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UNIVERSITY OF GHANA 
 
COLLEGE OF HEALTH SCIENCES 
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY 
 
 
 
ANTI-HYPERGLYCAEMIC AND ANTI-OXIDANT EFFECT OF 
SYNEDRELLA NODIFLORA (L) GAERTN  
IN STREPTOZOTOCIN-INDUCED DIABETES IN RATS. 
 
 
BY 
 
 
PHILIP ANTWI-ADJEI SENYAH 
(10601964) 
 
 
A THESIS SUBMITTED TO THE UNIVERSITYOF GHANA,  
LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR 
THE AWARD OF MASTER OF PHILOSOPHY 
 IN PHARMACOLOGY 
 
 
 
JULY, 2018 
 
 
 
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DECLARATION 
DECLARATION BY THE CANDIDATE 
I hereby declare that this is the product of my own research undertaken under supervision and 
has neither been presented either in whole or in part for another degree elsewhere. I am solely 
responsible for any residual flaws in the work. 
Signature………………….………………                                Date………………………………                                     
Philip Antwi-Adjei Senyah (10601964) 
 
DECLARATION BY SUPERVISORS 
 We hereby declare that the principal work and presentation of the thesis was supervised by us in 
accordance with guidelines on supervision of thesis laid down by the University of Ghana. 
Principal Supervisor 
Signature…………………………………                                 Date……………………………. 
(Dr. Patrick Amoateng) 
 
Co-supervisor 
Signature………………………………….                                Date……………………………..  
(Dr Kennedy Edem Kukuia) 
 
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ACKNOWLEDGEMENTS 
I will like to express my sincere gratitude to God and my Lord Jesus Christ for me seeing 
through this stage of my life till the end. What would I have done if YOU were not with me? I 
have never walked alone. 
As for Pharm. Dr. Patrick Amoateng, my supervisor, my mentor, my friend and my big brother, I 
have nothing but praises for your support, expertise and counsel. You have put in so much and 
God bless you so much. You were undoubtedly pivotal in bringing me this far.  
To my co-supervisor Dr. Kennedy Edem Kukuia thanks for being there, a warm smile and 
having a listening ear. God bless you sir. 
To all the Lecturers in the department of Pharmacology and Toxicology, especially Dr Kwame 
BN Banga (H.O.D), Dr. Isaac J Asiedu-Gyekye, Dr. Seth K. Amponsah, Prof. Kwasi A. Bugyei 
and Prof. Alexander K Nyarko, God bless you all so much. You are deeply appreciated. 
Also to my course mates, Mr. Joseph Torbi, Miss. Efua Koomson, Mr. Kwabena Oteng- Boahen, 
Mr.Richard N Lartey , Mr. Isaac Prah and Mr. Martin Akandawen etc , thank you all so much . I 
am really indebted to you all, God bless. 
To my lovely wife Rosemond, love you so much. Thanks for your patience and support. 
 
 
 
 
 
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ABSTRACT 
Background: Synedrella nodiflora is a common weed in Ghana that is boiled to treat epilepsy 
and threatened abortion. The ethanolic whole plant extract has been demonstrated to have anti-
inflammatory, antioxidant, hypoglycemic, anticonvulsant, sedative, antidepressant and 
antipsychotic effects in murine models. The study presents the anti-oxidant and anti-
hyperglycaemic effects of the ethanolic extract of the whole plant extract of Synedrella nodiflora 
(SNE) on a diabetic rat model. 
Method: Diabetes was induced using an intraperitoneal injection of streptozotocin (STZ) (55 
mg/kg). SNE (100, 300, 1000 mg/kg, p.o) and insulin (0.1, 0.3, 1.0 IU/kg, s.c.) were 
administered for 8 weeks. Bodyweight and fasting blood sugar were measured twice weekly. 
Serum biochemistry, haematological indices, organ histopathology, MDA and SOD assay were 
done. 
Results: SNE induced a significant decrease (P< 0.05) in hyperglycaemia, with a corresponding 
increase in weight of the rats. The extract also reduced significantly the liver enzymes AST, 
ALT, ALP and increased albumin levels compared to the vehicle group (P< 0.01). SOD and 
MDA tests both showed significant increase (P< 0.05) and decrease (P< 0.01) respectively for 
SNE high dose of 1000 mg/kg, indicating antioxidant activity. Haematological indices showed a 
decrease in RBC and an increase in eosinophil count in the vehicle group (P< 0.05). 
Conclusion: The ethanolic extract of the whole plant of Synedrella nodiflora possess anti-
oxidant and anti-hyperglycaemic effects on rat model of diabetes. 
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TABLE OF CONTENTS 
 
DECLARATION ............................................................................................................................. i 
ACKNOWLEDGEMENTS ............................................................................................................ ii 
ABSTRACT ................................................................................................................................... iii 
TABLE OF CONTENTS ............................................................................................................... iv 
LIST OF FIGURES ..................................................................................................................... viii 
LIST OF TABLES ....................................................................................................................... viii 
LIST OF ABBREVIATIONS ........................................................................................................ xi 
CHAPTER ONE ............................................................................................................................. 1 
1.0 GENERAL INTRODUCTION ............................................................................................. 1 
1.1 BACKGROUND ............................................................................................................... 1 
1.2 RESEARCH QUESTIONS ............................................................................................... 3 
1.3 PROBLEM STATEMENT ................................................................................................ 3 
1.4 JUSTIFICATION OF THE STUDY ................................................................................. 4 
1.5 AIM OF RESEARCH ....................................................................................................... 4 
1.6 OBJECTIVES OF RESEARCH ........................................................................................ 5 
CHAPTER TWO ............................................................................................................................ 6 
2.0 LITERATURE REVIEW ...................................................................................................... 6 
2.1 INTRODUCTION ............................................................................................................. 6 
2.2 DIABETES ........................................................................................................................ 6 
2.2.1 Types of diabetes. ....................................................................................................... 6 
2.2.2 Type1 diabetes (T1DM) .............................................................................................. 7 
2.2.2.1 Pathophysiology of TIDM ................................................................................... 7 
2.2.3 Type 2 Diabetes Mellitus (T2DM) ............................................................................. 7 
2.2.3.1 Pathophysiology of T2DM ................................................................................... 8 
2.2.3.2 Insulin resistance in skeletal muscles ................................................................... 8 
2.2.4 Gestational Diabetes Mellitus (GDM) ........................................................................ 9 
2.2.4.1 Pathophysiology of GDM .................................................................................... 9 
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2.2.5 Other specific types (Monogenic diabetes)............................................................... 10 
2.2.6 Symptoms of diabetes ............................................................................................... 10 
2.2.6.1 Clinical features of type I diabetes ..................................................................... 11 
2.2.6.2 Clinical features of Type II diabetes .................................................................. 11 
2.2.7 Pre-diabetes ............................................................................................................... 11 
2.2.8 Diagnosis................................................................................................................... 11 
2.2.8.1 Glycated haemoglobin ....................................................................................... 12 
2.2.8.2 Fasting Blood Sugar (FBS)/Random Blood Sugar (RBS) ................................. 12 
2.2.8.3 Oral Glucose Tolerance Test (OGTT) ............................................................... 12 
2.2.8.4 Anti-diabetic drugs ............................................................................................. 13 
2.2.9 The Economic burden of Diabetes. ........................................................................... 17 
2.2.10 Complications of diabetes ....................................................................................... 17 
2.2.10.1 Macrovascular complications ........................................................................... 17 
2.2.10.2 Microvascular complications ........................................................................... 18 
2.2.10.2.1 Diabetic nephropathy ................................................................................. 18 
2.2.10.2.2 Diabetic retinopathy .................................................................................. 18 
2.2.10.2.3  Diabetic neuropathy .................................................................................. 18 
2.3 DIABETES: THE HERBAL APPROACH. .................................................................... 19 
2.3.1 Diabetes Mellitus and Medicinal plants .................................................................... 19 
2.3.2 Diabetic complications and medicinal plants. .......................................................... 21 
2.4 SYNEDRELLA NODIFLORA (L) GAERTN ................................................................... 22 
2.4.1 Plants description ...................................................................................................... 22 
2.4.2 Folkloric uses ............................................................................................................ 23 
2.4.3 Phytochemical screening .......................................................................................... 24 
2.4.3 Pharmacological tests................................................................................................ 24 
2.6 ANIMAL MODEL IN DIABETES ................................................................................ 25 
CHAPTER THREE ...................................................................................................................... 27 
3.0 MATERIALS AND METHODS ........................................................................................ 27 
3.1 STUDY DESIGN ............................................................................................................ 27 
3.2 PLANT EXTRACT PREPARATION ............................................................................ 27 
3.2.1 Plant acquisition ........................................................................................................ 27 
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3.2.2 Preparation of the whole plant of Synedrella nodiflora ............................................ 27 
3.2.3 Phytochemical screening .......................................................................................... 28 
3.2.3.1 Alkaloids (Dragendorff‟s Test) .......................................................................... 28 
3.2.3.2 Saponins (Frothing test) ..................................................................................... 28 
3.2.3.3 Phytosterols (Lieberman-Burchard‟s test) ......................................................... 28 
3.2.3.4 Terpenoids (Salkowski test) ............................................................................... 29 
3.2.3.5 Glycosides (General test) ................................................................................... 29 
3.2.3.6 Tannins (Ferric Chloride test) ............................................................................ 29 
3.2.3.7 Phenolic  Compounds (Acetic acid test) ............................................................ 29 
3.2.3.7 Flavonoids (Kumar test) ..................................................................................... 30 
3.2.3.9 Volatile Oils ....................................................................................................... 30 
3.3 EXPERIMENTAL ANIMALS AND HUSBANDRY .................................................... 30 
3.4 INDUCTION OF DIABETES......................................................................................... 31 
3.4.1 Preparation of citrate buffer ...................................................................................... 31 
3.4.2 Process of diabetes induction with STZ.................................................................... 31 
3.4.3 Oral glucose tolerance test ........................................................................................ 32 
3.4.4 Grouping ................................................................................................................... 32 
3.4.5 Preparation of drugs .................................................................................................. 32 
3.4.6 Drug administration .................................................................................................. 33 
3.4.7 Fasting blood sugar (FBS) determination ................................................................. 33 
3.4.8 Determination of percentage change in bodyweight and relative organ weight. ...... 33 
3.5 PREPARATION OF SD RATS FOR BLOOD TESTS AND HISTOPATHOLOGY ... 34 
3.5.1 Sacrificing of rats. ..................................................................................................... 34 
3.5.2 Preparation of organs ................................................................................................ 34 
3.5.3 Preparation of blood .................................................................................................. 35 
3.5.4 Preparation of serum ................................................................................................. 35 
3.5.5 Kidney and liver Function tests ................................................................................ 35 
3.5.6 Haematological analysis ........................................................................................... 35 
3.6 HISTOPATHOLOGY ..................................................................................................... 36 
3.6.1 Processing of Organs into slides ............................................................................... 36 
3.6.2 Examination of slides ................................................................................................ 36 
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3.7 ANTIOXIDANT ACTIVITY .......................................................................................... 36 
3.7.1 Determination of Superoxide Dismutase (SOD) Activity ........................................ 37 
3.7.2 Procedure for SOD activity determination ............................................................... 37 
3.7.3 Malondialdehyde (TBARS) determination ............................................................... 38 
3.7.4 Procedure for MDA determination ........................................................................... 38 
3.8 ANALYSIS OF RESULTS ............................................................................................. 39 
CHAPTER FOUR ......................................................................................................................... 39 
4.0 RESULTS............................................................................................................................ 40 
4.1 PHYTOCHEMICAL SCREENING................................................................................ 40 
4.2 INDUCTION OF DIABETES......................................................................................... 40 
4.3 ANTIHYPERGLYCAEMIC ACTIVITY OF SNE ........................................................ 42 
4.4 WEIGHT PARAMETERS .............................................................................................. 46 
4.4.1 Percentage change in bodyweight ............................................................................. 46 
4.4.2 Relative Organ Weight ............................................................................................. 48 
4.5 BLOOD BIOCHEMISTRY ............................................................................................ 50 
4.6 HAEMATOLOGY .......................................................................................................... 52 
4.7 ANTIOXIDANT ACTIVITY .......................................................................................... 54 
4.7.1 SOD and MDA assay ................................................................................................ 54 
4.8 HISTOPATHOLOGY ..................................................................................................... 56 
4.8.1 Liver .......................................................................................................................... 57 
4.8.2 Kidney ....................................................................................................................... 58 
4.8.3 Lungs......................................................................................................................... 59 
4.7.4 Brain (cerebral cortex) .............................................................................................. 60 
4.7.5 Spleen ........................................................................................................................ 61 
CHAPTER FIVE .......................................................................................................................... 62 
5.0 DISCUSSION ..................................................................................................................... 62 
5.1 DISCUSSION .................................................................................................................. 62 
5.2 CONCLUSION ............................................................................................................... 69 
5.3 RECOMMENDATIONS................................................................................................. 70 
REFERENCES ...................................................................................................................... 70 
 
