SCREENING OF ANTI DIABETIC ACTIVITY

PRESENTED BY-Sanjiv Kumar Panda

GUIDED BY- Dr. Bimalendu Chowdhury,
M. Pharm, PhD (HOD of pharmacology)
M PHARM (1ST YEAR)
BRANCH-PHARMACOLOGY
ROLAND INSTITUTE OF PHARMACEUTICAL
SCIENCES, KHODASINGI,BERHAMPUR, ODISHA
What is diabetes ? PG -1
• Diabetes is a chronic metabolic disorder characterized by elevated levels of sugar
(glucose) in the blood.
• It happens when the body doesn’t make enough insulin or can’t use it effectively.
Diabetes mellitus
The main characteristic of diabetes mellitus is abnormalities in the metabolism of
carbohydrates, proteins, and fats brought on by either a relative or total lack of
insulin activity or secretions.
Two major types of diabetes are
• Type- I or IDDM (Insulin dependent diabetes mellitus)
• Type- II or NIDDM (Non insulin dependent diabetes mellitus)
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BLOOD GLUCOSE HOMEOSTASIS PG -3

Normal blood glucose level-

70- 108 mg/dL
or
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PATHOPHYSIOLOGY OF DIABETES MELLITUS

• Oral hypoglycemic drugs PG -5

2) Insulin sensitizers
1) Insulin secretagogues

Sulfonyl ureases Biguanides:- Metformin, phenformin

1st generation—Chlorpropamide, Tolbutamide Glitazones – Rosiglitazone, Pioglitazone
2nd generation-Glipizide, Glyburide, Glibenclamide
3rd generation- Glimepiride
Meglitinide analogs – Repaglinide, Nateglinide

4) GLP-1 Analog or incretin

3) DPP-4 Inhibitor- Sitagliptin, mimetics- Xenatide,
Saxagliptin, Alogliptin Acarbose, Voglibose, Miglitol etc.
Exenatide, Dulaglutide etc.
MODELS USED FOR SCREENING ANTI-DIABETIC ACTIVITY PG -6

Chemically Induced Diabetes

 Alloxan induced diabetes
IN VIVO METHODS (IDDM)
 Streptozotocin induced diabetes
 Hormone induced diabetes
 Neonatal Streptozotocin induced diabetes
 IN VIVO METHODS (NIDDM)
STZ and Nicotinamide Induced Diabetes
 Low Dose STZ With High Fat Diet
 Adrenaline induced acute hyperglycemia
Genetically Induced Diabetes PG -7

 Non obese diabetic mouse (NOD mouse) DEVELOPS TYPE-1 D.M

 Bio breeding rat (BB Rat)

 WBN/KOB RAT
 GOTO-KAKIZAKI RAT DEVELOPS TYPE-2 DIABETES
MELLITUS
 ZUCKER-FATTY RAT
 1) ALLOXAN INDUCED DIABETES PG -8

PRINCIPLE:-
• Alloxan is a urea derivative chemical agent which causes selective necrosis of the
Beta() cells of the pancreatic islets.
• Alloxan is a highly reactive molecule it produces hydrogen peroxide (H2O2) and
free radicals so it damage the pancreatic beta cells and induce permanent
diabetes.
Free radical formation destroy Cell Induced diabetes
PROCEDURE PG -9

• Albino rats of either sex 150-200 g are injected with a single dose of alloxan
monohydrate.
• 100-180 mg/kg body weight alloxan administered subcutaneously.
• A triphasic reaction is shown in blood glucose levels, with hypoglycaemia lasting an
2-4 hour, followed by hyperglycaemia for eight hours, and persistent
hyperglycemia for 24 hours and -cell destruction.
• Animals showing fasting blood glucose level above 180 mg/dl after 48 hour of
alloxan administration are considered diabetic.
• Animals are divided in to three groups i.e. positive control, standard and test
group.
• Blood glucose is measured at various intervals and the results were compared to
the control.
Evaluation:-
• The data was analyzed statistically using ANOVA followed by dunnett’s t-test to
2) STREPTOZOTOCIN INDUCED DIABETES MELLITUS PG -10

