Biochemical studies to evaluate the anti-diabetic, anti-oxidative and immunomodulatory potentials of Syzygium cumini (Jamun) & Pterocarpus marsupium (Vijaysaar) Thesis Submitted to University of Lucknow for the degree of Doctor of Philosophy in Biochemistry by Shalini Srivastava M. Sc. (Biochemistry) Department of Biochemistry University of Lucknow, Lucknow 2014
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Biochemical studies to evaluate the anti-diabetic, anti-oxidative and immunomodulatory potentials
of Syzygium cumini (Jamun) & Pterocarpus marsupium (Vijaysaar)
Thesis Submitted to
University of Lucknow for the degree of
Doctor of Philosophy
in
Biochemistry
by
Shalini Srivastava M. Sc. (Biochemistry)
Department of Biochemistry University of Lucknow, Lucknow
2014
Dedicated To
My parents
UNIVERSITY OF LUCKNOW LUCKNOW-226007
DR. DEEPAK CHANDRA DEPARTMENT OF BIOCHEMISTRY Associate Professor 0522-2740069(Office), 0522-2740132 (Fax) 94151-64388 (Mobile), e-mail:[email protected]
CERTIFICATE
This is to certify that the work embodied in this thesis entitled
“Biochemical studies to evaluate the anti-diabetic, anti-oxidative and
immunomodulatory potentials of Syzygium cumini (Jamun) &
Pterocarpus marsupium (Vijayasaar)” has been carried out by Ms.
Shalini Srivastava under my supervision. The work presented here is
original, carried out by the candidate herself and has not been submitted
so far, in part or full, for any other degree or diploma of any other
University/Institute. It is further certified that the candidate has fulfilled
the conditions laid down under the ordinance for the degree of Doctor of
Philosophy (Ph.D.) in Biochemistry by the University of Lucknow.
(Deepak Chandra)
Contents SN. Topic Page No.
1. Acknowledgment i-ii
2. Preface iii-iv
3. Abbreviations v-vii
4. Introduction 1-6
5. Review of Literature 7-56
6. Materials and Methods 57-71
7. Objectives 72
8. Chapter I:
Anti-diabetic and anti-oxidative potentials of crude Syzygium cumini and Pterocarpus marsupium extracts
73-107
9. Chapter II:
Anti-diabetic and anti-oxidative potentials of purified Syzygium cumini and Pterocarpus marsupium extracts
108-135
10. Chapter III:
Immunosuppression in diabetes and immunomodulatory properties of Syzygium cumini and Pterocarpus marsupium
136-151
11. Summary 152-165
12. Reference 166-203
13. List of Publications 204
i
ACKNOWLEDGEMENT
“To the Supreme, whose eternal blessings, divine love and spiritual guidance helps us to fulfil all our aspirations.”
Social beings in all conditions, achieve their goals with the assistance, co-operation and support of all the concerned people. I take this moment to express my most sincere appreciation, for the people who have knowingly or unknowingly played a role in my life as a teacher, friend, counsellor and supporter.
I am immensely grateful to my supervisor, Dr. Deepak Chandra, Associate Professor, Department of Biochemistry, University of Lucknow, Lucknow, for his meticulous efforts, inspiring guidance, unstinted co-operation and sentimental support which helped me to complete my thesis with ease. His great sense of humour with intellect, versatile personal qualities and critical appreciation influenced me to enjoy my work a lot. It was a wonderful experience to work under his guidance.
I express my gratitude to Prof. U. N. Dwivedi and Prof. S. K. Agarwal, former Heads, Department of Biochemistry, University of Lucknow, Lucknow, for providing me the necessary infrastructure and resources to accomplish my research work in the department and for their encouragement, moral support and keen interest in my progress.
I sincerely thank Prof. R. K. Mishra, Head, Department of Biochemistry, University of Lucknow, Lucknow, for his indispensable suggestions, constructive criticism, admirable teaching and encouragement. He will always be an inspiration and his passion for teaching will be cherished.
Few words cannot be sufficient to express my gratitude towards a few people. In this view I thank my teachers Prof. P.C. Mishra, Prof. M. K. Misra, Prof. Raj Khanna, Dr. Sudhir Mehrotra, Dr. Samir Sharma, Dr. Kusum Yadav and Dr. Minal Garg for their whole hearted support, consistent help and motivation.
I am grateful to Dr. Desh Deepak, Associate Professor, Department of Chemistry, University of Lucknow, Lucknow, for his help.
I am immensely thankful to my seniors: Dr. Umanath Tripathi, Dr. Ashita Gupta, Dr. Pratima Tripathi, Dr. Ankita Srivastava, Dr. Giti Verma, Mr. Veda Prakash Pandey and Mr. Sanjay Yadav for their wholesome support and substantial help. I thank my juniors: Mrs. Priyanka, Ms. Mandipika, Ms. Padminee, Ms. Nandini, Ms. Ambika and Mr. Bhanu for their constant cooperation and maintaining a healthy environment all along. The time shared with them during this period shall be an everlasting memory.
I take this great opportunity to owe the success in my life to my loving parents. I sincerely dedicate this piece of art & science to the efforts of my father,
Introduction
ii
Dr. D. C. Srivastava and my mother, Dr. (Mrs.) V. K. Srivastava and for their sacrifices which have enabled me to stand where I am and their constant encouragement.
I would do injustice if I do not thank my brothers Dr. Shekhar Srivastava and Mr. Sharad Srivastava, and sister-in-law, Dr. Swati Srivastava, for being my strength and support.
It’s my fortune to gratefully acknowledge my husband Dr. Prashant Kumar Saxena for his concern and support during the writing period of my thesis, which helped me to complete my work with ease. I also thank him for helping me out in statistics.
I thank the staff members of the Department of Biochemistry for their help and cooperation rendered at various stages of this work.
I would be ungrateful if I do not acknowledge all the animals sacrificed during the course of this study.
Shalini Srivastava
iii
PREFACE
Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in insulin
secretion, insulin action, or both. Insulin deficiency in turn leads to chronic
hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism. It is
estimated that 25% of the world population is affected by this disease. Oxidative
stress due to prolonged hyperglycaemia causes tissue damage leading to severe
diabetic complications such as retinopathy, neuropathy, nephropathy, cardiovascular
complications and ulceration. Hyperglycaemia is also one of the potential mediators
of altered defence mechanism and immunosuppression in diabetes. Thus, diabetes
covers a wide range of heterogeneous diseases. Diabetes mellitus may be categorized
into several types but the two major types are type 1, insulin dependent diabetes
mellitus (IDDM) and type 2, non-insulin dependent diabetes mellitus (NIDDM).
Sushruta (6th century BCE) identified diabetes and classified it as
Medhumeha. .He further identified it with obesity and sedentary lifestyle, advising
exercises to help cure it. The word diabetes comes from the Greek word diabenein
which means to pass through, in reference to the excessive urine passed as a symptom
of this disease.
Common symptoms of diabetes are hyperglycemia, polydipsia, polyurea,
weight loss, blurred vision and susceptibility to acquire infections. Drugs are used
primarily to save life and alleviate symptoms. Secondary aims are to prevent long-
term diabetic complications by eliminating various risk factors, to increase longevity.
Insulin replacement therapy is the mainstay for patients with type 1 DM while diet
and lifestyle modifications are considered the cornerstone for the treatment and
management of type 2 DM. Insulin is also important in type 2 DM when blood
glucose levels cannot be controlled by diet, weight loss, exercise and oral
medications. Oral hypoglycaemic agents are also useful in the treatment of type 2
DM. Oral hypoglycaemic agents include sulphonylureas, biguanides, alpha
glucosidase inhibitors, meglitinide analogues, and thiazolidenediones. The main
objective of these drugs is to correct the underlying metabolic disorder, such as
insulin resistance and inadequate insulin secretion.
Preface
iv
Despite considerable progress in the treatment of diabetes by oral
hypoglycemic agents, search for newer drugs continues because the existing synthetic
drugs have several limitations. The herbal drugs with anti-diabetic activity are yet to
be commercially formulated as modern medicines, even though they have been
acclaimed for their therapeutic properties in the traditional systems of medicine. The
plants provide a potential source of hypoglycemic drugs because many plants and
plant derived compounds have been used in the treatment of diabetes. Ayurveda and
other traditional medicinal system for the treatment of diabetes describe a number of
plants used as herbal drugs. Hence, they play an important role as alternative
medicine due to less side effects and low cost.
In the present study the anti-hypergylcemic, anti-oxidative and
immunomodulatory potentials of Syzygium cumini seed and Pterocarpus marsupium
bark have been studied. The crude extracts of these plant parts show anti-
hypergylcemic, anti-oxidative and immunomodulatory potentials. Attempts were
made to purify the active principles responsible for anti-diabetic activity, present in
these plant extracts and elucidate their mechanism of action. The active constituent(s)
present in these medicinal plants might cause pancreatic beta cells re-generation,
insulin release, increase peripheral glucose utilization and may also fight the problem
of insulin resistance. The outcomes of the study will not only be useful in isolating
newer and effective anti-hyperglycemic constituent(s) from these plant extracts but
the isolation of constituents with anti-oxidative potential will also be useful in
managing the pathogenesis of various oxidative stress mediated disorders including
diabetes. Modulation of the immune responses through the stimulatory or suppressive
activity of phyto-extracts may help to maintain a disease-free state in normal or
unhealthy people.
v
ABBRIVIATIONS
ADH Anti-diuretic hormone
ADP Adenosine diphosphate
AGE Advanced glycation end products
ALT Alanine aminotransferase
AMP Adenosine monophosphate
AP Alkaline phosphatase
AST Aspartate aminotransferase
ATP Adenosine triphosphate
α Alpha
β Beta
BSA Bovine serum albumin
b.w. Body weight
CAT Catalase
CDNB 1-chloro 2,4-dinitrobenzene
dl Deciliter
DNPH 2,4-dinitrophenylhydrazine
DPPH Diphenylpicrylhydrazyl
DTNB 5,5-Dithiobis-2-nitrobenzoic acid
EDTA Ethylenediamine tetracetate
ELISA Enzyme linked immune sorbent assay
FBG Fasting blood glucose
FCA Freund’s complete adjuvant
FIA Freund’s incomplete adjuvant
Fig. Figure
g Gram
G6PD Glucose 6-phosphate dehydrogenase
GPx Glutathione peroxidase
GSH Glutathione (reduced)
GSSG Glutathione (oxidized)
GST Glutathione S-transferase
h Hour
Abbreviations
vi
H2O2 Hydrogen peroxide
HK Hexokinase
HRP Horse radish peroxidase
IgG Immunoglobulin G
IgM Immunoglobulin M
IL Interleukins
1U International unit
KDa Kilo Dalton
Kg Kilogram
LDH Lactate dehydrogenase
LDL Low density lipoprotein
LMWA Low molecular weight antioxidants
LPO Lipid peroxidation
LPS Lipopolysaccharide
MDA Malondialdehyde
mg Mili gram
min Minute
ml Mili liter
µM Micromole
mM Milimole
NaCl Sodium chloride
NAD Nicotinamide adenine dinucleotide
NADH Nicotinamide adenine dinucleotide (reduced)
nm Nano meter
NO Nitric oxide
O2·¯ Superoxide radical
OH• Hydroxyl radical
PBS Phosphate buffer saline
P value Degree of significance
PEP Phosphoenol pyruvate
PFK Phosphofructokinase
PK Pyruvate kinase
PKC Protein kinase C
Abbreviations
vii
PM Pterocarpus marsupium
% Percentage
RBC Red blood cells
ROS Reactive oxygen species
rpm Rotation per minute
SC Syzygium cumini
SD Standard deviation
-SH Sulfhydryl group
SOD Superoxide dismutase
TBA Thiobarbituric acid
TBARS Thiobarbituric acid reactive substances
TD Thymus dependent
TEP 1,1,3,3-tetraethoxy propane
TI Thymus independent
TMB Tetra methyl benzidene
Tris Tris (hydroxymethyl) aminomethane
TT Tetanus toxoid
Wt Weight
Introduction
1
Introduction
The human population worldwide appears to be in the midst of an epidemic of diabetes. An estimated 285 million people, corresponding to 6.4% of the world's adult population suffer from diabetes. The number is expected to grow to 438 million by 2030, corresponding to 7.8% of the adult population. Despite the great strides that have been made in the understanding and management of diabetes, the disease and disease-related complications are increasing unabated. Parallel to this, recent developments in understanding the pathophysiology of the disease process have opened several new avenues to identify and develop novel therapies to combat the diabetic plague. Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in combating diabetes and diabetes-related complications. Therefore, as the disease is progressing unabated, there is an urgent need of identifying indigenous natural resources in order to procure them, and study in detail, their potential on different newly identified targets in order to develop them as new therapeutics.
Diabetes is a chronic disorder in which homeostasis of carbohydrate, protein and lipid metabolism is improperly regulated by insulin. It is characterized by elevated fasting and post prandial blood sugar levels. Diabetes mellitus is a complex metabolic disorder resulting from either insulin insufficiency or insulin dysfunction. Type I diabetes (insulin dependent) is caused due to insulin insufficiency because of lack of functional β cells. Patients suffering from this are therefore totally dependent on exogenous source of insulin while patients suffering from Type II diabetes (insulin independent) are unable to respond to insulin and can be treated with dietary changes, exercise and medication. Type II diabetes is the more common form of diabetes constituting 90% of the diabetic population (Modak et al., 2007). Tissues where glucose uptake is insulin independent (cardiac tissue, blood vessels, peripheral nerves, renal medulla and ocular lens) face severe and sustained hyperglycemia (Chandra et al., 2002a). The major complications of diabetes include atherosclerosis, retinopathy, nephropathy and neuropathy etc.
Introduction
2
Diabetes related complications
The major risks of the diabetes disorder are chronic complication affecting
multiple organ systems which eventually arise in patients with poor glycemic control.
Different symptoms, complications and possible risk factors of diabetes mellitus are
summarized in Table 1. Microvascular complications are peripheral neuropathy which
can lead to foot ulcer and possibly progressing to necrosis, infection, gangrene,
sometimes requiring limb amputation. Nephropathy is a progressive kidney disease
caused by angiopathy of capillaries in the glomeruli of kidney. Retinopathy (damage
to retina) is an ocular manifestation of systemic disease which can lead to blindness.
Macrovascular complications include atherosclerosis and myocardial infarction or
ischemic heart disease. Possible mechanisms implicated in hyperglycemia induced
and antibacterial. The fruits and seeds are used to treat diabetes, pharyngitis,
spleenopathy, urethrorrhea and ringworm infection. The leaves have antibacterial
properties and are used to strengthen the teeth and gums. The leaves have also been
extensively used to treat diabetes, constipation, leucorrhoea, fever, gastropathy,
dermopathy and to inhibit blood discharges in the feces (Jagetia and Baliga, 2002;
Pepato et al., 2001; Pari and Saravanan, 2002; Mitra et al., 1995). In addition,
pharmacological evaluation of this plant reveals its anti-diabetic, hypolipidemic,
antioxidant, anti-HIV, anti diarrhoeal, anti-inflammatory, antibacterial, antipyretic,
radioprotective and neuropsychopharmacological activity (Srivastava and Chandra,
2013; Jadhav et al. 2009). Pterocarpus marsupium (PM) belongs to the family
fabaceae and is commonly known as Indian Kino in English and Vijaysar in Hindi.
Pterocarpus marsupium shows anti-diabetic, hepatoprotective and cardiotonic
activity. Studies have also reported its ability as a COX-2 inhibitor (Devgun et al.,
2009).
Several reports are available regarding the anti-hyperglycemic effects of SC
and PM but the data about their active constituents, their mechanism of action is not
conclusive. Oxidative stress greatly contributes to the progression of diabetic
complications. The medicinal plants mentioned above are reported to exhibit
antioxidant potential but it is inconclusive that their anti-oxidative property is due to
Introduction
6
an independent activity associated to their constituent (s) or simply because of
normoglycemic condition achieved due to their consumption. Therefore, the present
study is planned to systematically evaluate the anti-hyperglycemic and anti-oxidative
potentials of SC and PM. The study is also aimed to isolate and characterize the active
constituents present in these plants and responsible for anti-hyperglycemic and anti-
oxidative potential, using alloxan induced diabetic rats as model. The effects of these
plant extracts on immune system in normal and diabetic rats were also tested in order
to evaluate their immunomodulatory potential.
Review of Literature
7
Review of Literature
DIABETES
Diabetes is characterized as an abnormal pathological condition resulting due to imbalance in secretion of hormone(s), primarily insulin secreted from β cells of pancreas. Diabetes is one of the major health problems not only in developing countries like India but throughout the world (Al Ali et al., 2013). Two types of diabetes are known:
Diabetes insipidus: Diabetes insipidus is often called as water diabetes. The symptoms of diabetes
insipidus are, excessive thirst, passing large volume of diluted urine and a general feeling of weakness, which are much similar to the other type of diabetes i.e. diabetes mellitus (sugar diabetes). Diabetes insipidus can be caused either by a defect in pituitary gland or a defect in kidney (nephrogenic diabetes insipidus). These defects cause either under secretion of the anti-diuretic hormones (ADH) or vasopressin. ADH is responsible for re-absorption of water in kidney but due to its under secretion in diabetes insipidus the body’s water retention capacity is reduced and the patient passes large volume of urine. Thus the symptoms observed include profound thirst, dehydration and low blood pressure.
Diabetes mellitus: Diabetes mellitus (DM) is a heterogeneous metabolic disorder characterized
by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both. Banting and Best isolated insulin in 1922 and treated a diabetic patient with it. Insulin is a peptide hormone composed of 51 amino acids and has a molecular weight of 5808 Da. In mammals, insulin is synthesized in the pancreas within the beta cells (β-cells) of the islets of Langerhans. In β cells, insulin is synthesized from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase E. These modifications of proinsulin remove the center portion of the molecule (i.e., C-peptide). The remaining polypeptides (51 amino acids in total), the B- and A- chains, are linked together by disulfide bonds to form insulin.
