental conditions for Atomic Absorption Spectrophotometer
(AAS)
Appendix 2: Standard Calibration Curve for Magnesium
Appendix 3: Standard Calibration Curve for Chromium
xvi
122
123
124
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
Diabetes mellitus is a chronic endocrinologic disorder caused by inherited and/or
acquired deficiency in production of insulin by the pancreas or by the ineffectiveness of
the insulin produced. It is characterized by high blood levels of glucose, which in turn
damages many of the body systems particularly the blood vessels and nerves (Chen,
1998; WHO, 2003). This is caused by disturbances in the regulatory systems responsible
for the storage and utilization of the chemical energy from food. This includes the
metabolism of carbohydrates, fats and proteins resulting from defects in insulin secretion,.
insulin action, or both (Shillitoe, 1988; Votey and Peters, 2004).
According to WHO, more than 190 million people suffer from diabetes mellitus
worldwide. The disease incidence is increasing rapidly and it is estimated that the figure
will double by the year 2025. Most people with diabetes in developed countries will be
aged 65 years or more by year 2025 yet, in developing countries the affected age bracket
will be in the 45-65 year and in their most productive years (WHO, 2003). It is expected
that the prevalence of diabetes will continue to increase in Africa and Asia as a result of
changes in lifestyles and urbanization (Diabetes Control and Complications Trial
Research Group, 1993). 49.0 million people suffer from the disease in South-East Asia;
India alone accounts for almost a quarter of all patients in this region, with an estimate of
15 million people (Agrawal, 2004).
1
\
In the European continent an estimated 32.2 million patients suffer from diabetes;
over a million of the cases being from United Kingdom alone (Effective Health Care,
1999). This figure accounts for over 2% of the UK population who suffer from the
disease (CaIman, 1998). Demographically, the Northern American continent has
approximately 21.4 million. Latin America has an estimated 12.6 million diabetic patients
(Harris et aI., 1987). In the United States diabetes mellitus is the third leading cause of
death after heart disease and cancer and it affects approximately 17 million adults (Voet
and Voet, 1995; Collins, 2002). In Australia it is a disease of modern society and it is
estimated that 700,000 people are diabetic (Murtagh, 1999). In 2000 there were 7.5
million cases of diabetes in Africa and the figure is expected to rise to around 18.2
million by the year 2030 with about 190,000 sufferers from Kenya alone (Mngola, 2004).
All African countries are struggling to care for a large number of diabetic patients, yet
more than 80% of the cases are undiagnosed (Mngola, 2004). In Kenya the prevalence
rate of diabetes is estimated at 3-10 % and 15% of this are people below 30 years.
Diabetes affects up to 10 % of Kenyans, a percentage that could be higher as most cases
of type 2 diabetes are diagnosed many years after onset (Njenga, 2005).,-
The effects of diabetes mellitus include long-term damage and failure of various
organs, progressive development of the specific complications such as retinopathy which
leads to blindness, nephropathy which may lead to renal failure, and/or neuropathy which
may cause foot ulcers, amputation, and autonomic dysfunction, including sexual
dysfunction. People with diabetes are at increased risk of cardiovascular, peripheral
vascular and cerebrovascular diseases. Several pathogenetic processes are involved in the
2
development of diabetes. These include destruction of f3-cells of the pancreas that lead to
decreased sensitivity to insulin action (WHO, 1999; Votey and Peters, 2004).
Economic aspects of diabetes and diabetes care currently attract considerable
attention as the world diabetes epidemic takes hold and the healthcare activities of
countries come under pressure to accomplish more within constrained resources
(Sobngwi et al., 2001). Diabetes mellitus is a very expensive disease and has profound
implications in terms of long-term microvascular and macrovascular complications and
their associated cost. These complications reduce both life expectancy and quality of life
(Ashcroft and Ashcroft, 1992; Collins, 2002; Votey and Peters, 2004).
Diabetes mellitus appears in two forms: type 1 diabetes mellitus or insulin
dependent diabetes mellitus (IDDM) and type 2 or non- insulin dependent diabetes
mellitus (NIDDM) (Voet and Voet, 1995). Type 1 is also referred to as juvenile-onset
diabetes, occurring frequently in the early teen years in many patients. Type 1 diabetes
mellitus is an inherited defect of the immune system triggered by environmental stimuli.
The disease is characterized by the presence of autoantibodies to the pancreatic f3-cells
that produce insulin. The f3-cells are inflammed and destroyed in this disease (Voet and
,-
Voet, 1995). Type 2 diabetes mellitus is a metabolic disorder resulting from the body's
inability to produce sufficient, or to properly use, insulin. Patients with type 2 diabetes
mellitus have either relatively low insulin production or insulin resistance, and
occasionally require insulin administration (WHO, 1999).
Type 1 diabetes mellitus requires treatment with insulin injections, which
involves injecting insulin under the skin in the fat for it to get absorbed into the blood
3
stream where it can then access all the cells of the body, which require it. The cost of
insulin in Kenya is very high with the average cost of insulin vials being Ksh 1,500 to
2,000, which is out of reach of the 10 -income earners (Foster, 1974; Baron et al., 2002;
Njenga, 2004)). In type 2 diabetes mellitus, oral hypoglycemic drugs are used to control
hyperglycemia.
In developing countries, traditional herbal medicines have continued to play an
important role side by side with modem medicine especially in primary health care in
poorer or rural areas. Herbal preparations constitute valuable natural resources from
which chemicals of great potential for agriculture and medicine are found (Seneader,
1985).
Plant derivatives with presumed hypoglycemic properties have been used in folk
medicine and traditional healing systems globally such as Native American Indian,
Jewish Chinese East Indian and Mexican (Yaniv et al., 1987; Covington, 2001). Many
modem pharmaceuticals used in conventional medicine have also natural plant origins.
Among them metformin, derived from the flowering plant, Galega officinalis, is a
common traditional remedy for diabetes (Pandey et al., 1995; Oubre et al., 1997).,-
Similarly, the use of vitamin and mineral supplements to treat or prevent primary or
secondary disease is of increasing interest (O'Connell, 2001)
There are other forms of diabetes that include gestational diabetes, brittle
diabetes, unstable diabetes and ketosis prone diabetes. Patients suffering from the ketosis
prone diabetes have a classic symptom of ketones present in blood and urine (Guthrie and
Guthrie, 1999)
4
Greater prevalence of diabetes complications has been reported in populations of
African origins compared to Caucasians; this is because of poor compliance and or
difficult access to appropriate care and affordability of treatment in difficult
socioeconomic environments (Cowie, 1993; Goldschrnid et al., 1995; Musey et aI., 1995;
Delamater et aI., 1999).
1.2 Epidemiology, Prevalence and Incidence of Diabetes Mellitus
Epidemiological studies show that diabetes mellitus develops from a complex
interaction between environmental and genetic factors. For example, the offspring and
siblings of diabetic parents are more likely to develop diabetes than those of nondiabetic
parents; the offspring of two diabetic parents is more likely to develop diabetes than an
offspring having only one diabetic parent; the lower than expected frequencies of
diabetes in identical twins and offspring and siblings of diabetics are suggestive of the
importance of environmental factors in the expression of the genetic component for
diabetes. A second event such as a viral infection or a disturbance of the immune system
occurring early in life is postulated to trigger type 1 diabetes in genetically susceptible
individuals. Type 2 occurs without such events though its expression is modulated by
"
factors such as obesity. Inheritance plays a more important role in development of type 2
diabetes than type 1 diabetes (Kaplan and Pesce, 1996).
There is a global trend towards increases in the incidence and prevalence of
diabetes mellitus in African populations (King et al., 1998). Global estimates of the
number of people with diabetes in Africa was approximately 3 million in 1994 and is
expected to increase 2-3 fold by the year 2010 (Amos et aI., 1997). The prevalence of
5
diabetes in African communities is increasing with ageing of the population and lifestyle
changes, which are associated with rapid urbanization and westernization (King et al.,
1999). Urban residents have a 1.5-4 fold higher prevalence of diabetes compared to their
rural counterparts. Urban lifestyles in Africa are characterized by changes in diet where
subjects consume high levels of refined sugars and saturated fat and reduction in fiber
intake coupled with reduced physical activity (Sharma et al., 1996; Gill et al., 1997;
Mennen et al., 2000).
Diabetes mellitus displays a geographic, ethnic and racial prevalence especially in
type 2 diabetes mellitus. The disease is more prevalent among Hispanics, Native
Americans, African Americans and Asians Pacific Islanders than in whites. The Pima
Indians living in the Arizona in the United States of America have the highest rate of type
2 diabetes mellitus in the world. South Eastern Asians as well as the Australian
Aborigines are at a higher risk of developing non-insulin dependent diabetes mellitus
when compared to the Caucasians (Osei, 1995; Gill et al., 1997; Votey and Peters, 2004).
Type 2 diabetes mellitus, is the predominant form (70-90%); this is due to high urban
I' growth rate, dietary changes, and reduction in physical activities and increasing obesity.
It is estimated that the prevalence of diabetes is due to triple within the next 25 years.
Type 1 diabetes mellitus also displays a geographical and regional prevalence.
Estimates show that 5.3 million people live with type 1 diabetes worldwide, of which
395,000 or 7.4% are children. The disease is common in areas near the equator (Leslie
and Gale, 1995). Consumption of large quantities of cow's milk during childhood may
increase the risk of developing type 1 diabetes in children who are already genetically
6
susceptile to the disorder. Children who have sibilings with diabetes are more than five
times as likely to develop the autoimmune disorder if they drink more than half a liter
(about three glasses) of cow's milk a day, compared with children who drink less milk
(New York Health Reuters, 2000). Type 1 diabetes tends to have fewer tendencies to
have other family members affected with diabetes than type 2. In the first large family
study of diabetes, less than 4% of parents and 6% of siblings of a person with diabetes
also had diabetes. The incidence of diabetes mellitus is equal in females and males in all
populations but it is greater for blacks than whites. It is estimated that slightlymore than
218,000 persons develop type 1 diabetes worldwide annually. Of these, 86,400, or 40%
are children. The Europeans contribute 60,000 new cases annually, while the Southeast
Asian region contribute 45,000 new cases, followed by the North American region with
36,000 new cases annually. The African region contributes the lowest number of cases,
6,900 persons annually. The proportion of children among the new cases ranges from
29% in the European Region to 54% in the African Region, reflecting the combined
effect of differences in age structure and incidence levels (Kaplan and Pesce, 1996).
Type 2 diabetes is becoming increasingly common because more people are
living longer as diabetes prevalence increases with age. It is also being seen more
frequently in younger people in association with the rising prevalence of childhood
obesity. Although type 2 diabetes still occurs most commonly in adults aged 40 years and
older, the incidence of disease is increasing more rapidly in adolescents and young adults
than in other age groups (Votey and Peters, 2004).
7
1.3 Types of Diabetes Mellitus
1.3.1 Type I Diabetes Mellitus
Type I diabetes or insulin dependent diabetes mellitus (IDDM) is a chronic
autoimmune disease in which the body's own immune system attacks the ~-cells in the
Islets of Langerhans of the pancreas, destroying them or damaging them sufficiently to
reduce insulin production. It is thought that the autoimmunity against ~-cells is induced
in a susceptible individual by a foreign antigen, such as a virus that immunologically
resembles some ~-cell component (Voet and Voet, 1995).
Environmental factors, such as a virus, initiates the process of beta cell
destruction in genetically susceptible individuals. This external influence precipitates an
inflammatory response in the pancreas known as insulitis. Activated T-lymphocytes
infiltrate the islet cells in the pancreas and cause ~-cell destruction via localized release of
cytokines. Cytotoxic amounts of nitric oxide and reactive oxygen intermediates are also
released, contributing to free radical damage to the ~-cells. The initial steps in free-
radical induced islet cell death involve breaks in DNA strands and the activation of the
enzyme poly (ADP-ribose) polymerase (PARP). PARP is involved in DNA repair and
consumes large amounts of NAD+ in the process. The depletion of intracellular NAD+
pools leads to islet cell death (Dahlquist, 1994).
Infectious viral agents may also stimulate the immune system to attack its own
cells. Viruses that trigger the immune system may also penetrate the ~-cells and cause
their destruction leading to a decrease in insulin secretion by the pancreas. Other than
viruses, other environmental stimuli may stimulate the immune system to attack the islets
8
of langerhan hence their destruction. This attack on the ~-cells leads to a decline in
insulin production (Lucassen and Bell, 1995; Kaplan and Pesce, 1996; Weiss, 1997).
Type I diabetes mellitus results from the gradual disappearance of insulin production.
Although age-related, type 1 diabetes mellitus is not confined to the young or even the
middle aged but may occur rarely in the elderly. The sex distribution is equal (Shillitoe,
1988;Greens, 1995).
Type I diabetes has a genetic component which must be present for susceptibility
to occur. Although the exact mechanism is unclear, transmission is believed to be
autosomal dominant, recessive or mixed. If a first-degree relative has the disease, the
child has a 5-10 % chance of developing type- I diabetes. The susceptibility gene resides
on the short arm on the sixth chromosome, either within or in close proximity to the
major histocompatibility complex, that is, the HLA region. The major alleles conferring
risk of type I are HLA-DR3, HLA-DR4, HLA-Dw3, HLA-Dw4, HLA DQ, HLA DP,
HLA-B8 and HLA-B15 (Hawkes 1997). However, no single HLA allele or combination
is specific for susceptibility to type I. Type I is strongly associated with particular HLA
I' DQ-encoded heterodimers. On the other hand, some alleles of the major
histocompatibility complex confer protection against the development of type I. These
are HLA-DR2 and HLA-DQ B1 and they appear to have dominance over susceptibility
alleles (Nepom and Ehrlich, 1991).
1.3.2 Type nDiabetes Mellitus
Type II diabetes mellitus is a syndrome characterized by disordered metabolism
of carbohydrate and fat. This leads to hyperglycemia with fasting plasma glucose level
9
>126 mg/dL (Diabetes Care, 1997). Insulin resistance and a relative ~-cell defect are the
underlying pathological problems leading to hyperglycemia (Shillitoe, 1988; Collins,
2002)
The disease is characterized by peripheral insulin resistance in insulin targeting
tissues such as the skeletal muscle and adipocytes with an insulin secretory defect and
deficiency of insulin receptors (Votey and Peters, 2004). The type 2 diabetes mellitus is
the most common form of diabetes and it accounts for over 90% of the diagnosed cases
of diabetes (Gursche and Rona, 2000). Type 2 diabetes usually develops in middle age or
later. This tendency to develop later in life has given rise to the term "adult onset
diabetes" (Kaplan and Pesce, 1996). The typical type 2 diabetes mellitus occurs in obese
individuals with a genetic pre-disposition for this condition although the genes
responsible for the disease causation are carried on different chromosomes from the type
1 diabetes. Ttype 2 diabetes mellitus strong familial aggressions. Family studies show
that the genetic component in causing diabetes mellitus is relatively strong. Several genes
have been suggested as markers for type 2. Obesity is associated with insulin resistance
I' (Shillitoe, 1988; Frogel, 1992; Voet and Voet, 1995; Votey and Peters, 2004).
NIDDM is a growing problem among the developing countries (Moshi et al.,
2000). There is probably a social class bias in the prevalence of the type 2 diabetes with
the disorder being more common in socially disadvantaged groups (Shillitoe, 1988). This
is due to the changing socio-economic factors, which in turn affect the dietary and living
conditions of the people (Moshi et al., 2000)
10
The genetic factor in addition to some environmental factors such as aging, excess
caloric intake followed by deficient caloric expenditure and obesity may result to insulin
resistance. This makes the body produce more insulin to overcome the resistance. The
resistance to the action of insulin is associated with excessive production of glucose by
the liver and impaired glucose utilization by peripheral tissues especially the muscles.
Overtime, chronic stimulation of insulin secretion diminishes and the islet ~-cell reserve
is exhausted. Clinical diabetes is as a result of a state of absolute insulin deficiency.The
production of more insulin to overcome resistance leads to either exhaustion of the ~-
cells resulting in insulin deficiency or a condition known as glucotoxicity. Sugar in high
amounts may be toxic to cells of the body. The toxicity damages the ~-cells and a decline
in insulin secretion ensues (De Fronzo, 1987; Seely and Olesky, 1993; Olesfsky, 1999;
Guthrie and Guthrie, 1999).
1.3.3 Other Forms of Diabetes
1.3.3.1 Brittle diabetes
Brittle diabetes is frequently characterized by very frequent and extreme oscillations
I' between hypo and hyperglycemia (Elber et al., 1997). It is an uncommon complicationof
type 1 diabetes but the seriousness of the complication and demands on the health care
systems warrants aggressive intervention. Brittle diabetes is always secondary to a
specific, identifiable etiology. There are many different causes of brittle diabetes, but the
most common is a psychological abnormality (Elber et al., 1997).
11
1.3.3.2 Gestational Diabetes Mellitus (GDM)
Gestational diabetes mellitus (GDM) IS defined as any degree of glucose
intolerance with onset of pregnancy (Votey and Peters, 2004). It occurs when the
pregnant woman's body cannot produce enough insulin, resulting in high blood sugar.
GDM affects an estimated 2-3 % of pregnant women (WHO, 1999;Wikipedia the free
encyclopedia, 2004). It is estimated that 39 % of women with gestational diabetes
manifest type 2 diabetes mellitus later in life (Kaplan and Pesce, 1996).
This disease also leads to higher rates of cesarean delivery and chronic
hypertension (Votey and Peters, 2004). Generally a test for gestational diabetes is
undertaken between the 24th and 28th week of pregnancy. Often, gestational diabetes can
be managed through a combination of diet and exercise. If that is not possible, it is treated
with insulin, in a similar manner to diabetes mellitus (Guthrie and Guthrie, 1999; WHO,
1999).
