PROJECT

ON
EFFECT OF ANTIOXIDANT THERAPY ON DIABETES MELLITUS

AT

DR.APJ ABDUL KALAM TECHNICAL UNIVERSITY

Submitted For the Partial Fulfilment of the requirement

For the Degree
Of
Bachelor Of Pharmacy

Submitted By Submitted To

Manoj Singh DR.Gangeshwar

(Department Of Pharmacy)

FUTURE INSTITUTE OF PHARMACY (850), BAREILLY,(U.P)

1
 CERTIFICATE

This is to certify that the project entitled, “EFFECT OF ANTIOXIDANT

THERAPY ON DIABETES MELLITUS” submitted to the Future Institute
Of Pharmacy, 850, Bareilly, U.P. in fulfillment of requirements for the
Degree of B.Pharm embodies the original project work carried out by
Ravi Shankar under the guidance and supervision of Dr. Gangeshwar

Prof. Dr. Rahul Shukla

Director Of Future Institute Of Pharmacy

2
 CERTIFICATE

This is to certify that the project entitled, “EFFECT OF ANTIOXIDANT

THERAPY ON DIABETES MELLITUS” submitted tothe Future Institute
Of Pharmacy, 850, Bareilly, U.P. is a bonafidework carried out by
Ravi Shankar (Roll No. 18850500xx) under the guidance and
supervision during the academic session 2021-2022

GUIDE

Prof. Dr. Gangeshwar

(Associate Professor)
Future Institute Of Pharmacy, Bareilly

3
 DECLERATION

I hereby, declare that this project work, “DIABETES THAT CAN

BE PREVENTED BY HERBAL DRUG “Tinosporacordifolia”
embodies the results of original research work carried out by
me in the Future Institute Of Pharmacy, 850, Bareilly, U.P.
under the supervision of Dr. Gangeshwar. The extent sources
of information wee derived from this existing literature and
have been indicated throughout this thesis at appropriate
places.
I further declare that. I have not submitted this desertion
previously for any degree /diploma to me.

Date: Manoj Singh

4
 ACKNOWLEDGEMENT
“Gratitude makes a sense of our past, brings peace for today
and creates a vision for tomorrow.”
First and Foremost, praises and thanks to the God, the
Almighty, for His showers of blessings through-out thesis to
complete the thesis successfully.
I am extremely grateful to my parents for providing me
resources and sacrifices for education.
I also pay obeisance to my academic supervisor
Dr.Gangeshwar. for valuable advice guidance and patience
given throughout and also for his unfailing faith in me is one of
the major reason for my strength and courage which motivates
me to select this work as per my intellectual pursuits.
I bow my had to Dr. Rahul Shukla, Director of Future Institute
Of Pharmacy Bareilly and also grateful to all my colleagues and
lab mates for providing an enjoyable working hand or a listener.
I would like to express my deep appreciation and gratitude to
the people for helping me complete this thesis.

Date: Manoj Singh

5
 Content
1. Definition
2. EPIDEMIOLOGY
3. ETIOLOGY
4. CLASSIFICATION
5. THERAPY
6. FREE RADICALS
7. THE REACTIONS IN CELL DUE TO FREE
RADICALS
8. Antioxidants
9. Enzymes
10. Recycling of Vit E.
11. Conclusion

DEFINITION
 

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Diabetes mellitus is a group of metabolic disorders characterised by hyperglycemia;
associated with abnormalities in carbohydrate, fat and protein metabolism and
resulting in chronic microvascularmacrovascular and neuropathic complications.

Diabetes mellitus (Madhumeha) has been known since ages and the sweetness of
diabetic urine has been mentioned in Ayurveda by Sushruta.

 Its pharmacotherapy however is just over 80 yrs old.

The word DIABETES (to flow through) has coined by the Greek physician Aeretaeus
in the 1st century AD. In the 17th century, Willis observed that the urine of diabetics
was "Wonderfully Sweet as if imbued with honey or sugar"

The presence of sugar in urine of diabetics was demonstrated by Dobson in 1755. It

is characterized by either a deficiency in insulin, or resistance to insulin. Diabetic
patients also suffer from a wide variety of complications due to their disease, such
as atherosclerosis, retinopathy, poor circulation, and liver and kidney problems.
Diabetes also seems to be accompanied by a shortage of antioxidants and an
increase in free radicals, the end result being oxidative stress. Research is being
done to determine if the incurring oxidative stress is just another complication or if it
may be a cause, in part or in full, of some of the diabetic complications. This paper
will discuss the role of free radicals in the pathology of diabetes and its
complications.