Review of Literature
8
Insulin is a hormone that is central to regulating carbohydrate and fat
metabolism in the body. The stimuli for insulin secretion include ingested protein and
glucose in the blood produced from digested food. Insulin binds to the extracellular
portion of the alpha subunits of the insulin receptor which in turn causes a
conformational change in the insulin receptor that activates the kinase domain that
resides on the intracellular portion of the beta subunits. This further activates a
cascade of protein kinases and a whole series of enzyme
phosphorylation/dephosphorylation reactions which account for the effects of
insulin. Insulin causes cells in the liver, muscle, and fat tissues to take up glucose and
amino acids from the blood, activate protein synthesis from amino acids and glycogen
and triglyceride synthesis from glucose. Insulin stops the use of fat as an energy
source by inhibiting the release of glucagon. In the absence of insulin, glucose is not
taken up by body cells and the body begins to use fat as an energy source
or gluconeogenesis. Thus failure in the control of insulin levels, results in diabetes
mellitus.
Two types of diabetes mellitus have been reported, a) Type I or insulin
dependent diabetes mellitus (IDDM), found among 10% of the total diabetes mellitus
patients (Ejaz and Wilson, 2013), b) Type II or non insulin dependent diabetes
mellitus (NIDDM) and represents about 90% of the total diabetes mellitus patients
(Booth et al., 2013).
Type I DM also known as juvenile diabetes is the classical threatening form of
diabetes characterized by the autoimmune destruction of insulin producing β-cells of
islets of langerhans leading to absolute insulin deficiency. Type I DM is the result of
an unfavorable interaction between environmental factors and an inherited
predisposition of the disease. The environmental factors that might lead to type I
diabetes includes viral infections specially those caused by the coxsackie and other
enterovirus , mycobacterium, chemical toxins in the food and neonates exposure to
cow’s milk constituents, which may cross react with specific β-cell antigens (Virtanen
et al., 1993; Tisch and McDevitt, 1996). Type I diabetes is managed with insulin
injection, life style adjustments and careful monitoring.
Review of Literature
9
Type II diabetes mellitus is also known as adult-onset diabetes. Insulin
resistance and disorders of insulin secretion represent core elements in the
pathogenesis of type II diabetes mellitus (Giorgino et al., 2005). The natural history of
type II diabetes usually begins with obesity due to insulin resistance which ultimately
led to under secretion of insulin which may be attributed to several metabolic effects
including increased hepatic glucose production. The transition from normal to
impaired glucose tolerance is associated with a decline in insulin stimulated glucose
disposal and a decline in the acute insulin secretory response. This decrease in the
first phase insulin response is responsible for post-prandial hyperglycemia (Lin and
Sun, 2010; Panunti et al., 2004). Fasting hyperglycemia, on the other hand, is caused
by unrestrained basal hepatic glucose output, primarily a consequence of hepatic
resistance to insulin action. Chronic hyperglycemia further impairs β-cell secretory
kinetics and tissue sensitivity to insulin, a phenomenon known as glucotoxicity
(Dailey, 2004).
Biochemistry of diabetes
The plasma glucose levels rarely exceed 120 mg/dl in normal humans, but
higher levels are routinely found in patients with deficient insulin action. When the
plasma glucose level is higher than 180 mg/dl (the maximum limit of renal tubular
absorption of glucose), sugar is excreted in urine (glycosuria). The urine volume is
increased owing to osmotic diuresis and coincident obligatory water loss (polyurea)
and this in turn leads to dehydration (hyperosmolarity), increased thirst and excessive
drinking of water (polydipsia). Protein synthesis decreases in the absence of insulin
partly because the transport of amino acids into muscles is diminished (O’Neill et al.,
2010). Thus the insulin deficient persons are in negative nitrogen balance. The
deficiency of insulin leads to lipolysis resulting in increased plasma fatty acid levels.
When the capacity of the liver to oxidize fatty acids to CO2 is exceeded, β-hydroxy
butyric acid accumulates (ketosis) (Fex et al., 2004). The organism initially
compensates for the accumulation of these organic acids by increasing respiratory
losses of CO2 but if unchecked by the administration of insulin, severe metabolic
acidosis supervenes and the patient dies in diabetic coma.
Review of Literature
10
Effect on glucose metabolism
Insulin influences the intracellular utilization of glucose in a number of ways.
In normal person about half of the glucose ingested in converted to energy through
glycolytic pathway and about half is stored as fat or glycogen. In the absence of
insulin, glycolysis is decreased and the anabolic process of gluconeogenesis is
decreased (Magnusson et al., 1992). Only 5% of an ingested glucose load is converted
to fat in insulin deficient diabetics. Insulin increases hepatic glycolysis by increasing
the activity and amount of several key enzymes including glucokinase,
phosphofructokinase and pyruvate kinase (Wu et al., 2005). The flux through the
glycolytic pathway is adjusted in response to conditions both inside and outside the
cell. The rate in liver is regulated to meet major cellular needs: (1) the production of
ATP, (2) the provision of building blocks for biosynthetic reactions, and (3) to lower
blood glucose, one of the major functions of the liver. When blood sugar falls,
glycolysis is halted in the liver to allow the reverse process, gluconeogenesis. In
glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate
kinase are effectively irreversible in most organisms. In metabolic pathways,
such enzymes are potential sites of control, and all three enzymes serve this purpose
in glycolysis. A balance between glucose production and its utilization is necessary to
maintain normal blood glucose levels. Diabetes is characterized by elevated
production and low utilization of glucose (Taylor and Agius, 1998). A number of
changes in several enzymes present in the liver and other tissues are known to occur
in diabetes mellitus e.g. activity of hepatic glucokinase is markedly decreased and
activity of glucose-6-phosphatase is almost doubled (Hinder et al., 2013). This
imbalance results in constant hyperglycemia in the diabetic state. In skeletal muscles
insulin promotes glucose entry through the transporter and also increases Hexokinase
II activity, which phosphorylates glucose and initiates glucose metabolism. Insulin
stimulates lipogenesis in adipose tissue (Polakof et al., 2011) 1- by providing the
acetyl CoA and NADPH required for fatty acid synthesis. 2- by measuring the normal
levels of the enzyme acetyle CoA-carboxylase, which catalyzes the conversion of
acetyl CoA to malonyl CoA. 3- by providing the glycerol involved in triglycerol
synthesis.
Review of Literature
11
The action of insulin on glucose transport, glycolysis and glucogenesis occur
within seconds or minutes since they primarily involve the activation or inactivation
of enzyme involved by phosphorylation or dephosphorylation (Denton et al., 1981).
The formation of glucose from non-carbohydrate precursor involves a series of
enzymatic steps, many of which are stimulated by glucagon (through C-AMP), by
glucocarticoid hormones and to a lesser extent by α and β adrenergic agents,
angiotensin II and vasopressin (Roth & Beaudoin, 1987; Kuchler et al., 2010). The
key gluconeogenic enzyme in the liver is phosphoenolpyruate carboxykinase
(PEPCK). Insulin decreases the amount of this enzyme by selectively inhibiting of the
gene that codes for the m-RNA for PEPCK (Scott at al., 1998). The net action of all
the above effects of insulin is to decrease the blood sugar level. In this action, insulin
stands alone against an array of hormones that attempt to counteract this effect.
Effect on lipid metabolism
In patient with insulin deficiency, lipase activity increases, resulting in
enhanced lipolysis and increased concentration of free fatty acids in plasma and liver
(Costabile et al., 2011). Glucagon levels also increases in these patients and this
enhances the release of free fatty acids. Free fatty acids are metabolized to acetyl CoA
and finally to CO2 and H2O via citric acid cycle. In patient with insulin deficiency the
capacity of this process is rapidly exceeded and the acetyl CoA is converted to
acetoacetyl CoA and then to acetoacetic and hyroxy-butyric acids (Bickerton et al.,
2008). Insulin apparently affects either formation or clearance of VLDL and LDL,
since levels of these particles and consequently the levels of cholesterol are often
elevated in poorly controlled hyperglycemia.
Effect on protein metabolism
Insulin generally has an anabolic effect on protein metabolism by stimulating
protein synthesis (Bonadonna et al., 1993; Kimball et al., 1994). Insulin stimulates the
uptake of neutral amino acids in muscles, an effect that is not linked to glucose uptake
or to a subsequent incorporation of amino acids into protein. The effect of insulin on
general protein synthesis in skeletal and cardiac muscles and in liver are exerted at the
level of m-RNA translation (Scheper et al., 2007). In recent years insulin has been
Review of Literature
12
shown to influence the synthesis of specific proteins by affecting changes in the
corresponding m-RNA.
OXIDATIVE STRESS
Oxidative stress results from an imbalance between radical-generating and
radical scavenging systems, i.e. increased free radical production or reduced activity
of antioxidant defenses or both. Oxygen is vital for aerobic life processes. However,
about 5% or more of the inhaled O2 is converted to reactive oxygen species (ROS)
such as O2¯, H2O2, and ·OH by univalent reduction of O2 (Harman, 1993). Therefore
under aerobic conditions the cells are always endangered by ROS, which are
efficiently taken care of by the highly powerful antioxidant systems of the cell. When
the balance between ROS production and antioxidant defenses is lost, ‘oxidative
stress’ results which through a series of events deregulates the cellular functions
leading to various pathological conditions including cardiovascular dysfunction,
neurodegenerative diseases, gastroduodenal pathogenesis, metasbolic dysfunction of
almost all the vital organs, cancer, and premature aging (Thomas and Kalyanaraman,
1997).
1) THE MECHANISM OF ROS FORMATION
Although O2 can behave like a radical (a diradical) owing to the presence of
two unpaired electrons of parallel spin, it does not exhibit extreme reactivity due to
quantum-mechanical restrictions. Its electronic structure results in formation of water
by reduction with four electrons, i.e:
O2 + 4H++ 4e- 2H2 O
In the sequential univalent process by which O2 undergoes reduction, several
reactive intermediates are formed, such as superoxide (O2¯), hydrogen peroxide
(H2O2), and the extremely reactive hydroxyl radical (·OH), which are collectively
termed as the reactive oxygen species (ROS). The process can be represented as:
O2 e-
O2¯ e- H2O2 e- ·OH e- H2O
These oxygen-derived pro-oxidants, can cause damage to biological targets
such as lipids, DNA, and proteins, and on the defending systems of the cell, which are
Review of Literature
13
composed of enzymes and reducing equivalents, or antioxidants. In general these pro-
oxidants are referred to as reactive oxygen species (ROS) that can be classified into 2
groups of compounds, radicals and nonradicals. The radical group, contains
compounds such as nitric oxide radical (NO·), superoxide ion radical (O2·¯), hydroxyl
radical (·OH), peroxyl (ROO·) and alkoxyl radicals (RO·), and one form of singlet
oxygen (Halliwell et al., 2000). These species are radicals, because they contain at
least 1 unpaired electron in the shells around the atomic nucleus and are capable of
independent existence (Halliwell and Gutteridge, 1999). The occurrence of one
unpaired electron results in high reactivity of these species by their affinity to donate
or obtain another electron to attain stability. The group of nonradical compounds
contains a large variety of substances, some of which are extremely reactive although
not radical by definition. Among these compounds produced in high concentrations in
the living cell are hypochlorous acid (HClO), hydrogen peroxide (H2O2), organic
peroxides, aldehydes, ozone (O3), and O2 as shown in table 1.
i) Some examples of ROS
Superoxide Ion Radical (O2·¯ /HO·
2)
This species possess different properties depending on the environment and
pH. Due to its pKa of 4.8, superoxide can exist in the form of either O2·¯or, at low pH,
hydroperoxyl (HO·2) (Sohal et al., 1989). The latter can more easily penetrate
Review of Literature
14
biological membranes than the charged form. Hydroperoxyl can therefore be
considered an important species, although under physiological pH most of the
superoxide is in the charged form. In a hydrophilic environment both the O2·¯ and
HO·2 can act as reducing agents capable, for example, of reducing ferric (Fe +3) ions to
ferrous (Fe +2) ions; however, the reducing capacity of HO·2 is higher. The most
important reaction of superoxide radicals is dismutation (reaction 1), in which a
superoxide radical reacts with another superoxide radical. One is oxidized to oxygen,
and the other is reduced to hydrogen peroxide (Fridovich, 1997).
HO·2 /O2
·¯ + HO·2 /O2
·¯ + H+ H2O2 + O2 (1)
Hydroxyl Radical (·OH)
The reactivity of hydroxyl radicals is extremely high (Halliwell and
Gutteridge, 1999). In contrast to superoxide radicals that are considered relatively
stable and have constant, relatively low reaction rates with biological components,
hydroxyl radicals are short-lived species possessing high affinity toward other
molecules. ·OH is a powerful oxidizing agent that can react at a high rate with most
organic and inorganic molecules in the cell, including DNA, proteins, lipids, amino
acids, sugars, and metals. ·OH is considered the most reactive radical in biological
systems and due to its high reactivity it interacts at the site of its production with the
molecules closely surrounding it.
Hydrogen Peroxide (H2O2)
The result of dismutation of superoxide radicals is the production of H2O2.
There are some enzymes that can produce H2O2 directly or indirectly. Although H2O2
molecules are considered reactive oxygen metabolites, they are not radical by
definition. They can, however, cause damage to the cell at a relatively low
concentration (10 µM). They are freely dissolved in aqueous solution and can easily
penetrate biological membranes. Their deleterious chemical effects can be divided
into the categories of direct activity, originating from their oxidizing properties, and
indirect activity in which they serve as a source for more deleterious species, such as
OH· or HClO. Direct activities of H2O2 include degradation of haem proteins; release
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15
of iron; inactivation of enzymes; and oxidation of DNA, lipids, SH groups, and keto
acids (Halliwell and Gutteridge, 1999).
Nitric Oxide (NO·), Peroxynitrite (ONOO¯), and Other Members of the
Family
The nitric oxide, or nitrogen monoxide, radical (NO·) is produced by the
oxidation of one of the terminal guanido nitrogen atoms of L-arginine. In this
reaction, catalyzed by the group of enzymes called nitric oxide synthase (NOS), L-
arginine is converted to nitric oxide and L-citrulline. Three types of the enzymes
exist: neuronal NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). One-
electron oxidation results in the production of nitrosonium cation (NO+), while one-
electron reduction leads to nitroxyl anion (NO¯), which can undergo further reactions,
such as interacting with NO· to yield N2O and OH· (Beckman and Koppenol, 1996).
NO· can react with a variety of radicals and substances like H2O2 and HClO to yield a
line of derivatives such as N2O3, NO2¯, and NO3¯. One of the most important
reactions under physiological conditions is that of superoxide and nitric oxide radicals
resulting in peroxynitrite (reaction 2). This reaction helps to maintain the balance of
superoxide radicals and other ROS and is also important in redox regulation (Czapski
and Goldstein, 1995).
NO· + O2·¯ ONOO¯ (2)
The protonated form of peroxynitrite (ONOOH) is a powerful oxidizing agent
that might cause depletion of sulfhydryl (-SH) groups and oxidation of many
molecules causing damage similar to that observed when OH· is involved. It can also
cause DNA damage such as breaks, protein oxidation, and nitration of aromatic amino
acid residues in proteins (Murphy et al., 1998).
The Role of Transition Metals
Most of the transition metals—those in the first row of the D block in the
periodic table contain unpaired electrons and can, therefore, with the exception of
zinc, be considered radicals by definition (Halliwell and Gutteridge, 1999). They can
participate in the chemistry of radicals and convert relatively stable oxidants into
powerful radicals. Among the various transition metals, copper and especially iron are
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16
most abundant, present in relatively high concentrations, and are major players in the
Fenton reaction (Fenton, 1894) and the metal-mediated Haber-Weiss reaction (Haber
and Weiss, 1934).
For the production of ·OH, except during abnormal exposure to ionizing
radiation, generation of ·OH in vivo requires the presence of trace amount of transition
metals like iron or copper. A simple mixture of H2O2 and Fe2+ salt forms ·OH, as
given by the following Fenton reaction:
Fe2+ + H2O2 Fe3+ + ·OH + OH¯
Traces of Fe3+ can react further with H2O2 to form the following products:
Fe3+ + H2O2 Fe2+ + O2¯ + H+
Thus, a free-radical mechanism for the generation of .OH may be deduced as
follows:
O2¯ + H2O2 OH¯ + ·OH + O2
Unfortunately, the rate constant for the above reaction is very low but can be
accounted for if the reaction is catalyzed by traces of transition metal ions – the metal-
catalyzed Haber–Weiss reaction. The various steps of this reaction are:
Fe3+ + O2¯ Fe2+ + O2
Fe2+ + H2O2 Fe3+ + ·OH + OH¯
and the net result is:
O2¯ + H2O2 O2 + ·OH + OH¯
However, redox-active free iron or copper do not exist in biological systems, as these
transition metal ions remain bound to proteins, membranes, nucleic acids or low-
molecular weight chelating agents like citrate, histidine, or ATP (Halliwell and
Gutteridge, 1984). However during ischemic condition, and cellular acidosis,
transition-metal ions may be released from some metalloproteins (Chevion et al.,
1993), resulting in generation of ·OH.
ii) Sources of ROS
The cell is exposed to a large variety of ROS and RNS from both exogenous
and endogenous sources (Figure 1). The former include, first, exposure to di-oxygen,
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17
which, although a nonreactive biradical, can independently cause oxidation and
damage to proteins and enzymes, exemplified by inhibition of aconitase and fumerase
in the Krebs cycle and glutamate decarboxylase, which results in decreased γ-
aminobutyric acid in the brain (Halliwell and Gutteridge, 1999). Ozone (O3) present
in the upper atmosphere acts as a damaging species to biological tissues. It can
damage lungs, and can serve as a powerful oxidizing agent that can oxidize biological
components directly (Rao and Davis, 2001). Exposure of living organisms to ionizing
and non ionizing irradiation constitutes another major exogenous source of ROS
(Shadyro et al., 2002). Air pollutants such as car exhaust, cigarette smoke, and
industrial contaminants encompassing many types of NO derivatives constitute major
sources of ROS that attack and damage the organism either by direct interaction with
skin or following inhalation into the lung (Koren, 1995). Drugs are also a major
source of ROS (Rav et al., 2001). There are drugs, such as belomycinem and
adreamicine, whose mechanism of activity is mediated via production of ROS, those
like nitroglycerine that are NO· donors, and those that produce ROS indirectly.
Narcotic drugs and anesthetizing gases are considered major contributors to the
production of ROS (Chinev et al., 1998). A large variety of xenobiotics (eg. toxins,
pesticides, and herbicides such as paraquat) and chemicals (eg,mustard gas, alcohol)
produce ROS as a by-product of their metabolism in vivo (Elsayed et al., 1992). One
of the major sources of oxidants is food for a large portion of the food we consume is
oxidized to a large degree and contains different kinds of oxidants such as peroxides,
aldehydes, oxidized fatty acids, and transition metals (Ames, 1986). Food debris that
reaches the intestinal tract places an enormous oxidative pressure on the intestinal-
tract mucosa (Srigirdhar et al., 2001).