Pregnancy may favor the occurrence or aggravation of diabetic retinopathy and
neuropathy in patients with pre-existing diabetes (Boivin et al., 2002). The mother is also
I'
at a risk of developing toxemia and eclampsia (Guthrie and Guthrie, 1999). Fetuses from
pregnancies with gestational diabetes have a high risk of macrosomia, asphyxia, neonatal
hypoglycemia and hyperinsulinaemia, excessive fat accumulation, insulin resistance,
pancreatic exhaustion and possible risk of child and adult obesity and type 2 diabetes
mellitus later in adult life (Glueck et aI., 2002).
The pathophysiology of gestational diabetes includes insulin resistance and
decreased insulin secretion (Boivin et aI., 2002). Although glucose tolerance normalizes
12
shortly after pregnancy, with gestational diabetes in the majority of women the risk of
developing type 2 diabetes is especially high (Vambergue et al., 2002). The risk factors
involved in gestational diabetes include a family history of type 2 diabetes. Maternal age
is also a risk factor, the risk increasing with the age of the woman (Vambergue et aI.,
2002).
1.3.4 Causes of Diabetes Mellitus
Diabetes mellitus can be caused by use of drugs that are toxic to the ~-cells and
cause drug-induced diabetes. These drugs include alloxan, streptozocin, dilantin, thiazide,
pentamidine and a-interferon therapy. High doses of glucocorticoides, such as steroids,
some cancer chemotherapeutic agents especially L-asparagines, antipsychotics and mood
stabilizers (phenothiazines) are also known to cause diabetes mellitus. Many drugs can
impair insulin secretion. They elevate blood sugar level through various mechanisms
(WHO, 1999).
These drugs may not, by themselves, cause diabetes but they may precipitate
diabetes in persons with insulin resistance (phelps et al., 1989; O'Byrne and Feely, 1990;
I'
Pandit et al., 1993; Votey and Peters, 2004). Certain toxins such as Vacor a rat poison
and pentamidine can permanently destroy pancreatic ~-cells (Gallanosa et al., 1981;
Esposti et aI., 1996; WHO, 1999). Hormones can also impair insulin action. Such
hormones include thyroid hormone, a-adrenergic agonists, ~-adrenergic agonists, growth
hormone, cortisol, glucagons and epinephrine, which antagonize insulin action. Diseases
associated with excess secretion of these hormones can cause diabetes, for instance,
acromegaly, Cushing's syndrome, glucagonoma and phaeochromocytoma (phelps et al.,
13
1989; Mac Farlane, 1997; WHO, 1999; Wikipedia the free encyclopedia, 2004). Diseases
of the exocrine pancreas and processes that diffusely injure the pancreas can cause
diabetes. Acquired processes include pancreatitis, trauma, infection, pancreatic
carcinoma, and pancreatectomy (Larsen et al., 1987; Gullo et al., 1994). With the
exception of cancer, damage to the pancreas must be extensive for diabetes to occur.
However, adenocarcinomas that involve only a small portion of the pancreas have been
associated with diabetes. Somatostatinoma, and aldosteronoma-induced hypokalaemia,
can cause diabetes, at least in part by inhibiting insulin secretion (Conn, 1965; Krejs et
al., 1979).
Certain viruses have been associated with p-cell destruction. Diabetes occurs in
some patients with congenital rubella viruses that include Coxsackie B, cytomegalovirus
and other viruses (Forrest, 1971). Adenovirus and mumps have been implicated in
inducing the disease (pak et al., 1988; King et al., 1998). Many genetic syndromes are
accompanied by an increased incidence of diabetes mellitus. These include the
chromosomal abnormalities of Down's syndrome, Klinefelter's syndrome and Turner's
I' syndrome. Wolfram's syndrome, an autosomal recessive disorder characterized by
insulin-deficient diabetes and the absence of p-cells at autopsy, also causes
hyperglycemic states and if the glycemic status is prolonged it leads to permanent
diabetes. Other genetic syndromes sometimes associated with diabetes are Friedreich's
ataxia, Huntington's chorea, Lawrence-Moon-Biedel syndrome, myotonic dystrophy,
porphyria and Prader-Willi syndrome (Barrett et al., 1995).
14
1.3.5 Symptoms of Diabetes Mellitus
Type 2 diabetes mellitus has a slow onset often taking years, but in type 1,
particularly in children, onset may be quite fast within weeks or months. Early symptoms
of type 1 diabetes are polyuria, polydipsia and consequent increased fluid intake. There
may also be weight loss despite normal or increased eating, increased appetite and
irreduceable fatigue. These symptoms may also manifest in type 2 diabetes in patients
who present poorly controlled diabetes. Thirst develops because of osmotic effects, that
is, sufficiently high glucose above the renal threshold in the blood is excreted by the
kidneys but this requires water to carry it and causes increased fluid loss, which must be
replaced. The lost blood volume will be replaced from water held inside body cells,
causing dehydration (WHO ,1999).
Another common presenting symptom is altered VISIon. Prolonged high blood
glucose causes changes in the shape of the lens in the eye, leading to blurred vision.
Especially dangerous symptoms in diabetics include the smell of acetone on the patient's
breath (a sign of ketoacidosis), a rapid, deep breathing, and an altered state of
consciousness or arousal. Hostility and mania are both possible, as is confusion and
lethargy. The most dangerous form of altered consciousness is the so-called "diabetic
coma" which produces unconsciousness. Early symptoms of impending diabetic coma
include polyuria, nausea, vomiting and obdominal pain, with lethargy and somnolence, a
later development, progressing to unconsciousness and death if untreated. Irritability,
skin infections or sores that take long to heal and vaginal infections in women are also
some common symptoms of diabetes mellitus (Dierkx et aI., 1996; WHO, 1999). These
15
symptoms affect the quality of life of an individual and when severe can lead to a
decrease in work performance in adults and an increase in the number of falls in the
elderly (Davison, 1991).
1.3.6 Complication of Diabetes Mellitus
Diabetes mellitus is a systemic disease that affects every organ of the body.
Cardiovascular and renal lesions are the most common abnormalities leading to death.
1.3.6.1 Diabetic retinopathy
Diabetic retinopathy is a leading cause of blindness and visual disability (WHO,
2003). It is the most common cause of blindness in people of working age in
industrialized countries (Williams, 1994). Up to 40% of people have some retinopathy
when type 2 diabetes is first diagnosed (Diabetic Retinopathy Study Research Group,
1998). Twenty years after diagnosis, all of those with type 1 diabetes and 60% with type
2 diabetes have some degree of retinopathy (Diabetic Retinopathy Study Research Group,
1998). In diabetic retinopathy, small blood vessels in the retina (back of the eye) become
blocked, swollen or leaky, causing edema, and new, fragile vessels grow haphazardly in
I'
the retina. This process continues for years without causing visual symptoms or visual
impairment; during this period, retinopathy is only detected by eye examination. If it is
left untreated, bleeding and scarring leads to progressive loss of vision (Early Treatment
Diabetic Retinopathy Study Research Group, 1991; Kaplan and Pesce, 1996; Diabetic
Retinopathy Study Research Group, 1998).
16
1.3.6.2 Diabetic ketoacidosis (DKA)
Diabetic ketoacidosis (DKA) is one of the severe complications of diabetes
mellitus. DKA is a medical emergency and if left without prompt proper treatment,
patients have substantial chance of death. Before the era of insulin, DKA was the leading
cause of patient deaths (Beigelman, 1971; WHO, 1999; Wikipedia the free encyclopedia,
2004). DKA is characterized by high blood sugar, or hyperglycemia. The complication
begins with physiologic stress that causes release of catecholamines, glucagon, and
cortisol. This stress may be emotional or physical, although the most common cause by
far is infection (e.g. , pneumonia) (American Diabetes Association, 1995.Diabetic
Retinopathy Study Research Group, 1998).
This complication is most common in type 1 diabetes mellitus where there is no
circulating insulin. In situations where there is a severe deficiency in insulin levels, the
body switches to fat metabolism, a mechanism which actually exists to protect the organs
during periods of starvation, as glucose is not available to be taken up due to lack of
insulin even though blood levels of glucose are high. Ketones are produced from fats,
I'
partly because the brain utilize ketones for energy as they can pass the blood-brain
barrier. As the level of available glucose for the brain and other organs runs low (due to
the persistent low levels of insulin despite the rising levels of serum glucose as a by
product of the fat metabolism,) more and more fats are metabolized releasing more and
more ketones (WHO, 1999).
Accumulation of these ketone bodies results in metabolic acidosis as pH buffers
in the serum are used up. Rising levels of glucose and ketones increase the osmolality of
17
the serum. the hyperglycemic state initially encourages the patient's kidneys to produce
more urine, causing the body to lose water and electrolytes such as potassium and
phosphate, leading to dehydration and hypokalemia. Treatment consists of hydration to
lower the osmolality of the blood, replacement of lost electrolytes, insulin to force
glucose and potassium into the cells, and eventually glucose simultaneously with insulin
in order to correct other metabolic abnormalities, such as elevated blood potassium
(hyperkalemia) and elevated ketone bodies. Survival is dependent on how badly deranged
metabolism is at presentation to a hospital, but the process is only occasionally fatal
(Beigelman, 1971; Lehninger et al., 1982; American Diabetes Association, 1995; Kaplan
and Pesce, 1996)
1.3.6.3 Angiopathy
Angiopathy is one of the complications of diabetes mellitus, which occurs after
the patient has had the disease for along time. It refers to the damage to linings (basement
membranes) of blood vessels. There are two types of angiopathy; one is macro-
angiopathy where fat and blood clots build upon the blood vessels and stick to the walls
,-
and block the flow of blood. The other type is micro-angiopathy where the walls of the
small blood vessels become so thick and weak that they bleed and therefore leak protein
and slow blood flow through the body. Cells such as those at the center of the eye do not
get enough blood and may be damaged. Angiopathy increases the risk of coronary heart
disease and stroke and can lead to retinopathy and nephropathy (Kaplan and Pesce, 1996;
WHO, 1999).
18
1.3.6.4 Nephropathy
Diabetes is among the leading causes of kidney failure, but the frequency varies
between populations and also due to the severity and duration of the disease (WHO,
2003). Nephropathy refers to the damage of the glomerulus and its associated capillaries.
Capillary damage is caused by angiopathy and the result is a reduction in the filtering
capacity of the kidneys. Proteinuria is the first sign of diabetes nephropathy (Kaplan and
Pesce, 1996).
1.3.6.5 Hyperglycemia
Acute hyperglycemia, even when not associated with DKA, is harmful to the body
because if the blood glucose level exceeds the renal threshold for glucose, an osmotic
diuresis ensues, with loss of glucose, electrolytes, and water. Hyperglycemia impairs
leukocyte function through a variety of mechanisms. As a result of high blood glucose
levels patients with diabetes have a low resistance to illness, an increased rate of wound
infection, and impaired wound healing (Gursche and Rona, 2000).
Management of hyperglycemia during medical illness and surgery IS important
I'
because serious medical illness and surgery produce a state of increased insulin
resistance. Blood glucose levels of 1000-2000 mg/L are maintained in medical and
surgical patients with diabetes to prevent electrolyte abnormalities and volume depletion
secondary to osmotic diuresis. This is also done to prevent the impairment of leukocyte
function and wound healing that occurs when blood glucose levels are elevated (Votey
and Peters, 2004).
19
1.3.6.6 Diabetic neuropathy
Diabetic neuropathy is the most common complications of diabetes mellitus
and occurs in 50 % of diabetics (WHO, 2003). Acute hyperglycemia decreases nerve
function and chronic hyperglycemia is associated with the loss of myelinate and
unmyelinated fibers and loss of nerve conduction. The complication is recognized by a
number of symptoms that include numbness, pain, tingling or burning sensations in the
extremities, dizziness and double vision. Other symptoms are decreased gastric motility,
erectile dysfunction, bladder dysfunction and impaired cardiac function (Kaplan and
Pesce, 1996; Didier, 2000; WHO, 2003)
1.3.6.7 Diabetic Foot Disease
At some time in their life, 15% of people with diabetes develop foot ulcers
associated with peripheral neuropathy and ischaemia, peripheral vascular disease and
superimposed infections (Apelqvist et aI., 1993; Boulton et al., 1995; WHO, 2003).
Diabetic foot disease is one of the most costly complications of diabetes especially in
communities with inadequate foot wear (WHO, 2003). Hyperglycemia is associated with
"rnild defects in nerve conduction and the feet become insensitive. Neuropathy also leads
to deformed foot secondary to tendon shortening which leads to decreased motility of the
foot. The combination of foot insensitivity and foot deformities allows undue stress to
small areas of the foot and promotes foot ulcers. Infection is a frequent complication of
both vascular and neuropathic ulcers (Wheat, 1980).
Recurrence rates for diabetic foot ulcers are 35-40% over three years and 70%
over five years (Apelqvist et aI., 1993). These ulcers can have serious consequences.
20
They are highly susceptible to infection, which may spread rapidly, causmg
overwhelming tissue destruction (Edmonds et al., 1986). 5-15% of people with diabetic
foot ulcers require lower extremity amputation, usually because of gangrene; foot ulcers
precede 85% amputations in people with diabetes (pecoraro et aI., 1990; Larsson, 1994).
Up to two thirds of non-traumatic amputations are in people with diabetes whose ulcers
have progressed to gangrene (peacock et aI., 1985; Bild et aI., 1989; Laing et aI.,
1991;Effective Health Care, 1999; WHO, 2003).
1.3.6.8 Metabolic disorders
Metabolic complications are more common in type 2 than in type 1 diabetes
mellitus. They include non-ketotic hyperglycemic hyperosmolar coma (NKHHC) and
hypoglycemia. NKHHC is most common in older patients with type 2 diabetes. It is a life
threatening often fatal complication that occur spontaneously in persons with
undiagnosed diabetes mellitus. It can also occur after long periods of uncontrolled
hyperglycemia. The predisposing factors range from the use of potentially hyperglycemic
inducing agents, surgical procedures, or acute or chronic diseases and infections.
I'
NKHHC is characterized by severe hyperglycemia (greater than 6000 mg/L), absence or
right ketosis, plasma hyperosmolality and profound dehydration (Foster, 1974).
1.3.6.8.1 Hypoglycemia
Hypoglycemia occurs in both type 1 and 2 diabetes and arises as a result of poor
management of the disease either from too much or poorly timed insulin or oral
hypoglycemics or too much exercise, not enough food, or poor timing of either.
21
Aggressive use of insulin treatment to maintain normal glucose levels can increase the
risk of hypoglycemia (Kaplan and Pesce, 1996).
Diabetics usually carry something sugary to eat or drink as these symptoms are
rapidly reduced if treated early enough. Other ways of treating hypoglycemia include an
injection of glucagon which causes the liver to convert its internal stores of glycogen to
be released as glucose into the blood. Oral or intravenous dextrose is also given. In most
cases, recovery is rapid and trouble free. Longstanding hypoglycemia require hospital
admission to allow supervised recovery and adjustment of diabetic medications (WHO,
1999 )
1.3.6.8.2 Arteriosclerosis
This is an occlusive vascular disease and it is the most common complication of
type 2 diabetes. The lesions of arteriosclerosis represent a series of highly specific
cellular and molecular responses that have many characteristics of an inflammatory
disease (Steiner, 1981). These lesions occur in medium and large sized arterials and lead
to ischemia of the heart, brain or extremities resulting in infarction, stroke or peripheral
I' extremity ischemia. The arteries accumulate plaque slowly over the years. The patient
remains asymptomatic, the process accelerates gradually until occlusion occurs and
ischemia and tissue death results (Wheat, 1980).
1.3.6.8.3 Lactic acidosis
Lactic acidosis is a serious condition characterized by excessive accumulation of
lactic acid and metabolic acidosis and this results from tissue hypoxia (oxygen
deficiency) (Kaplan and Pesce, 1996). The hallmark of lactic acidosis is the presence of
22
tissue hypoxemia that leads to enhanced anaerobic glycolysis with increased lactic
formation. In diabetes mellitus, lactic acidosis is seen in association with alcohol
intoxication, phenformin therapy and ketoacidosis. Pyruvic acid is converted into lactic
acid by lactic dehydrogense (LDH) in the presence of the reduced nicotinamide
dinucleotide (NADH), which in turn is converted into NAD. The reaction is reversible
and involves LDH in both directions. The conversion of acetoacetate to ~-
hydroxybutyrate also requires the oxidation of NADH. Lactic acidosis results from
decreased availability of NAD, which is secondary to lack of oxygen. The deficiency of
NAD also impairs the conversion of ~-hydroxybutyrate into acetoacetate. Thus lactic
acidosis predisposes to accumulation of ~-hydroxybutyrate. Like the accumulation of
keto acids, lactate accumulation causes increased blood hydrogen ions and therefore low
pH (Gabbay, 1975; Kaplan and Pesce, 1996).
1.3.6.8.4 Hyperosmotic coma
Hyperosmotic diabetic coma is another acute problem associated with improper
management of diabetes mellitus. It has some symptoms in common with DKA, but of a
I'different cause, and requires different treatment. With very high blood glucose levels
(>300 mg/dl) water is osmotically driven out of the cells into the blood. The kidneys also
dump glucose into the urine, resulting in concomitant loss of water, an increase in blood
osmolarity. The osmotic effect of high glucose levels combined with the loss of water
result in high serum osmolarity that the body cells become directly affected as water is
drawn out. Electrolyte imbalance is also common. This combination of changes, if
23
prolonged, results in symptoms similar to ketoacidosis, including loss of consciousness.
As with DKA, urgent medical treatment is necessary (WHO, 1999).
1.4 Diagnosis of Diabetes Mellitus
The diagnosis of diabetes should never be made on the basis of a single abnormal
blood glucose value. At least one additional plasma/blood glucose test result with a value
in the diabetic range is essential, either fasting, from a random (casual) sample, or from
the oral glucose tolerance test (OGTT). If such samples fail to confirm the diagnosis of
diabetes mellitus, it is advisable to maintain surveillance with periodic re-testing until the
diagnostic situation becomes clear. In these circumstances, the clinician takes into
consideration such additional factors as ethnicity, family history, age, adiposity, and
concomitant disorders, before deciding on a diagnostic or therapeutic course of action
(WHO, 1999).