 
Diabetic patients have an increased level of blood glucose, but many patients also
have secondary effects from the diabetes, such as poor circulation, ischemic brain
injury, platelet adhesion and aggregation, excess free radicals, shortage of
antioxidants, heart disease, cataracts and liver and kidney problems. Most of these
are caused, at least in part, by too much glucose. Excess glucose, hyperglycaemia,
can be toxic to cells in several ways, two of which are the formation of Advanced
Glycation End products (AGEp) and free radicals such as O2•-, •OH. Both types of
products can contribute to diabetic complications. Excessive free radicals can come
from several pathways; ischemia, hyperglycemia, increased mitochondria leak,
catecholamine oxidation and leukocytes. Diabetic subjects have been shown to
have increased levels of superoxide and hydrogen peroxide. Changes in antioxidant
enzymes and small antioxidant molecules have also been documented.

EPIDEMIOLOGY
 

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    About 3% of the world population, approximately 100 million people
suffer from diabetes, making this one of the most common non-
communicable diseases4. Type 1 diabetes DM usually develops in
childhood or early childhood, although some latent forms do occur. Type 1
DM accounts for up to 10% of all cases of DM. In northern Europe the
prevalence is approximately 0.3% in those under 30 years of age2. In
United States, about 5% of all diabetic patients have Type 1 DM with an
incidence of 18 per 100000 inhibitants per year.

 
    Type 2 DM is a heterogeneous disorder of glucose metabolism
 Type 2 DM accounts for as much as 90% of all cases of DM, and usually
results from defects in insulin sensitivity and a relative defect in insulin
secretion.
The overall prevalence of type 2 DM in the United States is about 6.6% in
person of age 20 to 74 years.

 Type 2 DM is much commoner than type 1, accounting for over 75% of all
patients with diabetes in population. The vast majority of diabetes patients
have type 2 DM

 The incidence of type 2 DM increases with age and with increase in

obesity.

In general non obese population the prevalence is 1 to 3%. In the more

obese secretion, there is a sharp increase in prevalence with figures of 6 to
8% in the USA increasing to values as high as 30% in Hindu Tamils in
south Africa.
 There are more than 125 million persons diabetes in the world today, and
by 2010 this number is expected to approach 220 million. Diabetes is 5
times more common among Asian immigrants in the UK than in the
indigenous population.

ETIOLOGY
 
    The aetiology of type 1 has been the subject of considerable research.
Genetic factors are important but do not explain fully development of type
8
1. There is a strong immunological component to type 1 and a clear
association with many organ specific auto immune disease.

Circulating islets cell antibodies (ICAs) are present in more than 70% of
type 1 at the time of diagnosis.

    Type 2 DM has a much stronger genetic relationship than type 1.

Identical twins have concordance rate approaching 100%. Obesity and
family history are risk factors for the development of NIDDM.

Type I diabetes is treated with insulin supplementation, while type II can

often be controlled with just diet and exercise.

If these measures are insufficient, there are also a multitude of oral

hypoglycaemic agents available for the treatment of this disease. Insulin is
used only when the previous measures fail.

CLASSIFICATION
 

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    The American Diabetes Association Expert Committee on the diagnosis
and classification of diabetes mellitus recommends the use of Type 1 and
Type 2 DM.

 
Aetiological classification of DM by WHO 1999 as follows

Type 1: (-cell destruction usually leading to absolute insulin deficiency)

(Insulin dependent DM, (IDDM) Juvenile onset DM]

 Autoimmune
 Idiopathic

Type 2: May range from predominantly insulin with relative insulin

deficiency to a predominantly secretory defect with or without insulin
resistance] [Non insulin dependent DM6 (NIDDM) Maturity onset DM].

OTHER SPECIFIC TYPES

 Genetic defects  cell function

 Genetic defects in insulin action
 Diseases of the exocrine pancreas
 Endocrinopathies
 Drug or chemical induced eg. Miotinic acid, glucocorticoids, high dose
thiazides, pentramidine, interferone alpha infections.
 Uncommon forms of immune mediated diabetes
 Other genetic syndromes associated with diabetes
 Gestational diabetes

THERAPY
 
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All patients with diabetes should receive healthy living advice. This includes
advice on appropriate physical activity, and lifestyle modificatin particularly
smoking cessation and healthy eating.