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LIST OF FIGURES 
Figure 1: Picture of Synedrella nodiflora showing leaves and flowers   ...................................... 24 
Figure 2: The effect of STZ injection on SD rats in diabetes induction.. ..................................... 42 
Figure 3: The effect of the administration of SNE and insulin in diabetic rats over 8 weeks.. .... 44 
Figure 4: Effect of diabetes on bodyweights of diabetic rats receiving vehicle and SNE……….47 
Figure 5: The effect of SNE on SOD activity (A) and MDA levels (B) in STZ-induced diabetic  
male SD rats after 8week of administration……………………………………………..……….55 
Figure 6: Photomicrographs of  liver isolated from rats after 8 weeks of continuous 
administration……………………………………………………………………………………57 
Figure 7: Photomicrographs of kidneys isolated from rats after 8 weeks of continuous 
administration …………………………………………………………………………………...58 
Figure 8: Photomicrographs of lung isolated from rats after 8-week continuous administration..
....................................................................................................................................................... 59 
Figure 9: Photomicrographs of cerebral cortex of brains isolated from rats after 8-week 
continuous administration ............................................................................................................. 60 
Figure 10: Photomicrographs of spleens isolated from rats after 8-week continuous 
administration ............................................................................................................................... 61 
 
 
 
 
  
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LIST OF TABLES 
Table 1: Diabetes drugs, mechanisms of action and side effects………………………………..14 
Table 2: Plants used in diabetes, mechanisms of action and model……………………………..20 
Table 3: Pure compound for treating diabetic complications and their mechanisms of action.....21 
Table 4: Animal models for diabetes and their limitations……………….……………………...26 
Table 5: Phytochemical screening results ……………………………………….…………...….40 
Table 6: ED50 of insulin and SNE………………… ……………….…………………………...44 
Table 7: Effect of diabetes on relative organ weights of diabetic rats receiving vehicle and SNE 
(100-1000 mg/kg, p.o) for 8 weeks…………………………………………………………...….48 
Table 8: The effect of SNE (100–1000 mg/kg, p.o) on blood biochemistries in STZ–induced 
diabetic SD rats after 8-week administration .........……………………………………………...50 
Table 9: The effect of SNE (100–1000 mg/kg, p.o) on haematological indices in STZ -induced 
diabetic SD rats after 8week administration compared to control and vehicle…………………..52 
 
 
 
 
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LIST OF ABBREVIATIONS 
ABBREVIATION  DEFINITION 
AGE Advanced Glycation Endproducts 
ALP Alkaline Phosphatase 
ALT Alanine Transaminase 
AST Aspartate Transaminase 
Bas Basophils 
DAG Diacylglycerol 
DN Diabetic Neuropathy 
Eos Eosinophils 
FBS Fasting Blood Sugar 
FPG Fasting Plasma Glucose 
HCT Haematocrit 
HDL High Density Lipoprotein 
HGB Haemoglobin 
LDL Low Density Lipoprotein 
Lymp. Lymphocytes 
MDA Malondialdehye 
MCV  Mean Corpuscular Volume 
MCH  Mean Corpuscular Haemoglobin 
MCHC Mean Corpuscular Haemoglobin Concentration 
NO-cGMP Nitric Oxide- Cyclic Guanidine Monophosphate 
PLT Platelets 
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PKC Protein Kinase C 
RAGE Receptor for Advanced Glycation Endproducts 
RBC Red Blood Cells 
SD Sprague Dawley 
SOD Superoxide Dismutase 
SNE Synedrella Nodiflora Extract 
STZ Streptozotocin 
TBARS Thiobarbituric Acid Reactive Substances 
Tot Chol. Total cholesterol 
Tot. Pro Total Protein 
T1DM Type 1 Diabetes Mellitus 
T2DM Type 2 Diabetes Mellitus 
WBC White blood Cells 
 
 
 
 
 
 
 
 
 
 
 
 
 
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CHAPTER ONE 
1.0 GENERAL INTRODUCTION 
1.1 BACKGROUND 
Diabetes is a well-known chronic disorder characterized by many metabolic changes in the body 
as a result of insufficiency in secretion of insulin and peripheral resistance to insulin (Georga et 
al., 2018). The worldwide prevalence of diabetes is on the rise due to obesity, poor feeding 
habits and sedentary lifestyle (Klonoff, 2009). There were about 518,400 known cases of 
diabetes in Ghana as at 2017 (International Diabetes Federation, 2017). Diabetes presents with 
long term complications that lead to damage of the retina, kidney, and liver. It can also lead to 
cardiovascular diseases (Dokken, 2008). Recent prospective studies have provided unequivocal 
evidence on the crucial role of prolonged hyperglycaemia in the development of chronic diabetic 
complications (Matuszewski et al., 2013). 
Hyperglycaemia causes tissue damage through multiple mechanisms including increased flux of 
glucose and other sugars through the polyol pathway, increased intracellular formation of 
advanced glycation end products (AGEs), increased expression of the receptor for AGEs and its 
activating ligands, activation of protein kinase C isoforms, and the over activity of the 
hexosamine pathway (Vinik et al., 2013). A lot of researches have shown that oxidative stress 
and inflammation in diabetes play key roles in inducing many secondary chronic complications 
(Cefalu, 2009; Kawano, 2014; Wellen and Hotamisligil, 2005). 
 In view of the many metabolic changes that occur in diabetes, many drugs are employed to 
tackle the cause, complications and symptoms at the same time. Treatment of diabetes is 
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challenging as the pathological mechanisms of diabetes and its complications are 
multidirectional and complicated (Zychowska et al., 2013).  
Diabetes is treated with drugs of many classes including, biguanides, sulfonylureas and alpha-
glucosidase inhibitors. These drugs may be used individually or in combination depending on the 
patients‟ response. This raises concerns about drug-drug interactions, compliance and cost of 
treatment. There are also concerns of long term overstimulation of the beta pancreatic cells by 
sulfonylureas which may lead to secretory failure (Ramedi and Nichols, 2008).  Side effects like 
lactic acidosis and weight gain have been well established for biguanides and sulfonylureas, 
respectively (BNF 68, 2014). All these create clinical challenges in the management of diabetes. 
Cost of managing diabetes has placed enormous economic burden on patients, family members 
and healthcare delivery as a whole (Zhang  and Gregg, 2017). 
Plants have been an alternate source of medicines for many over the years. Plants may have a 
significant role to play in diabetes (Matough et al., 2012; Tiwari et al., 2002). Apart from their 
ease of accessibility, plants are known to possess anti-oxidative, anti-inflammatory, analgesic 
and anti-hyperglycaemic activity (Arumugam et al., 2013). All of these properties will tackle key 
components of diabetes and possibly its complications (Bhagour et al., 2016; Tiwari et al., 
2018). 
Synedrella nodiflora is a plant that originated from the Americas, but is locally abundant around 
streams and rivers in West Africa. Traditionally it is used in the treatment of hiccup, epilepsy and 
threatened abortion by boiling and drinking of the whole plant (Mshana et al., 2000). It has been 
demonstrated to possess anticonvulsant, analgesic and anti-inflammatory properties (Amoateng 
et al., 2011; Haque et al., 2012). It has also been proven to have low toxicity even at high doses 
in rats (Adjei  et al., 2014; Amoateng et al., 2016). 
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Of much importance to this study is its anti-hyperglycaemic, anti-oxidant properties in addition 
to its anti-inflammatory and analgesic properties already established (Amoateng  et al., 2015; 
Haque et al., 2012; Woode et al., 2009). Oxidative stress, inflammation, and hyperglycaemia are 
the main factors that influence the progression of diabetes. Synedrella nodiflora may be a very 
useful plant in diabetes if it can significantly reduce these key factors that play major roles 
diabetes in a rat model. 
1.2 RESEARCH QUESTIONS 
 Can the extract significantly reduce hyperglycaemia in the diabetic rat model? 
 Will the anti-oxidative properties result in reduction of oxidative stress markers in vivo? 
 Does the extract have any organo-protective effects in diabetic rats? 
 What other effects will the extract have on blood biochemistry and haematology in 
diabetes?  
 
1.3 PROBLEM STATEMENT 
Diabetes causes a lot of complications. Diabetes can affect the heart, kidney, liver and eye. It 
also inculcates hyperglycaemia, inflammation and oxidative stress simultaneously to achieve its 
adverse outcomes (Giacco and Brownlee, 2010). These complications can be very distressing to 
patients. Diabetic neuropathy for example is characterized by pain, hypersensitivity, numbness of 
the limbs which can result in possible foot amputations. The total cost of treatment of diabetes 
and all its complications as well as symptoms continue to present a burdensome socio-economic 
challenge to patients, relatives, clinicians and healthcare systems (Zhang  and Gregg, 2017).  
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1.4 JUSTIFICATION OF THE STUDY 
Synedrella nodiflora is very common and easily accessible in Ghana. It has been proven to have 
low toxicity in chronic and acute administration of high doses in rats (Adjei  et al., 2014a; 
Amoateng et al., 2014b; Amoateng et al., 2016). The plant also possesses anti-inflammatory and 
in vitro antioxidant properties (Amoateng  et al., 2011; Haque et al., 2012). Also the methanolic 
extract of the plant has shown hypoglycemic activity in alloxan-induced Long-Evans diabetic 
rats, given by intra-peritoneal injection for 3 days (Zahan et al., 2012). This study however seeks 
to further study the effects of an ethanolic extract of the whole plant in streptozotocin-induced 
diabetic rats. This will provide further pharmacological information regarding the anti-diabetic 
potential of the plant towards drug discovery. The hypoglycaemic and in vitro antioxidant 
properties of the ethanolic extract from the plant provides the basis for further research into the 
possible anti-hyperglycaemic and in vivo anti-oxidant activity. In vitro antioxidant activity may 
not always translate into activity in vivo. An in vivo antioxidant activity could be beneficial since 
oxidative stress plays vital role in worsening of diabetes and its complication. An agent that 
could possibly reduce sugar levels, provide antioxidant protection, reduce inflammation and also 
reduce pain will be an excellent alternative to current drugs that do not have all these properties 
combined. It is also a cheaper alternative to current treatments. 
 
1.5 AIM OF RESEARCH 
This research aims at investigating the anti-oxidant and anti-hyperglycaemic effect of the 
ethanolic extract of Synedrella nodiflora (L.) Gaertn in diabetic rat models. 
 
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1.6 OBJECTIVES OF RESEARCH 
 Induce diabetes in Sprague-Dawley rats with streptozotocin (STZ) and evaluate the anti-
hyperglycaemic properties of the extract in comparison with a standard drug by 
comparing fasting blood sugar (FBS) levels. 
 Evaluate the effect the extract on oxidative stress by measuring SOD and MDA levels. 
 Assess the effect of the extract on blood biochemistry and heamatology in diabetes. 
 Assess the histopathological  effect of the extract on major organs in diabetic rats. 
 
 
 
 
 
 
 
  
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CHAPTER TWO 
2.0 LITERATURE REVIEW 
2.1 INTRODUCTION  
2.2 DIABETES 
Diabetes mellitus is a group of complex  metabolic disorder characterized by deficiency in 
insulin secretion, impaired insulin action, or a combination of both leading to hyperglycaemia 
(Crawford, 2017). Diabetes is a chronic illness requiring continuous medical care with 
multifactorial risk-reduction strategies targeting glycemic control. Insulin is a hormone 
manufactured by the beta cells of the pancreas, which is required to utilize glucose from digested 
food as an energy source (Le Roith and Zick, 2001). Chronic hyperglycaemia is associated with 
micro vascular and macro vascular complications that can lead to visual impairment, blindness, 
kidney disease, nerve damage, amputations, heart disease, and stroke (Goldenberg  and 
Punthakee 2013). Its long term complications include damage to the heart, retina, kidney and the 
peripheral nerves. Damage to the nerves lead to diabetic neuropathy (DN). It is reported that 
dyslipidemia  coupled with lipid peroxidation in diabetes induce many secondary chronic 
complications including atherosclerosis and neural disorders (Upadhyay et al., 2018). 
 