Diabetes screening is recommended for many types of people at various stages of life
or with several different risk factors. Many health care recommendations for adults
recommend universal screening at age 40 or 50 years, and sometimes occasionally
<thereafter. Earlier screening is recommended for those with risk factors such as obesity,
family history of diabetes, high risk ethnicity such as Hispanic, Indian American,
AfricanAmerican, Pacific Island, and South Asian ancestry (WHO,1999).
1.4.1 Functional Tests
1.4.1.1 Postprandial Plasma Glucose
Diabetes is more readily detected when the carbohydrates metabolic capacity IS
tested by stressing the system with a defined glucose load. Measurement of the rate at
24
which glucose load is cleared from the blood compared to the rate of glucose clearance in
healthy persons detects any impairment in glucose metabolism. A meal high in
carbohydrates is used as carbohydrate load though a 7Sg glucose drink is preferred over a
meal. In postpradial plasma test, blood is drawn two hours after ingestion of the meal or
drink. Levels of 1200 to 1400 mg/l are ambiguous and levels below 1200 mg/l are
normal. This test is highly inaccurate because several variables are difficult to control and
adjust. These include age, weight, previous diet, activity, illness, medication, time of the
day that the test is conducted and the actual size of the glucose dose. When a meal is used
as the load, the effective glucose load depends on the digestion of disaccharides and
polysaccharides and their subsequent absorption from the intestinal tract (Kaplan and
Pesce, 1996).
1.4.1.20' Sullivan Test
This test is used to detect gestational diabetes. A SOg load of glucose is given to a
fasting patient and blood drawn after one hour. Gestational diabetes is suggested by
plasma glucose levels above 1S00 mg/l (Kaplan and Pesce, 1996).
1"1.4.1.3 Oral Glucose Tolerance Test
This test evaluates glucose clearance from the circulation after glucose loading under
defined controlled conditions. The conditions include a minimum carbohydrate intake of
1S0g/day 3 days before the test among others. Daily carbohydrate intake less than this
lowers carbohydrate intolerance. There should be an 8-16 hour fasting before testing. The
patient should be active since inactivity decreases glucose tolerance. Exercise and
emotional stress should be avoided. For most subjects 7Sg total glucose load is
2S
sufficient. Blood samples are drawn at fasting and 1, 2 and 3 hours after ingestion of
glucose. WHO suggests impaired glucose tolerance or diabetes when both fasting and 2
hour plasma glucose levels exceed 1390 mg/l, when the 2 hour value is 1400 to 2000
mg/l, impaired glucose tolerance is suggested and when the 2 hour value exceeds 2000
mg/l diabetes mellitus is suggested (Kaplan and Pesce, 1996).
1.4.1.4 Intravenous Glucose Tolerance Test
This test is used for persons with malabsorption disorders or previous gastric or
intestinal surgery. A glucose load of 0.5g/kg body weight is used. Nondiabetics respond
with a plasma glucose level of 2000 to 2500 mg/I. Discontinuations of the glucose
loading decreases plasma glucose levels within about 90 minutes. Diabetics demonstrate
plasma glucose levels above 2500 mg/l during administration of the load. On
discontinuation of the loading, plasma glucose levels in diabetics also return to fasting
levels after about 90 minutes (Kaplan and Pesce, 1996).
1.5 Management of Diabetes MeUitus
1.5.1 Conventional Therapy
~1.5.1.1Insulin
Until the 1950s, insulin therapy was the only treatment available for diabetes
mellitus (Jahodar, 1993). Insulin is a polypeptide hormone that has the empirical formula
C254H377N65 075S6. It is synthesized in the endocrine pancrease by the beta cells of the
pancreas otherwise known as islets of Langerhans (Kaplan and Pesce, 1996 ;
http://en.wikipedia.org/wikilInsulin 2004) The hormone is responsible for the regulation
of carbohydrates and triaglyceride metabolism. Insulin acts mainly on muscle, liver and
26
adipose tissue cells to stimulate synthesis of glycogen, fats and proteins while inhibiting
the breakdown of these metabolic fuels (Voet and Voet, 2004). The stimulus for insulin
secretion is high blood glucose. The pancreas normally secretes low levels of insulin, but
the amount secreted into the blood increases as the blood glucose rises. Similarly, as
blood glucose falls, the amount of insulin secreted by the pancreatic islets goes down. In
response to insulin, target cells absorb glucose from the blood, having the net effect of
lowering the high blood glucose levels to the normal range. In diabetic patients there is an
aberration in the functioning of insulin (Guthrie and Guthrie, 1999).
As carbohydrates in food are rapidly metabolized to glucose, the peripheral
sugar in blood, insulin is produced by the p-cells in response to rising levels of glucose in
the blood after a meal. Insulin makes it possible for body tissues to utilize glucose for
fuel or conversion to other molecules or for storage. Insulin also controls the signal for
conversion of glucose to glycogen for storage in the liver and muscle cells. Higher insulin
levels increase many anabolic processes such as cell growth, cellular protein synthesis
and fat storage. Insulin is the peripheral signal in converting many bi-directional
,-processes of metabolism from a catabolic to anabolic direction. If the amount of insulin
produced is insufficient, the cells respond poorly to the effects of insulin or if the insulin
itself is defective, glucose is not utilized properly by the body cells nor stored
appropriately in the liver and muscles. The net effect is persistent high blood glucose
levels, poor protein synthesis and other metabolic derangements (Diabetes Control and
Complications Trial Research Group, 1993).
27
Plasma glucose levels principally regulate insulin secretion from P-cells.
Increased uptake of glucose by pancreatic p-cells leads to a concomitant mcrease m
metabolism. The increase in metabolism leads to an elevation in the ATP/ADP ratio.
This in turn leads to an inhibition of an ATP-sensitive K+ channel. The net result is a
depolarization of the cell leading to Ca2+ influx and insulin secretion. In fact, the role of
K+ channels in insulin secretion presents a viable theraputic agent for treating
hyperglycemia due to insulin insufficiency (pirola et al., 2004; Robinson-White and
Stratakis, 2002).
Like the receptors for other protein hormones, the receptor for insulin is
embedded in the plasma membrane. The insulin receptor is composed of two alpha
subunits and two beta subunits linked by disulfide bonds. The alpha chains are entirely
extracellular and house insulin binding domains, while the linked beta chains penetrate
through the plasma membrane. The insulin receptor is a tyrosine kinase. In other words,
it functions as an enzyme that transfers phosphate groups from ATP to tyrosine residues
on intracellular target proteins. Binding of insulin to the alpha subunits causes the beta
subunits to autophosphorylate, thus activating the catalytic activity of the receptor. The
I'
activated receptor then phosphorylates a number of intracellular proteins, which in turn
alters their activity, thereby generating a biological response (pirola et al., 2004).
In most non-hepatic tissues, insulin increases glucose uptake by increasing the
number of plasma membrane glucose transporters known as GLUTs. Glucose
transporters are in a continuous state of turnover. Increases in the plasma membrane
content of transporters stem from an increase in the rate of recruitment of new
28
transporters into the plasma membrane, deriving from a special pool of preformed
transporters localized in the cytoplasm. GLUT1 is present in most tissues, GLUT2 is
found in liver and pancreatic ~-cells, GLUT3 is in the brain and GLUT4 is found in heart,
adipose tissue and skeletal muscle (pirola et al., 2004).
All of the post-receptor responses initiated by insulin binding to its receptor are
mediated as a consequence of the activation of several signal transduction pathways.
These include receptor activation of phosphatidylinositol-3-kinase (pI3K). Activation of
PI3K involves a linkage to receptor activation of insulin receptor substrates, which
include lRS 1, lRS2, lRS3 and lRS4. Activated PI3K phosphorylates membrane
phospholipids, the major product being phosphotidylinositol 3, 4, 5 trisphosphate (PIP3).
PIP3 in turn activates various enzymes, which are thought to initiate many of the
metabolic actions of insulin (Shepherd, 2002).
Insulin also has profound effects on the transcription of numerous genes, effects
that are primarily mediated by regulated function of sterol-regulated element binding
protein (SREBP). These transcriptional effects include increases in glucokinase, pyruvate
"kinase, lipoprotein lipase (LPL), fatty acid synthase (FAS) and acetylCoA carboxylase
(ACC) and decreases in glucose 6-phosphatase, fructose 1,6-bisphosphatase and
phosphoenolpyruvate carboxykinase (PEPCK) (Shepherd, 2002).
Epinephrine diminishes insulin secretion by a cAMP-coupled regulatory path.
This hormone also counters the effect of insulin in liver and peripheral tissue, where it
binds to ~-adrenergic receptors, induces adenylate cycles activity, increases cAMP, and
activates PKA activity similarly to that of glucagons. In addition, epinephrine influences
29
glucose homeostasis through interaction with a-adrenergic receptors (Shepherd, 2002).
Hormonal control of insulin action is as a result of two gastrolintestinal hormones that
have significant effects on insulin secretion and glucose regulation. These hormones are
the glucagon-like peptides (principally glucagon-like peptide-I, GLP-l) and glucose-
dependent insulinotropic peptide (GIP). Both of these gut hormones constitute the class
of molecules referred to as the incretins. Incretins are molecules associated with food
intake-stimulation of insulin secretion from the pancreas (Robinson-White and Stratakis,
2002).
Insulin is injected into the fat under the skin where it is absorbed directly by
the blood. An alternative to injections is an insulin pump (worn outside the body) that
delivers insulin through a catheter in the tissue below the skin of the abdomen. The pump
eliminates the need for injections and offers better control of blood sugar (Anderson et
aI., 2001). Insulin is unavailable and unaffordable in many poor countries despite being
listed by World Health Organization as an essential drug. Access to insulin by those who
require it is a subject of special concern to International Health Agencies and Nation
I'
Health Authorities (WHO, 2003).
1.5.1.2 Oral Hypoglycemic Drugs
Type 2 diabetes is presently treated using five classes of oral hypoglycemic
drugs. These drugs are effective in some diabetics but they lose their effectiveness in a
significant percentage of patients. Oral hypoglycemic agents lower blood sugar by
promoting the pancreas to secrete insulin. These classes of compounds include
30
biguanides, sulfonylureas, a-glucosidase inhibitors, thiazolidinediones and meglitinides
(Jahodar, 1993; Kelly, 1995; Anderson et aI., 2001).
1.5.1.2.1 Sulfonylureas
They include drugs such as glipizide, glyburide, and glimepiride. They lower
blood sugar by promoting the pancreas to secrete insulin. These are most effective in
individuals who still have some working ~-cells in the pancreas.. The mode of action of
sulfonylureas could be chiefly explained by inhibition of KATP channels initiating
insulin secretion from the pancreatic ~-cells (Chakrabarti and Rajagopalan, 2002). Side
effects include weight gain. These drugs are also hypoglycemic (Kelly, 1995; Anderson
et al., 2001).
1.5.1.2.2 Biguanides
Metformin is a biguanide, which is derivered from Galega officinalis a medicinal
plant (Oubre et al., 1970). The drug acts by reducing plasma glucose through the
inhibition of hepatic glucose production and increases glucose uptake in the muscle. It
also lowers blood sugar by improving the response to insulin cells in the body. The
,-
muscle uses more glucose and the liver makes less glucose. The biguanides are also
known to reduce LDL. Weakness, weight loss, nausea, abdominal discomfort, and
diarrhea, shortness of breath, dizziness lactic acidosis and kidney toxicity are some of the
side effects associated with this class of drugs (Anon, 1998c; Anderson et al., 2001)
1.5.1.2.3 a-glucosidase inhibitors
This includes drugs such as acarbose, precose and miglitol, which are inhibitors
of intestinal a- glycosidase (Raing et at. 2000).These agents inhibit the breakdown of
31
complex carbohydrates and delay the absorption of monosaccharide from the
gastrointestinal tract (Campbell et at. 1996). These two actions decrease postprandial
glucose level from the intestines, thereby reducing the rise in blood glucose that occurs
after a meal. Side effects include flatulence and interference with the body's absorption of
iron, gas accumulation, bloating and diarrhea (Annon, 1998b; Anderson et al., 2001).
1.5.1.2.4 Meglitinides
These drugs include prandin, repaglinide and nateglinide. These drugs are taken
with meals and reduce the elevation of blood sugar that follows eating. They enhance
insulin secretion from the pancreas. These drugs when taken without meals lower the
blood sugar dramatically and inappropriately. Other side effects associated with
meglitinides include weight gain and gastrointestinal disturbances (Anderson et al .,
2001).
1.5.1.2.5 Thiazolidinediones
These are expensive oral hypoglycemic drugs. Examples include
rosiglitazone and pioglitazone. The drugs work to decrease insulin resistance and
I' improve the muscle ability to use insulin. They also decrease plasma triglycerides levels.
Molecular mechanisms of action of these agents are through binding to peroxisome
proliferator-activated receptor gamma (pP ARy). PP ARy is a member of a family of
nuclear receptors and is expressed in many tissues, including colon, skeletal muscle,
liver, and heart and activated macrophages and is most abundant in adipocytes
(Greenfield and Chisholm, 2004).
32
Thiazolidinediones are selective agonists of PP ARy. When activated by a ligand,
such as a thiazolidinedione, PP ARy binds to the 9-cis retinoic acid receptor (RXR
[retinoid X receptor]) to form a heterodimer. This binds to DNA to regulate the genetic
transcription and translation of a variety of proteins involved in cellular differentiation
and glucose and lipid metabolism (Greenfield and Chisholm, 2004).
Activation of PP ARy leads to enhanced differentiation and proliferation of
preadipocytes into mature fat cells, particularly in non-visceral (peripheral or
subcutaneous) fat depots. There is an up regulation of enzymes/transporters m
adipocytes like an increase in lipoprotein lipase, fatty-acid transporter 1 and glycerol
kinase to facilitate their uptake of fatty acids. Most of these consequences of PP ARy
stimulation are not seen in visceral adipocytes, even though these cells have abundant
PPARy receptors (Greenfield and Chisholm, 2004). Studies in animals and humans have
shown that thiazolidinediones only improve insulin action (and glycaemic control in
diabetes) in the presence of insulin resistance. This may be explained by the fact that the
effects of these drugs on lipid redistribution are only beneficial if there is excess tissue
"
lipid availability. The 'lipid-steal' effect of thiazolidinediones may therefore be a major
contributor to improved insulin action in muscle (enhanced glucose utilization) and liver
(reduced hepatic glucose output), as the direct effects of PPARy stimulation in muscle
and liver are unclear (Greenfield and Chisholm, 2004).
Thiazolidinediones are thought to improve insulin sensitivity by altering hormone
production by adipocytes. Adipocytes secrete a number of important hormones, referred
to as 'adipokines', including leptin, adiponectin, resistin and tumour necrosis factor-a
33
(TNF-a) (Furnsinn and Waldhausl, 2002). The thiazolidinediones, via PPARy activation,
increase the production of adiponectin, which has been shown to increase fat oxidation,
improve insulin action and to have anti-atherogenic properties. They also reduce the
secretion of substances which impair insulin action such as TNF-a and, possibly, resistin
(Gurnell et al., 2003). Thiazolidinediones are associated with weight gain and increase in
LDL-cholesterol, anemia, edema, and liver damage (Annon, 1998a; Anderson et al.,
2001).
1.5.1.3 Supplements with Glucose-Lowering Effects
Dietary options are available for people with diabetes mellitus. Supplying diabetic
patients with additional key nutrients improves sugar control and helps prevent many
major complications of diabetes. Metabolism of several trace elements is altered in
insulin-dependent diabetes mellitus and these nutrients have specific roles in the
pathogenesis and progress of the disease (Tuvemo and Gebre-Medhin, 1985). Oxyanions,
such as vanadate or vanadyl cause insulin-like effects on rats by stimulating the insulin
receptor tyrosine kinase. Tungstate and molybdate show the same effects on rat
,,-
adipocytes and hepatocytes (Matsumoto, 1994).
Since these oxyanions naturally exist in organisms, oxyanion therapy, which is
the oral administration of vanadate, vanadyl, molybdate, or tungstate, is considered to be
orthomolecular medicine. Therefore, these oxyanions provide an alternative to
chemotherapy. Many diseases in addition to diabetes mellitus are also treated since
tyrosine kinases are involved in a variety of diseases. Selenium is involved in processes
which protect the cell against oxidative damage by peroxides produced from lipid
34
metabolism. Increased urinary loss of zinc is a commonly encountered feature of diabetes
(Tuvemo and Gebre-Medhin, 1985; Matsumoto, 1994; Singh, 1995; Frank et aI., 2000).
Some of the minerals used are chromium, vanadium, magnesium, zinc and manganese
among others (Mertz, 1969; Offenbacher et aI., 1980; Sjogren et aI., 1988).
1.5.1.3.1 Zinc
Zinc is an essential element in mammals involved in the healing processes,
hormone metabolism and immune responses (Walsien, 1976). People with type 1
diabetes (IDDM) are zinc deficient, which may impair immune function. Zinc
supplements lower blood sugar levels in people with IDDM. People with type 2 diabetes
(NIDDM) have low zinc levels, caused by excess loss of zinc in their urine. High-dose
oral zinc enhances wound healing in diabetics (Tuvemo and Gebre-Medhin, 1985). Zinc
is essential to the proper functioning of insulin in the body and is necessary for the
binding of insulin to the islets of Langerhans in the pancreas, which is necessary for the
functional and morphological integrity of P-cells. One of zinc's main uses is regulating
the immune system, which is responsible for fighting off harmful viruses and bacteria.
~ Problems with the circulatory and immune systems leads to poor wound healing and
dangerous infections in diabetics.
Zinc increases the number and activity of certain types of immune system
cells that are especially important for fighting infections. In addition, zinc helps control h
blood sugar. Since diabetic patients also have low zinc levels, it is important that they get
plenty of zinc in their diets. Calf liver, crimini mushrooms and spinach are three very
good sources of zinc (Lehman and Spinas, 1996).