Although the exposure of the organism to ROS is extremely high from
exogenous sources, the exposure to endogenous sources is much more important and
extensive, because it is a continuous process during the life span of every cell in the
organism.
For the production of O2¯, normally the tendency of univalent reduction of O2
in respiring cells is restricted by cytochrome oxidase of the mitochondrial electron
transport chain, which reduces O2 by four electrons to H2O without releasing either O2
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18
– or H2O2. However, O2 – is invariably produced in respiring cells (Fridovich, 1983).
This is due to the probable ‘leak’ of single electron at the specific site of the
mitochondrial electron transport chain, resulting in inappropriate single electron
reduction of oxygen to O2¯ (Loschen et al.,1974). When the electron transport chain
is highly reduced, and the respiratory rate is dependent on ADP availability; ‘leakage’
of electrons at the ubisemiquinone and ubiquinone sites increases so as to result in
production of O2¯ and H2O2 (Turrens and Boveris, 1980).
For the production of H2O2, peroxisomal oxidases and flavoproteins, as well
as D-amino acid oxidase, L-hydroxy acid oxidase, and fatty acyl oxidase participate.
Cytochrome P-450, P-450 reductase and cytochrome b-5 reductase in the endoplasmic
reticulum under certain conditions generate O2¯ and H2O2 during their catalytic cycles
(Bast et al., 1991). Likewise, the catalytic cycle of xanthine oxidase has emerged as
an important source of O2¯ and H2O2 in a number of different tissue injuries.
Xanthine oxidase, produced by proteolytic cleavage of xanthine dehydrogenase
during ischemia, upon reperfusion in presence of O2, acts on xanthine or
hypoxanthine to generate O2¯ and H2O2 (McCord, 1987).
The phagocytic cells, such as neutrophils, when activated during phagocytosis,
generate O2¯ and H2O2 through activation of NADPH oxidase. Neutrophil
accumulation in inflammated tissue is one of the major reasons of oxidative damage
due to generation of ROS. In addition, spontaneous dismutation of O2 at neutral pH or
dismutation by superoxide dismutase, results in H2O2 production (Morel et al., 1991).
Figure 1: Exogenous and endogenous sources of reactive oxygen species (ROS).
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19
2) CONSEQUENCES OF FREE RADICALS GENERATION
Reactive oxygen species can attack vital cell components like polyunsaturated
fatty acids, proteins, and nucleic acids. To a lesser extent, carbohydrates are also the
targets of ROS. These reactions can alter intrinsic membrane properties like fluidity,
ion transport, loss of enzyme activity, protein cross-linking, inhibition of protein
synthesis, DNA damage: ultimately resulting in cell death (Halliwell and Gutteridge,
1990). Some of the well-known consequences of generation of the free radicals in
vivo are: DNA strand scission (Brawn and Fridovich, 1981), nucleic acid base
modification (Moody and Hussan, 1982), protein oxidation (Pacifici et al., 1993) and
lipid peroxidation (Halliwell and Gutteridge, 1990).
Lipid peroxidation
Oxygen radicals catalyse the oxidative modification of lipids (Gardner, 1989).
This peroxidation chain reaction is illustrated in figure 2. The presence of double
bond adjacent to a methylene group makes the methylene C–H bonds of
polyunsaturated fatty acid (PUFA) weaker and therefore the hydrogen becomes more
prone to abstraction. While lipid peroxidation is not initiated by O2¯ and H2O2, ·OH,
alkoxy radicals (RO·), and peroxy radicals (ROO·) result in initiating the lipid
peroxidation (Turrens and Boveris, 1980). This can lead to a self perpetuating process
since peroxy radicals are both reaction initiators as well as the products of lipid
peroxidation. Lipid peroxy radicals react with other lipids, proteins, and nucleic acids;
propagating thereby the transfer of electrons and bringing about the oxidation of
substrates. Cell membranes, which are structurally made up of large amounts of
PUFA, are highly susceptible to oxidative attack and, consequently, changes in
membrane fluidity, permeability, and cellular metabolic functions result.
Figure 2: Mechanism of lipid peroxidation by ROS.
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DNA damage
ROS can cause oxidative damages to DNA: both nuclear and mitochondrial.
The nature of damages includes mainly base modification, deoxyribose oxidation,
strand breakage, and DNA– protein cross-links. Among the various ROS, ·OH
generates various products from the DNA bases which mainly include C-8
hydroxylation of guanine to form 8-oxo-7,8 dehydro-2’- deoxyguanosine, a ring-
opened product; 2,6-diamino-4-hydroxy-5- formamimodipyrimidine, 8-OH-adenine,
2-OH-adenine, thymine glycol, cytosine glycol, etc. (Wiseman and Halliwell, 1996).
ROS-induced DNA damages include various mutagenic alterations as well. For
example, mutation arising from selective modification of G : C sites specially
indicates oxidative attack on DNA by ROS. The action of 8-oxodeoxy- guanosine as a
promutagen, as well as in altering the binding of methylase to the oligomer so as to
inhibit methylation of adjacent cytosine has been reported in cases of cancer
development (Weitzman et al., 1994). ROS have also been shown to activate
mutations in human C-Ha-ras-1 protooncogene, and to induce mutation in the p53
tumour-suppressor gene (Hussain et al., 1994). Besides, ROS may interfere with
normal cell signalling, resulting thereby in alteration of the gene expression, and
development of cancer by redox regulation of transcriptional factors/activator and/or
by oxidatively modulating the protein kinase cascades. The oxidative damage of
mitochondrial DNA also involves base modification and strand breaks, which leads to
formation of abnormal components of the electron transport chain. This results in the
generation of more ROS through increased leakage of electrons, and therefore further
cell damage. Oxidative damage to mitochondrial DNA may promote cancer and
aging, eventually (Richter, 1988).
Oxidative damage of proteins
During mitochondrial electron transport chain, free radicals are produced
which can stimulate protein degradation. Oxidative protein damage may be brought
about by metabolic processes which degrade a damaged protein to promote synthesis
of a new protein. In the process of cataractogenesis, oxidative modification plays a
significant role in cross-linking of crystalline lens protein, leading to high-molecular-
weight aggregates, loss of solubility, and lens opacity (Guptasarma et al., 1992).
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21
Lipofuscin – an aggregate of peroxidized lipid and proteins – accumulates in
lysosomes of aged cells, Alzheimer’s disease brain cells, and ironoverloaded
hepatocytes (Wolf et al., 1986). On the basis of extensive studies on aging processes,
it has been established that catalytically inactive or less active, more thermolabile
forms of enzyme accumulate in cells during aging, and show a dramatic increase in
the level of protein carbonyl content: an index of metal-catalysed oxidation of
proteins (Stadtman and Oliver, 1991). In human erythrocytes, levels of
glyceraldehyde-3-P-dehydrogenase, aspartate aminotransferase, and phosphoglycerate
kinase decline with age together with an increase in protein carbonyl content. The
carbonyl content of protein in rat hepatocytes also increases with age along with
decrease in the activities of glutamine synthetase and glucose-6-P-dehydrogenase,
without any loss in the total enzyme protein (Oliver et al., 1987). An oxidative
inactivation of glutamine synthetase occurs during ischemic-reperfusion injury of
gerbil brain (Oliver et al., 1990) (Figure 3).
Figure 3: An overall picture of the metabolism of ROS and the mechanism of oxidative tissue damage leading to pathological conditions.
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3) ANTIOXIDANT SYSTEMS INVOLVED IN THE
SCAVENGING PROCESS
a) Primary defense against ROS: Catalytic removal of ROS by
antioxidant enzymes
Superoxide dismutase (SOD), catalase, and peroxidases constitute a mutually
supportive team of defense against ROS. While SOD lowers the steady-state level of
O2¯, catalase and peroxidases do the same for H2O2.
Superoxide dismutase
The first enzyme involved in the antioxidant defense is the superoxide
dismutase: a metalloprotein found in both prokaryotic and eukaryotic cells (Fridovich,
1983). The iron-containing (Fe-SOD) and the manganese-containing (Mn-SOD)
enzymes are characteristic of prokaryotes. In eukaryotic cells, the predominant forms
are the copper-containing enzyme and the zinc-containing enzyme, located in the
cytosol. The second type is the manganese containing SOD found in the
mitochondrial matrix. The biosynthesis of SOD is mainly controlled by its substrate,
the O2– (Fridovich, 1986). Induction of SOD by increased intracellular fluxes of O2
–
has been observed in numerous microorganisms, as well as in higher organisms
(Crapo and McCord, 1976).
Glutathione peroxidase and Glutathione reductase
Glutathione peroxidase catalyses the reaction of hydroperoxides with reduced
glutathione (GSH) to form glutathione disulphide (GSSG) and the reduction product
of the hydroperoxide (Figure 4). This enzyme is specific for its hydrogen donor, GSH,
and nonspecific for the hydroperoxides ranging from H2O2 to organic hydroperoxides
(Meister and Anderson, 1983). It is a seleno-enzyme; two-third of which (in liver) is
present in the cytosol and one-third in the mitochondria. Glutathione disulfide is
recycled back to glutathione by glutathione reductase, using the cofactor NADPH
generated by glucose 6- phosphate dehydrogenase (Freeman and Crapo, 1982).
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23
Heme peroxidase
Heme peroxidases such as horseradish peroxidase, lactoperoxidase, and other
mammalian peroxidases have been studied most extensively. The enzyme catalyses
the oxidation of a wide variety of electron donors with the help of H2O2 and thereby
scavenges the endogenous H2O2 (Dawson, 1988).
Catalase
Catalase present in almost all the mammalian cells is localized in the
peroxisomes or the microperoxisomes. It is a hemoprotein and catalyses the
decomposition of H2O2 to water and oxygen and thus protects the cell from oxidative
damage by H2O2 and .OH (Deisseroth and Dounce, 1970).
Figure 4: Catalytic removal of ROS by antioxidant enzymes.
b) Secondary defense against ROS: Free-radical scavengers
In addition to the primary defense against ROS by antioxidant enzymes,
secondary defense against ROS is also offered by small molecules which react with
radicals to produce another radical compound, the ‘scavengers’. When these
scavengers produce a lesser harmful radical species, they are called ‘antioxidants’.
For example, α-tocopherol, ascorbate, and reduced glutathione (GSH) may act in
combination to act as cellular antioxidants (Figure 5). α-tocopherol, present in the cell
membrane and plasma lipoproteins, functions as a chain-breaking antioxidant. Once
the tocopherol radical is formed, it can migrate to the membrane surface and is
reconverted to α-tocopherol by reaction with ascorbate or GSH. The resulting
ascorbate radical can regenerate ascorbate by reduction with GSH, which can also
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24
directly scavenge ROS, and the resulting GSSG can regenerate GSH through
NADPH-glutathione reductase system.
Figure 5: Mechanism of free-radical scavenging action of cellular low-molecular weight antioxidants- α-tocopherol, ascorbate, and reduced glutathione (GSH), through NADPH-glutathione reductase (GR) system.
This cooperative activity may explain the synergism obtained when several
scavengers are involved and the beneficial use of large combinations of low molecular
weight antioxidants (LMWA) in antioxidant therapy (figure 6).
Examples of LMWA
1) Glutathione
It is a low-molecular-mass, thiol-containing tripeptide, glutamic acid-cysteine-
glycine (GSH) in its reduced form and GSSG in its oxidized form, in which 2 GSH
molecules join via the oxidation of the -SH groups of the cysteine residue to form a
disulphide bridge. It acts as a cofactor for the enzyme peroxidase, thus serving as an
indirect antioxidant donating the electrons necessary for its decomposition of H2O2. It
is also involved in many other biochemical pathways and cellular functions like
metabolism of ascorbic acid, maintenance of communication between cells,
prevention of oxidation in -SH groups of protein, and copper transport (Chance et al.,
1979). Glutathione can act as a chelating agent for copper ions and prevent them from
participating in the Haber-Weiss reaction, it serve as a cofactor for several enzymes,
such as glyoxylase and those involved in leukotriene biosynthesis, and play a role in
protein folding, degradation, and cross-linking. In addition to its biochemical
functions, it can scavenge ROS directly. GSH can interact with OH·, ROO·, and RO·
radicals as well as with HCLO and ‘O2 Upon reaction with ROS, it becomes a
glutathione radical, which can be regenerated to its reduced form (Gul et al., 2000).
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2) Melatonin
It is the hormone synthesized by the pineal gland, that helps to regulate
circadian rhythms that also possesses a powerful antioxidant capacity in vitro, as it
scavenges a variety of ROS, mainly through donation of the hydrogen atom by the (-
NH) group. Indirectly it alters the antioxidant activity of the cell, for example, by
induction of synthesis of antioxidant enzymes or modulating other cellular responses
leading to secretion and accumulation of other antioxidants. High local concentrations
of melatonin in the brain may explain its great protective effect against head injury in
rats (Reiter et al., 2002).
3) Histidine dipeptides
It includes the compounds (carnosine, homocarnosine, and anserine) that are
synthesized in the brain and skeletal muscles that have anti-oxidative potentials. They
are considered multifunctional antioxidants, because they can act in many ways to
destroy and remove ROS. They can scavenge directly ·OH, ROO·, and RO· radicals;
bind H2O2; quench efficiently ‘O2; and bind transition metals and prevent them from
participating in the metal-mediated Haber- Weiss reaction. In vivo they diminish
oxidative damage in many systems, including the ischemic process. These compounds
do not exert pro-oxidant effects as other reducing antioxidants sometimes do. They
also act as endogenous buffers and thus prevents protein glycosylation (Boldyrev,
1993).
4) Uric acid
Uric acid provides an excellent example of the adaptation of the organism to
oxidative stress. It is a cellular waste product originating from the oxidation of
hypoxanthine and xanthine by xanthine oxidase and dehydrogenase. Urate, the
physiological state of uric acid, reacts with hydroxyl radicals producing a stable urate
radical that can be regenerated by ascorbate to its prior state, urate. This compound
can act with peroxyl radicals, 1O2, O3, NO·, and other nitrogen oxygen radicals. Urate
also protects protein from nitration; it can chelate metal ions, such as copper and iron,
and prevent them from participating in redox reactions (Ames et al., 1981).
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5) Dietary source antioxidants
Antioxidant molecules like ascorbic acid, tocopherols, polyphenols and
carotenoids present in green vegetables, fruits, fish and other food items. Both
lipophilic and hydrophilic compounds possess different oxidation potentials reflecting
their ability to donate electrons and act as antioxidants.
a) Ascorbic acid (ascorbate, vitamin C)
It is an example of a water-soluble antioxidant. Like other antioxidants,
ascorbate, which at physiological pH exists as a mono anion, possesses many
biochemical functions in addition to its activity as a scavenger. It is required as a
cofactor for many enzymes, such as proline hydroxylase and dopamine β-
hydroxylase. As an antioxidant, ascorbate is an efficient scavenger, or reducing
antioxidant, capable of donating its electrons to ROS and eliminating them. It can
donate 2 electrons; following donation of 1 electron, it produces the ascorbyl
(semidehydroascorbate or ascorbate) radical, which can be further oxidized to
produce dehydroascorbate. Because the ascorbyl radical is relatively stable, it makes
ascorbate a powerful, important antioxidant. This radical can lose its electron and be
transformed to dehydroascorbic acid or regenerated to the reduced form by obtaining
an electron from another reducing agent, such as GSH or NADH, via the mediation of
an enzyme like NADH-semidehydroascorbate reductase (Carr and Frei, 1999).
The oxidation product, dehydroascorbic acid, can also be regenerated by the
enzyme dehydroascorbate reductase at the expense of 2 molecules of GSH. The
compound dehydroascorbate is not stable and is decomposed to di-keto- L-gulonic
acid and then to oxalic and L-threonic acids, which can be further decomposed to
oxalic acid. In vitro, ascorbate can act as an efficient antioxidant and scavenge a
variety of ROS including hydroxyl, peroxyl, thyil, and oxosulphuric radicals.
Ascorbate is also a powerful scavenger of HClO and peroxynitrous acid and can
inhibit the peroxidation process. It can react with 1O2 and act synergistically with
other antioxidants to regenerate, for example, the tocopherol radical to its reduced
form. Indirect evidence has shown that, in vivo, ascorbate acts directly as an
antioxidant (Arrigoni and De Tullio, 2002).
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b) Vitamin E (Tocopherol)
It is a chain breaking antioxidant that scavenges ROO. to inhibit the lipid
peroxidation process in biological membranes . There are eight naturally occurring
substances that are known to be members of the vitamin E family. These compounds
have 3 asymmetric carbon atoms, giving 8 optical isomers. Although all possess
antioxidant activity, the RRR-a -tocopherol or d-a -tocopherol is considered the most
effective one, as the others are not retained and absorbed well in body tissues. Other
members of the family consist of d-β, d-γ, and d-δ-tocopherols and d-α-, d-β-, d-γ-,
and d-δ-tocotrienols. These compounds can scavenge other ROS, such as 1O2.
Following interaction, tocopherol is converted to tocopherolquinone and subsequently
to tocopherylquinone. As with other scavengers, a -tocopheryl radical can be recycled
to its active form. Other roles for tocopherol also exist, such as those of a membrane-
stabilizing agent and a potential pro-oxidant compound in some systems when
transition metals are present (Herrera and Barbas, 2001).
Figure 6: The sources and cellular responses to reactive oxygen species (ROS).
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DIABETES INDUCED OXIDATIVE STRESS AND ITS
COMPLICATIONS
Glucose in chronic excess causes toxic effects on structure and function of
organs. There are many potential mechanisms whereby excess glucose metabolites
traveling along these pathways might cause cell damage. Multiple biochemical
pathways and mechanisms of action for glucose toxicity have been suggested (Figure
7). These include glucose autoxidation, protein kinase C activation, methylglyoxal
formation and glycation, hexosamine metabolism, sorbitol formation, and oxidative
phosphorylation. However, all these pathways have in common the formation of
reactive oxygen species that, in excess and over time, cause chronic oxidative stress,
which has been suggested to be involved in the pathogenesis and progression of
diabetic tissue damage. This is particularly relevant and dangerous for the islet, which
is among those tissues that have the lowest levels of intrinsic antioxidant defenses
(Robertson, 2004).