2.2.1 Types of diabetes. 
The common types of diabetes include; Type1 Diabetes Mellitus (T1DM), Type 2 Diabetes 
Mellitus (T2DM), Gestational Diabetes Mellitus and  Other Specific Types also known as 
Monogenic Diabetes. 
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2.2.2 Type1 diabetes (T1DM)   
Type1 diabetes (T1DM)  results from pancreatic   beta cell destruction. Most childhood diabetes 
are of type 1. This type of  diabetes is due to an autoimmune process for which the etiology of 
the beta cell destruction is unknown.T1DM is a chronic disorder of glucose homeostasis, leading 
to a absolute deficiency in insulin resulting in hyperglycemia. 
2.2.2.1 Pathophysiology of TIDM 
T1DM begins with a predisposition at birth with the inheritance of genetic risk factors. 
Development of autoimmune type 1 diabetes is typically divided into stages, starting with 
genetic susceptibility and ending with complete loss of the β-cells of the pancreatic islets 
(Georga et al., 2018). The β-cells are destroyed by autoantibodies which  are produced by the 
presentation of autoantigens to autoreactive lymphocytes (Brinkman, 2017). Environmental 
factors such as infections may trigger autoimmune destruction of the beta pancreatic cells by 
inciting cross-reactivity against antigens on the beta-cells that bear a similar molecular structure 
(Rewers and Ludvigsson, 2016). The process involving immune activation to clinically relevant 
islet cell destruction may take years but is marked early by the presence of serum autoantibodies.  
These may include: Glutamic Acid Decarboxylase Autoantibodies (GADA), Tyrosine 
phosphatases IA-2 and IA-2b, Zinc transporter (ZnT8), Insulin Autoantibodies (IAA) (Chahine, 
2017). 
2.2.3 Type 2 Diabetes Mellitus (T2DM) 
Type 2 diabetes mellitus (T2DM) has become an epidemic burden in worldwide. Diabetes 
mellitus is a common metabolic disorder associated with chronic complications such as macro-
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micro vascular complications. The onset of T2DM is often silent and many years may pass 
before diagnosis (Barrett, 2017). 
 
2.2.3.1 Pathophysiology of T2DM 
In type 2 diabetes, the two main pathological defects are impaired insulin secretion through 
dysfunction of pancreatic β-cell and impaired insulin action through insulin resistance. T2DM 
leads to a decrease in pancreatic beta-cell function over a period of time. This may occur 
concurrently with insulin resistance and hyperglycemia (Khanam et al., 2017). 
The pathophysiology of type 2 diabetes includes aberrant secretion of glucagon, resulting in 
elevated glucagon concentrations in both the fasting state and after a meal (Girard, 2017). 
Postprandial glucagon suppression may also be flawed. Some T2DM patients can also have an 
increase in glucagon secretion in response to a meal. It is also known that such patient‟s alpha 
pancreatic cells maybe resistant to the glucagon suppressive effects on glucose and insulin. The 
resulting hyperglucagonemia may increase hepatic glucose output, all leading to persistent 
hyperglycemia (Haedersdal et al., 2018). 
 
2.2.3.2 Insulin resistance in skeletal muscles 
Insulin resistance in skeletal muscle manifests itself primarily as a reduction in insulin stimulated 
glycogen synthesis due to reduced glucose transport. Ectopic lipid accumulation in skeletal 
muscles and liver plays an important role in inducing insulin resistance (Baynest, 2015). 
Multiple defects in insulin signaling are responsible for impaired glucose metabolism in insulin 
resistance. Increased lipids promote insulin resistance through the activation of signaling 
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pathways like protein kinase C (PKC), ceramide, and other lipid molecules and the accumulation 
of lipids in target tissues can induce insulin resistance (Gustafson et al., 2015). Lipid 
accumulation within myocytes and hepatocytes is strongly associated with insulin resistance in 
type 2 diabetic patients, in non-diabetic offspring of type 2 diabetic subjects, in subjects with 
impaired glucose tolerance, and in obese subjects (Sesti, 2006). 
Furthermore, excess adipose tissue secretes the metabolites leptin, adiponectin, and tumor 
necrosis factor-alpha and these can decrease cell sensitivity to insulin (Caselli, 2014). 
 
2.2.4 Gestational Diabetes Mellitus (GDM) 
Gestational diabetes mellitus identifies with women who develop diabetes mellitus during 
pregnancy. Women who develop Type 1 diabetes mellitus during pregnancy and women with 
undiagnosed asymptomatic Type 2 diabetes mellitus that is discovered during pregnancy are 
classified as GDM. In most women who develop GDM, the disorder may start in the second or 
third trimester of pregnancy (Piper et al., 2017). 
 
2.2.4.1 Pathophysiology of GDM 
In pregnancy, women develop some insulin resistance, which most likely evolved as a means to 
facilitate energy delivery to the fetus. Insulin resistance occurs as a response to placental 
hormones released during pregnancy. Several pregnancy hormones are thought to disrupt the 
usual action of insulin as it binds to its receptor, most probably by interfering with cell signalling 
pathways, leading to increase in insulin resistance (Mack and Tomich, 2017). Insulin resistance 
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may progress to GDM by revealing autoimmune-mediated or genetic abnormalities in pancreatic 
beta-cell function and/or worsening of chronic insulin resistance in pregnant women (Evensen, 
2012). 
 
2.2.5 Other specific types (Monogenic diabetes) 
Certain types of diabetes mellitus of various known etiologies are grouped together to form the 
classification called “Other Specific Types” (Punthakee et al., 2018). Individuals in this group 
include  persons  with genetic defects of  their beta-cell function (this type of diabetes was 
formerly called MODY or maturity-onset diabetes in youth), persons with diseases affecting 
their  exocrine pancreas, such as pancreatitis or cystic fibrosis, persons with dysfunction 
associated with other endocrinopathies (e.g. acromegaly), and persons with pancreatic 
dysfunction from  drug use, chemicals or infections . This group comprise less than 10% of DM 
cases (Goldenberg  and Punthakee, 2013). 
 
2.2.6 Symptoms of diabetes 
The symptoms of both T1DM and T2DM are similar. They may however develop more  rapidly 
and clearly in type 1 diabetes. Some of the symptoms include weight loss, polyurea, polydipsia, 
polyphagia, constipation fatigue, cramps, blurred vision, and candidiasis. Diabetic patients may 
become susceptible to microvascular complications (neuropathy, retinopathy and nephropathy) 
and macrovascular disease (coronary artery, heart, and peripheral vascular diseases) after 
longstanding hyperglycemia (Baynest, 2015). 
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2.2.6.1 Clinical features of type I diabetes 
In addition to the above symptoms, T1DM also present with diabetic ketoacidosis (DKA), 
accompanied with vomiting, abdominal pain, and lethargy. It can also present as an incidental 
finding performing a urine or blood test for other reasons. DKA can be life-threatening when it 
presents as an acute complication of T1DM (Brinkman, 2017). 
 
2.2.6.2 Clinical features of Type II diabetes 
Most cases are diagnosed because of complications or accidentally.  There is also a high risk of 
large vessel atherosclerosis commonly associated with hypertension, hyperlipidemia and obesity. 
Most patients with type 2 diabetes die from cardiovascular complications and end stage renal 
disease (Zappas and Granger, 2016). 
 
2.2.7 Pre-diabetes  
Individuals with impaired glucose tolerance (IGT) and impaired fasting glycaemia (IFG) have 
intermediate conditions in the transition between normality and diabetes. People with IGT or IFG 
are at high risk of progressing to type 2 diabetes. 
2.2.8 Diagnosis  
A positive test result for any of the below tests isnt conclusive. It should be repeated to exclude  
laboratory error, unless history and clinical signs do not leave any doubt about the diagnosis. 
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Preferably, the same test method should be used. In case of conflicting results from two different 
tests, the test being positive for diabetes should be repeated. 
 
2.2.8.1 Glycated haemoglobin 
The HbA1C test is recommended for diagnosis of diabetes based on a threshold of ≥ 6.5%. The 
HbA1C test may be influenced by ethnicity, haemoglobinopathies,acute/chronic blood loss , 
anemia and becomes invalid in conditions with abnormal red cell turnover (Nitin, 2010). This 
test is relatively expensive and not yet readily available. 
 
2.2.8.2 Fasting Blood Sugar (FBS)/Random Blood Sugar (RBS) 
Fasting blood sugar is the level of sugar in the blood after an 8 hour period fasting. This test is 
usually done in the early morning when the patient has eaten nothing. The normal range for a 
non-diabetic should be between 4-6 mmol/l. Chronic  Fasting blood sugar (FBS) levels greater 
than 7 mmol/l (126 mg/dl) could be indicative of diabetes. Random blood sugar is also the sugar 
level of the  blood 2 hours after eating a meal .With the classic symptoms of hyperglycaemia or a 
hyperglycaemic crisis, a random plasma glucose ≥11.1 mmol/l (200 mg/dl) maybe be diagnostic 
of diabetes. 
 
2.2.8.3 Oral Glucose Tolerance Test (OGTT) 
OGTT is also  used clinically to diagnose impaired glucose tolerance and as a standardized test 
of carbohydrate metabolism and insulin secretion. It requires an oral administration of glucose 
and an observation plasma level of glucose and insulin levels over time, usually 2hours. A 
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prolonged elevation even after 2 hours of administration in both plasma glucose and insulin will 
imply an impaired glucose tolerance and insulin resistance. An OGTT of ≥11.1 mmol/l (200 
mg/dl) is suggestive of diabetes. The test should be performed according to the WHO 
instructions with an equivalent of 75 g anhydrous glucose dissolved in water. 
 
2.2.8.4 Anti-diabetic drugs 
Over the years many anti-diabetic drugs have been developed to lower blood sugar. The drugs 
are varied and act via various mechanisms. Despite the great strides made in anti-diabetic drug 
development, contemporary orthodox drugs are still present with various challenges. These 
include side effects, compliance issues and cost. The table below provides examples of orthodox 
drugs, brand names, their mechanism of action and side effects. 
 
 
 
 
 
 
 
 
 
 
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Table 1: Diabetes drugs, mechanisms of action and side effects. 
 Drug classes  Examples of drugs Mechanism of action Side effects 
Sulphonylureas Gliclazide (Diamicron®) Stimulate the pancreas by binding to Hypoglycemia  
Glimepiride (Amaryl®) Weight gain 
specific site of on the β-cell K
Glyburide (Diaβeta®) ATP  
channel complex and inhibiting its 
activity. 
     
Act  as ligands for proxisome Foot swelling due to 
Thiazolidinediones Pioglitazone (Actos®) water retention, weight 
proliferating activation factor ɣ(PPARɣ) gain. 
Rosiglitazone Regulates of genes that control glucose 
Pioglitazone : increased 
and lipid metabolism. Leads to increased 
(Avandia®) risk of bladder cancer  
glucose uptake in liver increased 
Rosiglitazone : 
sensitivity to insulin. 
increased risk of non-
fatal heart attack  
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Meglitinides Nateglinide (Starlix®) Stimulate insulin release by closing cell Hypoglyceamia  
 KATP  channel complex and inhibiting its 
activity. This causes calcium influx into 
the β-cell and lead to depolarization and 
release of insulin.  
  
Biguanides Metformine Increase the activity of AMP-dependent Diarrhoea, metallic 
kinases causing increased  glucose aftertaste , lactic 
(Glucophage®) 
uptake, decrease in gluconeogenesis  and acidosis 
lipogenesis, increased glucose sensitivity 
and decreased peripheral resistance  
 
Alpha- Acarbose (Glucobay®) Inhibits enzymes in the intestine that Bloating and flatulence. 
glucosidases metabolize carbohydrates. Slows down 
inhibitors 
absorption of carbohydrates. 
Dipeptidyl- Linagliptine (Trajenta®) Increase insulin secretion from β-cells Pharyngitis, headache. 
peptidase-4 (DPP- and decrease glucagon secretion from α-
Saxagliptine 
4) inhibitors cells by increasing glucagon-like 
(OnglyzaMC) 
peptide-1. 
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 Glucagon-like Dulaglutide (Trulicity®) Suppress hepatic glucose production Nausea, diarrhea, 
peptide-1 (GLP-1) Increase insulin secretion from β-cells vomiting. 
                                                 L   i r a  g  l u  t i d  e    ( V   i c  t o  z  a  ®   )                     
agonist and decrease glucagon secretion from α-
Exenatide (Byetta®) cells. 
Sodium glucose Dapagliflozine Lowers renal threshold for glucose, and Vulvovaginitis,balanitis 
cotransporter 2 (Forxiga®) reduce renal reabsorption of filtered urinary tract infections, 
(SGLT2) inhibitors glucose from tubular lumen leading to hypoglycemia. 
 
increased urinary glucose excretion. 
    
Insulins  Short acting- insulin Bind to plasma membrane receptor  and 
lispro(Humalog®), initiate a cascade of signaling events that 
results in the utilization of glucose and 
insulin aspart 
glycogen synthesis. 
(Novolog®). 
Intermadiate acting - 
NPH(Humulin N®). 
Long acting – insulin 
glargine(lantus®). 
(www.diabete.qc.ca, 2017) 
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2.2.9 The Economic burden of Diabetes. 
Diabetes and its complications impose direct or indirect economic stress on individuals,families, 
health care systems and countries (Aronson et al., 2018). People living with diabetes and their 
families  are impacted negatively as the cost of treatment is usually high and can affect family 
income and may also affect the work of the diabetic individual. Many strategies have been 
employed  to prevent  the increase in type 2 diabetes and to reduce its negative economic impact. 
These include cost reductions in healthy foods, taxation or reformulation of unhealthy foods, 
promoting physical activity, and nutritional education and informative food labelling. Also 
health care policies that allow for early detection of diabetes, ease and accessibility to affordable 
diabetes medication are also essential (Zhang  and Gregg, 2017). 
 