35
1.5.1.3.2 Manganese
Manganese is a trace element that is essential for human health. Manganese (Mn)
plays an important role in a number of physiologic processes. It is a constituent of some
enzymes and an activator to others (Keen, 2001). Low dietary manganese or low levels of
manganese in blood or tissue is associated with several chronic diseases such as
osteoporosis, epilepsy and diabetes mellitus (Nicoloft et al., 2004). A number of
manganese-activated enzymes play important roles in the metabolism of carbohydrates,
amino acids, and cholesterol (Institute of Medicine, 2001).
Pyruvate carboxylase, a manganese-containing enzyme, and phosphoenolpyruvate
carboxykinase (PEPCK), a manganese-activated enzyme plays critical roles in
gluconeogenesis the production of glucose from non-carbohydrate precursors (Leach and
Harris, 1997).
Manganese is a key element in bone development and its deficiency results in
abnormal skeletal muscle development in a number of animal species. It is the preferred
cofactor of enzymes called glycosyltransferases, which are required for the synthesis of
proteoglycans that are needed for the formation of healthy cartilage and bone (Keen and
I'
Zidenberg-Cherr, 1996). Manganese metal is used in the making and activation of
superoxide dismultase an antioxide enzyme that help to protect the cell membranes and
tissue from degeneration and disruption (Nicoloft et al., 2004). Wound healing is a
complex process that requires increased production of collagen. Manganese is required
for the activation of prolidase, an enzyme that functions to provide the amino acid,
proline, for collagen formation in human skin cells (Keen, 2001).
36
1.5.1.3.3 Chromium
It is a trace element required for the maintenance of normal glucose metabolism.
Chromium is found in a variety of foods and supplements, including liver, brewer's
cheese, meats, fish, fruits, vegetables and whole grains (Anderson et al., 2001).
Chromium helps insulin pull glucose from the bloodstream into the cells for energy
(O'Connell, 2001). Chromium supplements also reduce blood glucose levels in
individuals with type 2 diabetes and reduce the need for insulin in those with type 1
diabetes. Joint administration of doses of biotin and chromium picolinate combats insulin
resistance, improve p-cell function, enhance postprandial glucose uptake by both liver
and skeletal muscle, and inhibit excessive hepatic glucose production (McCarty, 1999).
1.5.1.3.4 Vanadium
Vanadium is an essential trace mineral present in the soil and in many foods. It
mimics the action of insulin and in a number of human studies; vanadyl sulfate (a form of
vanadium) increases insulin sensitivity in those with type 2 diabetes. Vanadium lowers
blood glucose to normal levels (reducing the need for insulin) in diabetics (Tuvemo and
Gebre-Medhin, 1983; Matsumoto, 1994)
I'
1.5.1.3.5 Magnesium
Hypomagnesemia IS common m patients with diabetes especially those with
glycosuria, ketoacidosis, and excess urinary magnesium losses. Hypomagnesemia
increases the risk of ischemic heart disease and severe retinopathy (Tuvemo and Gebre-
Medhin, 1985). A strong association exits between low levels of magnesium in the blood
and type 2 diabetes. Magnesium has no effect on hepatic glucose output and nonoxidative
37
glucose disposal (O'Connel, 2001; Mooradian et aI., 1994). Increased deficiency of
magnesium potentially causes states of insulin resistance. Magnesium has a role in the
evolution of such complications as neuropathy, retinopathy, thrombosis, and
hypertension. Low magnesium levels worsen blood sugar control and that food rich in
magnesium (such as whole grains, green leafy vegetables, bananas; legumes, nuts, and
seeds or magnesium supplements) promote healthy blood glucose levels. Taking
magnesium supplements improves the action of insulin and decrease blood sugar levels,
particularly in the elderly (Anderson et al., 2001).
1.5.1.3.6 Potassium
Potassium supplementation yields improved insulin sensitivity, responsiveness
and secretion in diabetics; insulin administration induces a loss of potassium; and a high
potassium intake reduces the risk of heart disease, atherosclerosis, and cancer (Norbiato
et al., 1984;Khaw and Barrett-Connor, 1984).
1.5.1.3.7 Inositol
Inositol is needed for normal nerve function. Diabetes causes nerve damage, or
<diabetic neuropathy. Some of these abnormalities are reversed by inositol
supplementation (Gegersen et aI., 1999).
1.5.1.3.8 a-lipoic acid and y-linolenic acid
a-lipoic acid (ALA) is a powerful natural antioxidant and is used to improve
diabetic neuropathies and reduces pain. y-linolenic acid (GLA) found in black currant
seed oil, borage oil, and evening primrose oil, is also helpful in improving damaged nerve
function common in diabetes (Jacob et aI., 1999).
38
1.5.1.3.9 Carnitine
Carnitine is a substance needed by the body for proper utilization of fat for
energy. When diabetics are given carnitine at 1 mg per kg of body weight, high blood
levels of fats is reduced by 25-39% in ten days. In addition, carnitine improves the
breakdown of fatty acids, and therefore prevents diabetic ketoacidosis (Giancaterini et al.,
2000).
1.5.1.3.10 Fiber
A high-fiber diet helps prevent development of type 2 diabetes. High fiber diet also
improves cholesterol and triglyceride levels in those with diabetes (Anderson et al.,
2001). High fiber diets are uniformly recommended for diabetics. Insoluble fiber is more
characteristic of brans and husks of whole grains, nuts, and seeds. Soluble fibers include
pectins, fruits, vegetables gums, and mucilages, which act to increase the viscosity of
food in the intestine, thus slowing or reducing the absorption of glucose (Broadhurst,
1997)
1.5.1.3.11 Antioxidants
Antioxidants such as ~-carotene, vitamin E and vitamin C are scavengers of free
radicals, which are unstable, and potentially damaging molecules generated by normal
chemical reactions in the body. Because insulin resistance is associated with
cardiovascular disease, people with diabetes benefit from nutrients that help manage
elevated blood lipid levels, high blood pressure, or congestive heart failure. In addition,
to lowering blood glucose, antioxidants improve cholesterol levels in people with type 2
diabetes. Elevated levels of free radicals cause damage to nerves and blood vessels
39
leading to neuropathy. Antioxidant supplements improve nerve communication In
damaged areas and reduce the symptoms of diabetic neuropathy (Charalambous, 1994;
Duke, 1997).
1.5.1.3.12 Biotin
This is a vitamin B-complex that is helpful for both type 1 and type 2 diabetes and
is found in brewer's yeast. Administration of high-dose biotin improves glycemic control.
Biotin can substantially lower fasting glucose in type 2 diabetics, without side-effects
(Mc Carty, 1999).
1.5.1.4 Exercise
Exercise plays an important role in controlling diabetes because it lowers blood
sugar and helps insulin to work more efficiently in the body. Exercise also enhances
cardiovascular fitness by improving blood flow and increasing the heart's pumping
power. It also promotes weight loss and lowers blood pressure.
Exercise has value, only when it is done regularly at least three to four sessions
per week for 30 to 60 minutes per session. People with type 2 diabetes who exercise
I' regularly lose weight and gain better control over their blood pressure, thereby reducing
the risk for cardiovascular disease (a major complication of diabetes). People with type 1
diabetes who regularly exercise reduce their need for insulin injections. Despite the
benefits of exercise, however, many people have difficulties in sticking with an exercise
program for a long period of time.
Healthcare practitioners can help develop suitable routines as well as strategies
that may improve adherence to such routines. Anyone with long-standing diabetes should
40
undergo a thorough screemng before beginning an exercise program and should be
monitored carefully by his or her physician. Exercise therapy is usually individualized to
account for the patient's interest, motivation and physical status (American Diabetes
Association, 1995). Exercise should be started at a low level and gradually increased to
avoid adverse effects such as injury, hypoglycemia or cardiac problems (Alexandria,
1994;Anderson et aI., 2001)
1.5.1.5 Acupuncture
Acupuncture is a physical intervention therapy that is used in diabetes mellitus
management. This practice is widely practiced as an alternative therapy for chronic pain.
Therefore, relatively few people have turned to acupuncture for treating diabetes.
Increasingly, people with pain and other health problems for which acupuncture is
selected also have diabetes (lIui, 1995; Dharmananda, 2003). Acupuncture may trigger
the release of natural painkillers and reduce the debilitating symptoms of neuropathy
(nerve damage) a complication of diabetes. In one study of diabetics suffering from
chronic painful neuropathy, acupuncture reduced pain and improved sleep in 77% of the
I' participants and eliminated the need for pain medications in 32% of the participants.
Acupuncture may therefore be a reasonable option for diabetics with neuropathy who
either find no symptom relief or develop side effects from conventional drug treatment.
The therapy has been observed experimentally and clinically and experiments
show that acupuncture can activate glucose -6- phosphatase and affect the hypothalamus.
The practice can also act on the pancreas to enhance insulin synthesis, increase the
41
number of receptors on target cell and accelerate the utilization of glucose, resulting in
lowering of blood sugar (Chen and Wei, 1985; Hui, 1995; Huang, 1996).
1.5.1.6 Herbal Management of Diabetes Mellitus
From the earliest times medicinal plants have been crucial in sustaining the
health and well being of mankind. They form an indispensable source of both preventive
and curative medicinal preparations (Dery et aI., 1999; Chevalier, 2001; Musila et al.,
2003). Ayurvedic literature shows that since the time of Charak and Sushrut, many herbal
medicines in different formulations have been used to manage diabetes mellitus
(Chattopadjay et al., 1993).
Many traditional cultures practice customs that involve traditional or folk
remedies. The cultures that are commonly known include the Ayurveda, which is a
healing practice rooted in India. Japanese, Chinese, Korean and Southeast Asian
traditions involve herbal powders and teas. Other practices which involved uses of folk
remedies involving herbs and heavy metals include Hispanic and Caribbean healing
practices. The ancient Mayan, Aztec and Inca civilizations had a profound understanding
I'
of local medicinal plants (Keen et al., 1994; Nuttfall and Flores, 1997; Borins, 1998;
Chevallier, 2001).
Modem oral hypoglycemic drugs were originally derived from herbs. Other
drugs such as digitalis; narcotics, atropine and senna have their origins from plants
(Vann, 1998). Traditional medicine is used by a large proportion of the population in
many countries. The World Health Organization (WHO) estimates that up to 80% of the
world's population, mostly in developing countries, relies on traditional medicine
42
practices for its health care needs. This is particularly true for the poorer sections of the
population in developing countries because natural remedies are not only cheaper than
modern medicines, but are often the only medicines available in remote rural regions
(Geoffrey, 1996; GTZ, 2001).
In Africa, in particular, traditional medicine has always existed and has been
practiced since time immemorial. Besides serving medical and cultural functions,
medicinal plants in Africa and other developing countries frequently provide
economically disadvantaged groups such as small holders and landless people with their
only form of cash income (Hirt and Pia, 1995; GTZ, 2001).
In Kenya, traditional medicine continues to play a major role in Primary Health
Care (PRC). More than 70% of the Kenyan population relies on traditional medicine as
its primary source of health care (Odera, 1997). It is more accessible than modern health
facilities for most of the population in the country. It is relatively inexpensive, locally
available, and usually accepted by the local communities compared to modern
conventional medicine. The substantial contribution to human health and well being of
,.
medicinal plant species is now widely appreciated and understood. Indeed, there is a
growing demand for many of the species and an increasing interest in their use. Medicinal
plants have the advantage of having little or no side effects. The many side effects
associated with conventional treatment of diabetes with oral hypoglycemic drugs and
insulin, the inadequacy of other forms of therapies such as diet, exercise, mineral
supplement and physical interventions such as acupuncture have necessitated patients
suffering from diabetes mellitus to seek more reliable and comfortable form of therapy,
43
and herbal medicine is the most commonly used alternative medicine therapy (Eisenberg,
1997; Brody, 1998).
Medicinal plants are also important sources of therapeutic agents in the industrial
production of pharmaceuticals (Lambert et al., 1997). Many of these plants were used for
many centuries and some times as regular constituents of the diet; it is assumed that they
do not have many side effects. However, chronic consumption of large amounts of
traditional remedies must always be taken with caution, as toxicity studies have not been
conducted for most of these plants (Shnkar et al., 1980).
The technical inadequacies of modem medicines and the concern over the side
effects which has made many feel that the medicine is unnatural coupled with their rising
cost have made many patients turn to more gentle alternative herbal medicines
(Kokwaro, 1993; Vann, 1998; Angell and Kassirer, 1998; Dey et a/., 2002).
Medicinal herbs used in the management of diabetes are known to contain natural
compounds with antidiabetic activity. These compounds include complex carbohydrates,
alkaloids, glycopeptides, terpenoids peptides and amines steroids, flavonoids, sulfur
,.
lipids, coumarins, inorganic compounds and ions. These natural compounds are said to
play an important role in maintaining the normal blood glucose level (WHO, 1985;
Maries and Farnsworth, 1994). Crude extracts from plants such as Allium cepa, Allium
sativum, Azadirachta indica, Ocimum sanctum and Vinca rosea have been shown to have
hypoglycemic activity (Chattopadhyay et a/. , 1993).
Trigonella foenum graecum (fenugreek) is another Ayurvedic traditional plant
with proven activity. In addition to mucilage, fenugreek also contains protein, saponins,
44
and the hypoglycemic phytochemicals coumann, fenugreekine, nicotinic acid, phytic
acid, scopoletin and trigonelline (Marles and Farnsworth, 1994; Duke, 1997)
Onions and garlic have been used in many cultures to treat diabetes. Garlic, a
member of the lily family is most commonly used for flavorful cooking worldwide
(Shane, 200 1; Sheela and Augusti, 1992). Onions and garlic have blood sugar lowering
effects (Sharma et aI., 1977; Sheela and Augusti, 1992). The volatile oils in raw onion
and garlic have been shown to lower fasting glucose concentration in both diabetic
animals and human subjects (Jain et aI., 1973).
The active components are allypropyl disulfide (APDS) and diallyl disulfide oxide
respectively. The mechanism of action is thought to be by competing with insulin for
insulin non-activating sites in the liver, which results in an increase of free insulin
(Sheela and Augusti, 1992).
Ocimum sanctum (holy basil) is another commonly used herb in Ayurveda
(related species include Ocimum album and Ocimum basilicum). Studies in animal
models have demonstrated that it has hypoglycemic activity (Chattopadhyay, 1993). Holy
I'
basil has effects that include enhanced ~-cell function and insulin secretion. Clinical trial
of Ocimum sanctum showed positive effects on both fasting and postprandial glucose in
patients with type 2 diabetes using a local preparation of fresh leaf powder mixed in
water for 4 weeks (Agrawal et aI., 1996).
Ficus carica (fig leaf) is a popular plant used for patients with diabetes in Spain
and other areas in Southwestern Europe. Its active component is unknown. Several
studies in animal models with diabetes have shown it to have both the short and long-
45
term hypoglycemic effects. Potential hypolipdemic effects in diabetic rats have also been
demonstrated (Campillo et al., 1991; Torres et aI., 1993; Perez et al., 1996). Its effects on
glucose are unknown; however, some studies suggest that it facilitates the uptake of
glucose peripherally (Serraclara et al., 1998).
Opuntia streptacantha (nopal) or the prickly pear cactus is found in arid regions
throughout the Western hemisphere. It has a high soluble, fiber and pectin content, which
may affect intestinal glucose uptake, partially accounting for its hypoglycemic actions
(Shapiro and Gong, 2002). Animal model studies have reported decreases in postprandial
glucose and HbA1c with synergistic effects with insulin (Frati et aI., 1991).
Gymnema sylvestre is another commonly used herb in Ayurveda. The plant is a
woody climber that grows in tropical forests of central and southern India. According to
common folklore, chewing the leaves causes a loss of sweet taste. Studies of an ethanol
leaf extract, GS4, in diabetic rat and rabbit models have reported regeneration of islets of
Langerhans, decreases in blood glucose, and mcreases of serum insulin
(Shanmugasundaram et al., 1990). Mechanism of action is unknown although it is
I'
postulated that it causes an increase in glucose uptake and utilization, increases insulin
release through increased cell permeability, increase in ~-cell number, and stimulation of
~-cell function (persand et aI., 1999; Shane, 2001).
Ackee fruit (Blighia sapida) is a tropical fruit belonging to the Sapindaceae
family. It has its origin in West Africa but has traversed the Atlantic to the Carribean. The
trivial name ackee, is derived from the terms "anke" and "akye-fufuo" which are used to
describe the fruit in West Africa. Consumption of the ackee is mainly in Jamaica, Haiti
46
and some parts of West Africa. In Jamaica, the fruit serves as a major component of the
national dish ackee and codfish. The ackee has been the cause of widespread epidemics
both in Jamaica and in West Africa. From as early as the 19th century there were
speculations that the fruit may be toxic. It was not until 1955, however, that the actual
causative factor of its toxicity was elucidated. A non-proteinogenic amino acid,
hypoglycin A, so named due to its ability tolnduce severe hypoglycemia (Goldson,
1992). Its chemical structure was elucidated in 1958 and scientifically it is referred to as
L-alpha-amino-beta-methylene cyclopropane propionic acid. Hypoglycin A is found
predominantly in the immature fruit. Concentrations within the arilli ranges from over
1000 ppm in the immature fruit to less than 0.1 ppm in the fully mature fruit. III effects
occur only when the immature fruit is consumed. Hypoglycin A is transaminated to
methylenecyclopropyl-alanine (MCP A) and subsequently undergoes oxidative
decarboxylation to form MCP A-CoA. MCP A-CoA exerts its effect by inhibiting several
coenzyme A dehydrogenases, which are essential for gluconeogenesis. Depletion of
glucose reserves and the inability of cells to regenerate glucose lead to hypoglycemia
~ (Goldson, 1992).