 
The therapy for diabetes can be classified into two major classes.
 Non-pharmacotherapy
o Dietary therapy
o Physical activity

 Pharmcotherapy

o Insulin therapy
o Oral hypoglycaemic agents

 Stimulation of insulin by  cells

eg. Sulphonylureas,  meglitinides

 Inhibitors of hepatic gluconeogenesis5

e.g. biguanides

 Inhibitors of intestinal  glucosidases
 eg. Acarbose, miglitol

 Drugs which reduce insulin resistance

     eg. Thiozolidinediones

FREE RADICALS
 

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    A free radical is an item or molecule with one or more unpaired
electrons or free radicals are chemical species with a single unpaired
electron in an outer orbit. Such chemical states are extremely unstable and
readily react with inorganic or organic chemicals. These oxidants are
generated during the normal metabolic reactions in the body. Small atoms
of reactive oxygen are continually found in the body in the cell membrane
and close to the cell organells. Free radicals initiate autocatalytic reactions;
molecules that react with free radicals are in turn converted into free radials
further propagating the chain of damage. Free radicals damage underlies
chemicals and radiation injury, toxicity from oxygen and other gases
cellular ageing, microbial killing by phagocytic cells, inflammatory cell
damage, tumour destruction by macrophages and other injurious process.
Free radicals and other oxidants may be involved in the pathogenesis of
cancer, DM, cardiovascular and neurological disorders.
REACTIVE OXYGEN SPECIES

 
Symbol  Name 
O2 Singlet O2
O. Superoxide free radical 
OH. Hydroxide free radical 
RO. Alkoxyl free radical 
ROO. Peroxyl free radical 
H2O2 Hydrogen peroxide 
LOOH  Lipid peroxide 

FREE RADICALS GENERATION IN THE BODY

 
    The redox reactions that occur during normal physiological process, the
free radicals are produced. During normal respiration,

e.g. Molecular oxygen is sequentially reduced in mitochondria by the

addition of four electrons to generate water.

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In the process, small amounts of toxic intermediate species are generated.

These includes
 Superoxide radicals O2
 H2O2
 OH.
  Some intracellular oxides (such as Xanthine oxidases) generate
superoxide radicals as a direct consequence of their activity.
Transition metals such as copper and iron also accept or donate free
electrons during certain intracellular reactions and thereby catalyse
free radicals formation as in the Fenton reaction.

 
 Fe2+ + H2O2
 Fe3+ + OH. + OH–
 
 Since most intracellular free iron is in the Fe3+ state it must first be
reduced to the Fe2+ form to participate in the Fenton reaction. The reduction
step is catalysed by superoxide ion and thus iron and
superoxidessynergise to elicit maximal oxidate cell injury.

 
     Nitric oxide (NO) an important chemical mediator normally
synthesised by a variety of cell types that can act as a free radical or can
be converted into highly reactive nitrite species.

     The absorption of radiant energy (eg. UV or X-rays) ionizing

radiation can hydrolyse water into hydroxyl (OH.) and (H.) free radicals.
     The enzymatic metabolism of exogenous chemicals
e.g. CCl4
     Cycloxygenation
     Lipoxygenation
     Lipidperoxidation

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     Neutrophills stimulated by exposure to microbes
     Reperfusion of ischemic organs
 Metabolism of xenobiotics (foreign chemicals) including, alcohol,
cigarette smoke, motor car exhaust

THE REACTIONS IN CELL DUE TO

FREE RADICALS
 
Lipid peroxidationof membranes
Double bonds in membrane polyunsaturated lipids are vulnerable to be attacked by
oxygen derived free radicals. Lipid radicals interaction yield peroxides which are
themselves unstable and reactive and an autocatalytic chain reaction ensures.

 
DNA fragmentation: Free radicals reactions with thymine in nuclear and
mitochondrial DNA produce single strand breaks such DNA damage has been
implicated in both cell killing and the malignant transformation of cells.
14
 
Cross linking of proteins: Free radicals promote sulphahydryl mediated proteins
cross linking resulting in enhanced rates of degradation or loss of enzymatic
activities. Free radicals reactions may also directly cause poly peptide
fragmentation.