2.2.10 Complications of diabetes  
Complications of diabetes can be classified into microvascular and microvascular complications. 
 
2.2.10.1 Macrovascular complications 
Diabetes can affect the brain in various ways including the risk of stroke, cerebrovascular 
diseases like cognitive impairment. There is also an increased risk of coronary heart disease. 
Peripheral vascular disease may occur as blood vessels begin to narrow. This can also culminate 
in reduced blood flow and oxygen to the periphery. Lack of oxygen and blood flow can cause 
nerves to malfunctiona  and  increase numbness in the limbs. Lack of sensitivity  predisposes 
patients to injury. Lack of blood /oxygen supply cause wounds to heal slowly. Gangrene may  set 
in and  amputation if the limb maybe required (Melendez-Ramirez et al., 2010). 
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2.2.10.2 Microvascular complications 
Microvascular complications include diabetic nephropathy, diabetic retinopathy and diabetic 
neuropathy. 
2.2.10.2.1 Diabetic nephropathy 
Specific  renal functional and morphological changes  lead to diabetic nephropathy and are as a 
result of hyperglycemia. The high sugar content causes changes in the structure of the basement 
membrane of the glomerulus. The altered structure causes hyperfiltration, albuminuria, renal 
hypertrophy etc. Glomerular hypertension in diabetes,  renal insufficiency and the defective 
kidney  may eventually lead to kidney failure (Surya et al., 2014). 
 2.2.10.2.2 Diabetic retinopathy 
 Hyperglycemia  is a leading cause of blindness in diabetic patients. Hyperglycemia and blood 
pressure can damage capillaries in the eye leading to retinopathy, cataracts and glaucoma 
(Khalil, 2017).  
2.2.10.2.3 Diabetic neuropathy 
Diabetic neuropathy are a heterogenous group of  nerve disorders that results from perpetual  
hyperglycemia associated with diabetes. It is one of the major complications of diabetes believed 
to affect which approximately half of all diabetes patients (Tiwari et al., 2018) . DN can affect 
various  nerves over the body. Diabetic neuropathy  treatment is cumbersome. This is  because  
there are no specific  medication, and the pathomechanism of  the development of diabetic 
neuropathy  and its associated pain is multidirectional and complicated. It also leads to 
morbidity, mortality and  diabetic amputations (Vinik et al., 2013). 
 
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2.3 DIABETES: THE HERBAL APPROACH 
There are varied approaches in modern medicine formulation to ameliorate the harmful effects of 
diabetes and its subsequent complications. The use of herbal formulations as alternative is 
gaining more attention because they are thought to have fewer side effects and cost less (Parikh 
et al., 2014). Also, some of these plants may possess antioxidant, anti-inflammatory, analgesic 
and anti- hyperglycaemic properties all in one plant giving them an advantage over most 
orthodox drugs. A lot of current research is focusing on herbal drug preparations and plants as 
potential substitutes or alternatives in the treatment of diabetes mellitus and their complications. 
Moreover the cost of treatment of diabetes is still a burden to many around the world (Zhang  
and Gregg, 2017). 
2.3.1 Diabetes Mellitus and Medicinal plants 
Herbal medicines have been used for management of diabetes for decades. Ethnobotanical 
studies have reported about 800 plants that are claimed to possess antidiabetic properties around 
the world (Alarcon-Aguilara et al., 1998). Many plants have been successfully tested for their 
antidiabetic/antihyperglycaemic effects in STZ-induced and alloxan- induced diabetic animal 
models (Arulselvan et al., 2014; Arumugam et al., 2013). Plants act by various mechanism to 
exert their anti-hyperglycemic activity. A few anti-diabetic plants and their possible mechanisms 
of action are listed in the table below. 
 
 
 
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Table 2: Plants used in diabetes, mechanisms of action and model 
Plant name/part        Mechanism of Action                           Model      Reference  
Artemisia afra          Regeneration of pancreatic beta cells,  STZ  (Sunmonu and Afolayan, 2013) 
(Leaves)                   Enhances glucose utilization. 
  
Dioscorea spp.         Improves glucose utilization in           STZ             (Niu et al., 2010)   
(Whole plant)            skeletal muscle  
 
Kigelia pinnata         Improve lipid profile, body weight.     STZ          (Kumar et al., 2012) 
(Flower)                    Stimulates GLUT4 expression 
                              
Psidium guajava       Inhibits  activity of α-glucosidase       Alloxan     (Huang et al., 2011) 
(Leaves)                   Stimulates glucose metabolic enzymes 
 
Sapindus trifoliatus     Has insulin-like activity. Increase      Alloxan      (Sahoo et al., 2010)  
(Fruits)                        glucose usage peripherally. 
  
Terminalia bellerica     Augments secretion of insulin by     STZ       (Latha and Daisy, 2013) 
(Fruits)                         modulation of cAMP and calcium 
 
 Scoparia dulcis             Antioxidant,stimulates insulin          STZ          (Latha et al., 2009) 
(Whole plant)                secretion, secretagogue activity 
                                                (Arulselvan et al., 2014; Bhagour et al., 2016) 
 
 
 
 
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2.3.2 Diabetic complications and medicinal plants. 
Generally plants that have anti-hyperglycaemic properties may improve diabetic complications. 
However plants that have analgesic, anti-inflammatory and anti-oxidant properties in addition are 
preferred. Other plants have also shown different pathways to inhibit diabetic neuropathy. A few 
pure herbal compounds have been isolated, and have been shown to improve diabetic 
complication like neuropathy and retinopathy. These compounds are shown in table below:   
Table 3: Pure compound for treating diabetic complications and their mechanisms of 
action. 
Compound                 Mechanism of Action                         Reference 
Quercetin                   Central analgesic activity                  (Ferreira et al., 2013) 
 
Rutin                          Metal chelating property                   (Oh, 2016) 
                                   Anti-oxidant, anti-inflammatory 
                                   anti- hyperglycaemic.                        (Tian et al., 2016) 
 
Baicalein                    Anti-oxidant, anti-inflammatory,      (Stavniichuk et al., 2011)  
                                   Inhibits sorbitol pathway. 
                                   
Naringenin                 Anti-oxidant                                       (Al-Rejaie et al., 2015) 
 
Hesperidin                Anti-oxidant, anti-inflammatory,      (Visnagri et al., 2014) 
                                   analgesic 
Diosmine                   Anti-oxidant, anti-inflammatory        (Jain et al., 2014; Wojnar et al., 2017)                                                                                                   
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2.4 SYNEDRELLA NODIFLORA (L) GAERTN 
Synedrella nodiflora (L.) Gaertn (asteraceae) also known as node weed is a herb native to 
tropical America. It is however found throughout the West-African sub region, especially in 
marshy or cool and moist places. It is the plant of interest in this study. 
2.4.1 Plants description  
Synedrella nodiflora is an erect branched seasonal shrub usually 30-80 cm tall and it belongs to 
the Asteraceae family (USDA 2010). The plant has a shallow root system that is strongly 
branched. The stems are sometimes woody in nature and may branch directly from the base of 
the plant. The plant normally has long internodes with a hairy swollen node. The plant can grow 
roots on the lower parts of the stem in moist environment. The leaves appear in opposite pairs, 
around 4-9 cm long, elliptic to ovate in shape, commonly displaying three veins and margins that 
are finely toothed. Its petioles are short and finely hairy, and joined by a ridge across the stem. 
It flowers in small bunches of about 2 to 8, at the node and tips throughout the upper parts of the 
plant. The inflorescence normally presents with yellow coloured petals. It produces dimorphic 
dark brown seeds. Ray floret seeds are flat, oblong, 3-5 mm long, teeth pointing upward along 
the paler marginal wing. Disc floret seeds are thicker, elongate, 3-4 mm long, with 2-4 stiff 
bristles at the apex. Both seeds produce the same plant (Adjibode et al., 2015). 
 
 
 
 
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2.4.2 Folkloric uses 
In Ghana, it is known in the Akan dialect as „tutumrika ko hwἐ ἐpo’ (run to watch the sea). The 
aqueous extract of the full plant is used orally to treat epilepsy. The leaves are also used to treat 
hiccup and also to treat threatened abortion (Mshana et al., 2000).The leaves of Synedrella 
nodiflora leaves are usually eaten as food by horses, rabbits, guinea pigs and pigs (Hidayat, 
2001). Some humans are known to eat it without any toxicity reported so far (Li et al., 2014). In 
Ghana farmers sometimes use the plant leaves as post-harvest protectants (Cobbinah et al., 
1999). In Nigeria, some local tribes use the whole plant to treat cardiac problems and to stop 
wounds from bleeding (Idu and Onyibe, 2007). In countries like Malaysia and Indonesia, the 
plant is used as a poultice for sores, headaches, ear aches, stomach aches and to treat rheumatism 
(Burkill, 1985; Sumi et al., 2011).  
 
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Figure 1: Picture of Synedrella nodiflora showing leaves and flowers (CABI, 2018).   
 
 
2.4.3 Phytochemical screening  
Phytochemical screening has been done using solvents like chloroform, methanol, butanol, 
ethanol, hexane and water. The plant possesses many phytochemical constituents including 
flavonoids,  phenolics, steroids, triterpenoids, saponins, alkaloids, phytosterol  glycosides, 
tannins, saponins, reducing sugars and phytosterols (Adjibode et al., 2015). It also had a high 
total phenolic content (Dutta et al., 2012). 
2.5.3 Pharmacological tests 
The plant has shown anti-oxidant / anti-lipid peroxidative properties in vitro (Amoateng  et al., 
2011; Onocha et al., 2016). The constituents may also be responsible for other proven activities 
of Synedrella nodiflora extracts including anti-convulsant activity (Amoateng et al., 2011), anti-
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inflammatory activity (Haque et al., 2012), analgesic (Amoateng et al., 2017), antimicrobial 
(Bhogaonkar et al., 2011) and hypoglycemic effects (Adjibode et al., 2015). Its ethanolic extract 
also show no toxicity on biochemical, haematological and histopathological parameters in sub-
acute, acute and long term administration in male Sprague – Dawley rats (Adjei  et al., 2014; 
Amoateng et al., 2014; Amoateng et al., 2016). Finally the ethanolic extract has also exhibited 
analgesic effect in paclitaxel and vincristine induced neuropathy (Amoateng  et al., 2015; 
Amoateng  et al., 2017). These properties have opened the door for much research to be done on 
this plant for its potential medicinal benefits in managing diabetes for humans. 
 
2.5 ANIMAL MODEL IN DIABETES 
Animal models have been used extensively in diabetes to study the pathophysiological changes 
that take place in the condition and to evaluate the effect of drugs/potential drugs on the disease. 
Diabetes can be induced in animal models by chemical, viral, genetic or by spontaneous 
autoimmune mechanisms. Certain models may also present limitations that may not be found in 
other models. The STZ- induced diabetic model is one of the most widely used and reliable 
methods. Below is a table of a few examples of animal models in diabetes, induction mechanism, 
main features and possible limitations. 
 
 
 
 
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Table 4: Models for diabetes and their limitations 
Induction mechanism                  Model                              Limitations 
Chemical                                     Streptozotocin                 May affect other internal organs  
         Alloxan 
 
Spontaneous          NOD mice                        Gender differences.  
Autoimmune         BB rats                             Unpredictable 
 
Genetic          AKITA mice                    Not validated by antineuropathic drugs 
 
Obese model OLETF rats Exhibit renal complications 
(Polygenic)         
 
Viral                                           Cocksackie B virus           Outcome is dependent on replication  
                                                   Kilham rat virus                 levels of the virus as well as timing  
                                                                                              of the infection 
 
Induced obesity      High fat feeding                 Genetic difference is response to high  
                                                                                             fat diet 
 
(King, 2012)
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CHAPTER THREE 
3.0 MATERIALS AND METHODS  
3.1 STUDY DESIGN 
The study was an experimental study design. 
3.2 PLANT EXTRACT PREPARATION 
3.2.1 Plant acquisition 
The whole plant samples (stems, leaves and roots) of Synedrella nodiflora were harvested in 
University of Ghana at the Botanical Gardens (Latitude, 5.6599, longitude, -0.1840) in the month 
of October, 2017. Harvesting was done in the rainy season of the year when the plant was in 
abundance, in the cool of the morning. The plant was authenticated in the Ghana Herbarium of 
Department of Botany in the University of Ghana, Legon. A voucher specimen labeled 
(PA01/UGSOP/GH17) was kept. 
 