Momordica charantia is a vegetable indigenous to tropical areas, including India,
Asia, South America, and Africa. It is also known as balsam pear, karela (karolla), and
bitter melon. Common preparations of the herb range from injectable extracts to fruit
juice and fried melon bits (Leatherdale et aI., 1981; Welhinda et aI., 1986; Srivastava et
al., 1993; Shane, 2001). Its active components include charantin, vicine, and
polypeptide-p, an unidentified insulin-like protein similar to bovine insulin. The
47
mechanism of action includes increased insulin secretion, tissue glucose uptake, liver and
muscle glycogen synthesis, glucose oxidation, and decreased hepatic gluconeogenesis.
Studies in alloxan-induced diabetic rabbits demonstrated hypoglycemic activity (Akhatar
et al., 1981).
Aloe vera is the most well known species of Aloe, a desert plant resembling the
cactus in the Liliaceae family. It is popularly used to treat burns and promote wound
healing. The dried sap of the Aloe vera is a traditional remedy for diabetes in the Arabian
Peninsula (pandey et al., 1995).
Pterocarpus marsupium is used in India as a treatment for diabetes. The flavonoid
epicatechin extracted from the bark of this plant, has been shown to prevent ~-cell
damage in rats. Further, both epicatechin and a crude alcohol extract of Pterocarpus
marsupiums have been shown to regenerate functional pancreatic ~-cells in diabetic
animals (Chakravarthy et al., 1981; Chakravarthy et al., 1982).
Vaccinium myrtillus (bilberry) is a perennial plant that grows in the woods and
forest meadows. Bilberry leaf tea has a long history of folk use in the treatment of
I' diabetes. Oral administration of Bilberry leaf tea reduces hyperglycemia in normal and
diabetic dogs, even when glucose is concurrently injected intravenously (Bever and
Zahnd, 1979). The anthocyanoside myrtillin, extracted with a slightly acidic ethanol
solution, is the most active constituent. It is weaker in activity than insulin but it is less
toxic, even at 50 times the therapeutic dose. A single dose produces a beneficial effect
lasting for several weeks.
48
Vaccinium myrtillus anthocyanosides provide other benefits in diabetics.
Specifically, they have been shown to increase intracellular vitamin C levels, decrease the
leakiness and breakage of small blood vessels, prevent easy bruising, and exert potent
antioxidant effects. These effects are important in dealing with the microvascular
abnormalities of diabetes (passariello et aI., 1979; Caselli 1985).
Medicinal herbs used in indigenous medicines for the management of diabetes
mellitus contain both organic and inorganic constituents. Some of these inorganic trace
elements possess antidiabetic properties, which accounts for the activity of medicinal
herbs.
Evaluation of a traditional medicine is carried out by identification, which is done
by acquiring ethnomedical clinical data, which include pharmaceutical information.
Ethnobotanical data, which includes botanical, ecological and pharmacognostical
information, is also needed. Chemical data that includes phytochemistry and
chemotaxonomy too is required. Information from the practicing herbalist is also an
important aspect in evaluating traditional medicine. There is need to further investigate
" these plants so as to ensure their safety and efficacy. Laboratory animal experimentation
are used to determine the safety of the medicine to a high degree of accuracy and confirm
the information gathered from the traditional medicinal practitioneer.
The seven plants assessed for blood glucose lowering effect and their safety in
this study are:
Caesalpinia volkensii (Caesalpiniaceae) is known locally as Mubuthi in Mbeere.
It is a woody climber with recurved sharp prickles. The plant has pinnate leaves, yellow
49
flowers and flat pods. It is used to treat many diseases, the most common being malaria.
Pounded leaves are used in soups and tea mainly for malaria. Roots are used for
gonorrhea and bilharzias while seeds are used for stomach ulcers. Buds are crushed for
eye troubles and the Shambaa and Bondeni communities use it as an aphrodisiac
(Gacathi, 1989; Kokwaro, 1993). Aqueous decoctions, ethanol macerates, and petroleum
ether, methanol and water Soxhlet extracts of Caesalpinia volkensii are active against
malaria (Kuria et al., 2001).
Shrub of Vernonia Iasiopus (Compositae), which grows in the wild reaches up to
2 metres. The local name for Vernonia Iasiopus is Mucatha in Mbeere. The plant has
toothed leaves and purple flower heads. A decoction from the leaves and roots is used in
the treatment of intestinal worms in sheep (Gacathi, 1989). Its leaves are chewed or
boiled to cure stomach ache (Kokwaro, 1993). Methanol extracts of Vernonia Iasiopus
are active against Pifalciparum singly and in combination with chloroquine (Muregi et
aI., 2003).
Carissa edulis (Apocynaceae) belongs to the family Apocynaceae and its
I' vernacular name is Mukawa in Mbeere. This is a common plant of dry areas (Gacathi,
1989; Beentje, 1994). Carissa edulis is an evergreen shrub common in most districts in
Kenya. The shrub is branched spiny and grows to a height of up to 4 m high. Flowers are
sweetly scented; pinkish white fruits. They are edible and dark purple when ripe. The
bark produces milky latex on cutting. Carissa edulis is used traditionally for the treatment
of headache, chest complaints, rheumatism, gonorrhea, syphilis, and rabies and as a
diuretic (Nedi et al., 2004). Root decoction is also used for malaria treatment and also for
50
indigestion and lower abdominal pains when one is pregnant (Kokwaro, 1993). Oral
administration of the ethanolic extracts of Carissa edulis leaves on streptozotocin (STZ)
induced diabetic rats reduced the blood glucose level in STZ diabetic rats during the first
three hours of treatment (EI-Fikyet aI., 1996).
Ficus sycomorous (Moraceae) trees grow up to about 25m in height. Its
vernacular name is Mukuyu in Mbeere. Figs which are red when ripe are produced from
the trunk. The milkyjuice is used to relieve toothache. The bark is used in the treatment
of liver troubles and diarrhea, while bark decoction is taken for the treatment of general
abdominal pains and stomach disorder (Kokwaro, 1993; Gacathi, 1989).
Kleinia squarrosa (Asteraceae) grows in Mbeere and Machakos districts of
Eastern province in Kenya. The vernacular name is Mungendia Nthe'nge in Mbeere. The
plant is used locally in the treatment of asthma, amenorrhea, syphilis, jaundice, and
malaria. The stem is usually boiled or pounded and soaked and the mixture taken as a
decoction or infusion (Musila et al., 2003).
Azandirachta indica (Meliaceae) whose common name is neem has been reputed
to possess cardiovascular, antimicrobial, immunomodulatory, hypoglycemic and a,-
number other effects .A bitter principle, nimbi din, isolated from seeds of neem tree is
effective in reducing fasting blood glucose at a dose of 200mgl kg in alloxan diabetic
rabbits Aqueous extract of tender neem leaves have been reported to reduce blood
glucose and this effect is due to blocking the action of epinephrine on glycogenolysisand
peripheral utilization of glucose . Studies have also shown that neem has moderate
51
reversal of diabetic retinopathy in streptozotocin induced diabetic rats (Eshrat and
Hussain, 2002; Khosla et al., 2000).
Helichrysum odoratissimum (Compositae) belongs to the family compositae. The
local name is Mutaa in Mbeere. The plant is a weak shrub and is common along the forest
edges and clearings (Gacathi, 1989). Leaves of this plant are used as anthelmintic;
pounded leaves are applied over wounds resulting from burns. Young branches are used
as eye drops for conjunctivitis while roots are crushed and steeped in hot or cold water
and the extract used as a purgative (Kokwaro, 1993).
1.6 Justification
Diabetes mellitus is a predominant public health concern. WHO projects a 170 %
growth in the number of people with diabetes in the developing countries by 2025. This is
equivalent to 84 to 228 million people (WHO, 1998). It causes substantial morbidity,
mortality, and long-term complications and remains an important risk factor for
cardiovascular disease. With increasing rates of childhood and adult obesity, diabetes is
I'
likely to become even more prevalent over the coming decade. The combination of
rising prevalence of diabetes and the high rate of long term complication especially in
Africa will lead to drastic increase of the burden of diabetes on the health systems of
African counties most of which face economic difficulties. Possible prevention strategies
have been hindered by the scarcity of data on diabetes in Africa. Many of the
conventional drugs that are available for the management of the disease are not only
expensive but also have numerous side effects, which lead to complications. Herbal
medications on the other hand are cheaper and locally available. Many of the plant
52
species growing throughout the world contain active constituents that offer benefits that
conventional drugs lack, helping combats illness while supporting the body's efforts to
regain good health.
Many plants have been traditionally used to manage diabetes without
authentication on their antidiabetic properties and assessment of their safety. The use of
crude extracts without pharmacological and toxicological evaluation could lead to serious
complications and even death. Since many of the conventional drugs have originated
from medicinal herbs, a study of these plants may lead to the discovery of drugs with
higher efficacy and low toxicity.
1. 7 Hypothesis
The aqueous extracts from medicinal plants as used by traditional medicine practitioneers
(TMP) have hypoglycemic properties.
1.8 Main Objective
To evaluate seven Kenyan medicinal plants for their potential to manage diabetes
mellitus and their safety.
1.8.1 Specific Objectives
1. To screen aqueous extracts from some plants used in traditional medicine for in
vivo hypoglycemic activity.
11. To determine the trace element composition and content in water extracts from
the plants used in traditional medicine to manage diabetes mellitus.
111. To determine the classes of compounds present in the plants used in traditional
medicine to manage diabetes mellitus
53
IV. To evaluate preliminary toxicity of the water extracts from the seven plants used
m traditional medicine to manage diabetes mellitus through observation of
histological sections.
54
CHAPTER TWO
MATERIALS AND METHODS
2.1 Collection of Plant Materials
Seven medicinal plants were collected from their natural habitat in various parts
of Mbeere district in Eastern province, Kenya based on the folklore reports from
practicing herbalists on their anti-hyperglycemic activity. An acknowledged authority
identified the plants and voucher specimens were deposited at the National Museum of
Kenya herbarium. The seven plants investigated for antidiabetic activity included
Helichrysum odoratissimum, Azandirachta indica, Ficus sycomorus, Carissa edulis,
Vernonia lasiopus, Caesalpinia volkensii and Kleinia squarrosa.
The various parts of the plants collected were root barks, leave and stem barks.
The stem barks, root barks and roots were harvested and cut into small pieces and air-
dried at 25±3°C for one month. The dried materials were then crushed into powder by use
of an electric mill (Christy and Norris Ltd England). The material that was not
immediately used was stored in plastic bags and kept at room temperature away form
I' direct sunlight.
2.2 Preparation of Extracts
Plant extracts were prepared by boiling 100 g of crushed material in one liter of distilled
water for two hours with frequent stirring. The mixture was left to cool at room
temperature and then decanted into a dry clean conical flask through folded cotton gauze
stuffed into a funnel. The decanted extract was then filtered using filter papers by use of a
vacuum pump. The filtrate was freeze-dried in 150 ml portion using a freeze drier for 72
55
bours. Afterwards tbe powder was pooled together and stored at 4°C In airtight
containers.
2.2.1 Preparation of Extracts for Injection into Mice
Physiological saline was prepared by dissolving 0.8Sg of analytical grade sodium
chloride in 100 rnl of distilled water. The extracts for injection were prepared as follows:
the SOmglkg body weight dose was prepared by dissolving 12.Smg in lrnl of
physiological saline; the 100mglkg body weight dose was prepared by dissolving 2Smg
in lrnl of physiological saline; and the IS0mglkg body weight dose was prepared by
dissolving 37.Smg in lrnl of physiological saline. The animals were given 0.1 ml of the
extract solutions. Insulin was also reconstituted and animals in group 2 were given 0.1
rnl. The animals in groups 1 and 3 were also injected with 0.1 rnl physiological saline
intraperitoneally.
2.3 Pharmacological Testing
2.3.1 Animals
Health Swiss albino male mice were used for the study. The animals were 4-6
weeks old and weighed 23-27 g, and were fed on the standard mice diet and allowed free
access to water ad libitum. For experimental purposes, the animalswere fasted overnight
and allowed free access to water. The animals were divided into 4 groups of four animals
each. The animals in group 2, 3 and 4 were alloxanized using alloxan 4-6 days before the
start of the experiment. Group 1 was not alloxanized and were given O.lrnl of normal
. \
saline; Group two was given 0.1 rnl of normal saline; the third group of animals were
given insulin at the dose of 1 unit per kg body weight; group 4 animals was treated with
S6
plant extracts at three dose levels: 50mg/kg body weight, 100mg/kg body weight and 150
mg/kg body weight. Each dose level consisted of 4 animals. Group 1 and 2 served as the
experimental controls while group 3 served as the reference. Alloxan is a chemical that
kills the pancreatic islets cells that are responsible for insulin production. Once the ~-
cells are destroyed, the pancreas cannot produce insulin and the animal becomes diabetic.
The alloxan monohydrate 10 g used in this experiment was obtained from Fluka chemie
Gmbh ch 9471 Switzerland and injected intraperitoneally to mice at a dose of 150mg/kg
body weight. Insulin was given to mice at a dose of one unit per kglbody weight
(IIU/kgbw) intraperitoneally.
Before administration of the different treatments the animals were bled and blood
glucose level in the animals was measured. This was the initial measurement at time zero
hour. The animals were again bled hourly until the fourth hour.
2.3.2 Collection of Blood Samples
Blood samples were collected from the tails of the animals after wiping the tail
with surgical spirit. The tail was nibbed by use of a pair of sharp scissors; a drop of blood
I' was squeezed into a Supreme hypo guard meter. After collection of blood, the nibbled
side of the tail was rubbed with cotton wool soaked in absolute alcohol to protect the
animal from infection and to arrest bleeding.
2.3.3 Blood Glucose Determination
The principle of the test is based on a glucose oxidase/peroxidase reaction, which is
specific for ~-D-glucose. The hypoguard machine was used together with GB Supreme
blood glucose test strips. The Supreme Test Strip is a disposable plastic strip containing a
57
chemicallytreated test area used to measure the amount of blood glucose. The test area is
designed in such a way that when a drop of blood is placed on the top surface, color
change occurs which is determined by a Supreme hypoguard meter. The supreme Test
Strip was fully inserted into the meter before applying a drop of blood to fully cover the
test area inside the grey target. The supreme test strips and the supreme hypoguard meter
were obtained from hypoguard Ltd, United Kingdom through ChemoquipLtd, Kenya.
2.4 Phytochemical Screening
Phytochemical screening was undertaken to determine the class of compounds
present in the plants extracts using the method described by Houghton and Raman
(1998). Plants extracts were screened for the presence of alkaloids, saponins, tannins,
terpenoids, sterols, flavonoids and anthraquinones.
2.4.1 Alkaloids
0.5 g of each plant extract was stirred in 2 ml of 1% aqueous HCI and heated in a
boiling water bath for 10 minutes. The mixture was then filtered while hot and treated
with Dragendorff's reagent. Orange precipitate indicates the presence of alkaloids.
I'
2.4.2 Sterols and Terpenoids
0.5g of the extract was stirred with hexane to defat it. The residue was extracted
with 1 ml dichloromethane. The dichromethane solution was dried over anhydrous
sodium sulphate. 1 ml portion of the dichloromethane was mixed with 0.5 ml acetic
anhydride followed by two drops of sulphuric acid. A gradual appearance of green to
blue colors is taken as indicative of sterols. A color change from pink to purple indicates
the presence of terpenoids.
58
2.4.3 Saponins
0.5 g of the plant extract was shaken with 2 rnl water in a test tube. Frothing
which persisted for at least half an hour was a positive test for saponins (Rukunga, 1984).
2.4.4 Flavonoids
0.5 g of the extract was defatted by several washing in hexane. The defatted
residue was washed in 4 rnl 80% methanol and filtered. The filtrate was treated as
follows: To one 2 rnl portion of the filtrate, 1 rnl of 1% aluminum chloride in methanol
was added. Development of yellow color was indicative of the presence of flavonols,
flavones and chalcones. To another 2 rnl portion of the filtrate, 1 rnl of 1% potassium
hydroxide was added. Development of a dark yellow color was indicative of the presence
of flavonoids.
2.4.5 Tannins
1 g of extract was stirred in 2 rnl of distilled water and then filtered. Ferric
chloride was added into 2 rnl of the filtrate. The formation of a blue black to green
precipitate was evidence of the presence of tannins.
I'
2.4.6 Anthraquinone
In one sample, 0.5 g of the plant extract was shaken with 2 rnl of benzene and
filtered. A 10% ammonium hydroxide solution (1 rnl) was added and the mixture shaken.
A red color if observed in the ammoniac phase was indicative of the presence of free
anthraquinones.
In another sample, 0.5 g of a benzene washed plant extract was boiled with 2 ml
of 1 % Hel and filtered while hot. The filtrate was then shaken with 1 rnl benzene. The
59
benzene layer was removed and 10% ammonia hydroxide added. A red color if observed
was indicative of the presence of bound anthraquinones.
2.5 Trace Elements Determination
2.5.1 Energy Dispersive X-ray Fluorescence (EDXRF)
The different trace elements present in the extracts were investigated by use of
Energy Dispersive X-ray fluorescence analytical technique. 0.3g of the freeze-dried
material was weighed and made into pellets of2.5 em in diameter and weighing 100-200
rng/cm". The pellets were made using a pellet press machine. The pellets were weighed
and their weights recorded. The EDXRF system consists of an X-Ray spectrometer and a
radioisotope excitation source. The radiation from the radioactive source, Cd109 (T112 =
453 days and activity = 10 mCi) are incident on the sample, which emits the
characteristic X-Rays. These X-Rays are detected by Si (Li) detector (EG&G Ortec,
30mm2x10mm sensitive volume, 25~ Be window) with energy resolution of 200eV at
5.9keV Mn Ka. - line. The spectral data analysis was collected using personsl computer
based Cariberra S-100 multichannel analyser (MeA). The acquisation time applied in the,-
EDXRF measurements was 1000seconds. The X-Ray spectrum analysis and
quantification was done using IAEA QXAS software (QXAS, 1992), which is based on
on the Fundamental Parameters Method (FPM) (Sparks, 1975;Giauque et al., 1973,1977).