HYPERGLYCAEMIA AND FREE RADICALS

Hyperglycaemia can increase the levels of free radicals through protein glycation,
autoxidation glycation, protein kinase and an increase in the polyol pathway.
Autoxidation of glucose is the process by which it enolizes.

 
The process of autoxidation of a monosaccharide, showing how free radicals are produced when
excess glucose is present

    This process entails the reduction of oxygen, producing oxidizing intermediates,

such as Q2#~, #OH and H2O2, and alpha-ketoaldeydes.

These molecules can damage important biomolecules such as DNA, proteins and
lipids. The oxidizing intermediate formed by autoxidation is proposed to be a cause
for some of the structural damage seen in diabetes.

This reaction is often catalyzed by transition metals, and even with the catalyst, the
reaction is very slow.
These ketoaldehyde products may attach to proteins, in a process is called labile
glycation. Protein fragmentation and labile glycation due to glucose autoxidation can
be reduced by the use of a chelating agent, like DETAPAC.

Glucose can also undergo glycation directly, where the glucose molecule covalently
bonds to a protein to form a Schiff base. These molecules can then undergo
rearrangement to form Amadori products. Amadori products can then decompose to
form deoxyglucones, which are considerably more reactive than the sugar they
derived fromGlycation process and subsequent degradation of glycation products
    These more reactive ketoaldeyhdes may go on to react with other proteins to form
Advanced GlycationEndproducts (AGE) or Maillard products.

The Maillard products lead to the "browning" of the protein, the protein also
becomes fluorescent and crosslinked. Glycation is a reversible process. When
glycation follows autoxidation, also called glycoxidation, the products tend to be
more permanent modifications such as protein crosslinking.

15
Haemoglobinglycation is commonly used clinically to monitor the blood sugar level
over several weeks. The amount of haemoglobinglycation can help doctors and
patients monitor the glycaemia control.

 
An increase in the concentration of glucose contributes to an enhanced activity of
the two enzymes used in the polyol pathway, aldose reductase and sorbitol
dehydrogenase. With the increased activity of these two enzymes, the concentration
of both sorbitol and fructose increase.

This increased activity also causes the NADPH: NADP+ ratio to decrease and the
NADH: NAD+ ratio to increase. The change in these ratios can cause changes
throughout various systems in the cell.

The increase in the NADH:NAD+ ratio, also called hyperglycaemic pseudo-hypoxia,

may cause an increase in free radical production which may lead to ischemia. It may
also produce a reduction in glycolysis, which results in reduced pyruvate levels11.
The reduction in the amount of NADPH may cause an inhibition in enzymes which
are NADPH-dependent and lead to a shortage of the NADPH available for the many
pathways it is involved in.

 
Glucose, once phosphorylated to glucose-6-phospate, is metabolized through two
main processes in the cell, glycolysis and the pentose phosphate pathway.
Glycolysis results in the production of pyruvate, which then goes on to react in the
tricarboxylic acid cycle, among others and is also known to scavenge H2O2 and
other hydroperoxides.
The pentose phosphate pathway produces NADPH, which is the primary source of
reducing equivalents for the glutathione reductase system, among many other
oxidizing species.

So not only is glucose a primary energy source, it is also a means of removing toxic
H2O2 and hydroperoxides from cells.

Diabetic Complications and Free Radicals

Hyperglycaemia can produce a wide variety of secondary diabetic complications.
Oxidative stress plays an integral role in the development of complications due to
excess glucose.

16
 

 
 Excessive glucose can cause multiple secondary complications through a variety of pathways,
which are appearing to lead to oxidative stress.

 
Diabetic patients have been shown to have platelets with increased adhesiveness
and aggregation, increased concentrations of thromboxane A2, platelet factor 4 and
|3-thromboglobulin.

Increase in platelet aggregation can cause a variety of effects: vasoconstriction,

anoxia, ROS, atherosclerotic plaques, retinopathy, nephropathy and CV disease. It
shows how hyperglycaemia can be related to oxidative stress and further
complications.

Antioxidants
 
Antioxidants are the substances which prevent oxidation either by inhibiting the
chain propagation step of free radical reaction (oil soluble antioxidants)

e.g. butyrated - hydroxyl toluene or undergoing self oxidation (water soluble

antioxidants) eg. Vitamin-C.