1. Increased polyol pathway flux
The first enzyme in the polyol pathway is aldose reductase (alditol: NAD(P)+
1-oxidoreductase, EC 1.1.1.21). It is a cytosolic, monomeric oxidoreductase that
Figure 7: Biochemical pathways along which glucose metabolism can form ROS
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catalyses the NADPH-dependent reduction of a wide variety of carbonyl compounds,
including glucose. Its crystal structure has a single domain folded into an eight-
stranded parallel a/b-barrel motif, with the substrate-binding site located in a cleft at
the carboxy-terminal end of the b-barrel (Wilson et al., 1992). Aldose reductase has a
low affinity (high Km) for glucose, and at the normal glucose concentrations found in
non-diabetics, metabolism of glucose by this pathway is a very small percentage of
total glucose use. But in hyperglycaemic conditions, increased intracellular glucose
results in its increased enzymatic conversion to the polyalcohol sorbitol, with
concomitant decreases in NADPH. In the polyol pathway, sorbitol is oxidized to
fructose by the enzyme sorbitol dehydrogenase, with NAD+ reduced to NADH. Flux
through this pathway during hyperglycaemia varies from 33% of total glucose use in
the rabbit lens to 11% in human erythrocytes. A number of mechanisms have been
proposed to explain the potential detrimental effects of hyperglycaemia-induced
increases in polyol pathway flux. These include sorbitol-induced osmotic stress,
decreased (Na+&K+) ATPase activity, an increase in cytosolic NADH/NAD+ and a
decrease in cytosolic NADPH. Sorbitol does not diffuse easily across cell membranes,
and it was originally suggested that this resulted in osmotic damage to microvascular
cells.Thus, the contribution of this pathway to diabetic complications may be very
much species, site and tissue dependent (Figure 8) (Lee and Chung, 1999).
Figure 8: Involvement of polyol pathway in diabetic complications.
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2. Increased intracellular formation of advanced glycation end-
products
AGEs are found in increased amounts in diabetic retinal vessels (Stitt et al.,
1997) and renal glomeruli (Horie et al., 1997). They were originally thought to arise
from non-enzymatic reactions between extracellular proteins and glucose. But the rate
of AGE formation from glucose is orders of magnitude slower than the rate of AGE
formation from glucose-derived dicarbonyl precursors generated intracellularly, and it
now seems likely that intracellular hyperglycaemia is the primary initiating event in
the formation of both intracellular and extracellular AGEs (Degenhardt et al., 1998).
AGEs can arise from intracellular auto-oxidation of glucose to glyoxal (Wells-Knecht
et al., 1995), decomposition of the Amadori product (glucose-derived 1-amino-1-
deoxyfructose lysine adducts) to 3-deoxyglucosone (perhaps accelerated by an
amadoriase), and fragmentation of glyceraldehyde- 3-phosphate and
dihydroxyacetone phosphate to methylglyoxal. These reactive intracellular
dicarbonyls — glyoxal, methylglyoxal and 3-deoxyglucosone — react with amino
groups of intracellular and extracellular proteins to form AGEs. Methylglyoxal and
glyoxal are detoxified by the glyoxalase system (Thornalley, 1990). All three AGE
precursors are also substrates for other reductases (Suzuki et al., 1998).
Production of intracellular AGE precursors damages target cells by three
general mechanisms (Figure 9). First, intracellular proteins modified by AGEs have
altered function. Second, extracellular matrix components modified by AGE
precursors interact abnormally with other matrix components and with the receptors
for matrix proteins (integrins) on cells. Third, plasma proteins modified by AGE
precursors bind to AGE receptors on endothelial cells, mesangial cells and
macrophages, inducing receptor-mediated production of reactive oxygen species. This
AGE receptor ligation activates the pleiotropic transcription factor NF-kB, causing
pathological changes in gene expression.
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31
Figure 9: Mechanisms by which intracellular production of advanced glycation end-product (AGE) precursors damages vascular cells.
3. Activation of protein kinase C
The PKC family comprises at least eleven isoforms, nine of which are
activated by the lipid second messenger DAG. Intracellular hyperglycaemia increases
the amount of DAG in cultured microvascular cells and in the retina and renal
glomeruli of diabetic animals. It seems to achieve this primarily by increasing de novo
DAG synthesis from the glycolytic intermediate dihydroxyacetone phosphate, through
reduction of the latter to glycerol-3-phosphate and stepwise acylation. Increased de
novo synthesis of DAG activates PKC both in cultured vascular cells and in retina and
glomeruli of diabetic animals (Koya and King, 1998). The β- and δ-isoforms of PKC
are activated primarily, but increases in other isoforms have also been found, such as
PKC-α and - ε isoforms in the retina and PKC- α and - β in glomeruli of diabetic rats.
Activation of PKC has a number of pathogenic consequences by affecting expression
of endothelial nitric oxide synthetase (eNOS), endothelin-1 (ET-1), vascular
endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and
plasminogen activator inhibitor-1 (PAI-1), and by activating NF-kB and NAD(P)H
oxidases (Koya et al., 1997) (Figure 10).
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32
Figure 10: Consequences of hyperglycaemia-induced activation of protein kinase C (PKC).
4. Increased flux through the hexosamine pathway
Shunting of excess intracellular glucose into the hexosamine pathway might
also cause several manifestations of diabetic complication. In this pathway, fructose-
6-phosphate is diverted from glycolysis to provide substrates for reactions that require
UDP-N-acetylglucosamine, such as proteoglycan synthesis and the formation of O-
linked glycoproteins (Figure 11). The glycolytic intermediate fructose-6-phosphate
(Fruc-6-P) is converted to glucosamine-6-phosphate by the enzyme glutamine:
fructose-6-phosphate amidotransferase (GFAT). Intracellular glycosylation by the
addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalysed by the
enzyme O-GlcNAc transferase (OGT). Increased donation of GlcNAc moieties to
serine and threonine residues of transcription factors such as Sp1, often at
phosphorylation sites, increases the production of factors as PAI-1 (Du et al., 2000)
and TGF-β1(Kolm-Litty et al., 1998).This pathway is also important role in
hyperglycaemia induced and fat-induced insulin resistance (Hawkins et al., 1997).
Thus, activation of the hexosamine pathway by hyperglycaemia may result in many
changes in both gene expression and protein function, which together contribute to the
pathogenesis of diabetic complications.
Review of Literature
33
Figure 11: The hexosamine pathway.
5. Oxidative phosphorylation
Electron flow through the mitochondrial electron-transport chain (Figure 12)
is carried out by four inner membrane-associated enzyme complexes, plus
cytochrome c and the mobile electron carrier ubiquinone. NADH derived from both
cytosolic glucose oxidation and mitochondrial TCA cycle activity donates electrons to
NADH: ubiquinone oxidoreductase (complex I). Complex I ultimately transfers its
electrons to ubiquinone. Ubiquinone can also be reduced by electrons donated from
several FADH2-containing dehydrogenases, including succinate: ubiquinone
oxidoreductase (complex II) and glycerol-3-phosphate dehydrogenase. Electrons from
reduced ubiquinone are then transferred to ubiquinol: cytochrome c oxidoreductase
(complex III) by the ubisemiquinone radical-generating Q cycle. Electron transport
then proceeds through cytochrome c, cytochrome c oxidase (complex IV) and, finally,
molecular oxygen (O2). Electron transfer through complexes I, III and IV generates a
proton gradient that drives ATP synthase (complex V). When the electrochemical
potential difference generated by the proton gradient across the inner mitochondrial
membrane is high, the lifetime of superoxide-generating electron-transport
intermediates such as ubisemiquinone is prolonged. There seems to be a threshold
value above which superoxide production is markedly increased (Korshunov et al.,
Review of Literature
34
1997). Thus hyperglycaemia increases the proton gradient above this threshold value
as a result of overproduction of electron donors by the TCA cycle. This, in turn,
causes a marked increase in the production of superoxide.
Figure 12: Production of superoxide by the mitochondrial electron-transport chain.
6. Glyceraldehyde Autoxidation
Glyceraldehyde 3-phosphate is a phosphorylation product formed from
glucose during anaerobic glycolysis. The partner product, dihydroxyacetone
phosphate, also contributes to intracellular glyceraldehyde concentrations via
enzymatic conversion by triose-phosphate isomerase. Thereafter, glyceraldehyde 3-
phosphate is oxidized by glyceraldehyde-phosphate dehydrogenase (GAPDH).
Continuance of glycolysis yields pyruvate, which enters the mitochondria where it is
oxidized to acetyl-CoA, and the processes of the tricarboxylic acid cycle and
oxidative phosphorylation begin. One alternative to this classic pathway of glucose
metabolism is the less familiar route of glyceraldehyde autoxidation (Figure 7,
pathway 1). The potential relevance of this pathway to diabetes mellitus was pointed
out by Wolff and Dean. (Wolff and Dean, 1987), who emphasized that autoxidation of
α-hydroxyaldehydes generates hydrogen peroxide (H2O2) and α-ketoaldehydes. In the
presence of redox active metals, H2O2 can form the highly toxic hydroxyl radical.
This pathway, therefore, forms two potentially toxic substances, α-ketoaldehydes,
Review of Literature
35
which contribute to glycosylation-related protein chromophore development, and the
hydroxyl radical, a reactive oxygen species that can cause mutagenic alterations in
DNA.
PHARMACOLOGICAL INTERVENTIONS IN THE
TREATMENT OF DIABETES MELLITUS
Oral hypoglycaemic agents
There are five classes of oral pharmacological agents available to treat
4. Fourth injection was given in Freund′s Incomplete Adjuvant (FIA). (0.5ml
FIA + 0.5ml LPS)
The adjuvant and antigen were mixed properly to form a water in oil emulsion
before injecting.
TT injection were procured from Serum Institute of India Ltd., Pune, and
protein content was estimated. Four immunizations were done at an interval of 15
days.
Blood sample collection
Seven days after each immunization (LPS and TT) blood samples were
collected from rat’s eye. Blood samples were kept at room temperature for 30 minutes
for serum separation and then centrifuged at 5000 rpm for 10 minutes. Serum was
collected and stored at -20�C.
Serum dilution
Sera collected after all four immunizations were serially diluted with 1% BSA
in PBS for ELISA testing.
Testing of antibody titer
Sera of rats were tested for the presence of IgM and IgG antibodies by ELISA.
Levels of IL-4 were also tested for all groups of rats.
Protocol for ELISA to detect IgG and IgM:
Coating Antigen to microtiter plate
The antigens (TT and LPS) were diluted to a final concentration of 1µg/100µl
in phosphate buffer saline (PBS; 50mM pH=7.4) and wells were coated with 100µl
antigen preparation i.e. 1µg antigen. The plate was covered and incubated overnight at
4�C. The coating solution was removed and the plate was washed 4 times with PBS.
Materials and Methods
69
Blocking
The remaining protein-binding sites in the coated wells were blocked by
adding 400µl blocking solution (1% BSA in PBS) per well. The plate was covered
and incubated for four hours at 4°C. The plate was washed 4 times with PBS.
Incubation with the primary and secondary antibodies
100µl of primary antibody (appropriately diluted rat serum) was added to the
well. The plate was covered and incubated for 2½ hrs at room temperature. The plate
was washed four times with PBS. The 100µl of appropriately diluted secondary
antibodies (anti-rat IgG or IgM, HRP labelled) were added.
Detection
100µl of the substrate solution [Tetramethylbenzidine (TMB) + hydrogen
peroxide] was added. Then 100µl of stop solution (10N H2SO4) was added to the
wells. The absorbance of each well was recorded at 450nm.
Detection of IL-2 levels:
The levels of IL-2 in normal, diabetic and treated rat serum were estimated
using Invitrogen Rat IL-2 ELISA kit. The invitrogen rat IL-2 kit is supplied with rat
IL-2 antibodies coated onto the wells of the ELISA plate. The estimation was
performed as per manufacturer’s protocol. Briefly, to each well 50 µl of standard IL-2
solution at appropriate dilutions, 50 µl of unknown serum were added and incubated
for 2 hrs at room temperature. The plate was washed thoroughly and 50 µl of
biotinylated rat anti IL-2 antibody was added to each well and incubated for 2 hrs at
room temperature. After that the plate was washed thoroughly and 50 µl of
streptavidin-HRP conjugate was added to each well. The plate was incubated for 1 hr
at room temperature. After washing, the chromogen (50 µl) solution supplied with the
kit was added to each well and incubated for 20-30 min followed by addition of stop
solution (50 µl). The blue colour turned to yellow which was read at 450nm. Standard
curve was plot and concentration of IL-2 was calculated.
Materials and Methods
70
Detection of IL-4 levels:
The levels of IL-4 in normal, diabetic and treated rat serum were estimated
using Invitrogen Rat IL-4 ELISA kit. The invitrogen rat IL-4 kit is supplied with rat
IL-4 antibodies coated onto the wells of the ELISA plate. The estimation was
performed as per manufacturer’s protocol. Briefly, to each well 50 µl of standard IL-4
solution at appropriate dilutions, 50 µl of unknown serum were added and incubated
for 2 hrs at room temperature. The plate was washed thoroughly and 50 µl of
biotinylated rat anti IL-4 antibody was added to each well and incubated for 2 hrs at
room temperature. After that the plate was washed thoroughly and 50 µl of
streptavidin-HRP conjugate was added to each well. The plate was incubated for 1 hr
at room temperature. After washing, the chromogen (50 µl) solution supplied with the
kit was added to each well and incubated for 20-30 min followed by addition of stop
solution (50 µl). The blue colour turned to yellow which was read at 450nm. Standard
curve was plot and concentration of IL-4 was calculated.
Detection of IL-6 levels:
The levels of IL-6 in normal, diabetic and treated rat serum were estimated
using Invitrogen Rat IL-6 ELISA kit. The invitrogen rat IL-6 kit is supplied with rat
IL-6 antibodies coated onto the wells of the ELISA plate. The estimation was
performed as per manufacturer’s protocol. Briefly, to each well 50 µl of standard IL-6
soltion at appropriate dilutions, 50 µl of unknown serum were added and incubated
for 2 hrs at room temperature. The plate was washed thoroughly and 50 µl of
biotinylated rat anti IL-6 antibody was added to each well and incubated for 2 hrs at
room temperature. After that the plate was washed thoroughly and 50 µl of
streptavidin-HRP conjugate was added to each well. The plate was incubated for 1 hr
at room temperature. After washing, the chromogen (50 µl) solution supplied with the
kit was added to each well and incubated for 20-30 min followed by addition of stop
solution (50 µl). The blue colour turned to yellow which was read at 450nm. Standard
curve was plot and concentration of IL-6 was calculated.
Materials and Methods
71
Detection of IL-10 levels:
The levels of IL-10 in normal, diabetic and treated rat serum were estimated
using Invitrogen Rat IL-10 ELISA kit. The invitrogen rat IL-10 kit is supplied with rat
IL-10 antibodies coated onto the wells of the ELISA plate. The estimation was
performed as per manufacturer’s protocol. Briefly, to each well 50 µl of standard IL-
10 solution at appropriate dilutions, 50 µl of unknown serum were added and
incubated for 2 hrs at room temperature. The plate was washed thoroughly and 50 µl
of biotinylated rat anti IL-10 antibody was added to each well and incubated for 2 hrs
at room temperature. After that the plate was washed thoroughly and 50 µl of
streptavidin-HRP conjugate was added to each well. The plate was incubated for 1 hr
at room temperature. After washing, the chromogen (50 µl) solution supplied with the
kit was added to each well and incubated for 20-30 min followed by addition of stop
solution (50 µl). The blue color turned to yellow which was read at 450nm. Standard
curve was plot and concentration of IL-10 was calculated.
Objectives
72
OBJECTIVES
1) To evaluate anti-hyperglycemic activity of Syzygium cumini & Pterocarpus
marsupium.
2) To evaluate the effect of Syzygium cumini & Pterocarpus marsupium extracts
on activities of key glycolytic enzymes.
3) To isolate and identify the active component(s) present in the plant extracts of
Syzygium cumini & Pterocarpus marsupium having anti diabetic potential.
4) To evaluate the role of these plant extracts in management of diabetes
associated oxidative stress.
5) To evaluate the immunomodulatory effect of these plant extracts.
Chapter-I
Anti-diabetic and anti-oxidative potentials of crude Syzygium cumini and
Pterocarpus marsupium extracts
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
73
Diabetes mellitus is a common and very prevalent disease affecting the
citizens of both developed and developing countries. It is estimated that 25% of the
world population is affected by this disease. Diabetes mellitus is caused by the
abnormality of carbohydrate metabolism which is linked to low blood insulin level or
insensitivity of target organs to insulin (Maiti et al., 2004). Despite considerable
progress in the treatment of diabetes by oral hypoglycemic agents, search for newer
drugs continues because the existing synthetic drugs have several limitations. The
herbal drugs with anti-diabetic activity are yet to be commercially formulated as
modern medicines, even though they have been acclaimed for their therapeutic
properties in the traditional systems of medicine (Chan et al., 2012). The plants
provide a potential source of anti-hyperglycemic drugs because many plants and plant
derived compounds have been used in the treatment of diabetes. Many Indian plants
have been investigated for their beneficial use in different types of diabetes. Ayurveda
and other traditional medicinal system for the treatment of diabetes describe a number
of plants used as herbal drugs. Hence, they play an important role as alternative
medicine due to less side effects and low cost. The active principles present in
medicinal plants have been reported to stimulate pancreatic beta cells re-generation
and insulin release (Kavishankar et al., 2011). Hyperglycemia is involved in the
etiology of development of diabetic complications. Anti-hyperglycemic herbs
increase insulin secretion, enhance glucose uptake by adipose or muscle tissues and
inhibit glucose absorption from intestine and glucose production from liver (Hui et
al., 2009). Insulin and oral hypoglycemic agents like sulphonylureas and biguanides
are still the major players in the management but there is quest for the development of
more effective anti-diabetic agents.