2.5.2 Atomic Absorption Spectrophotometry (AAS)
This technique was used for analysis of magnesium, chromium and vanadium.
The machine used was Buck model 210 VGP Atomic absorption spectrophotometer. The
VGP AAS is designed to measure the concentration of elemental metals in solution. It
60
provides integrated measurements in absorbance or emission intensity as well as sample
concentration in comparison to standard solutions. Readings can be integrated over a
period from 0.5 seconds to 10 seconds.
2.5.2.1 Preparation of Standard Solutions
Standard stock solutions of 1000 parts per million (ppm) for AAS were used as
supplied by the manufacturers (Aldrich Chemical Co. Inc).
2.5.2.2 Preparation of Working Standards
Suitable aliquots of standard stock solutions of each element were taken in a
series of 100ml volumetric flasks. The solutions were diluted to volume using distilled-
deionised water, mixed thoroughly and transferred into plastic beakers. This was done for
each element when its analysis was to be undertaken. For each element, working standard
solutions were prepared within a given range (1 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm,
and 25 ppm). The relationship between concentration and absorbance was linear. In case
of magnesium, 2 ml of 5% Lanthanum solution was added to each series of the working
standard solution before diluting them to volume. Standard blank reagents for each
I' element were prepared by adding all the used reagents, except the target element being
determined.
2.5.2.320% Hydrochloric Acid (w/w)
The solution was prepared by transferring 548 ml of concentrated hydrochloric
acid (36%) of analar grade carefully into 300ml of distilled- deionised water contained in
1 litre volumetric flask. The solution was then diluted to one litre, mixed thoroughly and
kept in plastic bottles.
61
2.5.2.4 Lanthanum Solution
Lanthanum solution (SOmg/ml) was prepared by dissolving 12.6263g of
Lanthanum chloride in distilled-deionised water. The solution was then diluted to 250ml
with distilled-deionised water in a volumetric flask. After mixing thoroughly, the solution
was kept in clean plastic bottle and used during the determination of magnesium in the
plant materials.
2.5.3 Digestion of Plant Materials
Each plant material that was collected for the study was brought to solution by
wet oxidation/digestion. The procedure was repeated two times. Wet oxidation for
determination of magnesium (Mg), vanadium (V), and chromium (Cr) was done as
follows:
The dried samples of known weights were transferred into 100 ml Pyrex beakers
and to each beaker, 10 ml of concentrated Nitric acid (lIN03) was added, then allowed to
soak thoroughly. 3 ml of 60% Perchloric acid (HCI04) was added to each beaker, then
warmed on hot plate slowly at first, until frothing ceased. Heating was then intensified
" until all Nitric acid was evaporated. When charring occurred, the mixture was cooled, 10
ml of Nitric acid was added and heating continued until white fumes of Perchloric acid
were observed. The final solution was cooled and 25 ml of 20% Hydrochloric acid (HCI)
was added. The solution was then transferred into 100 ml volumetric flask by filtering
through Whatman filter paper NO.1. The solutions were then made to volume and shaken
well to allow proper mixing before the contents were transferred to plastic sample bottles.
The samples were kept in a freezer awaiting analysis.
62
2.5.4 Total Elemental Content Determination
Wet digests of the plant materials were analyzed for Mg, V and Cr. Working
standards were prepared according to the procedure given in section 2.5.2.2. Sample
solutions for analysis of magnesium were prepared by withdrawing 1 ml of the digested
sample solution into 100 ml volumetric flasks. 5% Lanthanum solution was added in each
flask and the mixture diluted to volume using distilled-deionised water. However, for
analysis of vanadium and chromium, the digested sample solutions were analyzed
without dilution.
After setting the AAS instrument to the right conditions for each element, the
respective standard and sample solutions were aspirated into the flame in turns to
determine their respective absorbance. At least four standard solutions were aspirated
between 6-10 samples to monitor the stability of the working conditions. Distil1ed-
deionised water was always flushed into the flame to re-establish the zero absorbance.
For each sample and element, the above procedure was repeated two times. The mean
absorbance for each sample solution and standard solutions were calculated and recorded.
To prepare a calibration curve for each element, a graph of mean absorbance
against corresponding concentrations of the standard solutions was plotted. In all cases,
the graphs were linear; the best fitting straight line was obtained by using Microsoft
Excel computer software (Microsoft Office 2000), which also helped to convert
absorbance readings to concentrations of elements in each sample analyzed with better
accuracy than manual graphical method.
63
The programme gave concentrations of the diluted and undiluted samples directly.
Concentration values obtained for the diluted samples were corrected by multiplying with
the respective dilution factors. The final values were expressed as Ilg/g dry matter, were
recorded. These values were obtained by using the expression below:
Elemental content = a/w x 1000
where, a is the amount of element (mg) in 100 ml sample analyzed
w is the dry weight (g) of the material analyzed
2.6 Preliminary in vivo Toxicity
Twenty four mice were divided into 8 groups of three animals each for in vivo
toxicity. The first three animals were treated with saline and served as controls. The other
groups were treated with 450 mg /kg body weight of the ~xt~acts. The animals were
injected with the extracts daily for 30 days and kept under close observation and fed on
standard mice pellets and tap water ad libitum. Any animal that died or showed signs of
death was sacrificed. The animals that were still alive after 30 days were put to sleep
using dry ice and sacrificed. The animals were dissected and pieces of pancreas, heart,
kidney, muscle and livers were removed and preserved in 10 % formalin for histological,.,
preparation and observation.
2.6.1 Histopathological Examination of Tissues
The tissues were removed from formalin and trimmed and then labeled using a
pencil. To remove the excess formalin, the tissues were washed in running water
overnight. Dehydration was then done in an automatic tissue processor for 3 hours for
64
each stage starting from 50%, 70%, 80% then 90%. The sections were then washed in a
bath of absolute alcohol twice to ensure that there was no trace of water.
The next step was to clear the alcohol from the sections, which was done twice by
using xylene. The next step involved infiltration with paraffin wax which was carried out
at 2 0 C below the melting point of wax for 3 hours. The tissues were then embedded in
molten paraffin wax and allowed to dry. The embedded tissues were then sectioned at 4-
5urn thickness and floated in warm water bath to spread out the tissues, which were then
attached to clean microscopic slides. After holding in hot oven for at least 15 minutes, the
sections were dewaxed in xylene and then stained with haematoxylin and eosin dyes
using standard histological procedures. The stained tissues were coverslipped with DPX,
dried and examined microscopically for pathological changes.
2.7 Data Analysis
In the in vivo hypoglycemic assays, student's t-test was used to evaluate the
significance between means of extract treated animals and the diabetic control, insulin
control and the normal control. The data was represented as means ± SEM. P<0.05 was
-considered statistically significant. Instat statistical computer package was used
65
CHAPTER THREE
RESULTS
3.1 In Vivo Hypoglycemic Activity Assay
3.1.1 Effect of Ficus sycomorus on Blood Glucose in Alloxan-Induced Diabetic Mice
The percent reductions of blood glucose levels in mice by the aqueous stem bark
extract of Ficus sycomorus at the three dose levels (50 mg/kg body weight, 100mgikg
body weight and 150 mg/kg body weight) during the 1st hour was 30%, 28 % and 49%,
respectively. At this hour the plant extract lowered the blood glucose levels but not to
normal (P<0.05)(Figure 1). The doses, however, significantly lowered blood glucose
levels as compared to the diabetic control ep<0.05). During the 2nd hour the glucose
lowering effect by the three dose levels was also observed, as the percentage reduction of
blood glucose was 54%, 58% and 61% respectively. The extract lowered blood glucose
levels to normal ("F> 0.05). In the 3Tdhour the extract lowered blood glucose levels by
59%, 65% and 70% respectively. At this hour the extract lowered blood glucose levels as
effectively as insulin ("F<0.05) especially by the 150 mg/kg body weight dose range.
"The same trend was observed during the 4th hour where the three dose levels lowered
blood glucose levels lower than even insulin. The percentage blood glucose reductions
were 72%, 73% and 78% respectively (bp<0.05).
The aqueous stem bark extract of Ficus sycomorus caused a steady decrease in
blood glucose levels in the diabetic control mice during the 1st and 2ndhours and then a
steep decrease during the 3Tdand 4thhours for all the doses.
66
Table l:Effects of Ficus sycomorus extracts on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group Treatment o hr 1 hr 2 hr 3 hr 4 hr
Normal Saline 59.3±2.6 57.3±lA 60.5±1.5 56.3±4.5 53.8± 2.2
Diabetic Saline 177.5± 8.3 192.0±7.6 206.8±5.2 216.0±5.6 225.5± 7.1
Diabetic Insulin lTIJlkgbw 139.5±11.6 51.0±2.3 48.3±3.0 51.8±1.0 54.8±IA
Diabetic Fsycomorus 178.0±20.1 133.3±3Ab* 80.3±3.8ab* 70.3±6.2ab 45.8±1.8ab*
50mg/kgbw
Diabetic Fsycomorus 156.8±10A 112.S±4.6ab* 6S.0±3.2ab 5S.0±3Aa 43.S±2.0ab*
100mglkgbw
Diabetic F.sycomorus 166.8±13.S 8S.3±2.8ab* 64.3±2.3ab 50.0±2.9a 36A±1.6ab*
lS0mglkgbw
*P<O.OSwith respect to normal control; ap<O.05 with respect to diabetic control; b P<O.OSwith respect to insulin. The
data was analyzed using student's t- test. All the results were expressed as mean ± SEM. All blood glucose levels were
recorded in mg/dl.
,
180
160-v.>
Q 1401..1
=='Cil 120"C
Q
Q 100-.Q= 80....~OIl= 60~.c::
U 40~e
20
0
Ohr 1hr 2 hrs
Time
3 hrs 4 hrs
--+- Mean 50 -II- Mean 100 -.- Mean 150 -II- Mean Insulin --.- Mean Saline --- Mean Normal
Figure 1: Percentage reduction in blood glucose by varying doses of Ficus sycomorus in diabetic mice
*P<O.05 with respect to normal control; ap<O.05 with respect to diabetic control; b P<O.05 with respect to insulin.
The data was analyzed using student's t-test
"
3.1.2 Effect of Caesalpinia volkensii on blood glucose in alloxan-induced diabetic
mice
At the three dose levels (50, 100, and 150mg/kg body weight) of the aqueous
leave extracts of Caesalpinia volkensii lowered blood glucose levels appreciably (Table
2; Figure 2). In the first hour, the three doses lowered blood glucose levels by 5%, 38%,
and 51%, respectively. The 50mg/kg body weight dose insignificantly lowered blood
glucose levels as opposed to the other two doses. The 100 and 150 mg/kg body weight
doses significantly lowered blood glucose levels at the 1st hour but not as effectively as
insulin ep<0.05; bp<0.05). The 150mg/kg body weight dose range lowered the blood
sugar level to normal. In the 2nd hour, the percent reduction of blood sugar levels at the
three dose levels was 29%, 48%, and 63%, respectively. At this hour, the two higher
doses lowered the blood glucose levels significantly to normal. The 50mg/kg body
weight dose lowered blood glucose levels of the extract treated animals when compared
to the diabetic controls but not as effectively as insulin·p<0.05). The 150mg/kg body
weight dose range exhibited hypoglycemic activity as effectively as insulin. During the
3rd hour, the percent glucose level reduction by the three dose ranges was 63%, 54%, and
69%, respectively. At this time all the three dose levels lowered blood glucose to normal
and as effectively as insulinbp<0.05). This trend was repeated during the 4th hour, the
extract lowered glucose levels by 58%, 63%, and 72 %, respectively. Here, the three
dose ranges lowered blood sugar levels to normal and as effectively as insulinbp<0.05).
69
Table 2: Effects of Caesalpinia volkensii extract on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group Treatment o hr 1 hr 2 hr 3 hr 4 hr
Normal Saline 59.8±1.9 56.5±2.0 56.0±2.4 54.8±1.1 54.8±3.8
Diabetic Saline 130.0±3.5 142.8±3.8 181.3±5.3 195.8±3.6 210.0±3.1
Diabetic Insulin lIU/kgbw 135.0±28.1 50.3±6.2 54.0±1.4 54.5±1.3 51.8±2.7
Diabetic C. volkensii 140.5±19.7 123.3±12.3b* 93.3±6.2ab* 49.8±O.3ab* 56.3±2.5a
50mg/kgbw
Diabetic C. volkensii 145.5±17.8 84.8±7.7ab* 72.O±5.Oab* 63 .8±3.1 ab* 52.3±1.Ia
100mglkgbw
Diabetic C. volkensii 171.0±16.1 86.3±12.2ab 63.5±6.4a 53.0±2.9a 47.5±2.2a
150mglkgbw
·P<O.05 with respect to normal control; ap<O.05 with respect to diabetic control; b P<O.05 with respect to insulin. The
data was analyzed using student's '1'- test. All the results were expressed as mean ± SEM. All blood glucose levels
were recorded in mg/dl.
,
180
r;; 160
QC.J 140=lilg 120- 100,&:J= 80..~~= 60~.c
40U
~ 20=
0
Ohr Ihr 2 hrs 3 hrs 4 hrs
Time
-tI- Mean 50 --.- Mean 100 -4- Mean 150 -8- Mean Insulin Mean Saline --- Mean Nonnal
Figure 2: Percentage reduction in blood glucose by varying doses of Caesalpinia volkensii in diabetic mice
*P<O.05with respect to normal control; 3p<O.05with respect to diabetic control; bP<O.05with respect
to insulin. The data was analyzed using student's t- test
\.
3.1.3 Effect of Carissa edulis on Blood Glucose in Alloxan-Induced Diabetic Mice
The aqueous root bark extract of Carissa edulis was also hypoglycemic (Table 3;
Figure 3). The three dose ranges of the extract lowered blood glucose levels by 23%,
42%, and 19%, respectively during the l " hour. As table 3 shows, the three dose ranges
lowered blood glucose levels when compared to the diabetic controlf'p-cu.Oc). In the 2nd
hour, the three dose levels lowered the blood sugar level by 48%, 60%, and 28%,
respectively. At this hour, the 50 and 100mglkg body weight dose range lowered blood
glucose levels to normal and as effectively as insulin in contrast to the 150mg/kg body
weight dose range. In the 3rd hour, the percent reduction of blood glucose levels by the
three doses was 53%, 62%, and 65%, respectively. As figure (hp<0.05). 3 shows, all the
three dose ranges significantly lowered blood sugar level as effectively as insulin. During
4th hour the extract lowered blood glucose levels as effectively as insulin. The percentage
reduction by the three dose ranges was 60%, 62 %, and 68%, respectively. The three dose
levels restored normoglycemic state and as effectively as insulin (hp<0.05).
72
Table 3: Effects of Carissa edulis extract on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group Treatment o hr 1 hr 2 hr 3 hr 4 hr
Normal Saline 62.0±3.2 63.0±3.2 55.3±3.4 55.8±1.9 55.3±2.1
Diabetic Saline 156.8±1.9 164.3±2.6 173.3±4.3 179.0±5.O 189.8±4.4
Diabetic Insulin lIU/kgbw 162.3±5.2 52.5±1.3 49.8±O.9 5O.5±1.0 51.8±O.9
Diabetic C.edulis 126.0±O.8 96.5±4.9ab* 65.8±10.0a 59.8±9.5a 5O.O±6.1a
5Omg/kgbw
Diabetic C.edulis 127.5±13.8 74.5±13.3a 5O.8±O.8a a* 46.8±2.9a47.8±1.7
100mglkgbw
Diabetic C.edulis 159.3±16.8 132.0±21.1 b* 117.8±19.6ab* 55.3±2.8a 51.3±2.3a
150mglkgbw
·P<O.05 with respect to normal control; ap<O.05 with respect to diabetic control; b P<O.05 with respect to insulin. The
data was analyzed using student's '1'- test. All the results were expressed as mean ± SEM. All blood glucose levels
were recorded in mgldl.
,
180
;;; 160
Q~ 140=-ell
"0 120
Q
Q 100-,Q
.5 80~ell= 60~.c
U 40
~ 20
0
Ohr 1hr 2 hrs 3 hrs 4 hrs
Time
-+- Mean 50 Mean 100 -.- Mean 150 -tt-Mean Insulin Mean Saline Mean Normal
Figure 3: Percentage reduction in blood glucose by varying doses of Carrisa edulis in diabetic mice
*P<O.05 with respect to normal control; Rp<O.05with respect to diabetic control; b P<O.05 with
respect to insulin. The data was analyzed using student's t- test.
\.
3.1.4 Effect of Kleinia squarrosa on Blood Glucose in Alloxan-Induced Diabetic Mice
Table 4 and Figure 4 show the pattern of blood glucose reduction by the aqueous
stem bark extracts of Kleinia squarrosa. In the 1st hour after administration of the extract
at the three dose levels (50,100, and 150mg/kg body weight), the blood sugar level was
lowered by 28%, 29%, and 14% respectively. At this point, the three dose ranges
significantly lowered the blood glucose levels but not as effectively as insulin ep<O.05;
bp<O.05). The same trend was repeated in the second hour where the three dose levels
produced hypoglycemic condition by 23%, 41%, and 21%, respectively. The percentage
decrease in blood sugar level by the three dose ranges in the 3rd hour was 54%, 51%, and
32% respectively. At this hour, the 50 and 100mg/kg body weight dose range lowered
blood glucose level to normal with the former being as effective as insulin. On the other
hand, the 150mg/kg body weight dose range showed similar hypoglycemic action as in
the second hour. In the 4th hour, the three dose ranges lowered blood glucose levels by
45%, 60%, and 32%, respectively. As Table 4 shows, the three dose ranges lowered the
blood sugar levels to normal.