3.2.2 Preparation of the whole plant of Synedrella nodiflora 
The collected plant material was air dried for 7 days and powdered using a hammer mill 
(Liming®, China). A 3 kg quantity of the sample was weighed, extracted with 70 % (v/v) 
ethanol by cold maceration for 3 days and concentrated in a rotary evaporator (Rotavapor R-210, 
Buchi, Switzerland) at 60 °C. It was finally oven dried (Gallenkamp®, UK) at 50 °C to a 
constant weight. A dark greenish slurry residue of 31.3 g was obtained and refrigerated until 
reconstituted into a solution for administration. This extract was labelled as SNE and stored in a 
refrigerator for later use . A yield of 10.4 % w/w was obtained. 
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3.2.3 Phytochemical screening 
Screening of SNE for the presence of alkaloids, tannins, saponins, triterpenoids, flavonoids, 
volatile oils, phenolic and glycoside was done according to various tests specified in literature 
(Evans and Evans, 2009; Sofowora, 1982).  
3.2.3.1 Alkaloids (Dragendorff’s Test) 
A 0.5 g quantity of SNE was extracted with 20 ml of ammoniacal alcohol and filtered. The 
residue obtained after evaporating the filtrate was shaken with 1 % H2SO4 and filtered. The 
filtrate was made alkaline with dilute ammonia solution and shaken with chloroform; the 
chloroform layer was separated and evaporated to dryness. The residue was dissolved in 1 % 
H2SO4 and one drop of Dragendorff‟s reagent (Sigma Aldrich Co. Ltd. Irvine, UK) was added. 
An orange-red precipitate shows alkaloids are present. 
3.2.3.2 Saponins (Frothing test) 
A 1 g quantity of SNE was dissolved in distilled water and filtered. The filtrate was given a  
vigorous shake and left to stand for 5 minutes. The presence of persistent  froth on standing 
indicated the presence of saponins in this extract. 
3.2.3.3 Phytosterols (Lieberman-Burchard’s test) 
Using 5 ml chloroform, 0.5 g of SNE was extracted with and filtered into a test tube. Several 
drops of acetic anhydride (Sigma Aldrich Co. Ltd. Irvine, UK) were added and mixed carefully. 
Two (2) drops of concentrated H2SO4 (BDH Laboratories, England) was added to test tube 
gently through the side. Formation of violet to blue coloured ring at the junction of two liquids 
showed the presence of steroid moiety. 
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3.2.3.4 Terpenoids (Salkowski test) 
With 5 ml chloroform, 0.5 g of SNE was extracted and filtered into a test tube. The careful 
addition of concentrated H2SO4 (BDH Laboratories, England) to test tube wall produced a violet 
colouration showing the presence of terpenoids. 
3.2.3.5 Glycosides (General test) 
A 0.5 g quantity SNE was extracted by warming with 5 ml of dilute H2SO4 on a water bath for 2 
minutes and filtered. The filtrate was turned alkaline by adding 2-5 drops of 20 % NaOH (BDH 
Laboratories, England). 1 ml of Fehling‟s solution A and B (Sigma Aldrich Co. Ltd. Irvine, UK) 
was added to the filtrate and dried on a water bath for 2 minutes. The presence of a brick-red 
precipitate indicated the presence of glycosides. 
3.2.3.6 Tannins (Ferric Chloride test) 
A 0.5 g quantity of SNE was boiled with distilled water for 5 minutes. The boiled extract was 
cooled, filtered and made up to 25 ml. To 1 ml of the extract, 10 ml of distilled water was added 
followed by 2-10 drops of 1 % Ferric chloride solution (Sigma Aldrich Co. Ltd. Irvine, UK). A 
blue-black or blue-green colouration showed a positive test for tannins. 
3.2.3.7 Phenolic  Compounds (Acetic acid test) 
A 0.5 g quantity of SNE was boiled with distilled water for 5 minutes. The boiled extract was 
cooled, filtered and made up to 25 ml. To 1 ml of the extract, 10 ml of distilled water was added 
followed by 3-4 drops of acetic acid (Sigma Aldrich Co. Ltd. Irvine, UK). The solution turned 
red indicating the presence of phenolic compounds. 
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3.2.3.7 Flavonoids (Kumar test) 
A 0.5 g quantity of SNE was dissolved in water and filtered; to this 2 ml of 10 % NaOH (BDH 
Laboratories, England) solution was added to produce a yellow colouration. A change in colour 
from yellow to colourless on addition of dilute HCl indicated the presence of flavonoids. 
3.2.3.9 Volatile Oils 
To a 0.3 g quantity of the extract a 5 drops of alcoholic solution of Sudan III was added and 
mixed thoroughly. No reddish coloured globules were obtained, indicating the absence of 
volatile oils. 
3.3 EXPERIMENTAL ANIMALS AND HUSBANDRY 
Male Sprague-Dawley rats (160-200 g) 8 weeks old were obtained from the Centre for Plant 
Medicine Research, Mampong, Ghana, and transported to Animal House Section of the Noguchi 
Memorial Institute. The animals were allowed to acclimatize and adapt to the new environment a 
week before the study commenced. They were put in cages of five rats each in stainless steel 
cages (34 cm × 47 cm × 18 cm) with wood shavings as bedding. The room for the cages was 
adequately ventilated at normal room temperature (22–26 °C). The relative humidity of 60–70 
%with a natural light-dark cycle was maintained during the period. Good sanitation practices 
were observed with regular cleaning of cages and change of bedding. The rats were fed with rat 
feed (Agricare Ltd, Kwadaso, Kumasi, Ghana) and given clean water ad libitum. The processes 
and procedures employed during the experiment stayed in agreement with the methods approved 
by the Scientific and Technical Committee (STC) of the Noguchi Memorial Institute for Medical 
Research (N.M.I.M.R) [reference number STC-6 (International Diabetes Federation) 2012–13] 
and also by the Noguchi Institutional Animal Care and Use Committee (N.I.A.C.U.C), College 
of Health Sciences, University of Ghana with protocol number NIACUC-2017-01-1E.The 
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procedures for the research work was permitted by the Department of Pharmacology, University 
of Ghana. 
 
 
3.4 INDUCTION OF DIABETES MELLITUS 
3.4.1 Preparation of citrate buffer 
To prepare the buffer solution, 1.76 g of sodium citrate (Mwt- 294, Zigma-Aldrich, St Loius, 
USA) was dissolved in 60 ml of distilled water to obtain a 0.1M solution.  1.05grams of citric 
acid monohydrate powder (Mwt-210, Zigma-Aldrich, St Loius, USA) was also dissolve in 50 
mls of water to obtain 0.1M solution. 44.5mls of 0.1M citric acid was combined with 55.5mls 
sodium citrate to obtain 100 ml of citrate buffer pH of 4.5. The amount of streptozotocin needed 
was calculated in relation to the total weight of all the rats, weighed and dissolved in the citrate 
buffer. 
3.4.2 Process of diabetes induction with STZ 
Induction of diabetes was done 1 week after arrival of rats (after acclimatization). Animals to be 
induced were fasted for 16 hours and subsequently weighed and given a single intra-peritoneal 
injection of STZ (55 mg/kg) (streptozotocin, Zigma-Aldrich, St Loius, USA) already dissolved 
in a freshly prepared citrate buffer of pH 4.5. Rats in control group were given only citrate buffer 
of equal volume. The rats were allowed access to feed and water ad libitum after the injection.  
Three (3) days after the injection, FBS (fasting blood sugar) was measured for all the animals 
using a blood glucose monitor (One Touch Select Plus®, Lifescan, Gubelstrasse, Switzerland). 
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FBS was measured at 7 am in the morning, one hour before feeding. Animals with values greater 
than 11 mmol/L were considered to be diabetic. 
3.4.3 Oral glucose tolerance test 
All STZ injected Sprague-Dawley rats were fasted overnight and orally administered with bolus 
glucose load of 2 g/kg at 08:00 AM. Blood samples were periodically measured from the tail 
vein at 0, 30, 60, 90, and 120 min after administration of the glucose. The levels of blood glucose 
were evaluated by using a glucose monitor (One Touch Select Plus®, Lifescan, Gubelstrasse, 
Switzerland). All SD rats with a blood glucose level above 11 mmol/L after 2 hours were 
considered to be diabetic. This test is a confirmatory test for diabetes (Ernsberger and Koletsky, 
2012). 
3.4.4 Grouping  
The Sprague Dawley rats were divided into 8 groups of 5 each. The first group comprised of rats 
that received no STZ. The rats injected with STZ, with FBS and OGTT >11 mmol/L were 
divided into 7 groups containing 5 animals each. The vehicle (citrate buffer) group was not 
treated with any drug. Subsequently there were three groups for insulin (0.1 IU/kg, 0.3 IUkg and 
1 IU/kg bodyweight) and SNE (100 mg/kg, 300 mg/kg and 1000 mg/kg bodyweight).  
3.4.5 Preparation of drugs                                                    
Insulin human (rDNA) (100 IU/ml solution, Actrapid®, novo nordisk, Denmark) was 
administered as reference drug was serially diluted with Normal saline (500 mls, Sanbao 
Pharmaceticals Ltd, Tema, Ghana) to obtain the minute quantities needed. This solution was 
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freshly prepared daily before administration subcutaneously. SNE was mixed with distilled water 
and administered orally by gavage. 
3.4.6 Drug administration 
The control and diabetic control group received no treatment except food and water. Insulin 
group received a subcutaneous injection of Insulin human (rDNA) twice daily. SNE group 
received a daily gavage at 7:30 am, 30 minutes before feeding. Drug administration was done 
every day for 8weeks before animals were sacrificed.  
3.4.7 Fasting blood sugar (FBS) determination 
FBS of animals were measured twice weekly and the weekly average calculated throughout the 
eight (8) weeks. Fasting blood sugar was taken at 7am every morning one hour before feeding 
using the OneTouch Select plus glucometer. 
 
3.4.8 Determination of percentage change in bodyweight and relative organ weight. 
 The first and final weights of the rats were recorded on the day of induction of diabetes and on 
the last day of the experiment when the rats were sacrificed. The percentage changes of body 
weight of each group were calculated using the formula below: 
             –               
Percentage change in body weight =   × 100 
              
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The harvested liver, spleen, kidney, brain and heart were washed with normal saline, dried with 
tissue paper, and weighed. The weight of the excised organs with respect to the body weight of 
the rat at the end of the experiment was calculated from the formula below: 
                         
Relative organ weight =   × 100 
                                   
 
3.5 PREPARATION OF SD RATS FOR BLOOD TESTS AND HISTOPATHOLOGY  
3.5.1 Sacrificing of rats. 
th
On the last day of the 8  week all animals were weighed. They were then chloroformed and 
sacrificed after 8 weeks. Blood was collected from the jugular vein to into serum separating gel 
tubes (BD Vacutainer® blood collection Tube Product, USA) and EDTA tubes (Mediplus 
vacutainer K3, Sunphoria Co. Ltd., Taiwan) for serum preparation and haematological analysis 
respectively. 
3.5.2 Preparation of organs 
 Rat organs (liver, kidney, lungs, brain and spleen) were harvested, cleaned and dried with tissue. 
The organs were then weighed. All organs were examined by trained veterinary doctors for any 
pathological lesions and placed in labeled containers containing 10 % neutral buffered formalin. 
The organs were then transported to a pathology laboratory for processing and examination. 
 
 
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3.5.3 Preparation of blood 
About 2 mls of blood was collected into EDTA tubes and carefully turned back and forth by 
rubbing them in the palms to allow even mixing of the blood with EDTA to prevent clotting. 
After 2 minutes of swiveling, the samples were transported on ice in thermos flask for 
haematological analyses. 
3.5.4 Preparation of serum 
To obtain the serum 3-4 ml of blood were allowed to clot at room temperature (27 °C) in the gel 
tubes after they were collected. The tubes were then centrifuged (Heraeus Labofuge 300, UK) at 
3000 rpm for 20 minutes. A clear supernatant obtained was collected by pipetting into plain 
tubes. These tubes were labeled accordingly and were cold-chain transported immediately to the 
lab for biochemical analysis. 
3.5.5 Kidney and liver Function tests 
From the serum prepared using the above procedure, the values for the level liver transaminases 
[aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase (ALP)], total protein 
(TP) and albumin (ALB), kidney urea (Ur) and creatinine (Cr) were determined. Lipid profile 
(HDL, LDL, total cholesterol) and were made using an automated clinical chemistry analyzer 
(ABX Pentra C200, Horiba Medical, USA).  
3.5.6 Haematological analysis 
An automated haematology analyser (Sysmex XP -300 TM Automated Analyser, USA) was 
employed in the assessment of the following haematological parameters from blood collected 
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above. The parameters included white blood cells (WBC), neutrophils (NEU), lymphocyte 
(LYM), red blood cell (RBC), haemoglobin (HGB), mean corpuscular volume (MCV), 
haematocrit (HCT), mean corpuscular haemoglobin, mean corpuscular haemoglobin 
concentration (MCHC), basophils and eosinophils, platelet (PLT). 
 