 
These antioxidants act against the free radicals and reduce the damaging properties
of free radicals and act as a free radical scavenging units.

Cells have developed several enzymatic and nonenzymatic systems to inactivate

free radicals.

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The enzymes are:
 Superoxide dismutase (SODs)
 Glutathione peroxidase (GSH)
 Catalase

Nonenzymatic antioxidants
 Endogenous or exogenous antioxidants.

e.g. Vitamin E, A, C and  carotene

Free ionised iron and copper catalyse the formation of reactive oxygen species
these elements are usually segmented by storage aid and/or transport protein.
(e.g. transferin, ferritine, ceruloplasmin).

 
    These antioxidants have been implicated in atherosclerosis (oxidised LDL is more
altherogenic) cancers, neurodegenerative diseases and inflammatory diseases.

Enzymes
Superoxide dismutase (SODs)
    The rate of spontaneous decay in significantly increased by the actions of
superoxide dismutase found in many cell types.

 
Glutathione Peroxidase (GSH)
    GSH also protects against injury by catalysing free radicals breakdown .
 
    The intracellular ratio of oxidised GSSG to reduce to GSH Glutathione is a
reflection of the oxidative state of the cell and an important aspect of the cell's ability
to catabolise free radicals.

Catalase
    Catalases present in peroxisomes direct the degradation of H2O2

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Endogenous and Exogenous antioxidants (Nonenzymatic antioxidants)

    Vitamin E, A, C and  carotene may either block the formation of free radical or

scavenge them once they have formed.
Vitamin E is an antioxidant that resides in the lipid layer of the cell membrane. Its
purpose is to stop the chain reaction begun by lipid radicals. Vitamin C then reacts
with the octocpherol radical, thereby removing the radical from the lipid bilayer and
moving it into the cytosol where it can be dealt with resident enzymes

Recycling of Vit E.
Alpha Lipoic Acid (ALA) is an antioxidant that is currently approved for diabetic
neuropathy in Germany. A 50% reduction in nerve blood flow has been found in
streptozotocin experimental diabetic animals. This reduction reverted to normal after
one month of treatment with alpha-lipoic acid (100 mg/kg).

The increase in nerve blood flow after ALA treatment appears to be dose-
dependent. While this effect of ALA on experimental diabetic rats is impressive, it is
yet to be seen if ALA will have such a substantial result on human diabetes.

 
 The nerve blood flow and vascular resistance are shown for control animals, streptozotocin treated
animals, which have had different doses of ALA over a month period (0-100 mg/kg)

LITERATURE EVALUATION
 
1. Effect of vitamin E supplementation on diabetic induced oxidative stress in
experimental diabetes in rats demonstrates that that vitamin E supplementation
augments the antioxidant defence mechanism in diabetes and provides evidence
that vitamin E may have a therapeutic role in free radical mediated diseases.

2. Chronic oral administration of vitamin C to diabetic or clinical research

volunteers who are deficient in vitamin C will improve insulin sensitivity and
endothelial function. Vitamin C levels in diabetic subjects and may suggest a
potential therapy to significantly improve endothelial dysfunction and insulin
resistance.

3. From a viewpoint of molecular mechanisms, HMG-CoA reductase inhibitors

(statins) might inhibit the high glucose-induced NADPH oxidase activation through
inhibition of Rac activity and finally prevent the increase in ROS production in
diabetes. Actually, recent clinical trial suggested that statins prevent several
19
vascular events in patients with type 2 diabetes without a high concentration of LDL-
cholesterol. These pleiotropic effects of statins can be expected to improve
endothelial dysfunction through nitric oxide production and/or an anti-oxidant effect
on diabetic patients.

4. A cohort of 2,285 men and 2,019 women 40–69 years of age and free of
diabetes at baseline (1967–1972) was studied. Food consumption during the
previous year was estimated using a dietary history interview. The intake of vitamin
C, four tocopherols, four tocotrienols, and six carotenoids was calculated. During a
23-year follow-up, a total of 164 male and 219 female incident cases occurred.
Vitamin E intake was significantly associated with a reduced risk of type 2 diabetes
which supports the hypothesis that development of type 2 diabetes may be reduced
by the intake of antioxidants in the diet.