75
Table 4: Effects of Kleinia squarrosa extract on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group Treatment o hr 1 hr 2 hr 3 hr 4 hr
Normal Saline 68.3±3.2 65.5±1.9 62.3±2.7 65.3±0.3 67.5± 1.0
Diabetic Saline 177.5±9.5 178.3±10.2 182.5±12.6 191.3±13.3 199.8± 14.9
Diabetic Insulin 123.5±15.2 47.3±8.3 49.8± 6.2 54.3± 2.3 62.5± 1.9
lIU/kgbw
Diabetic K squarrosa 142.0±14.6 102.9± 15.Oab* 112.3±12.9ab* 62.8±6.3a 75.3± 5.5a
50mg/kgbw
Diabetic Ksquarrosa 161.0 ±16.3 114.8±9.4ab* 92.8±3.3ab* 76.3±4.7ab 61.0± 7.5a
100mglkgbw
Diabetic Ksquarrosa 151.5 ±6.4 130.3±4.1 ab* 120.8±11.7ab* 103.5±8.5ab* 96.2± 6.2ab
150mglkgbw
\.
160
r;.; 140e
~ 120-ell
"C 100ee-,.Q
= 80....~ell 60==-=U 40
'?!-
20
0
Ohr 1hr 2 hrs 3 hrs 4 hrs
--+- Mean 50 Mean 100 --it- Mean 150 Mean Insulin Mean Saline --- Mean Normal
Figure4: Percentage reduction in blood glucose by varying doses of Kleinia squarrosa in diabetic mice
*P<O.05 with respect to normal control; °P<O.05 with respect to diabetic control; b P<O.05 with respect
to insulin. The data was analyzed using student's t- test.
3.1.5 Effect of Helichrysum odoratissimum on blood glucose in alloxan-induced
diabetic mice
In the 1st hour, the aqueous leaf extracts of Helichrysum odoratissimum at the
three dose ranges did not significantly lower blood glucose levels (-2%, 5%, and -11 %,
respectively)(Figure 5). In the 2nd hour, the three dose levels lowered the blood sugar
levels by 30%, 54%, and 54%, respectively. At this hour, the extract lowered blood
glucose levels to normal but not as effectively as insulin ("p<O.05; bp<O.05). In the 3rd hour,
the percent reduction of blood glucose level by the three dose levels was 26%, 62%, and
70%, respectively. The 50 and 100mg/kg body weight dose range lowered the blood
glucose level as they did in the 2nd hour. On the other hand, the I50mg/kg body weight
dose range lowered blood sugar level to below normal and as effectively as insulin
(Figure 5). In the fourth hour, the extract continued producing the hypoglycemic activity
in a dose dependent manner by 38%, 67% and 75%, respectively. At this point, the 100
and I50mg/kg body weight dose ranges lowered blood glucose levels to below normal
and the hypoglycemic potential of the dose of I50mg/kg body weight was comparable to
,-
that of insulin. The 50 and 100mg/kg body weight doses significantly lowered blood
glucose levels but not as effectively as insulin.
78
Table 5: Effects of H. odoratissimum extract on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group
63.8± 2.4Normal
Diabetic
Diabetic
Diabetic
Diabetic
Diabetic
Treatment
Saline
Saline
Insulin
lIU/kgbw
H. odoratissimum
50mg/kgbw
H. odoratissimum
100mglkgbw
H. odoratissimum
150mglkgbw
o hr 1 hr 2 hr
63.8±2.7
188.0±14.3
44.8±2.4
83.5±10.0ab
79.8±10.0ab
69.8±8.1 ab
3 hr
59.5±1.6
200.3±14.8
39.3±2.4
93.3±21. 9ab
58.8±3.6ab
45.5±3.6a*
4 hr
221.8± 15.9
37 .O± 1.4
77.3± 4.3ab*
54.5± 2.7ab*
38.8± 1.1a*
67.5±4.0 68.0± 2.9
·P<O.05 with respect to normal control; ap<O.05 with respect to diabetic control; b P<O.05 with respect to insulin.The
data was analyzed using student's 't'- test. All the results were expressed as mean ± SEM. All blood glucose levels
were recorded in mgldl.
155.8±11.0
191.0±7.5
171.0±9.4
59.5±1.9
121.0±12.1 122.3±23.8b*
175.0±24.9 170.5±29b*
150.5±5.4 167.0± 4.6b*
160
;;.; 140eCJ= 120-Of)
"0e 100e-.c 80=....~ 60Of)=co: 40.cU
~ 20
0
Ohr
--------- ----------- ---------,
--.a.-ab--- __ -z
1hr 3 hrs 4 hrs2 hrs
Time
-+- Mean 50 --- Mean 100 -4- Mean 150 -+- Mean Insulin -+- Mean Saline -- Mean Normal
Figure 5: Percentage reduction in blood glucose by varying doses of H.odoratissimum in diabetic mice
*P<O.05 with respect to normal control; ap<O.05 with respect to diabetic control; b P<O.05 with respect
to insulin. The data was analyzed using student's t- test
\.
3.1.6 Effect of aqueous leaf extracts of Azandirachta indica on blood glucose in
alloxan-induced diabetic mice
The aqueous leaf extracts of Azandirachta indica at all dose levels did not
appreciably show hypoglycemic activity during the first two hours (Table 6; Figure 6).
The percent reduction of the blood glucose level by the three dose ranges (50, 100, and
150mg/kg body weight) in the 1st hour was 3%, -6%, and -15%, respectively.Thus at this
hour, the 50mg/kg body weight dose lowered the blood sugar level when compared to the
diabetic control ep<0.05). In the 2nd hour, the three dose levels insignificantlylowered
blood glucose level by 6%, 12%, and -8%, respectively. The 50 and 100 mg/kg
bodyweight lowered blood glucose when compare to the diabetic control ep<0.05). In the
3rd hour, the percent reduction in blood glucose of the extract treated diabetic mice by the
three dose ranges was 22%, 22%, and 27%, respectively. The 150mglkg body weight
dose range had a higher potential in reducing blood sugar level than the two lower doses,
contrary to the 1st and 2nd hours (Figure 6). The three doses lowered blood glucose levels
when compared to the diabetic control. The case was different in the 4thhour where the
three dose levels significantly lowered blood glucose but not as effectively as insulin .At
I'this hour, the percentage blood glucose reduction by the three dose levels was 43 %,
26%, and 39%, respectively.
81
Table 6: Effects of Azandiracta indica extract on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group Treatment o hr 1 hr 2 hr 3 hr 4 hr
Normal Saline 61.8±3.4 57.3±2.3 59.5±2.1 51.8±2.8 53.5±2.3
Diabetic Saline 157.8±4.1 176.0±6.6 192.5±8.0 207.8±4.4 216.3±4.1
Diabetic Insulin 164.0±3.0 52.0±2.6 55.5±2.0 54.3±0.6 52.3±1.3
lIU/kgbw
Diabetic A.indica 169.0±8.0 164.5±10Jb* 159.O±9.7ab* 133.0±6.8ab* 96.8±2.3ab*
50mg/kgbw
Diabetic A.indica 150.8±7.6 160.0±9. 1b* 133.8±10.5ab* 116.3±10.4ab* 111.0±3 .1ab*
100mg/kgbw
Diabetic A.indica 133.8±1O.0 151.3±5.4ab* 143J±7.3ab* 103.0±26.1 ab* 82.5±5.1 ab*
150mg/kgbw
·P<0.05 with respect to normal control; ap<0.05 with respect to diabetic control; b P<0.05 with respect to insulin. The
data was analyzed using student's 't' - test. All the results were expressed as mean ± SEM. All blood glucose levels
were recorded in mg/dl.
,
180..,
Qji 160~.., 140Q
C.I='& 120
'0
~~ 100&;Q'-'.5 80
~=: 60~
&;
C.I.•.. 40=:~~ 20~~
0
Ohr 2 hrs 3 hrs 4 hrsIhr TIme
-+- Mean 50 -- Mean 100 -.- Mean 150 -e-- Mean Insulin ~ Mean Saline --.- Mean Normal
Figure 6: Percentage reduction in blood glucose by varying doses ofAzandiracta indica in diabetic mice
*P<O.05with respect to normal control; ap<O.05with respect to diabetic control; b P<O.05with respect
to insulin. The data was analyzed using student's t- test
3.1.7 Effect of aqueous leaves extract of Vernonia lasiopus on blood glucose in
alloxan-induced diabetic mice
The aqueous leaves extract of Vernonia lasiopus was shown to lower blood
glucose levels when compared to the diabetic control but not significantly at 1st and 2nd
hour (Table 7; Figure 7). The percentage reduction of blood glucose level of the diabetic
mice by the three dose ranges in the 1st hour was -29%, 22%, and 11%, respectively. The
50mglkg body weight dose did not show any hypoglycemic activity and the other doses
insignificantly lowered the blood glucose level (bp< 0.05). The 100mg/kg body weight
dose lowered the blood glucose of the diabetic mice by the biggest percentage (Figure 7).
The same trend continued in the 2nd hour where the three dose levels lowered blood sugar
level of the diabetic mice by -26%, 9%, and 0%, respectively ep< 0.05). In the 3rd hour,
the three dose levels lowered the blood glucose levels by 9%, 64%, and 20%,
respectively. At this hour, the 100mg/kg body weight dose lowered the blood glucose
level to normal but not as effectively as insulin (bp<0.05). This trend was the same in the
4th hour where the three dose levels lowered blood glucose level of the diabetic mice by
I'
41%, 59%, and 28%, respectively (bp< 0.05). At this hour, the dose level of 50mg/kg
body weight lowered blood sugar level to normal but also not as effectively as insulin .
.,
84
Table 7: Effects of Vernonia lasiopus extract on blood glucose levels in alloxan induced diabetic mice (mg/dl)
Animal Group Treatment o hr 1 hr 2 hr 3 hr 4 hr
Normal Saline 68.0±3.5 65.8±1.7 65.0±1.7 62.0±1.7 58.5± 1.0
Diabetic Saline 201.3±6.4 216.5±7.6 226.8±6.9 244.0±7.5 260.3± 13.3
Diabetic Insulin 186.8±8.5 64.0±3.9 56.3±1.8 51.0±1.8 43.5± 2.6
1IU/kgbw
Diabetic V./asiopus 136.5±1O.0 174.0±2.7ab* 168.8± 11.3ab* 119.3±20.9ab* 78.5± ll.4ab
50mg/kgbw
Diabetic V./asiopus 196.5±4.9 157.8±9.1ab* 179.0±6.4ab* 71.8±9.5ab 81.3± 12.0ab
100mg/kgbw
Diabetic V./asiopus 195.5±21.0 177.0±25.1 b* 200.8±33.1 b* 158.3±25.8ab* 141.0± 21.1 ab*
150mg/kgbw
·P<O.05 with respect to normal control; ap<O.05 with respect to diabetic control; b P<O.05 with respect to insulin. The
data was analyzed using student's 't' - test. All the results were expressed as mean ± SEM. All blood glucose levels
were recorded in mgldl.
,
160
CI,j 140~eCoO 120='OIl-= 100ee-.c 80=...
~ 60
~-= 40U
~ 20~
0
Ohr
*ab
*ab
*ab
ab *ab
Ihr 2hrs
Time
3hrs 4hrs
-+- Mean 50 --- Mean 100 --.- Mean 150 --I:r- Mean Insulin ~ Mean Saline -e-Mean Nannal
Figure 7: Percentage reduction in blood glucose by varying doses of Vernonia lasiopus in diabetic mice
*P<O.05with respect to normal control; ap<O.05with respect to diabetic control; b P<O.05with respect
to insulin. The data was analyzed using student's t- test.
\.
3.2 Trace Metal Analysis
The different trace elements in the aqueous extracts of Ficus sycomorus,
Caesalpinia volkensii, Carissa edulis, Kleinia squarrosa, Helichrysum odoratissimum,
Azandiracta indica and Vernonia lasiopus were investigated using EDXRF and AAS
technique. The trace elements analyzed by EDXRF techique were Manganese, Iron,
Nickel, Copper, Zinc, Strontium, Molybdenum and Lead (Table 8). Manganese
concentrations were below the limit of detection in all the plant extracts apart from in the
aqueous leave extracts of C.volkensii and A indica, which had 8.4±1.2~g/g and
9.41±O.3~g/g, respectively. In contrast Iron occurred in all plant extracts: 103.0±9.5~g/g
in Vlasiopus; 365.0±34.0~glg in Ksquarrosa; 109.0±18.0~glg in Ficus sycomorus;
210.0±27.0~g/g in Caesalpinia volkensii; 229.0±35.0~g/g in Carissa edulis; 151.0±14.0
ug/g in Helichrysum odoraiisstmum; and 110.0±18.01lg/g in Azandiracta indica. Nickel
was below the detection limit in all extracts apart from C.edulis, which had 8.8±1.5~g/g.
Copper was in six plant extracts: 8.8±2.2~g/g in Vlasiopus; 35.3±2.7~g/g in
Ksquarrosa; 1l.9±2.8~g/g in F. sycomorus; 13.2±2.2~g/g in C.volkensii; 18.5±2.4~g/g
I'
in Carissa edulis; and 7.9±O.8~glg in H.odoratissimum apart from in Aindica where it
was below the detection limit of the EDXRF technique. However, Zinc was present in all
plant extracts: 61.5±4.1~g/g m Vlasiopus; 235.0±21.0~g/g in Ksquarrosa;
79.0±1l.5~g/g in F.sycomorus; 20.2±3.3~g/g in C.volkensii; 40.5±6.1~g/g in C.edulis;
and 78.6±6.9~g/g in H.odoratissimum; and 65.3±4.2~g/g in A.indica. Strontium was
below the limit of detection of the EDXRF technique in all extracts except in
F.sycomorus which had 2.8±O.7~g/g. Molybdenum was present in four plant extracts:
87
24.4±2.81lg/g in C volkensii; 6.6±1.11lg/g in Cedulis; 7.6±O.7f1g/g in K.squarrosa; and
I.Bug/g in A.indica but below the limit of detection by the EDXRF technique in extracts
from Fsycomorus, Hiodoratissimum, and V.Zasiopus. Lead was in all plant extracts in
low concentrations: 6.4±O.11lg/g in Cedulis; 40.8±3.5 ug/g in K. squarrosa;
17.6±1.61lg/g in Y.Zasiopus; 7.3±O.51lg/g in C volkensii; 9.1±O.61lg/g in Fsycomorus and
11.8±1. 21lg/g in Hiodorcuissimum; and 1O.1±O.31lg/gin A.indica (Table 8).
Magnesium, Chromium and Vanadium were analyzed by Atomic Absorption
Spectrophotometry (AAS). Magnesium concentrations were 471.1±19.11lg/g in C.edulis;
846.6±1.9f1g/g in K. squarrosa; 831.4±3.7J.lg/g in Y.Zasiopus; 762.1±5.91lg/g in
Cvolkensii; 738.6±3.81lg/g in Fsycomorus and 950.6±1.11lg/g in Hiodoratissimum; and
848.3±O.81lg/g in A.indica while those for chromium were 112.6±63.0J.lg/g in Cedulis;
l Ou.Oug/g in K.squarrosa; 112.6±6.31lg/g in Y.Zasiopus; l05.1±2.91lg/g in C.volkensii;
103.8±2.4Ilg/g in Fsycomorus and l05.1±2.9J.lg/g in Hiodoratissimum; and
l03.8±1.31lg/g in A.indica. Vanadium concentrations were below the limit of detection by
ASS in all the plant extracts.
88
Table 8: Trace metals present in the hypoglycemic plants as analyzed by EDXRF (Jig/g)
Plants
Elements F.sycomorus C volkensii C. edulis K squarrosa H. odoratissimum A. indica V. lasiopus
t
Mn <5 8.4±1.2 <5 <5 <5 9.4±O.2 <5
Fe 109.0±18.0 210.0±27.0 229.0±35.0 365.0±34.0 151.0±14 110.0±18.0 103.0±9.5
Ni <5 <5 8.8±1.5 <5 <5 <5 <5eu 11.9±2.8 13.2±2.2 18.5±2.4 35.3±2.7 7.9±O.8 <5 8.8±2.2
Zn 79.0±11.5 20.2±3.3 40.5±6.1 235.0±21.0 78.6±6.9 65.3±4.2 61.5±4.1
Sf 2.8±O.7 <1 <1 <1 <1 <1 <1
Mo <5 24.4±2.8 6.6±1.1 7.6±O.7 <1 1.8±O.O <1
Pb 9.1±O.6 7.3±O.5 6.4±O.1 40.8±3.5 11.8±1.2 10.1±O.3 17.7±1.6
"
Table 9. Trace metals present in the hypoglycemic plants as analysed by AAS big/g)
Elemental Quantity «JiWg)
Plants Vanadium (V)Magnesium (Mg) Chromium (Cr)
Caesalpinia volkensii
Ficus sycomorus
Carissa edulis
Kleinia squarrosa
H. odoratissimum
Azandiracta indica
Vernonia lasiopus
762.1±5.9 105.1±2.9
738.6±3.8 103.8±2.4
471.1±19.1 112.6±63.0
846.6±1.9 100.0±0.0
950.6±1.1 105.1±2.9
848.3±0.8 103.8± 1.3
<1
<1
<1
<1
<1
<1
831.4 ±3.7 112.6±6.3 <1
3.3 Classes of Compounds in the aqueous Plant extracts
Phytochemical screening for the aqueous extracts of Ficus sycomorus, Caesalpinia
volkensii, Carissa edulis, Kleinia squarrosa, Helichrysum odoratissimum, Azandiracta
indica, and Vernonia lasiopus was undertaken to determine the class of compounds
present in them. Results show that Ficus sycomorus plant extract had only saponins and
tannins. Sterols, flavonols/flavones or chalcones and flavonoids were present in the leaf
extract of Caesalpinia volkensii. Carissa edulis roots contained flavonolsl chalcones or
flavones, flavonoids and tannins. Saponins, flavonolsl flavones or chalcones and tannins
were present in Kleinia squarrosa. Helichrysum odoratissimum leaves had alkaloids,
,. terpenoids and tannins. Azandiracta indica plant extract had alkaloids, terpenoids,
flavonesl flavonols or chalcones, flavonoids, and both free and bound anthraquinones.