Oxidative stress plays a pivotal role in the development of diabetes
complications. Oxidative stress and oxidative damage to the tissues are common end
points of chronic diseases, such as atherosclerosis, diabetes and rheumatoid arthritis
(Baynes and Thorpe, 1999). Oxidative stress is currently suggested as mechanism
underlying diabetes and diabetic complications (Kangralkar et al., 2010). During
diabetes, persistent hyperglycemia causes increased production of free radicals,
especially reactive oxygen species (ROS), in all tissues due to glucose auto-oxidation
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
74
and protein glycosylation. The increase in the level of ROS in diabetes could be due
to their increased production and/ or decreased destruction by nonenzymic
antioxidants, eg. reduced glutathione (GSH) and enzymic antioxidants like catalase
(CAT), glutathione S-transferase (GST), , and superoxide dismutase (SOD). The level
of these antioxidant enzymes critically influences the susceptibility of various tissues
to oxidative stress and is associated with the development of complications in diabetes
(Lipinski, 2001).
EXPERIMENTAL DESIGN
1- To evaluate anti-hyperglycemic effect of aqueous and alcoholic extracts of
SC seed powder at different doses.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + aq. SC treated (1.5 g/kg b.w./day)
Group III Normal + aq. SC treated (3 g/kg b.w./day)
Group IV Normal + aq. SC treated (5 g/kg b.w./day)
Group V Normal + alc. SC treated (50 mg/kg b.w./day)
Group VI Normal + alc. SC treated (100 mg/kg b.w./day)
Group VII Normal + alc. SC treated (200 mg/kg b.w./day)
Group VIII Diabetic control
Group IX Diabetic + aq. SC treated (1.5 g/kg b.w./day)
Group X Diabetic + aq. SC treated (3 g/kg b.w./day)
Group XI Diabetic + aq. SC treated (5 g/kg b.w./day)
Group XII Diabetic + alc. SC treated (50 mg/kg b.w./day)
Group XIII Diabetic + alc. SC treated (100 mg/kg b.w./day)
Group XIV Diabetic + alc. SC treated (200 mg/kg b.w./day)
Group XV Diabetic + Metformin (100 mg/kg b.w./day)
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
75
2- To evaluate anti-hyperglycemic effect of aqueous and alcoholic extracts of
PM bark powder at different doses.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + aq. PM treated (100 mg/kg b.w./day)
Group III Normal + aq. PM treated (200 mg/kg b.w./day)
Group IV Normal + aq. PM treated (400 mg/kg b.w./day)
Group V Normal + alc. PM treated (150 mg/kg b.w./day)
Group VI Normal + alc. PM treated (300 mg/kg b.w./day)
Group VII Normal + alc. PM treated (500 mg/kg b.w./day)
Group VIII Diabetic control
Group IX Diabetic + aq. PM treated (100 mg/kg b.w./day)
Group X Diabetic + aq. PM treated (200 mg/kg b.w./day)
Group XI Diabetic + aq. PM treated (400 mg/kg b.w./day)
Group XII Diabetic + alc. PM treated (150 mg/kg b.w./day)
Group XIII Diabetic + alc. PM treated (300 mg/kg b.w./day)
Group XIV Diabetic + alc. PM treated (500 mg/kg b.w./day)
Group XV Diabetic + Metformin (100 mg/kg body b.w./day)
3- Duration dependent effect of alcoholic extract of SC seed and aqueous
extract of PM bark on fasting blood glucose levels.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + alc. SC treated (100 mg/kg b.w./day)
Group III Normal + aq. PM treated (200 mg/kg b.w./day)
Group IV Diabetic control
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
76
Group V Diabetic + alc. SC treated (100 mg/kg b.w./day)
Group VI Diabetic + aq. PM treated (200 mg/kg b.w./day)
Group VII Diabetic + Metformin (100 mg/kg b.w./day)
4- Effect of aqueous and alcoholic extracts of SC seed on body weight.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Diabetic
Group III Normal + aq. SC treated (3 g/kg b.w./day)
Group VI Normal + alc. SC treated (100mg/kg b.w./day)
Group V Diabetic + aq. SC treated (3 g/kg b.w./day)
Group VI Diabetic + alc. SC treated (100mg/kg b.w./day)
Group VII Diabetic + Metformin (100 mg/kg b.w./day)
5- Effect of aqueous and alcoholic extracts of PM bark on body weight.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Diabetic
Group III Normal + aq. PM treated (200mg/kg b.w./day)
Group IV Normal + alc. PM treated (300mg/kg b.w./day)
Group V Diabetic + aq. PM treated (200mg/kg b.w./day)
Group VI Diabetic + alc. PM treated (300mg/kg b.w./day)
Group VIII Diabetic +Metformin (100 mg/kg b.w./day)
6- Evaluation of toxic effects of SC seed alcoholic extract and PM bark
aqueous extract doses in rats after 2 month exposure.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
77
Group II Normal + alc.SC treated (3 g/kg b.w./day)
Group III Normal + alc.SC treated (15 g/kg b.w./day)
Group IV Normal + aq. PM treated (200 mg/kg b.w./day)
Group V Normal + aq. PM treated (1 g/kg b.w./day)
7- To evaluate anti-oxidative effect of aqueous and alcoholic extract of SC in
alloxan induced diabetic rats.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + aq. SC treated (3 g/kg b.w./day)
Group III Normal + alc. SC treated (100 mg/kg b.w./day)
Group IV Diabetic control
Group V Diabetic + aq. SC treated (3 g/kg b.w./day)
Group VI Diabetic + alc. SC treated (100 mg/kg b.w./day)
Group VII Diabetic + Metformin (100 mg/kg b.w./day)
Group VIII Diabetic + Vitamin C (150 mg/kg b.w./day)
8- To evaluate anti-oxidative effect of aqueous and alcoholic extract of PM
in alloxan induced diabetic rats.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + aq. PM treated (200 mg/kg b.w./day)
Group III Normal + alc. PM treated (300 mg/kg b.w./day)
Group IV Diabetic control
Group V Diabetic + aq. PM treated (200 mg/kg b.w./day)
Group VI Diabetic + alc. PM treated (300 mg/kg b.w./day)
Group VII Diabetic + Metformin (100 mg/kg b.w./day)
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
78
Group VIII Diabetic + Vitamin C (150 mg/kg b.w./day)
RESULTS
A- To evaluate the anti-hyperglycemic effect of aqueous and alcoholic
extracts of Syzygium cumini at different doses.
Experiments were conducted to evaluate anti-hyperglycemic effect of aqueous
and alcoholic extracts of SC seeds. Rats were divided into 15 groups according to the
experimental design. The rats were fed with 3 different doses of aq. SC extract 1.5,
3.0 and 5.0 g/kg body weight/day and 3 different doses of alc. SC extract 50, 100 and
200 mg/kg body weight/day. An intraperitonial dose (150 mg/kg body weight) of
alloxan increased FBG levels in groups VIII-XV after 4-5 days of injection. FBG
levels were monitored on day 0 (when rats were confirmed for diabetes) and day 30
(end of experiments). In diabetic control group (VIII), higher FBG level (>270 mg/dl)
was maintained throughout the period of study. On the other hand the oral dose of aq.
extract of SC resulted in significant decrease in FBG levels in diabetic rats. The doses
1.5, 3.0 and 5.0 g/kg body weight/day resulted in decrease in FBG levels from
445.9±18.7 to 317.1±14.4 mg/dl, 460.4±23.6 to 160.6±6.7 mg/dl and 476.8±21.9 to
158.1±4.9 mg/dl respectively, in 30 days treatment (Table 1). Better effects were
observed when diabetic rats were administered orally different doses of alc. SC seed
extracts viz. 50, 100 and 200 mg/kg body weight/day. The FBG levels were decreased
from 455.9±12.9 to 300.8±18.3 mg/dl, 427.6±18.8 to 100.0±10.1 mg/dl and
437.7±19.2 to 103.8±9.8 mg/dl, respectively, in 30 days treatment (Table 1). Normal
rats treated with different doses of aq. and alc. extract of SC seeds did not show any
significant changes in FBG levels as compared to normal control (group I). The 3.0
and 5.0 g/kg body weight doses of aq. SC extract and 100 and 200mg /kg body weight
doses of alc. SC extract showed significant decrease in FBG levels which was better
than metformin (group XV), standard anti-diabetic agent (Table 1).
B- To evaluate the anti-hyperglycemic effect of aqueous and alcoholic
extracts of Pterocarpus marsupium at different doses.
Experiments were conducted to evaluate anti-hyperglycemic effect of aqueous
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
79
and alcoholic extracts of PM bark. Rats were divided into 15 groups according to the
experimental design. The rats were fed with 3 different doses of aq. PM extract 100,
200 and 400 mg/kg body weight/day and 3 different doses of alc. PM extract 150, 300
and 500 mg/kg body weight/day. An intraperitonial dose (150 mg/kg body weight) of
alloxan increased FBG levels in groups VIII-XV after 4-5 days of injection. FBG
levels were monitored on day 0 (when rats were confirmed for diabetes) and day 30
(end of experiments). In diabetic control group (VIII), higher FBG level (>270 mg/dl)
was maintained throughout the period of study. On the other hand the oral dose of aq.
extract of PM resulted in significant decrease in FBG levels in diabetic rats. The doses
100, 200 and 400 mg/kg body weight/day resulted in decrease in FBG levels from
403.1±16.2 to 335.6±7.7 mg/dl, 373.4±13.6 to 175.6±4.7 mg/dl and 388.3±16.1 to
170.5±5.2 mg/dl respectively, in 30 days treatment (Table 2). Treatment of diabetic
rats with different doses of alc. PM bark extracts viz. 150, 300 and 500 mg/kg body
weight/day decreased the FBG levels from 398.5±13.8 to 350.8±6.1 mg/dl,
355.5±10.8 to 300.3±6.1 mg/dl and 378.9±13.8 to 310.3±7.3 mg/dl, respectively, in
30 days treatment (Table 2). Normal rats treated with different doses of aq. and alc.
extract of PM bark did not show any significant changes in FBG levels as compared
to normal control (group I). The 200 and 400 mg/kg body weight doses of aq. PM
extract showed significant decrease in FBG levels which was better than metformin
(group XV), standard anti-diabetic agent (Table 2).
C- Duration dependent effect of alcoholic extract of SC seed and aqueous
extract of PM bark on fasting blood glucose levels.
The rats were divided into 7 groups according to experimental design. FBG
levels were monitored at day 0 (when rats were confirmed for diabetes), 15th and day
30th. The FBG levels of normal rats groups (group I-III) remained unchanged during
experimental period. Treatment of diabetic rats with alcoholic SC extract (group V)
showed decrease in FBG levels from 427.6±18.8 to 297.6±11.5 and 100.0±10.1 mg/dl
on the 15th and 30th day, respectively. Diabetic rats treated with aqueous PM extract
(group VI) showed decrease in FBG levels from 373.4±13.6 to 223.5±12.4 and
175.6±4.7 mg/dl on the 15th and 30th day, respectively. The anti-hyperglycemic effect
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
80
shown by alc. SC seed extract and aq. PM bark extract was better than metformin
(group VII) (Table 3).
D- Effect of Syzygium cumini and Pterocarpus marsupium extracts (aq. and
alc.) on body weight.
The change in body weight in the rats during the experimental period was
observed. For each plant extract, the rats were divided into 7 groups. The rats in all
the 7 groups were having almost similar body weight. Gain in body weight was
monitored on the 0 day (rats were confirmed for diabetes), and 30th day. Normal rats
treated with aqueous and alcoholic extracts of SC showed 17.9% and 20.4% increase
(Table 4), whereas those treated with aqueous and alcoholic extracts of PM showed
18.4% and 16.3% increase (Table 5) in body weight respectively. Diabetic (group II)
rats showed 10.4% decrease in body weight on the 30th day (table 4 and 5). Diabetic
rats treated with aqueous and alcoholic extracts of SC showed a total 14.2% and
17.6% increase in body weight respectively at the end of study (Table 4), whereas
those treated with aqueous and alcoholic extracts PM showed a total 15.8% and
13.2% increase in body weight, respectively (Table 5). Diabetic rats treated with anti-
hyperglycemic drug, metformin (group VII) showed 16.5% increase in body weight
on the 30th day.
E- Evaluation of toxic effects of alcoholic extract of Syzygium cumini and
aqueous extract of Pterocarpus marsupium.
Experiments were conducted to establish non-toxic nature of alcoholic SC
seeds extract and aqueous PM bark powder extract doses chosen for the study. Rats
were divided into 5 groups according to the experimental design. The rats were fed
with the dose of alcoholic extract of SC seeds and aqueous extract of PM bark via oral
route for 60 days. The minimum dose of SC extract was 100 mg/kg body weight/day
and maximum dose chosen was 500 mg/kg body weight/day. The minimum dose of
PM extract was 200 mg/kg body weight/day and maximum dose chosen was 1 g/kg
body weight/day. There was no morbidity and the rats of all the groups showed
normal growth as observed by similar gain in body weight (~32-37%) at the end of
experiment, in groups II to V, comparable to that of group I. Effect of these plants
extracts at two doses were observed on liver and kidney function. No significant
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
81
deviations from control values were observed in serum urea and serum creatinine
values in all the groups treated with SC (seeds) and PM (bark) extracts. No significant
changes from normal control rats were observed in serum transaminases (ALT and
AST) and alkaline phosphatase levels (Table 6).
F- To evaluate anti-oxidative effect of SC and PM extracts (aq. and alc.) in
alloxan induced diabetic rats.
Experiments were conducted to evaluate the effect of aqueous and alcoholic
extracts of SC (seeds) and PM (bark) on antioxidant (SOD, catalase and GST)
activities, reduced glutathione content and malondialdehyde levels in heart, liver and
kidney issues. Rats were divided into eight groups according to the experimental
design. The rats were fed with aqueous and alcoholic extracts of SC and PM at doses
of 3g and 100 mg/kg body weight/day, and 200 and 300 mg/kg body weight/day
respectively for 30 days.
Cardiac MDA levels
MDA levels were significantly increased from 39.44±1.68 to 105.3±10.93
(p<0.001) in cardiac tissue of diabetic rats. However, the treatment of SC and PM
extracts did not show any significant change in normal rats. Treatment of diabetic rats
with aq. and alc. SC seed extracts resulted in significant decrease (p<0.001) in
elevated MDA levels from 105.30±10.93 to 72.67±7.13 nM/g tissue, and from
105.30±10.93 to 51.08±5.99 nM/g tissue, respectively. Similarly, treatment of
diabetic rats with aq. extract of PM bark resulted in decrease in MDA levels from
105.30±10.93 to 82.87±7.81 nM/g tissue, whereas treatment with alc. PM bark extract
showed significant decrease (p<0.001) in MDA levels from 105.30±10.93 to
65.15±6.63 nM/g tissue. However, there was no significant change observed in
normal rats treated with SC and PM. The beneficial effects of SC and PM on MDA
levels were comparable to metformin (a standard anti-diabetic agent) and vitamin C
(Table 7, 8).
Cardiac GSH Content
GSH content was significantly decreased from 0.93±0.06 to 0.49±0.08 µg/mg
protein (p<0.001) in cardiac tissue of diabetic rats when compared with normal rats.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
82
Treatment of diabetic rats with aq. and alc. SC seed extracts resulted in significant
increase (p<0.001) in GSH content from 0.49±0.08 to 0.77±0.07 µg/mg protein, and
from 0.49±0.08 to 1.10±0.11 µg/mg protein, respectively. Similarly, treatment of
diabetic rats with aq. extract of PM bark resulted in increase in GSH content from
0.49±0.08 to 0.71±0.09 µg/mg protein, whereas treatment with alc. PM bark extract
showed significant increase (p<0.001) in GSH content from 0.49±0.08 to 0.89±0.15
µg/mg protein. However, there was no significant change observed in normal rats
treated with SC and PM. The beneficial effects of SC and PM on GSH content were
comparable to metformin (a standard anti-diabetic agent) and vitamin C (Table 9, 10).
Activities of cardiac antioxidant enzymes
Cardiac antioxidant enzyme activities are shown in fig. 1-6. The activities of
antioxidants (SOD, Catalase and GST) were significantly decreased (p<0.001,
p<0.001 and p<0.001) in cardiac tissue of diabetic rats (group IV) when compared
with normal rats (group I). The diabetic rats that received aq. and alc. extracts of SC
showed a significant (p < 0.001) increase from 8.52±1.82 (diabetic control) to
19.55±1.33 and 23.15±1.88 U/mg protein, respectively, in SOD activity (fig. 1).
Treatment of diabetic rats with aq. and alc. PM bark extract increased the SOD
activity from 8.52±1.82 (diabetic control) to 15.67±1.34 and 20.12±1.23 U/mg protein
(fig. 2). The diabetic rats treated with aq. and alc. SC extracts showed a significant (p
< 0.001, group V and VI) reversal of decreased catalase activity from 74.09±5.5
(diabetic control) to 100.7±4.8 and 119.54±5.5 U/mg protein respectively (fig. 3).
Treatment of diabetic rats with aq. and alc. PM bark extract increased the catalase
activity from 74.09±5.5 (diabetic control) to 101.23±5.3 and 118.93±5.1 U/mg protein
(fig. 4). The GST activity in diabetic rats was significantly (p<0.001) decreased to
1.27±0.09 from 2.39±0.15 U/mg protein observed in normal rats. The treatment of
diabetic rats with aq. and alc. SC extracts could result in significant improvement in
decreased GST activity, as it was increased from 1.27±0.09 to 1.59±0.12 and
2.25±0.07 U/mg protein when treated aq. and alc. SC extracts, respectively (fig. 5).
Treatment of diabetic rats with aq. and alc. PM bark extract increased the GST
activity from 1.27±0.09 (diabetic control) to 1.61±0.1 and 1.92±0.08 U/mg protein
(fig. 6). Normal rats treated with aq. and alc. extracts of SC and PM did not show any
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
83
significant effect on antioxidant enzyme activities when compared to normal control
(group I). On the other hand administration of metformin and vitamin C to diabetic
rats showed significant (p<0.001) increase in antioxidant activities (SOD, catalase and
GST) as compared with diabetic control rats.
Hepatic MDA levels
MDA levels were significantly increased from 194.30±9.96 to 418.07±27.48
(p<0.001) in hepatic tissue of diabetic rats. However the treatment of SC and PM
extracts did not show any significant change in normal rats. Treatment of diabetic rats
with aq. and alc. SC seed extracts resulted in significant decrease (p<0.001) in
elevated MDA levels from 418.07±27.48 to 299.66±19.50 nM/g tissue, and from
418.07±27.48 to 227.59±25.31 nM/g tissue, respectively. Similarly, treatment of
diabetic rats with aq. extract of PM bark resulted in decrease in MDA levels from
418.07±27.48 to 335.76±28.22 nM/g tissue, whereas treatment with alc. PM bark
extract showed significant decrease (p<0.001) in MDA levels from 418.07±27.48 to
259.87±18.49 nM/g tissue. However, there was no significant change observed in
normal rats treated with SC and PM. The beneficial effects of SC and PM on MDA
levels were comparable to metformin (a standard anti-diabetic agent) and vitamin C
(Table 7, 8).