5. .In the current study, intra-arterial administration of the antioxidant vitamin C

restored endothelium-dependent vasodilatation in patients with insulin-dependent
diabetes mellitus. This result supports the notion that oxygen-derived free radicals
may contribute to abnormal vascular function in patients with diabetes mellitus. It is
not certain that comparable effects would be observed after oral intake of vitamin C,
because comparable plasma concentrations would be difficult to achieve. Further
studies of the effects of long-term oral antioxidant therapy on vascular function are
warranted as a means of reducing vascular disease in patients with diabetes

 
1. The alterations of retinal glutamate, oxidative stress and NO appear to be
inter-related in diabetes, and antioxidant therapy may be a suitable approach to
determine the roles of these abnormalities in the development of diabetic
retinopathy.

2. In diabetics, there is increased superoxide release. With regard to diabetes,

antioxidants such as alpha-tocopherol, alpha-lipoate, and ascorbic acid
supplementation have been shown to be beneficial. Most importantly, alpha-
tocopherol therapy, especially at high doses, clearly shows a benefit with regard to
low-density lipoprotein oxidation, isoprostanes, and monocyte superoxide release.
Thus, it appears that, in diabetes, antioxidant therapy could alleviate the increased
attendant oxidative stress and emerge as an additional therapeutic modality.

3. The antioxidant treatment suppressed apoptosis in β-cells without changing

the rate of β-cell proliferation, supporting the hypothesis that in chronic
hyperglycaemia, apoptosis induced by oxidative stress causes reduction of β-cell
mass. The antioxidant treatment also preserved the amounts of insulin content and
insulin mRNA, making the extent of insulin degranulation less evident. Furthermore,
expression of pancreatic and duodenal homeobox factor-1 (PDX-1), a β-cell-specific

20
transcription factor, was more clearly visible in the nuclei of islet cells after the
antioxidant treatment. Observations indicate that antioxidant treatment can exert
beneficial effects in diabetes, with preservation of in vivo β-cell function. This finding
suggests a potential usefulness of antioxidants for treating diabetes and provides
further support for the implication of oxidative stress in β-cell dysfunction in diabetes

 
1. Data suggest that Cr supplementation was an effective treatment strategy to
minimize increased oxidative stress in type 2 diabetes mellitus patients whose
HbA1C level was >8.5%, and the Cr in EU groups might act as a pro-oxidant.

 
2. It has been shown that oxidative stress has an adverse effect on
glucose metabolism. Development of the disabling chronic complications
of diabetes mellitus (DM) has also been attributed to oxidative stress. It has,
been recommended that high doses of micronutrient antioxidant vitamins should
be administered in combination rather than as single supplements. The use of
certain antioxidant vitaminand mineral supplements may be beneficial as an
adjunct therapy in the management of DM and its complications.

 
1. Accumulating evidence indicates that increased antioxidant
defense systems reduce the susceptibility to IDDM in animal
models or in human study. It is suggested that pancreas-specific
ROS productions play a critical role in signaling the cellular
autoimmune/inflammatory response by activating the transcription
factor, NFB.

2. Various diabetogenic factors may lead to an increase

in ROS production, which activates the redox-sensitive NFB.
This may be the initial event for the expression of cytokines
and chemotactic agents involved in the autoimmune/inflammatory
response.

21
 It is believed that this cascade results in a cyclic
amplification of ROS and eventually leads to apoptosis and/or
necrosis of ß cells.
The specificity of antioxidants to
inhibit NFB activation and the hyperglycemic response emphasizes
the importance of selectivity in antioxidant therapy.

Conclusion
 
I believe the evidence, though circumstantial in many ways, points to a
distinct connection between free radicals and diabetic complications. An
increase in free radical concentration has been shown for diabetic
individuals. The increase in radicals can be caused by a variety of different
factors. Accompanying this radical production is a decrease in many of the
antioxidant defences of the cell. Antioxidant enzymes, such as SOD and
catalase, are generally decreased, as are several small antioxidant
molecules like vitamin C and vitamin E. This decrease in antioxidants and
increase in free radicals indicates a potentially dangerous situation for the
cells and the specimen as a whole. So from the current pre-clinical and
clinical studies and experiments it can be concluded that the antioxidant
therapy can be given to the DM patients for treating the complications and
stress.

22
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