3.6 HISTOPATHOLOGY 
3.6.1 Processing of Organs into slides 
Portions of the organs were selected into labeled tissue processing cassettes and processed into 
paraffin bocks. Each was passed through ascending grades of alcohol (70%, 80%, 90% and 
absolute) and further two changes of absolute alcohol for dehydration, cleared in three changes 
of xylene and finally infiltrated and embedded in paraffin wax. Five micron sections were cut 
from each block, mounted on microscope slides and stained by the haematoxylin and eosin 
method. 
3.6.2 Examination of slides 
The sections of the liver, spleen, lung, brain and kidney samples of the sacrificed animals were 
examined by a qualified histopathologist who was blind to the experimental profiles under a light 
microscope after they have been stained with eosin and haematoxylin.  
 
 
 
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3.7 ANTIOXIDANT ACTIVITY 
3.7.1 Determination of Superoxide Dismutase (SOD) Activity 
Superoxide anion (O2-) is produced as a by-product of oxygen metabolism and, if not regulated, 
causes many types of cell damage through oxidative stress. SOD reacts with superoxide anion to 
form hydrogen peroxide and oxygen in the body. This is a natural protective action against 
oxidative stress in the body. Hence the presence of Superoxide anion in a sample could be 
indicative of SOD depletion and high oxidative stress. The anion‟s presence can be detected by 
using the SOD Assay Kit because it reacts with highly water-soluble tetrazolium salt, WST-1 (2-
(4-Iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2Htetrazolium, monosodium salt) that 
produces a water-soluble formazan dye. Measurement of the IC50 (50% inhibition activity of 
SOD or SOD-like materials) can be determined by a colorimetric method.  
3.7.2 Procedure for SOD activity determination 
19160 SOD determination kit (Sigma-Aldrich, 3050 Spruce Street, St.Louis MO 63103, USA) 
was used for SOD assay. WST (water soluble tetrazolium salt) working solution was prepared by 
diluting 1 ml of WST solution with 19 ml of buffer solution. The enzyme working solution was 
centrifuged for 5 seconds and mixed by pipetting. It was then mixed with 2.5ml of the dilution 
buffer. Each sample was assigned four blank wells. The wells were labelled blank 1 blank 2 and 
blank 3 respectively. Twenty microliters (20 µ) of sample solution was added to each sample and 
blank 2 well.  20 µl of ddH2O (double distilled water) was also added to each blank 1 and blank 
3 well.  200 µl of WST Working Solution was subsequently added to each well and mixed. Then 
20 µl of Dilution Buffer was added to each blank 2 and blank 3 wells. Finally 20 µl of enzyme 
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working solution was added to each sample and blank 1 well and mixed thoroughly. These wells 
were incubated in the plate at 37 °C for 20 min. The absorbance at 450 nm was read using a 
microplate reader. SOD activity (inhibition rate %) was calculated using the following equation: 
                               
SOD activity (inhibition rate %) =   × 100 
                
This method is similarly employed by (Chiari-Andréo et al., 2017; Ukeda et al., 1997). 
 
3.7.3 Malondialdehyde (TBARS) determination 
Biological cells when posed to high levels of oxidative stress accumulate mixture of aldehydes 
which are derived from the decomposition polyunsaturated fatty acids. Aldehydes can be 
cytotoxic. Malondialdehyde (MDA) and MDA-like products are the most common lipid 
peroxidation products formed. The analysis of MDA by the classical TBA (2-thiobarbituric acid) 
test is still a simple but dependable method for determining oxidative stress.  
3.7.4 Procedure for MDA determination 
  Rat serum sample of 0.5 ml was mixed with 0.5 ml of ice-cold 10% Trichloacetic acid and 
incubated for 30 minutes to deproteinize it. Then 1.0 ml of 0.67% Thio-Barbituric Acid was 
subsequently added to the mixture mixed properly and heated at 100 ºC for 30 minutes. The 
mixture was then allowed to cool. 4.0 ml of  n-butanol was added to the mixture and  vortexed  
for 30 seconds. It was then centrifuged at 500 g for 10 minutes. The supernatant obtained was 
drawn into 96- well plates using a micropipette. Each sample was duplicated. The absorbance 
was then measured at 535 nm using the spectrophotometer (GloMax Explore GM35000, 
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Promega Corporation, Madison,Wisconsin, USA). The results were expressed in umol/L using 
5 -1
the extinction coefficient of 1.56 X10  Lmmol cm (Kamal et al., 1989).  
 
3.8 ANALYSIS OF RESULTS 
GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA) was used 
for all statistical analyses and ED50 determination. P < 0.05 was considered statistically 
significant in all analysis. The graphs were plotted with Sigma Plot for the Window version 11.0 
(Systat Software Inc., Germany) 
 
 
 
 
 
 
 
 
 
 
 
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CHAPTER FOUR 
4.0 RESULTS 
4.1 PHYTOCHEMICAL SCREENING 
Table 2: Results of Phytochemical screening of the ethanolic extract of Synedrella nodiflora. 
+ indicates presence and - indicates absence of constituent. 
CONSTITUENT                                                                        RESULT  
Alkaloids                                                                                               + 
Saponins                                                                                                + 
Taninns                                                                                                  + 
Phytosterols                                                                                           + 
Phenolics                                                                                                + 
Volatile oils                                                                                            - 
Terpinoids                                                                                              + 
Flavonoids                                                                                              + 
Glycosides                                                                                              + 
Absent:-       Present:+ 
 
 
 
 
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4.2 INDUCTION OF DIABETES 
The Fasting blood sugar of both control and treatment group were measured before and 72 hours 
after the injection of STZ (55 mg/kg bodyweight). There was a significant increase in fasting 
blood sugar levels for the STZ-treated group (P< 0.0001, F3,68 = 22.36, Fig. 2A) in comparison to 
the control group. Subsequently the vehicle group (untreated) maintained a high fasting blood 
sugar over the 8 week period significantly higher than the control group (P< 0.0001, F4,25= 27.56, 
Fig. 2B). 
 
 
 
 
 
 
 
 
 
 
 
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A. B.
Vehicle
35 Control
40  †††  †††
Control 30
 †††  †††
*** STZ- Treated  †††  †††  †††
30 25  †††
20
20
15
10 10
5
0
Day 1 Day 4 0
0 2 4 6 8
Time (Days)
Time (Weeks)
 
Figure 2: The effect of STZ injection of SD rats in diabetes induction. The FBS levels of control 
and STZ-treated rats (55 mg/kg, i.p) before and 72 hrs after treatment (A). The FBS levels of 
vehicle group and control group from week 1 to week 8 of experiment (B). Values are means ± 
s.e.m. (n=5). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; compared to control group (one-way 
 † †† †††
anova followed by bonferroni‟s post hoc test) for A. P ≤ 0.05; P ≤ 0.01; P≤ 0.001; 
compared to control group (two-way ANOVA followed by Newman-keuls post hoc test) for B. 
 
 
 
 
 
 
42 
 
Fasting blood sugar (mmol/L)
Fasting Blood Sugar (mmol/L)
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4.3 ANTIHYPERGLYCAEMIC ACTIVITY OF SNE 
The administration of SNE 1000 mg/kg gave a significant reduction in fasting blood sugar levels 
th  
on the 4  week (P< 0.05, F4,38 = 21.14, Fig.3C). Other SNE doses gave no significant reduction   
over the 8weeks (P> 0.05). The total fasting blood sugar activity (calculated as AUCs) showed 
that the doses of SNE gave no significant reduction (P< 0.05, F4,32 = 32.82, Fig.3B) in insulin 
over 8 weeks. The administration of insulin (0.1-1.0 IU/kg) showed a significant reduction in the 
rd th
FBS   for only 1IU/kg dose of insulin on the 3  and the 8  week (P< 0.05, F3,19 = 27.56, Fig.3A). 
All other doses of insulin did not give any significant reduction in fasting blood sugar compared 
to the control (P> 0.05). The total fasting blood sugar activity (calculated as AUCs) showed that 
the highest of insulin (1 IU/kg) gave a significant reduction (P< 0.05, F3,68 = 20.82, Fig.3B) in 
insulin over 8 weeks. An ED50 of 187.9 mg/kg and 0.156 IU/kg were obtained for the SNE 
extract and insulin respectively. 
 
 
 
 
 
 
 
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Vehicle
A SNE100mg /kg B
SNE 300 mg /kg 
35
SNE 1000mg /kg 200
180
30
160
25 140
120
20
100
15 80
   † 60
10
40
5 20
0
0
Vehicle 100 300 1000
0 2 4 6 8
Time (Weeks)
SNE(mg/kg)
              
C Vehicle D
Insulin 0.1IU /kg
40 250
Insuln 0.3 IU /kg
Insulin 1.0 IU /kg
35
200
30
25
150
20
15 100
   †
10  †  †
50
5
0
0 2 4 6 8 0
Vehicle 100 300 1000
Time (Weeks)
Insulin (IU /kg)  
Figure 3: The effect of the administration of SNE (100 mg/kg, 300 mg/kg and 1000 mg/kg) and 
insulin (0.1 IU /kg, 0.3 IU/kg and 1 IU/kg) in diabetic rats over 8 weeks. The  graphs on the left  
(A and C) show the time course of effects over the 8-week period and the right (B and D) show 
the total anti-hyperglycaemic activity calculated from the AUCs for the test duration. Values are 
† †† †††
means ± SEM (n=5). P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 compared with vehicle-treated group 
(two-way analysis of variance followed by Bonferroni‟s post hoc test). One way analysis of 
variance was employed for AUCs. 
44 
 
Fasting Blood Sugar (mmol/L)
Fasting Blood Sugar (mmol/L)
Antihyperglycaemic activitiy (calc. as AUCs)
Antihyperglycemic activity (calc. as AUCs)
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Table 6: ED50 of insulin and SNE. 
 
Drug                                                                                                    ED50 
 
Insulin (human (rDNA) 0.156 IU/ kg
 
SNE (Synedrella nodiflora ethanolic Extract)                                     187.9 mg/kg
 
 
80
INSULIN
SNE
60
40
20
0
-4 -2 0 2 4 6
Log Dose
 
 Log dose-response curves showing the effect of SNE (100 - 1000 mg/kg) and insulin (0.1 IU-1.0 
IU/kg) on hyperglycaemia in STZ-induced diabetic rats. Each point is the mean ± S.E.M. of 5 
animals.  
 
 
 
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 4.4 WEIGHT PARAMETERS 
4.4.1 Percentage change in bodyweight 
The percentage change in bodyweight was calculated by finding the difference between final and 
the initial weight and expressing it as a percentage of the initial weight. Induction of diabetes 
caused a significant weight loss (P< 0.001, F3,68 = 13.86, Fig 4.) in the vehicle and SNE doses 
(100 and 300 mg/kg) compared to the control after 8 weeks of treatment. There was a less 
significant loss in weight for   SNE 1000 mg/kg compared to the control (P< 0.01, F4,54 = 15.66, 
Fig 4.) after 8 weeks.  
 
 
 
 
 
 
 
 
 
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4.
50
40
30
20
   ††
10
0
-10  †††  †††
 †††
-20
Control Vehicle 100 300 1000
                    
                  SNE (mg/kg)
 
Figure 4. Effect of diabetes on bodyweights of diabetic rats receiving vehicle and SNE (100-
 † †† †††
1000 mg/kg, p.o).Values are means ± SEM (n=5). P≤ 0.05, P ≤ 0.01, P ≤ 0.001 compared 
with control group (one-way analysis of variance followed by Newman–Keul‟s post hoc test) 
 
 
 
 
 
 
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 Change in bodyweight (%)
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 4.4.2 Relative Organ Weight 
For relative organ weights, a significant increase was seen in only liver weights for the vehicle 
group (P< 0.05, F4,25 =14.32, Table 7). All other groups had no significant difference in relative 
organ weights for all organs (P> 0.05, Table 7). 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Table 7: Effect of diabetes on relative organ weights of diabetic rats receiving vehicle and 
SNE (100-1000 mg/kg) for 8 weeks. 
Organs  Relative organ weight (%)  
   
Vehicle                SNE 100 mg     SNE 300 mg     SNE 1000 mg P- VALUE 
      
 
6.00 ±0.5 8  †
     
  
Liver  4.17±0.20 4.45±0.38 4.28 ±0.31 0.02 
  
Kidney  0.52±0.02 0.42±0.02 0.52±0.03 0.48±0.03 0.35 
  
Lungs 1.33±0.38 1.03±0.03 0.92±0.11 0.88±0.08 0.12 
  
Brain 0.97±0.03 0.95±0.05 0.90±0.06 1.00±0.06 0.23 
  
Spleen 0.23±0.03 0.23±0.03 0.23±0.03 0.26±0.02 0.28 
 
† ††  †††
Values are means ± SEM (n=5). For P-value, P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 compared with 
vehicle-treated group (B) (one-way analysis of variance followed by Bonferroni‟s post hoc test) 
 
 
 
 
 
 
 
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4.5 BLOOD BIOCHEMISTRY  
Blood biochemistry after 8 weeks of treatment showed that there was a  significant decrease in 
creatinine level for all diabetic groups i.e. (P< 0.01, F4,13=9.497, Table 8) for vehicle and 300 
  
mg/kg SNE and (P< 0.05, F3,17=3.233, Table 8) for 100 and 1000 mg/kg SNE group. Albumin 
levels also decreased significantly for the vehicle group (P< 0.01, F4,25=4.343, Table 8). There 
were also significant increases in AST (P< 0.01, F3,18=3.429, Table 11), ALT and ALP (P< 0.05, 
F4,22=8.215, Table 8) for the vehicle group in comparison with the control. The SNE groups 
(100-1000 mg/kg) gave no significant change (P> 0.05, Table 8) in AST, ALT, ALP and 
albumin in comparison with the control. There were no significant changes in lipid profile, urea, 
total protein and globulin (P> 0.05, Table 8) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Table 8: The effect of SNE (100–1000 mg/kg, p.o) on blood biochemistries in STZ –induced  diabetic SD rats after 8-week 
a dministration .  
 