Hepatic GSH Content
GSH content was significantly decreased from 1.05±0.06 to 0.64±0.03 µg/mg
protein (p<0.001) in hepatic tissue of diabetic rats when compared with normal rats.
Treatment of diabetic rats with aq. and alc. SC seed extracts resulted in significant
increase (p<0.001) in GSH content from 0.64±0.03 to 0.79±0.05 µg/mg protein, and
from 0.64±0.03 to 1.03±0.09 µg/mg protein, respectively. Similarly, treatment of
diabetic rats with aq. extract of PM bark resulted in increase in GSH content from
0.64±0.03 to 0.83±0.08 µg/mg protein, whereas treatment with alc. PM bark extract
showed significant increase (p<0.001) in GSH content from 0.64±0.03 to 0.97±0.04
µg/mg protein. However, there was no significant change observed in normal rats
treated with SC and PM. The beneficial effects of SC and PM on GSH content were
comparable to metformin (a standard anti-diabetic agent) and vitamin C (Table 9, 10).
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
84
Activities of hepatic antioxidant enzymes
Hepatic antioxidant enzyme activities are shown in fig. 1-6. The activities of
antioxidants (SOD, Catalase and GST) were significantly decreased (p<0.001,
p<0.001 and p<0.001) in hepatic tissue of diabetic rats (group IV) when compared
with normal rats (group I). The diabetic rats that received aq. and alc. extracts of SC
showed a significant (p < 0.001) increase from 23.13±1.47 (diabetic control) to
39.30±1.55 and 45.90±1.43 U/mg protein, respectively, in SOD activity (fig. 1).
Treatment of diabetic rats with aq. and alc. PM bark extract increased the SOD
activity from 23.13±1.47 (diabetic control) to 30.89±1.22 and 38.99±1.47 U/mg
protein (fig. 2). The diabetic rats treated with aq. and alc. SC extracts showed a
significant (p < 0.001, group V and VI) reversal of decreased catalase activity from
41.99±1.5 (diabetic control) to 62.61±3.8 and 67.25±2.6 U/mg protein respectively
(fig. 3). Treatment of diabetic rats with aq. and alc. PM bark extract increased the
catalase activity from 41.99±1.5 (diabetic control) to 55.64±3.3 and 65.25±3.1 U/mg
protein (fig. 4). The GST activity in diabetic rats was significantly (p<0.001)
decreased to 3.44±0.19 from 6.6±0.35 U/mg protein observed in normal rats. The
treatment of diabetic rats with aq. and alc. SC extracts could result in significant
improvement in decreased GST activity, as it was increased from 3.44±0.19 to
4.9±0.32 and 6.29±0.47 U/mg protein when treated aq. and alc. SC extracts,
respectively (fig. 5). Treatment of diabetic rats with aq. and alc. PM bark extract
increased the GST activity from 3.44±0.19 (diabetic control) to 4.37±0.21 and
4.81±0.58 U/mg protein (fig. 6). Normal rats treated with aq. and alc. extracts of SC
and PM did not show any significant effect on antioxidant enzyme activities when
compared to normal control (group I). On the other hand administration of metformin
and vitamin C to diabetic rats showed significant (p<0.001) increase in antioxidant
activities (SOD, catalase and GST) as compared with diabetic control rats.
Renal MDA levels
MDA levels were significantly increased from 35.81±6.87 to 80.32±7.63
(p<0.001) in renal tissue of diabetic rats. However the treatment of SC and PM
extracts did not show any significant change in normal rats. Treatment of diabetic rats
with aq. and alc. SC seed extracts resulted in significant decrease (p<0.001) in
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
85
elevated MDA levels from 80.32±7.63 to 60.05±4.12 nM/g tissue, and from
80.32±7.63 to 56.71±5.49 nM/g tissue, respectively. Similarly, treatment of diabetic
rats with aq. extract of PM bark resulted in decrease in MDA levels from 80.32±7.63
to 66.83±4.11 nM/g tissue, whereas treatment with alc. PM bark extract showed
significant decrease (p<0.001) in MDA levels from 80.32±7.63 to 58.87±5.23 nM/g
tissue. However, there was no significant change observed in normal rats treated with
SC and PM. The beneficial effects of SC and PM on MDA levels were comparable to
metformin (a standard anti-diabetic agent) and vitamin C (Table 7, 8).
Renal GSH Content
GSH content was significantly decreased from 0.79±0.05 to 0.46±0.04 µg/mg
protein (p<0.001) in renal tissue of diabetic rats when compared with normal rats.
Treatment of diabetic rats with aq. and alc. SC seed extracts resulted in significant
increase (p<0.001) in GSH content from 0.46±0.04 to 0.57±0.02 µg/mg protein, and
from 0.46±0.04 to 0.78±0.07 µg/mg protein, respectively. Similarly, treatment of
diabetic rats with aq. extract of PM bark resulted in increase in GSH content from
0.46±0.04 to 0.55±0.04 µg/mg protein, whereas treatment with alc. PM bark extract
showed significant increase (p<0.001) in GSH content from 0.46±0.04 to 0.76±0.07
µg/mg protein. However, there was no significant change observed in normal rats
treated with SC and PM. The beneficial effects of SC and PM on GSH content were
comparable to metformin (a standard anti-diabetic agent) and vitamin C (Table 9, 10).
Activities of renal antioxidant enzymes
Renal antioxidant enzyme activities are shown in fig. 1-6. The activities of
antioxidants (SOD, Catalase and GST) were significantly decreased (p<0.001,
p<0.001 and p<0.001) in renal tissue of diabetic rats (group IV) when compared with
normal rats (group I). The diabetic rats that received aq. and alc. extracts of SC
showed a significant (p < 0.001) increase from 8.51±1.16 (diabetic control) to
19.6±1.44 and 23.4±1.76 U/mg protein, respectively, in SOD activity (fig. 1).
Treatment of diabetic rats with aq. and alc. PM bark extract increased the SOD
activity from 8.51±1.16 (diabetic control) to 16.57±1.66 and 19.66±1.23 U/mg protein
(fig. 2). The diabetic rats treated with aq. and alc. SC extracts showed a significant (p
< 0.001, group V and VI) reversal of decreased catalase activity from 45.76±1.91
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
86
(diabetic control) to 50.98±1.2 and 65.79±3.01 U/mg protein respectively (fig. 3).
Treatment of diabetic rats with aq. and alc. PM bark extract increased the catalase
activity from 45.76±1.91 (diabetic control) to 56.77±2.33 and 57.85±2.31 U/mg
protein (fig. 4). The GST activity in diabetic rats was significantly (p<0.001)
decreased to 9.21±0.77 from 18.44±0.85 U/mg protein observed in normal rats. The
treatment of diabetic rats with aq. and alc. SC extracts could result in significant
improvement in decreased GST activity, as it was increased from 9.21±0.77 to
14.76±0.69 and 18.14±0.73 U/mg protein when treated aq. and alc. SC extracts,
respectively (fig. 5). Treatment of diabetic rats with aq. and alc. PM bark extract
increased the GST activity from 9.21±0.77 (diabetic control) to 15.11±0.87 and
15.89±0.88 U/mg protein (fig. 6). Normal rats treated with aq. and alc. extracts of SC
and PM did not show any significant effect on antioxidant enzyme activities when
compared to normal control (group I). On the other hand administration of metformin
and vitamin C to diabetic rats showed significant (p<0.001) increase in antioxidant
activities (SOD, catalase and GST) as compared with diabetic control rats.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
87
Table 1 Anti-hyperglycemic effect of aqueous and alcoholic extracts of SC
seed at different doses
Fasting blood glucose levels (mg/dl) Groups Treatments
Day 0 Day 30
I Normal control 98.8±3.7 100.6±6.1
II Normal+Aq SC (1.5g/kg b.w.) 81.2±4.5 93.2±3.8
III Normal+Aq SC (3g/kg b.w.) 83.1±4.6 95.2±3.9
IV Normal+Aq SC (5g/kg b.w.) 87.2±4.2 94.7±4.2
V Normal+Alc SC (50mg/kg b.w.) 96.2±4.1 92.2±3.1
VI Normal+Alc SC (100mg/kg b.w.) 92.8±3.8 98.4±3.5
VII Normal+Alc SC (200mg/kg b.w.) 97.2±4.5 99.8±3.8
VIII Diabetic control 453.6±22.9* 437.1±19.7*
IX Diabetic+Aq SC (1.5g/kg b.w.) 445.9±18.7* 317.1±14.4*
X Diabetic+Aq SC (3g/kg b.w.) 460.4±23.6* 160.6±6.7*
XI Diabetic+Aq SC (5g/kg b.w.) 476.8±21.9* 158.1±4.9*
XII Diabetic+Alc SC (50mg/kg b.w.) 455.9±12.9* 300.8±18.3*
XIII Diabetic+Alc SC (100mg/kg b.w.) 427.6±18.8* 100.0±10.1*
XIV Diabetic+Alc SC (200mg/kg b.w.) 437.7±19.2* 103.8±9.8*
XV Diabetic+Metformin (100mg/kgb.w.)
389.9±4.6* 230.6±3.2*
SC= Syzygium cumini; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group II - VII were compared to group I; group VIII was compared to group I; group IX - XV were compared to group VIII.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
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Table 2 Anti-hyperglycemic effect of aqueous and alcoholic extracts of PM
bark at different doses
Fasting blood glucose levels (mg/dl) Groups Treatments
Day 0 Day 30
I Normal control 98.8±3.7 100.6±6.1
II Normal+Aq PM (100mg/kg b.w.) 100.3±2.5 102.1±6.2
III Normal+Aq PM (200mg/kg b.w.) 110.3±3.6 112.2±5.3
IV Normal+Aq PM (400mg/kg b.w.) 115.8±3.7 114.9±5.9
V Normal+Alc PM (150mg/kg b.w.) 99.1±2.6 99.8±3.5
VI Normal+Alc PM (300mg/kg b.w.) 109.5±3.7 106.3±3.9
VII Normal+Alc PM (500mg/kg b.w.) 105.9±2.5 106.7±4.7
VIII Diabetic control 453.6±22.9* 437.1±19.7*
IX Diabetic+Aq PM (100mg/kg b.w.) 403.1±16.2* 335.6±7.7*
X Diabetic+Aq PM (200mg/kg b.w.) 373.4±13.6* 175.6±4.7*
XI Diabetic+Aq PM (400mg/kg b.w.) 388.3±16.1* 170.5±5.2*
XII Diabetic+Alc PM (150mg/kg b.w.) 398.5±13.8* 350.8±6.1*
XIII Diabetic+Alc PM (300mg/kg b.w.) 355.5±10.8* 300.3±6.1*
XIV Diabetic+Alc PM (500mg/kg b.w.) 378.9±13.8* 310.3±7.3*
XV Diabetic+Metformin (100mg/kg b.w.)
389.9±4.6* 230.6±3.2*
PM= Pterocarpus marsupium; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group II - VII were compared to group I; group VIII was compared to group I; group IX - XV were compared to group VIII.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
89
Table 3 Duration dependent effect of alcoholic extract of SC seed and aqueous extract of
PM bark on fasting blood glucose levels
Fasting blood glucose levels (mg/dl) Groups Treatments
Day 0 Day 15 Day 30
I Normal control 98.8±3.7 99.8±3.9 100.6±6.1
II Normal+SC (100mg/kg b.w.) 96.2±4.1 95.2±5.6 92.2±3.1
III Normal+PM (200mg/kg b.w.) 100.3±2.5 98.3±4.4 102.1±6.2
IV Diabetic control 453.6±22.9* 445.7±18.9* 437.1±19.7*
V Diabetic+SC (100mg/kg b.w.) 427.6±18.8* 297.6±11.5* 100.0±10.1*
VI Diabetic+PM (200mg/kg b.w.) 373.4±13.6* 223.5±12.4* 175.6±4.7*
VII Diabetic + Metformin (100mg/kg b.w.)
389.9±4.6* 318.3±7.3* 230.6±3.2*
SC= Syzygium cumini; PM= Pterocarpus marsupium; *p<0.001. All values are expressed as mean ± SD. Group II and III were compared to group I; group IV was compared to group I; group V-VII were compared to group IV.
Table 4 Effect of aqueous and alcoholic extracts of SC seed on body weight
Body weight (g) Groups Treatments
Day 0 Day 30 % Gain
I Normal control 98.4±3.1 118.5±2.5 20.4
II Diabetic 95.3±2.3 85.4±3.8* -10.4
III Normal+Aq SC (3 g/kg b.w.) 95.8±2.8 112.9±4.6 17.9
IV Normal+Alc SC (100mg/kg b.w.) 120.4±5.8 144.9±6.5 20.4
V Diabetic+Aq SC (3 g/kg b.w.) 85.2±2.1 97.3±3.5* 14.2
VI Diabetic+Alc SC (100mg/kg b.w.) 82.9±4.2 97.5±4.5* 17.6
VII Diabetic+Metformin (100mg/kg b.w.) 124.4±6.5 144.9±5.4* 16.5
SC= Syzygium cumini; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group III and IV were compared to group I; group II was compared to group I; group V-VII were compared to group II.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
90
Table 5 Effect of aqueous and alcoholic extracts of PM bark on body weight
Body weight (g) Groups Treatments
Day 0 Day 30 % Gain
I Normal control 98.4±3.1 118.5±2.5 20.4
II Diabetic 95.3±2.3 85.4±3.8* -10.4
III Normal+Aq PM (200mg/kg b.w.) 94.5±4.4 111.9±5.5 18.4
IV Normal+Alc PM (300mg/kg b.w.) 115.7±6.7 134.6±5.6 16.3
V Diabetic+Aq PM (200mg/kg b.w.) 88.5±4.2 102.5±5.2* 15.8
VI Diabetic+Alc PM (300mg/kg b.w.) 108.3±5.8 122.6±6.9* 13.2
VII Diabetic +Metformin (100mg/kg b.w.)
124.4±6.5 144.9±5.4* 16.5
PM= Pterocarpus marsupium; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group III and IV were compared to group I; group II was compared to group I; group V-VII were compared to group II.
Table 6 Evaluation of toxic effects of SC seed (alcoholic extract) and PM bark (aqueous
Aq=Aqueous; Alc=alcoholic; SC= Syzygium cumini; PM= Pterocarpus marsupium; b.w.=body weight; ALT=Alanine aminotransferase; AST= aspartate aminotransferase; AP= Alkaline phosphatase. All values are expressed as mean ± SD
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
91
Table 7 Effect of aqueous and alcoholic extracts of SC on MDA levels (nM/g tissue) in
alloxan induced diabetic rats
Gps Treatments Tissues
I II III IV V VI VII VIII
Normal control Normal+Aq SC treated Normal+Alc SC treated Diabetic control Diabetic+Aq SC treated Diabetic+Alc SC treatedDiabetic +Metformin Diabetic+Vitamin C
All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001.
Table 8 The effect of aqueous and alcoholic extracts of PM on MDA (nM/g tissue)
content in alloxan induced diabetic rats
Gps Treatments Tissues
I II III IV V VI VII VIII
Normal control Normal+Aq PM treated Normal+Alc PM treated Diabetic control Diabetic+Aq PM treated Diabetic+Alc PM treated Diabetic +Metformin Diabetic+Vitamin C
All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001, **p<0.01
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
92
Table 9 The effect of aqueous and alcoholic extracts of SC on GSH (µg/mg protein)
content in alloxan induced diabetic rats
Gps Treatments Tissues
I II III IV V VI VII VIII
Normal control Normal+Aq SC treated Normal+Alc SC treated Diabetic control Diabetic+Aq SC treated Diabetic+Alc SC treated Diabetic +Metformin Diabetic+Vitamin C
All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001
Table 10 The effect of aqueous and alcoholic extracts of PM on GSH (µg/mg protein)
content in alloxan induced diabetic rats
Gps Treatments Tissues
I II III IV V VI VII VIII
Normal control Normal+Aq PM treated Normal+Alc PM treated Diabetic control Diabetic+Aq PM treated Diabetic+Alc PM treated Diabetic +Metformin Diabetic+Vitamin C
All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001, **p<0.01
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
93
Figure 1: The effect of aqueous and alcoholic extracts of SC on SOD activity (U/mg protein) in alloxan induced diabetic rats.
Figure 2: The effect of aqueous and alcoholic extracts of PM on SOD activity (U/mg protein) in alloxan induced diabetic rats.
SOD=Superoxide dismutase; 1U of SOD= 50% inhibition of auto-oxidation of
epinephrine/min; Normal treated and diabetic rats were compared with normal rats;
Diabetic treated rats were compared with diabetic rats.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
94
Figure 3: The effect of aqueous and alcoholic extracts of SC on Catalase activity (µM H2O2 decomposed/min/mg protein) in alloxan induced diabetic rats.
Figure 4: The effect of aqueous and alcoholic extracts of PM on catalase activity (µM H2O2 decomposed/min/mg protein) in alloxan induced diabetic rats.
1U of Catalase= µmoles H2O2 decomposed/min; Normal treated and diabetic
rats were compared with normal rats; Diabetic treated rats were compared with
diabetic rats.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
95
Figure 5: The effect of aqueous and alcoholic extracts of SC on GST (U/mg protein) activity in alloxan induced diabetic rats.
GST= Glutathione-s-transferase; 1 U of GST= µM GSH-CDNB complex formed/min; Normal treated and diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
96
DISCUSSION
Alloxan and streptozotocin are the most prominent diabetogenic chemicals in
diabetes research. In 1838, Wöhler and Liebig (Wöhler and Liebig, 1838) synthesised
a pyrimidine derivative, which was later called alloxan (Lenzen et al., 1996). In 1943,
alloxan became of interest in diabetes research when it was reported that it could
induce diabetes in animals (Dunn and McLetchie, 1943) as a result of the specific
necrosis of the pancreatic beta cells (Peschke et al., 2000). The resulting insulinopenia
causes a state of experimental diabetes mellitus called ‘alloxan diabetes’ (McLetchie,
1982).