Vcrncnia }:;:;;:;P;;:J~~<1f extract <11:;0 contained all the phytochemical compounds tested
except free anthraquinones, alkaloids, terpenoidsand
90
.Table 10: Phytochemistry ofthe seven medicinal plants
Classes of Plants
compounds F. C C K H. A. V.
s comorus volkensii edulis s uarrosa odoratissimum Indica lasio us
Alkaloids + +
Sterols + +
Terpenoids + +
Saponins + + +
Flavonols/flavon + + + + +
est
Chalcones
Flavonoids + + + +
Tannins + + + + +
Free +
Anthraquinones
Bound + +
Anthraquinones
Key: +Ve--+ Compound present -Ve--+ Compound absent
3.4 Preliminary in vivo toxicity
In vivo toxicity assay was undertaken on 24 mice that were divided into 8 groups
of three mice each. The first group of animals was treated with saline and served as
controls for the other extract treated groups. The other groups were treated separately
with the aqueous extracts of Ficus sycomorus, Caesalpinia volkensii, Carissa edulis,
Kleinia squarrosa, Helichrysum odoratissimum, Azandiracta indica, and Vernonia
lasiopus at a dose of 450mg/kg body weight as explained in section 2.6. The results
obtained were:
Ficus sycomorus aqueous stem bark extract caused local irritation to the body
tissues at the point of injection. This was evident after histopathological examination of
skeletal muscle tissue near the point of injection, which demonstrated by
pyrogranulomatous inflammation of the soft tissue. The kidney cells were not damaged
but there was mild perirenal steatite. Liver, spleen and heart tissues were normal as there
were no signs of pathology.
1
Platel:Histological section of skeletal muscle of a mouse treated with an aq eons
stem bark extract of Ficus sycomorus (450mglkgbw). Inflammation of
muscle tissue (arrow). Exposure time: 30 days Magnification: X400
Aqueous leaf extract of Caesalpinia volkensii plant extract had no toxic effects on
tissues examined. The liver tissue showed no signs of damage; the hepatocytes were
intact apart from mild inflammation in the liver. Kidney examination showed isolated
perivascular accumulation of lymphocytes but the renal cells were normal. The spleen
and heart tissues showed no signs of pathology.
Aqueous root bark extract of Carissa edulis showed no pathology in the liver
apart from mild irritation on the liver surface and mild inflammation. Examination of the
kidney tissues showed mild perivascular accumulation but the kidney cells were intact.
The spleen and the heart tissues were normal.
Animals treated with the leaf extract of Helichrysum odoratissimum showed mild
perivascular inflammation in the kidneys but the renal cells were intact. The spleen
tissues cells were intact apart from mild lymphoid depopulation .The liver had no signs of
pathology, the hepatocytes were intact but there was mild perihepatitis. The heart muscle
had no pathology.
93
Plate 2:Histological section of normal spleen of a mouse treated with normal saline.
Follicles are densely populated (O.lml). Exposure time: 30 days.
Magnifi1tion: XIOO.
j
!
Plate3: Histological section of a spleen of a mouse treated with an aqueous stem
bark extract of Helichrysum odoratissimum (450mglkgbw). Reduction in
lymphoid population manifested by abse ee offoDicles (arro s) Exposure
time: 30 days. Magnification: XIOO
The body tissues of animals treated with the aqueous leaf extracts of Azandiracta
I' indica showed no pathology except mild lymphoid depopulation in the spleen and mild
inflammation in the liver tissues.
Vernonia lasiopus leaf extract had no toxicity on body tissues of mice after treatment
with the dose 45Omg/kg body weight apart from mild inflammation in the liver .The other
organs had no pathology.
Animals injected with 450mg/kg body weight of Kleinia squarrosa stem bark
extract shown no pathological lesions in the organs that were examined.
94
CHAPTER FOUR
DISCUSSION, CONCLUSION AND RECOMMENDA nONS
4.1 Discussion
The aqueous stem and root bark extracts of Ficus sycomorus and Kleinia
squarrosa and Carissa edulis respectively, leaf extracts of Caesalpinia volkensii,
Helichrysum odoratissimum, Azandiracta indica and Vernonia lasiopus showed
hypoglycemic activity in alloxan-induced diabetic mice. Similar work carried out by
Tsuneki et al. (2004) demonstrated hypoglycemic activity on streptozotocin induced
diabetic mice on administration of aqueous leave extracts of Camellia sinensis.
That Ficus sycomorus and Caesalpinia volkensii extracts demonstrated a dose
dependent response on blood glucose lowering effect on alloxan induced diabetic mice. A
dose dependent hypoglycemic activity was also observed by Jouad et al. (2004) on
studies carries out on streptozotocin induced diabetic rats orally administered with leave
extracts of Eucalyptus globules. In another related study Nammi et al. (2003)
demonstrated a dose dependent hypoglycemic activity on alloxan-induced diabetic
I' rabbits on administration of fresh leave extracts of Catharanthus rose us Linn.
Similar results were observed by Aguiyi et al. (2000) who demonstrated
hypoglycemic activity on normal and alloxan-induced diabetic rats intraperitoneally
injected with methanolic extracts of Ocimum gratissimum leaves. This indicates that
these p!«tit extracts might have been absorbed through the cell lipids membrane through
facilitated diffusion. The ions might have been transported in the direction of its
electrochemical gradient. This tread is in agreement with expectations seen in
95
administration of higher concentration of drug. Ficus sycomorus and C. volkensii also
lowered blood sugar to below insulin levels. This suggests the possibility of islet repair
or mimicry of insulin action by elements from the extract.
Carissa edulis, Helichrysum odoratissimum, Kleinia squarrosa and Vernonia
lasiopus plant extracts demonstrated a non dose dependent response. This trends might
suggest that the extract may have been absorbed in the cell system through active
transport where at a particular concentration saturation of the extract occurred resulting in
the rest of the extract being excreted Azadirachta indica did not have appreciable activity
during the first three hours but significant activity was observed in the 4th hour. This
suggests that the extract might have been a prodrug, which was biotransformed to an
active form.
Besides, studies done with ethanolic seed extracts of Luffa aegyptiaca and leaves
of Carissa edulis have been shown to significantly lower blood glucose levels in
streptozotocin-induced diabetic rats (EL-fiky et al., 1996). Ozbek et al. (2004) also
demonstrated a significant blood glucose lowering effect in alloxan- induced diabetic
I'
mice by aqueous root extracts of Rheum ribes. Ethanolic leaf extracts of Cassia kleinii
were also shown to lower blood glucose in streptozotocin-induced diabetic rats (Babu et
al., 2003).
The hypoglycemic effect of the plants under study can be attributed to
alkaloids, sterols, terpenoids, saponins, flavonols, flavones, chalcones, flavonoids,
tannins, free anthraquinones and bound anthraquinones observed in these plants. Several
studies have shown the hypoglycemic activity of such compounds. Saponins were shown
96
to lower blood glucose in studies undertaken on gmseng species m alloxan-treated
diabetic rats (Kimura and Suzuki, 1985). Besides, flavonols, sterols flavones, and
chalcones in Trigonella foenum graecum plant extract have been shown to lower blood
glucose in diabetic animals (Maries and Farnsworth, 1994; Duke, 1997). Methylhydroxy
chalcone polymer (MHCP) from the extract of cinnamon has been shown to increase
glucose metabolism of cells 20-fold in vitro in the epididymal fat cell assay
(Broadhurst, 1997).
Flavonoids present in Pterocarpus marsupim bark extracts have been shown to
prevent beta cell damage in rats (Chakravarthy et aI., 1981). The Ginseng species have
terpenoidal components, which cause hypoglycemic effects in streptozotocin rat models
(Shapiro and Gong, 2002). The roots of Abroma augusta (Linn) contain some alkaloid
bases, which have been shown to reduce blood glucose levels in streptozotocin induced
diabetic rats (Eshrat and Hussain, 2002). Plant extract containing alkaloids may have
lowered blood glucose levels through the mechanism of beta cell regeneration. Alkaloids
such as alkaloid 1- ephedrine promote regeneration of pancreatic islets following atrophy,
I'
restore the secretion of insulin and promote hypoglycemia (Elloit et aI., 2000).
Polygonum multiflorum plant extracts, which contain anthraquinones, are used in the
treatment of peripheral neuropathy, a complication associated with diabetes mellitus
(Broadhurst, 1997).
Flavonoids, tannins and saponins present in the aqueous extract of Boerhavia
diffusa leaves produced non-dose dependent related decrease in blood glucose levels in
alloxan induced rats (Chude et al .,2000). Methanolic root extracts of Clitoria ternatea
97
Linn contains flavonoids, steroids, triterpenoids, sapomns and tannins which are
responsible for the glucose lowering ability in both normal and streptozotocin induced
diabetic rats (Boominathan et aI., 2004). The methanolic extracts of Cleome viscosas
Linn and the pentacyclic triterpenoid betulin isolated from chloroform fraction of
methanol extract exhibited significant hypoglycemic activity in both normal and
streptozotocin induced diabetic rats. The effect was dose dependent (parimaladeviet aI.,
2004). Terpenoids from bitter gourd (Momordica charantia L) are responsible for the
plant's hypoglycemic properties. In a clinical trial improved glucose tolerance was
observed in 73 % of patients with NIDDM who were administered M charantia fruit
juice (HerbaIGram, 1997).
Alkaloids such as catharanthine, leurosine sulphate, lochnerine,
tetrahydroalstonine, vindoline and vindolinine were shown to lower blood sugar levels
(thus easing the symptoms of diabetes). Alkaloids isolated from the Madagascar
periwinkle (Catharanthus roseus (L.) and marketed in England under the proprietary
name Vinculin lower blood glucose in diabetic patients (HerbalGram, 1997). Antidiabetic
I' and antihyperlipidemiceffect of Zizyphus jujuba in alloxan induced diabetic rats is due to
the alkaloid barberine present in the leaves of the plant. The chemical constituents in Z.
jujuba may have the ability to release insulin from pancreatic ,B-cellsand also have the
potential to protect it from alloxan-induced damage in experimental animals (Tiwari and
Rao, 2002).
Medicinal herbs used in indigenous medicines for the management of diabetes
mellitus contain both organic and inorganic constituents. Some of these inorganic trace
98
elements possess antidiabetic properties, which could account for the activity of
medicinal herbs. The blood glucose lowering effect by the aqueous extracts of the plants
of this study could also have been caused by trace elements. Such trace elements were
isolated in appreciable amounts from the plants such as Manganese, Copper, Nickel,
Magnesium, Zinc, Molybdenum and Chromium present in these plants. It has been
shown that Manganese reduces glucose intolerance present in deficiency cases (Keen and
Zidenberg-Cherr, 1996). Similarly Copper deficiency is associated with disturbed
carbohydrate metabolism. Zinc is involved in all aspects of insulin metabolism: synthesis,
secretion and utilization, while Zinc deficiency plays a role in the development of
diabetes in humans. Zinc also has a protective effect against beta cell destruction, and has
well-known anti-viral effects (Lehman and Spinas, 1996).
Molybdenum stimulates glycolysis and accelerates glycogen degradation in the
hepatocytes. It also increases receptor autophosphorylation and phosphorylation of its
substrate and augment glucose transport (Li et aI., 1995). Magnesium deficiency is the
most evident disturbance of metal metabolism in diabetes mellitus. Hypomagnesemia
I'increases the risk of ischemic heart disease and severe retinopathy (Tuvemo and Gebre-
Medhin, 1985; Matsumoto, 1994; Singh, 1995; Frank et al., 2000).
Chromium enhances the body's sensitivity to insulin and increases the number of
insulin receptors, to enhance receptor binding. It also potentiates insulin action
(Anderson et al, 2001). Experimentally, Chromium deficiency is associated with
impaired glucose tolerance, which is improved with supplementation (Trow et al., 2000).
The chromium, manganese and magnesium salts present in the saltbush Atriplex halimus
99
L., are believed to prevent diabetes from occurring in sand rats who feed regularly on
the plant and who have a genetic predisposition to diabetes (Snyder, 1996; HerbalGram,
1997). Studies undertaken by Ravi et al . (2004) shown that inorganic trace elementssuch
as zinc, chromium and vanadium which were oresent in the seeds of E. jambolana had
hypoglycemic activity in streptozotocin-induced diabetic rats.
The aqueous plants extracts studied did not alter the normal cell structure of the
heart, kidney, liver and spleen as indicated. This suggested the safety of these plants
when used to manage diabetes mellitus. The inflammation at the site of injection may
also be attributed to drug induced reactions (paumgartten et a!., 1990). The surface
irritations on the kidney that were present in all plant extracts apart from in Azandiracta
indica and Vernonia lasiopus could also be attributed to tannins and saponins, which
were present in these extracts (Diwan, 2000). Saponins and tannins cause hemorrhage
and congestion of veins in tissues (Chung et a!., 1998; Diwan, 2000). Terpenoids may
also have been responsible for the inflammation since terpenoids such as p-myrcene are
toxic to the stomach, liver and are highly irritanting to the peritoneum (paumgartten et
ral., 1990). Alkaloids present in the plant extracts may also have been responsible for the
local inflammation. Zeinsteger et a!. (2003) showed that Senecio grisebachii alkaloids
such as pyrrolizidine alkaloids (PAS) when metabolized in the liver produce toxic
products which cause intense cellular alterations including megalocytosis. Further
metabolism of the alkaloids present in the plant extracts may have produced toxic
products that caused surface irritations on the kidney.
100
The range of doses used in this study were within the doses used by Tsuneki et
al. (2004) and Jouad et al. (2004). Tsuneki et al. (2004) while examining the
hypoglycemic effect of green tea on blood glucose levels used a dose range of 30-
300mg/kg body weights in rats. Jouad et al. (2004) used a dose range of 150 and 300
mg/kg body weight while evaluating the hypoglycemic activity of aqueous extract of
Eucalyptus globulus in normal and streptozotocin-induced diabetic rats. Jouad et al.
(2004) used a dose of 4.5 g/kg body weight of Eucalyptus globulus leaf extract to
evaluate toxicity of this plant extract.
4.2 Conclusion
All the seven aqueous medicinal plant extracts showed significant antidiabetic
property. The results of this study confirm the suitability of Ficus sycomorus,
Caesalpinia volkensii, Carissa edulis, Kleinia squarrosa, Helichrysum odoratissimum
Azandiracta indica and Vernonia lasiopus extracts for the management of diabetes
mellitus. However, the modes of their action are still obscure.
The different trace elements and phytochemical compounds present in these
I' plants were associated with the hypoglycemic activity. Histological responses induced by
the extracts indicated no overt pathology attributable to these plants after intraperitoneal
administration. This might indicate their low acute toxicity after intraperitoneal
administration. Further toxicity tests using the oral route of administration at stardardized
dosage levels will be needed to comprehensively evaluate the safety of these extracts in
animal models after administration through the conventionally used route of treatment for
the management of diabetes mellitus. Herbal medicines are complex mixtures of different
101
compounds that often act in a synergistic manner and exert their full beneficial effect as
total extracts. Since herbalists use the extracts in combination for the management of
diabetes mellitus, incidences of toxicity are greatly arrested. In this study, the null
hypothesis is accepted and the study justified.
4.3 Recommendations
This study could not be exhaustive owing to the time frame within which it was done.
For this reason, further studies require to be undertaken. Viz:
• The blood glucose levels were only measured until the fourth hour. It would
be desirable to measure the blood glucose levels for longer periods so as to
determine the duration of action of the extracts.
• Studies should also be undertaken using other routes of extract administration
such as the oral route, which is normally used by herbalists to administer the
medicines. This would establish whether the extracts are inactived by the
digestive enzymes.
• Further toxicology tests should be undertaken to determine the safety of the
extracts. Tests should be undertaken on other body organs apart from the liver,
kidney, spleen and heart. The brain and intestines should also be examined to
determine the effects of the extract on the brain cells and gastrointestinal tract.
Liver functional tests should be included in the toxicological studies.
• Further studies on healthy animals that are not diabetic but given extracts so
that one may be able to establish the mechanism of action of these extracts are
required.
102
• Isolation and structural elucidation of the bioactive compounds would also be
desirable.
• Combination therapy should also be undertaken as done by the traditional
herbalists to test whether they are still hypoglycemic.
• Further studies should also be undertaken with the same plant species using
the same doses from other geographical areas and different seasons to test
whether the hypoglycemic activity is geographically and seasonally related.
• Asses the potentialof trace elements in the extracts to lower blood glucose in
vitro
103
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Appendices
Appendix 1: Instrumental conditions for Atomic Absorption Spectrophotometer
(AAS)
Trace Elements
Parameter Chromium Magnesium Vanadium
Wavelength (nm) 357.9 285.2 318.4
Slit Width (nm) 0.7 0.7 0.2
Lamp Current (mA) 5.0 5.0 5.0
Flow Rate (llmin)
Air 7.0 7.0 7.0
Acetylene l.8 l.8 l.8
122
Appendix 2: Standard Calibration Curve for Magnesium
Concentration 1 5 10 15 20 25 30 35 40 45 50
(ppm)
Absorbance 0.08 0.29 0.49 0.63 0.81 0.88 1.16 1.39 1.51 1.72 1.92
2.5 ,----------------------------------------------------------,
0.5
2
y = 0.l8l2x - 0.0975
-+- Seriesl
-Linear
o +-----r----,----_,----~----~----r_--_.----_,----,_----,_--~
1 ppm 5 ppm 10 ppm 15ppm 20 ppm 25 ppm 30 ppm 35 ppm 40 ppm 45 ppm 50 ppm
Concentration
Appendix 3: Standard Calibration Curve for Chromium
Concentration (ppm) 1 5 10 15 20 25
Absorbance 0.002 0.018 0.044 0.060 0.080 0.100
0.12
0.1 Y= 0.019x - 0.019
0.084.1C.I=~,Q 0.06;..0
r.fj,Q-e
0.04
0.02
0
1 ppm 5ppm 10pmm 15 ppm 20ppm 25 ppm
Concentration
"
--+- Absorbance
-Linear (Absorbance)
-..