 Parameter Control  Vehicle  SNE 100 mg SNE 300 mg SNE 1000 mg P value 
 
  
Urea (mmol/L) 9.07±0.54 14.62±2.18 10.59±0.24 14.65±.36 15.68±2.62 0.06 
   
Creat(mmol/L) 75.08±9.81 34.80±2.22** 45.43±4.45* 38 .43±4.01** 48.60±5.02* 0.02 
  
 Tot. Chol. 2.10±0.28 2.17±0.21 2.21±0.22 2.71±0.16 2.25±0.30 0.53 
  
 Triglycerides 2.45±0.45 2.55±0.60 1.700±0.25 1.42±0.14 1.50±0.08 0.08 
  
HDL(mmol/L) 0.81±0.13 1.06±0.31 0.89±0.17 1.19±0.11 0.95±0.15 0.52 
   
LDL(mmol/L) 0.68±0.13 0.48±0.04 0.47±0.04 0.52±0.05 0.57±0.06 0.43 
  
 
Tot. Pro. (g/L) 85.58±2.52 76.60±4.16 89.93±5.66 85.65±1.76 83.62±1.76 0.13 
  
 Albumin(g/L) 39.56±1.07 29.07±1.51** 37.60±2.73 36.30±1.70 35.54±1.05 0.01 
  
Globulin (g/L) 46.04±2.10 47.53±4.33 52.33±3.36 49.35±1.82 48.36±1.67 0.53 
  
ALT(U/L) 106.70±26.00 613.70±190.60* 140.70±15.66 192.40±40.65 219.40±58.92 0.04 
 
 
AST(U/L) 
191.30±106.20 1336.00±395.40** 199.10±20.79 193.00±27.40 318.70±56.70 0.01 
               
 
ALP(U/L) 297.70±41.79 892.00±149.00* 558.00±51.80 651.90±197.70 449.70±249.90 0.02 
 
Data are mean ± SEM (n = 5). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 compared to control group (one-way ANOVA followed by a 
Bonferroni‟s post hoc test) 
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4.6 HAEMATOLOGY 
The vehicle group showed a significant increase in eosinophil count (P< 0.01, F4,20 = 17.15, 
Table 9) after 8 weeks. All the SNE groups showed no significant change in eosinophil count 
compared to the control (P> 0.05, Table 9).The vehicle group also gave a significant decrease in 
RBCs (P< 0.001, F3,20 = 11.11, Table 9) and haemoglobin level (P< 0.01, F4,20 = 13.133, Table 9). 
There was no significant change in the same parameters for the SNE groups (P> 0.05, Table 
9).There were also no significant changes in other parameters (MCV, MCHC, MHC, PLT etc) 
for all animal groups. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Table 9: The effect of SNE (100–1000 mg/kg, p.o) on haematological indices in STZ -
induced diabetic SD rats after 8 week administration compared to control and vehicle. 
 P-
Parameter  Control Vehicle 100 mg/kg 300 mg/kg 1000mg/kg value 
 
WBC # 2.29±0.09 2.640±0.36 4.81±1.67 8.03±1.02 8.29±1.68 0.01 
  Neut % 22.05±4.35 23.35±14.85 38.80±0.40 29.95±5.65 30.62±2.89 0.33 
 
Lymp% 75.60±4.60 48.55±0.55 57.10±1.40 68.00±6.08 66.24±2.46 0.04 
 
Eos% 1.15±0.05 8.00±2.00** 1.90±0.10 1.17±0.51 1.68±0.54 0.01 
 
Mon % 0.90±0.60 0.95±0.05 0.80±0.10 0.53±0.20 0.56±0.14 0.38 
 
Bas% 0.30±0.20 1.60±0.10 1.40±0.80 0.35±0.12 0.96±0.14 0.08 
 
Neut# 0.50±0.08 0.83±0.05 1.92±0.62 2.24±0.15 3.97±0.33* 0.01 
 
Lym#  1.74±0.18 1.21±0.09 2.81±0.81 5.64±1.17 8.90±1.05* 0.01 
 
Mon# 0.02±0.01 0.02±0.01 0.04±0.02 1.04±1.02 0.08±0.06 0.67 
 
Eos# 0.02±0.01 0.19±0.04 0.09±0.04 0.08±0.03 0.22±0.09 0.31 
 
Bas# 0.01±0.01 0.02±0.01 0.08±0.07 0.03±0.03 0.12±0.03 0.13 
 
RBC(×10^12) 6.59±0.16 2.49±0.16*** 7.99±0.48 7.92±0.31 7.74±0.61 0.01 
 
MCV (fL) 59.65±0.25 60.95±1.25 61.20±1.40 61.58±0.68 63.18±1.81 0.62 
 
MCH pg 19.15±0.35 19.60±0.60 18.95±0.65 18.88±0.16 20.08±0.76 0.58 
 
MCHC (g/dL)  32.15±0.45 32.10±0.30 30.95±1.85 30.68±0.48 31.72±0.29 0.41 
 
HGB (g/dL) 12.75±0.45 5.00±0.30** 15.15±1.45 14.95±0.61 15.38±0.6 0.01 
 
PLT#  879.50±20.50 157.00±8.00 476.50±8.50 737.00±321.2 880.60±132.20 0.28 
Data are mean ± SEM (n = 5). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 compared to control 
group (one –way ANOVA followed by a Bonferroni‟s posthoc test). NB # = (×10^9/L) for counts. 
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4.7 ANTIOXIDANT ACTIVITY 
4.7.1 SOD and MDA assay 
For SOD activity, there was dose dependent increase in SOD activity for extracts of SNE. The 
 
SNE 1000 mg/kg gave a significant increase (P< 0.05, F4,25 = 4.050, Fig 5A) in SOD activity 
 
compared to the vehicle. The increase in activity by 100 and 300 mg/kg was not significant (P 
>0.05). MDA also showed a dose dependent decrease in the amount of TBARS substances. The 
highest decrease was in the SNE 1000 mg/kg group which was the only significant decrease (P 
<0.01, F3,18 = 3.650, Fig 5B). The other groups showed no significant decrease (P >0.05). 
 
 
 
 
 
 
 
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5A 5B
120 140
   †
120
100
100   ††
80
80
60
60
40 40
20 20
0
0 Vehicle 100 300 1000
Vehicle 100 300 1000
SNE(mg/kg) SNE (mg/kg)  
 
Figure 5. The effect of SNE (100, 300 and 1000 mg/kg, p.o) on SOD activity (A) and  MDA 
levels (B) in STZ -induced diabetic male SD rats after 8week of administration. Data are mean ± 
† †† †††
SEM (n=5). P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 compared to vehicle group (one –way ANOVA 
followed by a Bonferroni‟s post hoc test) 
 
 
 
 
 
 
 
 
 
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SOD Activity (U/ml of Serum)
Plasma MDA content (nM/ml)
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4.8 HISTOPATHOLOGY  
Liver histopathology showed a distortion in the microstructure for the vehicle group. There were 
large interstitial spaces, dilated central veins and increased blood congestion compared to the 
SNE groups. These changes were not obvious in the SNE which had a few slightly dilated 
central veins and blood congestion.  
 In the kidneys there were mild leucocyte infiltrations and a few patches of blood congestion in 
the kidneys for both extract treated and vehicle groups. There was however no serious 
pathological conditions observed like tubular or glomerular necrosis. Similarly, lung 
histopathology showed thickened alveolar with mild leucocyte infiltrations for all groups. Brain 
histopathology showed mild astrogliosis for all groups treated and untreated. However there was 
no significant demyelination or any abnormalities seen in all subjects. 
 
 
 
 
 
 
 
 
 
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4.8.1 Liver  
A B 
H 
H 
J 
I 
J 
  
C D 
G 
F F 
G 
  
Figure 6: Photomicrographs of livers isolated from rats after 8 week continuous administration of 
(A) Vehicle, (B) SNE 100 mg/kg, (C) SNE 300 mg/kg and (D) SNE 1000 mg/kg. Arrows show 
normal central veins (G) and normal distribution of hepatocytes (F).The arrows also indicate 
distorted central vein(I), blood congestion (J) and increased interstitial spaces (H)  (H&E 
staining, 20×). 
 
 
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4.8.2 Kidney  
A B 
F 
G 
H I F 
H 
 
 
C D 
G 
I 
F 
G 
F 
  
Figure 7: Photomicrographs of kidneys isolated from rats after 8 weeks of continuous 
administration of (A) Vehicle, (B) SNE 100 mg/kg, (C) SNE 300 mg/kg and (D) SNE 1000 
mg/kg. Arrow F shows normal glomeruli, G shows blood congestion, I for normal tubules and H 
for mild leucocytic infiltrations (H&E staining, 40× for A and C, 20×for B and D). 
 
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 4.8.3 Lungs 
A B 
F 
F 
F 
  
C D H 
E 
E 
F 
  
 
Figure 8: Photomicrographs of lung isolated from rats after 8-week continuous administration of 
(A) Vehicle, (B) SNE 100 mg/kg, (C) SNE 300 mg/kg and (D) SNE 1000 mg/kg. The arrow 
shows normal lung tissue (E), slightly thickened alveolar (F) and mild leucocyte infiltrations (H) 
(H&E staining, 20×). 
 
 
 
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4.7.4 Brain (cerebral cortex) 
A B 
E 
E 
E 
  
C D 
E 
E 
  
Figure 9: Photomicrographs of cerebral cortex of brains isolated from rats after 8-week 
continuous administration of (A) Vehicle, (B) SNE 100 mg/kg, (C) SNE 300 mg/kg and (D) 
SNE 1000 mg/kg. The arrow shows the presence of astrocytes (mild astrogliosis) (E). (H&E 
staining, 40×). 
 
 
 
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4.7.5 Spleen 
A B 
F 
E E 
F 
  
C E D 
E 
F F 
  
Figure 10: Photomicrographs of spleens isolated from rats after 8-week continuous 
administration of (A) Vehicle, (B) SNE 100 mg/kg, (C) SNE 300 mg/kg and (D) SNE 1000 
mg/kg. 
The arrow shows normal spleen tissue with the white pulp (E), and red pulp (F) (H&E staining, 
20×). 
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CHAPTER FIVE  
5.0 DISCUSSION 
5.1 DISCUSSION 
The study described demonstrates that the ethanolic extract of Synedrella nodiflora possesses 
anti-hyperglycaemic, antioxidant and a likely anti-inflammatory activity in STZ-induced diabetic 
rats. This may corroborate previously discovered properties, that is anti-oxidant (in vitro), 
analgesic and anti-inflammatory properties (Amoateng et al., 2015; Amoateng et al., 2017; 
Woode et al., 2009). Male Sprague Dawley rats were injected with STZ (55 mg/kg) to induce 
diabetes. STZ acts as a nitroso-urea alkylating agent that attacks the DNA of beta pancreatic 
cells. This causes beta cell death leading to a decreased insulin level hence diabetes. After 8 
weeks of extract administration the animals were assessed to find out if the extract had any 
significant effects on biochemical markers for diabetes. A number of effects were observed. 
Phytochemical screening identified the presence of alkaloids, saponins, tannins, phenolics, 
terpinoids, flavonoids and glycosides in the ethanolic extract. This comes as no surprise as 
ethanol and water are able to extract a wide range of phytochemicals individually. Both solvents 
are polar and will be very effective in dissolving phytochemicals with hydroxyl groups (-OH) 
like tannins, phenolics and flavonoids. Thus the hydroethanolic solvent (i.e 30% water, 70% 
ethanol) hand the ability to absorb a lot of bioactive compounds from the whole plant mixture. 
These phytochemicals could be contributing individually of synergistically to give the observed 
effects. There are likely to be bioactive compounds which may have anti-oxidant, anti-
hyperglcaemic and anti-inflammatory effects in the extract  
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The SNE dose of 1000 mg/kg reduced fasting blood sugar levels significantly after four (4) 
weeks of administration. Anti-hyperglycaemic activity of plants as a result the presence 
phytochemical constituents like polyphenols is well known and well established fact in 
pharmacology (Abdallah et al., 2011; Manickam et al., 1997). Also an ED50 of 187.9 mg/kg was 
obtained for the extract. Considering the fact that the extract is a mixture of various 
phytochemicals, it is possible that the constituents responsible for the anti-hyperglycaemic 
activity maybe just a fraction of the ED50 value obtained. ED50 represents the dose of extract 
required to achieve 50% of the desired response. If one particular compound/phytochemical is 
responsible for this activity could be potent since the ED50  dose is not very high. 
 It is possible that the extract may be acting via an extra-pancreatic effect since in this 
experiment most of the β-cells were destroyed. This possible effect the extract may have without 
stimulating the pancreas has been made mention of (Zahan et al., 2012). The exact mechanisms 
by which this extract reduced blood sugar will have to be determined in later studies. 
 