Alloxan has two distinct pathological effects: it selectively inhibits glucose-
induced insulin secretion through specific inhibition of glucokinase, the glucose
sensor of the beta cell, and it causes a state of insulin-dependent diabetes through its
ability to induce ROS formation, resulting in the selective necrosis of beta cells.
These two effects can be assigned to the specific chemical properties of alloxan, the
common denominator being selective cellular uptake and accumulation of alloxan by
the beta cell.
Alloxan is a very unstable chemical compound with a molecular shape
resembling glucose (Lenzen and Munday, 1991). Both alloxan and glucose are
hydrophilic and do not penetrate the lipid bilayer of the plasma membrane. The
alloxan molecule is structurally so similar to glucose that the GLUT2 glucose
transporter in the beta cell plasma membrane accepts this glucomimetic and transports
it into the cytosol (Gorus et al., 1982). Alloxan does not inhibit the function of the
transporter (Elsner et al., 2002), and can therefore selectively enter beta cells in an
unrestricted manner (Malaisse et al., 2001).
Selective inhibition of glucose-induced insulin secretion is the major
pathophysiological effect of the thiol group reactivity of alloxan (Lenzen et al., 1987).
Alloxan has a central 5-carbonyl group that reacts very avidly with thiol groups.
Glucokinase (hexokinase IV) is the most sensitive thiol enzyme in the beta cell
(Tiedge et al., 2000), with a half maximal inhibitory concentration in the 1–10 µmol/l
range. At higher concentrations, alloxan can inhibit many functionally important
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
97
enzymes, as well as other proteins and cellular functions (Konrad and Kudlow, 2002).
Inhibition of glucokinase reduces glucose oxidation and ATP generation, thereby
suppressing the ATP signal that triggers insulin secretion (Gunnarsson and
Hellerström, 1973). Inhibition of glucokinase is achieved within 1 min of exposure to
alloxan. The inhibition of insulin secretion after exposure to alloxan (Weaver et al.,
1978) is restricted to that induced by glucose and its epimer, mannose, both of which
induce insulin secretion through interaction with glucokinase (Lenzen and Panten,
1988). Insulin biosynthesis is also inhibited by alloxan (Niki et al., 1976), most likely
through the same mechanism.
Thiols such as the tripeptide glutathione (GSH), cysteine and dithiothreitol
protect glucokinase against alloxan inhibition because they reduce alloxan to dialuric
acid, which is not thiol reactive (Lenzen et al., 1988). However, only dithiols such as
dithiothreitol (Lenzen and Mirzaie, 1991) can readily reverse alloxan-induced
glucokinase inhibition. They achieve this by reducing functionally essential cysteine
residues of the glucokinase enzyme, which are oxidised through alloxan action
(Lenzen et al., 1988; Lenzen and Mirzaie, 1991). Likewise, glucose protects against
alloxan-induced inhibition of glucose-induced insulin secretion because its binding to
the sugar-binding site of glucokinase prevents the oxidation of the functionally
essential thiol groups.
Alloxan can generate reactive oxygen species (ROS) in a cyclic reaction with
its reduction product, dialuric acid (Munday, 1988), as depicted in the text box
‘Chemical redox cycling reactions between alloxan and dialuric acid, and protective
actions of cytoprotective enzymes’ (reactions i–ii). In the beta cells the toxic action of
alloxan is initiated by free radicals formed in this redox reaction (Winterbourn et al.,
1989). Autoxidation of dialuric acid generates superoxide radicals (iii–iv) and
hydrogen peroxide (iii–iv), and in the Fenton reaction (v), in the presence of a suitable
metal catalyst (typically iron) (vi), hydroxyl radicals (v–vii). The autoxidation of
dialuric acid involves the intermediate formation of the alloxan radical (i–iv)
(Winterbourn and Muday, 1989).
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
98
The reduction of alloxan to dialuric acid in the cell requires the presence of a
suitable thiol, typically the tripeptide glutathione (GSH) to generate the redox cycling
partner, dialuric acid, and oxidised glutathione (viii) (Brömme et al., 2000). The
triketone structure of alloxan is vitally important for this two-step reaction with
glutathione (Elsner et al., 2008), which generates the alloxan radical as an
intermediate product (ix–x). Other thiols such as cysteine, which are present at lower
concentrations in the cell, dithiols and ascorbic acid are also suitable reducing agents
and may therefore contribute to alloxan reduction (Elsner et al., 2006). Alloxan can
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
99
also generate ROS by reacting with thiol groups on enzymes (Lenzen and Mirzaie,
1992) and albumin (Sakurai and Miura, 1989). During each typical redox cycle a
small amount of ‘Compound 305’, an alloxan–GSH adduct (Brömme et al., 2002), is
formed. The intracellular concentration of Compound 305 increases in a time-
dependent manner, which gradually decreases the amount of reduced GSH available
in the cell for redox cycling, thus producing a lower pro-oxidative ratio between
alloxan and GSH, rather than a higher antioxidative ratio (Munday, 1988).
Paradoxically the thiols cysteine and GSH have long been reported to protect
rats against the development of alloxan diabetes when injected together with alloxan
(Sen and Bhattacharya, 1952). This observation can now be explained in light of the
established molecular mechanism of alloxan action. When concentrations of reducing
agents in the blood stream or in the extracellular space are significantly increased
through injection of a thiol, more alloxan is reduced extracellularly so that less is
available for intracellular accumulation. Normally the capacity for alloxan reduction,
redox cycling and the generation of ROS in the circulation (Sakurai and Miura, 1989)
is not sufficient to prevent the alloxan molecule from reaching and entering the beta
cell.
The major oxidation pathway of dialuric acid, a chain reaction dependent upon
superoxide radicals, is inhibited by superoxide dismutase (SOD; xi). In the presence
of SOD, an autocatalytic process involving the interaction between dialuric acid and
alloxan becomes important (Munday, 1988), while in the presence of a transition
metal, a third oxidation mechanism, dependent upon hydrogen peroxide, has been
identified (Munday, 1988). This latter step is inhibited by the hydrogen peroxide
inactivating enzyme catalase (Munday, 1988) (xii; text box: Chemical redox cycling
reactions between alloxan and dialuric acid, and protective actions of cytoprotective
enzymes). The other hydrogen peroxide inactivating enzyme, glutathione peroxidase,
can principally act in a similar manner. But this enzyme requires GSH, which is
oxidised in this reaction (xiii). When kept in the oxidised form, alloxan does not
generate ROS (Elsner et al., 2006). Thus, alloxan is not cytotoxic in the absence of
thiols such as GSH or when restricted to the extracellular space (Elsner et al., 2006).
Thiols in the plasma membrane, with which alloxan could interact and generate ROS
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
100
in a redox cycle, are apparently not present or not accessible to a sufficient extent to
allow the generation of ROS and damage the cells (Elsner et al., 2006).
Thus, it can be concluded that the pancreatic beta cell toxicity and the resultant
diabetogenicity of alloxan are due to redox cycling and the generation of toxic ROS
within the β cells. Different doses of alloxan have been reported in literature to induce
diabetes in different animal models. 70 mg/kg body weight, i.v. dose and 200 mg/kg
body weight i.p. doses have been reported (Zhou et al., 2009). The most widely used
dose to make rats diabetic was found to be 150 mg/kg/body weight in literature
(Khushk et al., 2010). In present study, 150 mg/kg body weight resulted in
hyperglycemic rats. FBG levels were in the range 250 mg/dl to 450 mg/dl, rats
showing glucose levels >270 mg/dl were included in the present study.
The present study was designed to evaluate beneficial effect of two commonly
used natural products i.e. SC and PM in Indian population, on anti-oxidant status and
anti-hyperglycemic activity in alloxan induced diabetic rats. The anti-hyperglycemic
effect of aqueous and alcoholic extracts of SC was evaluated at 3 different doses. For
aqueous extract the 3 doses were 1.5, 3, 5 g/kg b.w./day and for alcoholic extract the 3
doses were 50, 100, 200 mg/kg b.w./day. Administration of aqueous SC extract at a
dose of 1.5 g/kg b.w./day by oral route resulted in 27.5% decrease in FBG levels in
diabetic rats after 30 days, however 3 g/ kg/b.w./day dose resulted in better
management of FBG, as the decreased levels (by 63.3%) observed were close to
normal value (Table 1). With 5 g/kg b.w./day dose the results were comparable to the
results obtained with 3 g/kg b.w./day dose. These doses (3 and 5 g/kg b.w./day) of SC
extracts have not shown any hypoglycemic effect in normal rats. On the other hand
administration of alcoholic SC extract at a dose of 50 mg/kg b.w./day by oral route
resulted in 31.2% decrease in FBG levels in diabetic rats after 30 days, however 100
mg/ kg/b.w./day dose resulted in better management of FBG, as the decreased levels
(by 77%) observed were close to normal value (Table 1). With 200 mg/kg b.w./day
dose the results were comparable to the results obtained with 100 mg/kg b.w./day
dose. These doses (100 and 200 mg/kg b.w./day) of SC extracts have not shown any
hypoglycemic effect in normal rats. The effect of different doses of SC extract on
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
101
FBG levels has been previously evaluated in various studies (Singh and Gupta, 2007;
Prince et al., 1998).
Similarly, the anti-hyperglycemic effect of aqueous and alcoholic extracts of
PM was evaluated at 3 different doses. For aqueous extract the 3 doses were 100, 200,
400 mg/kg b.w./day and for alcoholic extract the 3 doses were 150, 300, 500 mg/kg
b.w./day. Administration of aqueous PM extract at a dose of 100 mg/kg b.w./day by
oral route resulted in 23.2% decrease in FBG levels in diabetic rats after 30 days,
however 200 mg/ kg/b.w./day dose resulted in better management of FBG, as the
decreased levels (by 59.8%) observed were close to normal value (Table 2). With 400
mg/kg b.w./day dose the results were comparable to the results obtained with 200
mg/kg b.w./day dose. These doses (200 and 400 g/kg b.w./day) of PM extracts have
not shown any hypoglycemic effect in normal rats. On the other hand administration
of alcoholic PM extract at a dose of 150 mg/kg b.w./day by oral route resulted in
19.7% decrease in FBG levels in diabetic rats after 30 days, however 300 mg/
kg/b.w./day dose resulted in 31.3% decrease in FBG levels (Table 2). With 500 mg/kg
b.w./day dose the results were comparable to the results obtained with 300 mg/kg
b.w./day dose. These doses (300 and 500 mg/kg b.w./day) of SC extracts have not
shown any hypoglycemic effect in normal rats. The effect of different doses of PM
extract on FBG levels has been previously evaluated in various studies (Gayathri and
Kannabiran, 2008; Gupta and Gupta, 2009).
Metformin, is a biguanide that affects the intestinal glucose absorption, insulin
secretion and hepatic glucose production to manage the diabetes. In vivo and in vitro
studies have demonstrated that metformin stimulates the insulin-induced component
of glucose uptake into skeletal muscle and adipocytes in both diabetic individuals and
animal models (Klip and Leiter, 1990). In the present study, anti-hyperglycemic
effects resulted due to metformin, used as positive control, were comparable to the
results obtained with these plant extracts (Table 1 and 2). The possible mechanism of
anti-hyperglycemic potential of these plant extract could be due to any of following
reasons viz, increase in the release of insulin from pancreas or an increased uptake of
glucose by the peripheral tissues.
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
102
Duration dependent anti-hyperglycemic effect of alcoholic extract of SC (100
mg/kg b.w./day) and aqueous extract of PM (200 mg/kg b.w./day) was evaluated on
the 15th and 30th day. SC extract showed a significant decrease in FBG, 33.22% and
77.56% on day 15th and 30th, respectively. PM extract showed in 49.85% and 60.60%
decrease in FBG levels on the 15th and 30th day, respectively (table 3). The doses of
SC and PM extracts did not show any hypoglycemic effect in normal rats. The pattern
of duration dependent anti-hyperglycemic effect shown by of SC and PM were
comparable with positive control metformin.
Administration of alloxan is reported to be associated with loss in body weight
(Siddiqui et al., 2005). In spite of the increased food consumption, loss of body
weight may be due to defect in glucose metabolism and excessive breakdown of
protein in tissues is a characteristic of diabetes (Sikarwar and Patil, 2010). In present
study alloxan induced diabetic rats showed a significant decrease in body weight from
118.5±2.5 to 85.4±3.8 g. As shown in table 4 treatment with aqueous (3 g/kg
b.w./day) and alcoholic (100 mg/kg b.w./day) extracts of SC resulted in 14.2% and
17.6% gain in body weight respectively, on 30th day. Similarly aqueous (200 mg/kg
b.w./day) and alcoholic (300mg/kg b.w./day) extracts of PM showed in 15.8% and
13.2% gain in body weight on 30th day (table 5). Treatment with SC and PM extracts
improved body weight of diabetic rats, indicating control over polyphagia and muscle
wasting resulted due to hyperglycemic condition. The restoration of decreased body
weight in diabetic rats after plant extract treatment has been previously reported
(Singh and Gupta, 2007; Prince et al., 1998). Normal rats when treated with these
plant extracts of SC and PM showed gain in body weight which was comparable to
normal control rats. The result obtained with anti-diabetic drug metformin with
respect to gain in body weight was comparable to the effects observed with SC and
PM extracts in diabetic rats.
The present study includes experiments which were conducted to establish the
toxicity of alcoholic SC seed extract and aqueous PM bark extract when administered
for two months. Two doses of these extracts, SC seed (100 and 500 mg/kg b.w./day)
and PM bark extract (200 mg and 1 g/kg b.w./day), were given to normal rats. There
was no morbidity and all the rats showed normal growth (gain in body weight),
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
103
similar to that of normal rats. Effects of these plant extracts were observed on liver
and kidney function. No significant deviation from control values were observed in
serum urea and creatinine values in all the groups. No significant changes from
control rats were observed in serum transaminases ALT and AST and alkaline
phosphatase levels (Table 6). The data suggest that prolong use of these extracts is
safe. This non-toxic nature of plant extract has been reported previously (Gayathri and
Kannabiran, 2008).
Alloxan induced diabetic rats exhibit most of the diabetic complication
mediated by oxidative stress (Ozturia et al., 1996). The mechanism of diabetes
induction due to alloxan involves free radical mediated destruction of pancreatic β-
cells (Tomlinson et al., 1992). Aqueous and alcoholic extracts of SC and PM have
been reported to possess anti-hyperglycemic activity and are non-toxic. In the present
study, anti-peroxidative and anti-oxidative effects of aqueous and alcoholic extracts of
SC seeds and PM bark were evaluated in diabetic rats in order to establish their anti-
oxidative potential.
Oxidative stress plays an important role in development of complications of
diabetes and it is postulated to be associated with increased lipid peroxidation
(Elangovan et al., 2000). Oxidative stress in cells and tissues results from the
increased generation of reactive oxygen species and from decrease in antioxidant
defense potential (Gumieniczek et al., 2002). Elevated generation of free radicals
resulting in the consumption of antioxidant defense components may lead to
disruption of cellular functions and oxidative damage to membranes and may enhance
susceptibility to lipid peroxidation (Baynes, 1991). The present study showed a
significant (p<0.001) increase in MDA levels in heart, liver and kidney tissues of
diabetic rats (Table 7) suggesting that peroxidative injury may be involved in alloxan
induced diabetes and may lead to other secondary complications. Treatment of
diabetic rats with aqueous and alcoholic extracts of SC could significantly lower the
elevated MDA levels by 30.9% and 51.4% in heart, 28.3% and 45.6% in liver, 25.2%
and 29.4% in kidney, respectively (Table 7). These results are in accordance with the
previously reported anti-peroxidative effect of SC extracts (Prince et al., 2003;
Ahmed et al., 2010). Similarly aqueous and alcoholic extracts of PM could lower the
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
104
elevated MDA levels by 21.3% and 38.1% in heart, 19.7% and 37.8% in liver, 16.8%
and 27.7% in kidney tissue, respectively (Table 8). These results are in accordance
with the previous studies showing the anti-peroxidative effect PM extract
(Mohammadi et al., 2009; Maruthupandian and Mohan, 2011; Singh et al., 2012).
Reduced glutathione (GSH) is known to protect the cellular system against the
toxic effects of lipid peroxidation (Nicotera and Orrenius, 1986). GSH functions as
direct free radical scavenger, as a co-substrate for glutathione peroxidase (GPx), as a
cofactor for many other enzymes and forms conjugates in endo and xenobiotic
reactions (Gregus et al., 1996). Several studies support the hypothesis that prolonged
hyperglycemia up-regulate the polyol pathway as well as advanced glycation end
products formation and free radical generation rates. A relative depletion of NADPH
occurs due to aldose reductase (AR) activation in different tissues of diabetic rats.
Role of AR has also been implicated in detoxification of lipid peroxidation products
such as 4-hydroxynonenal (4- HNE) and malondialdehyde (MDA) as GSH-aldehyde
adducts (Srivastava et al., 1998). This led to depleted GSH levels at cellular level. Our
results are also in accordance to these findings as depleted GSH levels and elevated
MDA levels were observed in heart, liver and kidney tissues of diabetic rats. Diabetic
rats treated with aqueous and alcoholic extracts of SC and PM resulted in significant
(p<0.001) increase in GSH content when compared with diabetic controls (Table 9).
Diabetic rats treated with aqueous and alcoholic SC extracts resulted in increase in
GSH content by 57.14% and 124.5 % in heart, 23.4% and 60.9% in liver, 23.9% and
69.6% in kidney, respectively (Table 9). Similarly aqueous and alcoholic extracts of
PM resulted in increase in GSH content by 44.9% and 81.6% in heart, 29.7% and
51.6% in liver, 19.6% and 65.2% in kidney, respectively (Table 10). Thus, the
treatment of SC and PM extracts to the alloxan induced diabetic rats resulted in
attenuation in elevated levels of TBARS in different tissues and also increased the
depleted GSH content. These findings suggest that the administration of SC and PM
extracts to the diabetic rats could overcome the oxidative stress status in different
tissues of diabetics. The significant recovery of GSH content by treatment with SC
extracts indicated their protective effect on antioxidants (Prince et al., 2003; Ahmed et
al., 2010). Previously reported antioxidant activity of SC extracts has been attributed
to anthocyanin/ellagitannin present in the extract (Aqil et al., 2012). Pterocarpus
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
105
marsupium extract has been reported to possess strong in vitro antioxidant activity
and may serve as a potential source of natural antioxidant for treatment of diabetes
(Mohammadi et al., 2009). The ethanol extract of P.marsupium has been shown to
possess significant antidiabetic, antihyperlipidaemic and antioxidant effects in alloxan
induced diabetic rats. (Maruthupandian and Mohan, 2011; Singh et al., 2012).