Weight gain or loss is an important parameter in the diagnosis and assessment of the progression 
of the diabetes. Weight loss in early stages of diabetes, especially type 1 is attributed to the lack 
of insulin. When insulin is lacking, cells do not receive enough glucose for energy and other 
metabolic processes. Thus the cells send a feed back to the brain, which causes the body to feel 
hungry. This may cause the patient to eat more (polyphagia). The feedback also causes the body 
to mobilize glucose from other sources (gluconeogenesis) (Georga et al., 2018). Fats stores and 
proteins may be used to produce more glucose for the body. This normally causes the loss in 
weight normally seen in type -1 diabetics. Ketones which are by-products of gluconeogenesis 
tend to accumulate in the blood leading to diabetic ketoacidosis common in diabetes 
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(Goldenberg  and Punthakee, 2013). From the experiment it could be seen that compared to the 
control, the diabetic vehicle group and the extract groups had a significant loss in weight by end 
of the experiment. However the highest dose of the extract (SNE 1000 mg/kg) produced a mild 
gain in weight. SNE 1000 mg/kg was the only dose of the extract that showed a positive 
percentage change in bodyweight though significantly lower than the control. It‟s possible that 
the extract provided a form of nutritive/supplementary value. The plant has already been known 
to be used as food by both humans and in livestock (Adjibode et al., 2015). The slight weight 
gain could also be as a result of its anti-hyperglycaemic effects.  
In general all organs form a fraction of the total body weight of all organisms. An enlargement 
may signify organ inflammation or carcinoma. Hepatomegaly or enlarged liver is a known 
complication of poorly controlled diabetes (Jardim et al., 2013). It occurs as a result of 
glycogenosis or increased synthesis of glycogen in the liver due to hyperglycemia (Al-Hussaini 
et al., 2012; Chatila and West, 1996). Other conditions that may cause enlarged liver in diabetes 
include fatty liver disease (steatosis) and liver inflammation (hepatitis) (Van Steenbergen and 
Lanckmans, 1995). A relatively large liver was observed in the vehicle group, significantly 
higher in comparison with the SNE treated groups. The increase in weight of the liver is in direct 
correlation to an increase in liver size. It can also be inferred that the extract prevented 
hepatomegaly by preventing liver inflammation or reducing hyperglycemia or both. 
Diabetes has been proposed to cause chronic liver disease, liver steatosis (fatty liver), liver 
inflammation and carcinoma (El-serag et al., 2004). Non-alcoholic fatty liver disease (NAFLD) 
and diabetes are common conditions that regularly co-exist and can act synergistically to drive 
adverse outcomes. These damages may be as a result of oxidative stress (Cichoż-Lach et al., 
2014; Yaribeygi et al., 2018). Furthermore, the role of free radicals in streptozotocin-induced 
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tissue damages in diabetic rats has been demonstrated already (Murugana and Pari, 2006). 
Hepatocytic proteins, lipids and DNA are among the cellular structures that are primarily 
affected by reactive oxidative species (Brosnan et al., 2012). Liver enzymes (ALT, ALP, AST, 
and GGT) are also usually released into the blood when such damages occur, and have been 
measured to assess the ability of certain extracts to reduce liver damage in diabetes (Ashraf and 
Samad, 2015; Hamadi et al., 2012).  In this study there was a significant increase in the levels of 
these enzymes for the vehicle group. The extract groups however showed no significant enzyme 
increase, most likely due to the fact that the extract had the ability to ameliorate the danger posed 
by oxidative stress in the liver (Amoateng  et al., 2011). 
 Creatinine levels were also significantly low for all the diabetic groups both treated and 
untreated. The lowest level of creatinine was however found in the vehicle group. Creatinine is a 
by-product of the breakdown of creatine in the muscles. The liver plays a major role in the 
biosynthesis of creatine (Brosnan et al., 2011). It is entirely possible that the oxidative stress on 
the liver and a possible inflammation of the liver caused the vehicle group to have the lowest 
level of creatinine. Another possible explanation for the decrease of creatinine in all the diabetic 
rats is that the diabetes affected the muscle mass due to gluconeogenesis. The lower the muscle 
mass of the rats the lower the amount of creatine/creatinine produced. Gluconeogenesis leading 
to weight loss has already been mentioned above.  
A low albumin level is a condition that has been associated with liver inflammation and protein 
protein-losing nephropathy (Throop et al., 2004). Considering the fact that liver inflammation is 
a key constituent of chronic diabetes it comes as no surprise that there are reports of decreased 
serum concentrations of albumin in diabetes (Viswanathan et al., 2004). Microalbuminuria 
however is still one of the early predictors of nephropathy in diabetes (Yasuda et al., 2014). 
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Significantly low albumin levels were observed for the diabetic untreated vehicle group. Since 
the extract group had albumin levels close to normal it suggests that the extract was able to 
reduce inflammation of the liver. This may be further buttressed by the fact that haematology 
results also showed a significantly high level of eosinophils in the vehicle group. Eosinophils are 
associated with allergic and inflammatory reactions (Gleich, 2000; Sampson, 2000). In liver 
disorders eosinophils are known to release cytokines like IL- 1β and IL-18 that cause 
inflammation (Palacios-Macapagal et al., 2017). There was a markedly high amount of 
eosinophils in the serum of the diabetic vehicle group. 
For the haematological indices, the results showed decrease in RBCs and haemoglobin for the 
vehicle group. Diabetic patients especially T1DM are known to develop erythropoietin-
dependent anaemia (Thomas, 2007; Thomas et al., 2005). Erythropoietin is a hormone produced 
and released by the kidney and it controls the production of RBCs in the bone marrow. It is 
believed that diabetes may affect the level of erythropoietin in 2 ways. Diabetes may directly 
damage the kidney (diabetic nephropathy) leading to a decrease in the production of the hormone 
(Khoshdel et al., 2008). Secondly autonomic nerves are also susceptible to damage in diabetic 
neuropathy. The release of erythropoietin is controlled by the autonomic nervous system; hence 
diabetic neuropathic damage may reduce the secretion of erythropoietin (Hadjadj et al., 2001; 
Spallone et al., 2004). The extract group however showed no significant decrease in RBCs. The 
extract may be preventing or reducing damage to the nerves and the kidney by reducing 
hyperglycaemia and/or oxidative stress. 
Histopathology also showed a change in the tissue structure of the liver for the vehicle group. 
The central veins were slightly dilated and distorted. There were also large interstitial spaces 
seen in liver tissue. Increase in blood supply to the liver usually leads to an increase in 
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hydrostatic pressure of the blood flowing to the liver. Increase in blood flow to the liver can be 
associated with inflammation (Bernardi et al., 2015; Mehta et al., 2014). The fluid buildup was 
responsible for the large spaces seen in between the liver tissues and is characteristic of increased 
hydrostatic pressure seen in liver inflammation and cirrhosis (Henriksen, 1980; Recker et al., 
2015). 
There were mild leucocytic infiltrations in the kidneys for both extract treated and untreated 
diabetic (vehicle) groups. Leucocyte infiltrations in the kidney are usually attributed to 
inflammation (Adalid-Peralta et al., 2008; Akcay et al., 2009). There were also a few blood 
congestions seen. There was however no serious pathological conditions observed such as 
tubular or glomerular necrosis. Possible diabetic nephropathy could still be in the nascent stage. 
Similarly lung histopathology showed thickened alveolar and leucocyte infiltrations for all 
groups of diabetic rats. Allergic and inflammatory reactions usually results in lung leucocyte 
infiltrations (Schmiedl et al., 2005). The presence leucocytes in lungs however, may not be 
unusual as it is part of the natural defense system against infection. Brain histopathology showed 
mild signs of astrogliosis in all groups. Demyelination or any other abnormalities were however 
not prominent. Astrogliosis is the mechanism employed by the CNS for defense and repair 
against injury. It is characterized by an increase in astrocytes in the area of damage (Sofroniew, 
2015). Diabetes is known to play a role in CNS neuropathy and gliosis (Reske-Nielsen and 
Lundbaek, 1968; Reske-Nielsen et al., 1970). The mild astrogliosis seen in all groups may be a 
sign of developing CNS neuropathy. There was however no significant difference seen amongst 
all diabetic groups. There was also no significant pathological damage observed. 
Plants rich in polyphenols have been known to possess anti-inflammatory properties (García-
Pérez et al., 2017). In countries like Malaysia the Synedrella nodiflora plant is locally used to 
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treat inflammatory conditions like rheumatism (Burkill, 1985).The plants extract has shown anti-
inflammatory properties in a previous studies (Abad et al., 1996; Haque et al., 2012). Because of 
the ability of the extract to reduce inflammation at the site of injury, it has been proposed that the 
extract may have peripheral anti-inflammatory activities.  This activity was seen in carrageenan 
induced paw oedema models (Haque et al., 2012).  
SOD (superoxide dismutase) activity and MDA (malondialdehyde) content were also measured 
to ascertain the ability of the extract to exert its antioxidant activity in vivo. SOD activity showed 
a dose dependent increase in activity. However, only the 1000 mg/kg of SNE, the highest dose, 
was significantly higher than the control. In the MDA assay the extract‟s highest dose also gave 
the only significant reduction in MDA, even though the decrease was dose dependent. SOD and 
MDA assays are among the most reliable and widely used assays for in vivo antioxidant 
properties. Oxidative stress is a cellular phenomenon which occurs as a result of physiological 
imbalance between levels of antioxidants and oxidants (free radicals), where there are more 
oxidants. SOD is one of the primary antioxidant enzymes contained in mammalian cells that 
serve as a defensive mechanism against oxidative stress (McCord et al., 1971).  
-
SOD basically reacts with the highly reactive superoxide anion (O2 ) in a reaction known as 
dismutation to form a hydrogen peroxide and oxygen which are relatively harmless (Birben et 
al., 2012). The superoxide anion is generated by various biochemical processes and it can 
damage macromolecules (proteins, nucleic acids, etc.) by oxidizing them (Ighodaro and 
Akinloye, 2017; Perry et al., 2007). Anti-oxidants neutralize ROS by donating electrons to them 
and stabilizing them (Nimse and Pal, 2015). A high SOD activity usually means the presence of 
antioxidants to prevent the natural SOD activity from being overwhelmed by ROS. When SOD 
activity is low, it most likely means oxidative stress has overwhelmed the natural defense 
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systems and the proteins that produce SOD have possibly been damaged by oxidation (Fucci et 
al., 1983; Stadtman, 1990). This goes to show that high dose of the extract was able to protect 
the SOD activity, thus the significantly high activity observed in comparison with the vehicle 
group that had a relatively low activity of the enzyme. Preliminary in vitro tests have shown that 
the extract may possess free radical scavenging properties (Onocha et al., 2016). 
Free radicals can attack the polyunsaturated fatty acids (PUFA) of cell membranes. The oxidative 
damage of PUFA, also known as lipid peroxidation is particularly destructive, because it perseveres as 
self-sustaining chain reaction (Marnett, 1999). MDA is one of the byproducts of the reaction of the 
ROS with polyunsaturated fatty acids (Ayala et al., 2014). Hence an increase in serum aldehyde is 
directly proportional to the level of oxidative stress (Gawel et al., 2004). A significantly low amount 
of aldehydes was seen in the 1000 mg/kg SNE dose compared with the control, confirming the 
antioxidant and anti-lipid peroxidative effect of the extract in vivo. In general there was a dose-
dependent decrease in aldehydes for the extract treated animals, but only the 1000 mg/kg SNE was 
statistically significant. Synedrella nodiflora extract possesses polyphenols and flavonoids which 
may account for its antioxidant activity. 
5.2 CONCLUSION 
The ethanolic extract of SNE showed anti-hyperglycaemic, anti-oxidant  and a possible anti-
inflammatory activity in streptozotocin-induced diabetes in rats. It also exerted various effects on 
blood biochemistry, haematology and histopathology which corroborated its anti-
hyperglycaemic, anti-oxidant activity and anti-inflammatory activity. 
 
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5.3 RECOMMENDATIONS 
 Further investigations should be conducted to fully evaluate the extract for its 
hepatoprotective activity. 
 The potential of the extract to lower or reverse diabetic neuropathy should be evaluated. 
 
 
 
 
 
 
 
 
 
 
 
 
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