The initiation and propagation of oxidative stress by overproduction of O2 –
and H2O2 and their conversion to potent toxic oxidants led to cause change in tissue
pathology (Muzykantov, 2001). Therefore, interception and detoxification of O2 – and
H2O2 appear to represent an important therapeutic goal. Superoxide dismutase (SOD),
catalase, and peroxidases constitute a mutually supportive team of defense against
ROS. While SOD lowers the steady-state level of O2¯, catalase and peroxidases do the
same for H2O2.
Reduced activities of SOD and catalase in heart, liver and kidney tissues of
diabetic rats has been observed in the present study (Fig 1, 2, 3 and 4) which may be
due to increased production of reactive oxygen species that can themselves reduce the
activities of these enzymes (Mc-cord et al, 1976). The reduced activities of SOD and
catalase have been reported in poor glycemic control and the inactivation of these
enzymes may be due to glycation of these proteins (Sozmen et al., 2001). The reduced
activities of these enzymes in tissues may lead to number of deleterious effects
(Wohaieb and Godin, 1987). The treatment of diabetic rats with aqueous and
alcoholic extracts of SC resulted in significant (p<0.001) increase in the activities of
anti-oxidant enzymes (SOD, CAT, GST) in heart liver and kidney tissues (figures 1,
3, 5). Similarly treatment of diabetic rats with alcoholic extract of PM resulted in
significant (p<0.001) increase in the activities of anti-oxidant enzymes (SOD, CAT,
GST) in heart liver and kidney tissues, whereas the results obtained with aqueous
extract were not significant (figures 2, 4, 6).
On basis of these findings a possible mechanism to explain the anti-oxidative
protection by SC and PM could be proposed. The diabetics have decreased SOD and
GPx activities due to inactivation by reactive oxygen species or by glycation of
enzymes. The elevated superoxide anions have been reported to inactivate catalase
(Chance et al., 1952). Thus, the altered SOD activity plays an important role on the
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
106
activity of catalase. Reduced activity of GST observed in diabetic state may also be
due to the inactivation caused by ROS. GST works together with glutathione and GPx
in decomposition of H2O2 or other organic hydroperoxides to non-toxic products
(Freeman and Crapo, 1982). A significant increase/recovery in SOD, catalase and
GST activities in SC and PM extract (aq./alc.) treated diabetic subjects may be due to
following reasons: the anti-hyperglycemic effect observed by these extracts resulted
in decreased glycation of these enzyme proteins (less inactivation of enzymes) which
in turn potentiate their reduction capacity by improving their antioxidant activities.
Achievement of near normoglycemic conditions in diabetic rats treated with SC and
PM extracts resulted in decreased free radical/ROS formation in diabetic tissues
which led to regain the GSH levels and reverse the ROS mediated inactivation of
GST, SOD and CAT activities. The significant recovery of anti-oxidant enzyme
activities by treatment with SC extracts indicated their protective effect on oxidative
stress (Prince et al., 2003; Ahmed et al., 2010). Previously reported antioxidant
activity of SC extracts has been attributed to anthocyanin/ellagitannin present in the
extract (Aqil et al., 2012). Pterocarpus marsupium extract has been reported to
possess strong in vitro antioxidant activity and may serve as a potential source of
natural antioxidant for treatment of diabetes (Mohammadi et al., 2009). The ethanol
extract of P.marsupium has been shown to possess significant antidiabetic, anti-
hyperlipidaemic and antioxidant effects in alloxan induced diabetic rats.
(Maruthupandian and Mohan, 2011; Singh et al., 2012).
Metformin (a biguanide derivative) is considered as an anti-hyperglycemic
rather than a hypoglycemic agent which makes the metformin a drug of choice.
Various mechanisms have been proposed to account for anti-hyperglycemic action of
metformin like suppression of basal hepatic glucose production, increased peripheral
glucose up take, and increased non-oxidative glucose metabolism (Cusi and
DeFronzo, 1998). All these lead to oxidative protection in diabetics. In the present
study the anti-hyperglycemic and anti-oxidative effects observed with SC and PM
extracts (aq. and alc.) treatments were comparable or even better than that of
metformin. Long term use of metformin has been reported to be associated with
several toxic effects on renal and hepatic systems (Cusi and DeFronzo, 1998). The
present study demonstrates that prolonged use of SC and PM extracts did not result in
Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts
107
any such toxic effect and also had better or comparable anti-hyperglycemic and anti-
oxidative effects. Thus these findings suggest that developing newer anti-diabetic
agent from these plant extracts may be safe and preferred.
Chapter-II
Anti-diabetic and anti-oxidative potentials of purified Syzygium cumini and
Pterocarpus marsupium extracts
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
108
Diabetes mellitus is a fast growing medical problem which is characterised by
disordered metabolism and abnormally high blood sugar levels resulting from the
body’s inability to produce or properly use insulin. A balance between glucose
production and its utilization is necessary to maintain normal blood glucose levels.
Diabetes is characterized by elevated production and low utilization of glucose
(Taylor and Agius, 1998). A number of changes in several enzymes involved in
glucose metabolism present in the liver and other tissues are known to occur in
diabetes mellitus e.g. activity of hepatic glucokinase, phosphofrucokinase and
pyruvate kinase is markedly decreased and activity of glucose-6-phosphatase is
almost doubled (Cahill et al., 1959; Prince et al., 1997; Sharma et al., 2011). The
chronic hyperglycemia of diabetes is associated with long-term dysfunction and
damage to various organs. Hence, there is a need to search for a medication for
lowering glucose as well as modify the alteration of key enzymes involved in
carbohydrate metabolism.
Hyperglycemia is closely associated with increased production of free radical
species and increased oxidative stress (Matsunami et al., 2010). Persistant
hyperglycemic status in diabetes and increased oxidative stress is associated with
altered glucose and lipid metabolism. Hyperglycemia causes oxidative stress and
tissue damage through 5 major mechanisms: (1) increased flux of glucose and other
sugars through the polyol pathway; (2) increased intracellular formation of AGEs
(advanced glycation end products); (3) increased expression of the receptor for AGEs
and its activating ligands; (4) activation of protein kinase C (PKC) isoforms; and (5)
overactivity of the hexosamine pathway. Oxidative stress plays a pivotal role in the
development of diabetes complications, both microvascular and macroovascular
(Baynes, 1991). Lipid peroxide mediated tissue damage has been observed in the
development of both the types of diabetes. A sophisticated enzymatic and
nonenzymatic antioxidant defense system including catalase (CAT), superoxide
dismutase (SOD) and reduced glutathione (GSH) counteracts and regulates overall
ROS levels to maintain physiological homeostasis. Increased concentration of
TBARS and the simultaneous decline in antioxidative defence mechanisms observed
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
109
in diabetic patients promotes the development of late complications (Karunakaran
and Park, 2013).
The pathogenesis of diabetes and its management by oral hypoglycemic agents
has stimulated great interest in recent years. Despite considerable progress in the
management of diabetes mellitus by synthetic drugs, the search for indigenous natural
anti-diabetic agents is still going on (Kania et al., 2013). Before the development of
modern pharmaceutical treatments, therapeutic capacity was completely dependent on
the use of medicinal herbs for prevention and treatment of diseases (Patel et al.,
2012a). Ethnobotanical information also indicates that more than 800 plants are used
as traditional remedies for treatment of diabetes throughout the world (Patel et al.,
2012b). There is still an unmet need for scientific proof of the antidiabetic activity of
medicinal plants and phytopharmaceuticals with fewer side effects. In view of this,
present study was taken up to explore antidiabetic potential of Syzygium cumini seeds
and Pterocarpus marsupium bark, and also to reduce the risk of late complications
and negative outcomes of diabetes which requires not only to control blood glucose
level but also to control oxidative stress.
Purification of alcoholic SC seed extract The alcoholic extract was subjected to silica gel chromatography and the
adsorbed compounds (mostly phenolics) were batch eluted with 100% methanol. The
eluted fraction was further purified on sephadex LH 20 beads and batch eluted with
different ratios of water and methanol (100% water, 70:30, 30:70,100% methanol).
Four fractions were obtained and tested for anti-hyperglycemic and anti-oxidative
activities. The fraction IV showing best results was further subjected to HPLC
column. Elution profile is given as figure 10. The eluted fraction was characterized as
caffeic acid. The anti-diabetic potential of caffeic acid was evaluated and results are
given below.
Purification of PM extract
The bark powder of PM was extracted with ethyl acetate and subjected to
silica gel beads. The fractions (mostly polyphenols) batch eluted with benzene were
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
110
pooled and lyophilized. The lyophilized material was re-suspended in minimum
volume of ethanol. The ethanol was evaporated and material was suspended in water
and dosed.
EXPERIMENTAL DESIGN
1- To evaluate the effect of sephadex LH 20 purified fractions of alcoholic
extract of SC on FBG levels.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + SC LH 20 purified fraction I (1.1mg/kg b.w./day)
Group III Normal + SC LH 20 purified fraction II (1.1mg/kg b.w./day)
Group IV Normal + SC LH 20 purified fraction III (1.1mg/kg b.w./day)
Group V Normal + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)
Group VI Diabetic control
Group VII Diabetic + SC LH 20 purified fraction I (1.1mg/kg b.w./day)
Group VIII Diabetic + SC LH 20 purified fraction II (1.1mg/kg b.w./day)
Group IX Diabetic + SC LH 20 purified fraction III (1.1mg/kg b.w./day)
Group X Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)
Group XI Diabetic + Metformin (100 mg/kg b.w./day)
2- To evaluate the effect of purified PM extract on FBG levels in diabetic
rats.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + purified PM extract (1mg/kg b.w./day)
Group III Diabetic control
Group IV Diabetic + purified PM extract (1mg/kg b.w./day)
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
111
Group V Diabetic + Metformin (100 mg/kg b.w./day)
3- To evaluate the in vitro anti-oxidant activity of the crude and LH 20
purified extracts of SC by DPPH radical scavenging assay.
I Radical scavenging activity of metformin
II Radical scavenging activity of crude aq. SC extract
III Radical scavenging activity of crude alc. SC extract
IV Radical scavenging activity of SC LH 20 purified fraction I
V Radical scavenging activity of SC LH 20 purified fraction II
VI Radical scavenging activity of SC LH 20 purified fraction III
VII Radical scavenging activity of SC LH 20 purified fraction IV
VIII Radical scavenging activity of BHT
4- To evaluate the in vitro anti-oxidant activity of the crude and purified
extracts of PM by DPPH radical scavenging assay.
I Radical scavenging activity of metformin
II Radical scavenging activity of crude aq. PM extract
III Radical scavenging activity of crude alc. PM extract
IV Radical scavenging activity of purified PM extract
V Radical scavenging activity of BHT
5- To evaluate the anti-oxidative effect of purified SC and PM extracts on
alloxan induced diabetic rats.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Normal + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)
Group III Normal + purified PM extract (1mg/kg b.w./day)
Group IV Diabetic control
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
112
Group V Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)
Group VI Diabetic + purified PM extract (1mg/kg b.w./day)\
Group VII Diabetic + Metformin (100 mg/kg b.w./day)
Group VIII Diabetic + Vitamin C (150 mg/kg b.w./day)
6- To evaluate the effect of SC and PM purified extracts on serum insulin
levels.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Diabetic control
Group III Diabetic + SC LH 20 purified fraction III (1.1mg/kg b.w./day)
Group IV Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)
Group V Diabetic + purified PM extract (1mg/kg b.w./day)
7- To evaluate the effect of purified SC and PM extracts on the activities of
glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate
kinase) in heart, liver and kidney tissues of diabetic rats.
Rats were divided into following groups, each group contains five rats
Group I Normal control
Group II Diabetic control
Group III Diabetic + SC LH 20 purified fraction III (1.1mg/kg b.w./day)
Group IV Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)
Group V Diabetic + purified PM extract (1mg/kg b.w./day)
RESULTS
A- To evaluate the effect of sephadex LH 20 purified fractions of alcoholic
extract of SC on FBG levels.
Experiments were conducted to evaluate anti-hyperglycemic effect of
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
113
sephadex LH 20 purified fractions of alcoholic extract of SC seeds. Rats were divided
into 11 groups according to the experimental design. The rats were fed with the four
fractions purified from sephadex LH 20 at a dose of 1.1mg/kg b.w./day. An
intraperitonial dose (150 mg/kg body weight) of alloxan increased FBG levels in
groups VI-XI after 4-5 days of injection. FBG levels were monitored on day 0 (when
rats were confirmed for diabetes) and day 30 (end of experiments). In diabetic control
group (VI), higher FBG level (>270 mg/dl) was maintained throughout the period of
study. On the other hand the oral dose of SC purified fractions resulted in decrease in
FBG levels in diabetic rats. The decrease in FBG levels on administration of fractions
I, II and III was from 435.4±16.3 to 406.8±15.7 md/dl, 444.2±20.3 to 389.6±12.9
mg/dl and 436.5±20.1 to 352±14.2 mg/dl, respectively. Better results were obtained
with fraction IV, which significantly (p<0.001) decreased the FBG level from
445.2±11.9 to 102.9±4.8 mg/dl (Table 1). Normal rats treated with the four fractions
did not show any significant changes in FBG levels as compared to normal control
(group I). Fraction IV showed significant decrease in FBG levels which was better
than metformin (group XI), standard anti-diabetic agent (Table 1).
B- To evaluate the effect of purified PM extract on FBG levels.
Experiments were conducted to evaluate anti-hyperglycemic effect of purified
extract of PM bark. Rats were divided into 5 groups according to the experimental
design. The rats were fed with the purified fraction of PM at a dose of 1mg/kg
b.w./day. An intraperitonial dose (150 mg/kg body weight) of alloxan increased FBG
levels in groups III-V after 4-5 days of injection. FBG levels were monitored on day 0
(when rats were confirmed for diabetes) and day 30 (end of experiments). In diabetic
control group (III), higher FBG level (>270 mg/dl) was maintained throughout the
period of study. On the other hand the oral dose of PM purified fraction resulted in
decrease in FBG levels in diabetic rats. The FBG level was significantly (p<0.001)
decreased from 413.4±16.8 to 161.7±11.3 mg/dl after treatment (Table 2). Normal rat
treated with the purified fraction of PM did not show any significant change in FBG
level as compared to normal control (group I). PM purified fraction showed
significant decrease in FBG levels which was better than metformin (group XI),
standard anti-diabetic agent (Table 2).
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
114
C- To evaluate the in vitro anti-oxidant activity of the crude and purified
extracts of SC and PM by DPPH radical scavenging assay.
Alcoholic extract of SC seeds exhibited a strong antioxidant property which
was 2-3 fold better than aqueous extract. Better radical scavenging activity shown by
alcoholic SC extract as compared to the aqueous extracts, suggests that the anti-
oxidative component(s) are better extracted in alcoholic extract (fig. 1). All the four
SC LI, LII, LIII, LIV = Syzygium cumini LH 20 column purified fractions I-IV. *p<0.001.All values are expressed as mean ± SD. Group II - V were compared to group I; group VI was compared to group I; group VII - XI were compared to group VI.
Table 2 Effect of purified PM extract on FBG levels in diabetic rats
Groups Treatments Fasting blood glucose levels (mg/dl)
I II III IV V
Normal Normal + PM Pur. Diabetic. Diabetic + PM Pur. Diabetic+Metformin
Day 0 Day 30 98.8±3.7 100.6±6.1 100.2±3.9 103.1±5.9
PM Pur. = Purified Pterocarpus marsupium extract. *p<0.001. All values are expressed as mean ± SD. Group II was compared to group I; group III was compared to group I; group IV and V were compared to group III.
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Figure 1: In vitro anti-oxidant activity of the crude and purified extracts of SC by DPPH radical scavenging assay.
Figure 2: In vitro anti-oxidant activity of the crude and purified extracts of PM
by DPPH radical scavenging assay.
Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts
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Table 3 Effect of purified SC and PM extracts on MDA (nM/g tissue) levels in alloxan
induced diabetic rats
Gps Treatments Tissues
I II III IV V VI VII VIII
Normal Normal + SCLIV Normal + PM Pur. Diabetic Diabetic + SCLIV Diabetic + PM Pur. Diabetic + Metformin Diabetic + Vit C
All values are expressed as mean ± SD; Normal treated and diabetic rats were
compared with normal rats; diabetic treated rats were compared with diabetic rats;
*p<0.001.
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Figure 3: Effect of purified SC and PM extracts on SOD activity (U/mg protein) in alloxan induced diabetic rats.
SOD=Superoxide dismutase; 1U of SOD= 50% inhibition of auto-oxidation of
epinephrine/min; Normal treated and diabetic rats were compared with normal rats;
Diabetic treated rats were compared with diabetic rats.
Figure 4: Effect of purified SC and PM extracts on CAT activity (U/mg protein) in alloxan induced diabetic rats.
1U of Catalase= µmoles H2O2 decomposed/min; Normal treated and diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.
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Figure 5: Effect of purified SC and PM extracts on GST activity (U/mg protein) in alloxan induced diabetic rats.
GST= Glutathione-s-transferase; 1 U of GST= µM GSH-CDNB complex formed/min; Normal treated and diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.
Figure 6: Effect of purified SC and PM extracts on serum insulin levels.
Diabetic rats were compared with normal rats; Diabetic treated rats were compared
with diabetic rats.
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Figure 7: Effect of purified SC and PM extracts on the activity of hexokinase (mU/mg protein) in heart, liver and kidney tissues of diabetic rats.
Diabetic rats were compared with normal rats; Diabetic treated rats were compared
with diabetic rats.
Figure 8: Effect of purified SC and PM extracts on the activity of phosphofructokinase (mU/mg protein) in heart, liver and kidney tissues of
diabetic rats.
Diabetic rats were compared with normal rats; Diabetic treated rats were compared
with diabetic rats.
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Figure 9: Effect of purified SC and PM extracts on the activity of pyruvate kinase (mU/mg protein) in heart, liver and kidney tissues of diabetic rats.
N=Normal, D=Diabetic, DSCLIII=Diabetic+SC LH 20 purified fraction III treated, DSCLIV=Diabetic+SC LH 20 purified fraction IV treated, DPMP=Diabetic+PM purified extract treated. Diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.