Principle:
• It is a mono functional nitrosourea derivative it is obtained
from streptomyces achromogenes.
• Streptozotocin (broad spectrum antibiotic) is a naturally
occurring chemical agent that produce type-I diabetes in
animal model and type-II with multiple low doses or with
combination with nicotinamide.
• Streptozotocin is a alkylating agent that damages DNA and
which is particularly toxic to islet of β cells.
• STZ separates into its glucose and methylnitrosourea
components upon absorption into beta cells.
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• The methylnitrosourea moiety, due to its alkylating properties,

modifies biological macromolecules, fragments DNA, and destroys beta
cells.
• Protein glycosylation may be an additional damaging factor,
o In the attempt to repair DNA, poly ADP-ribose polymerase (PARP) is
overstimulated.
oThis diminishes cellular NAD+, and subsequently ATP stores. The
depletion of the cellular energy stores ultimately results in beta cell
necrosis.
Procedure:-
Streptozotocin is dissolved in 100 mM citrate buffer at PH-4.5 and
calculated amount of the fresh solution is administered to overnight
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The previously mentioned solution is intraperitonially administered into albino

rats of both sexes weighing 150- 200gm.
After injecting streptozotocin for 48 hours, animals with fasting blood glucose
levels more than 200 mg/dl are classified as diabetic.
Three groups of animals are used ; the positive control group, standard group &
the test group.
Blood samples are collected from animals that have fasted for 6 hours via the
retroorbital vein following a six weak course of therapy.
Centrifuging at 3000 rpm while cooling to 2-40c for 5 minutes and separate the
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• Using an autoanalyzer the glucose peroxidase method(GOD-POD) kit is used to

estimate serum glucose levels.
• Decrease in blood glucose level in test is compared with the standard.
Evaluation:-
The data was analyzed statistically using ANOVA followed by dunnett’s t-test to
conform about the statistical significance of the result.
3) Hormone induced diabetes PG -14

PRINCIPLE:-
o Steroid dexamethasone triggers an immunological response in the
islets of Langerhans, resulting in IDDM in animals.
PROCEDURE:-
• Dexamethasone 2–5 mg/kg BW, IP is injected twice daily into adult rats weighing
150–200 g.
• The condition known as IDDM is brought on by repeatedly injecting the same
dose level over the course of 20–30 days.
• Diabetic if fasting blood glucose level is greater than 150 mg/dL.
• Animals are divided in to three groups i.e. positive control, standard and test
group.
• Decrease in blood glucose level in test is compared with the standard.
4)Neonatal streptozotocin induced diabetes PG -15

Principle:-
• When newborn rats are given streptozotocin, their pancreatic beta cells are
severely destroyed, and their pancreatic insulin reserves are reduced and their
blood glucose levels rise.
Procedure:
• At birth or within the first five days after birth, neonatal rats are given a
treatment of streptozotocin (90 mg/kg BW) formulated in citrate buffer (pH 4.5) IP
infusion.
•Following that, a single daily dose of 28IU of insulin is given subcutaneously.
• A triphasic reaction is shown in blood glucose levels, with hypoglycaemia lasting
an 2-4 hour, followed by hyperglycaemia for eight hours, and persistent
hyperglycemia for 24 hours and -cell destruction.
• Drug samples to be screen is administered by suitable root and blood glucose
level is analyzed to determine the activity.
5)STZ and Nicotinamide Induced Diabetes PG -16

Principle:-
• Rats have been shown to develop experimental diabetes when given both nicotinamide (NA) and
streptozotocin (STZ).
• Administering NA to rats can partially protect insulin-secreting cells from STZ, which is known to
induce harm to pancreatic -cells.
Procedure:-
•Rats were given a single intraperitoneal injection of NAD (120 mg/kg) and STZ (60 mg/kg) to cause
type-2 diabetes.
•NAD is an antioxidant that hunt free radicals to prevent the cytotoxic activity of STZ and only
slightly damages the mass of beta cells in the pancreas, resulting in Type-2 diabetes.
• After 48 hours, blood glucose levels were measured using a glucose strip, and animals classified as
diabetics had blood glucose levels between 140 and 270 mg/dl.
• Then the animals are divided into three group i.e control, standard and test.
• All the treatments are administered orally and monitor the blood glucose at different time interval.
Evaluation:
• Reduction in blood glucose in test is compared with standard.
6)Low Dose STZ With High Fat Diet PG -17

o It is the more stable animal model for type-II diabetes mellitus by combining a
high fat diet with several low doses of streptozotocin injections.
o For the moment the antidiabetic impact of berberine was assessed using this new
model.
Procedure:-
• A single injection of STZ (30 mg/kg BW) is given to the rats (Wistar or SD) along
with a high energy diet consisting of 20% sucrose.
•After 4 week period, changes in body weight are noted, and standard procedures
are used to analyse the levels of glucose, triglycerides, total cholesterol, and LDL.
• Three distinct sets of diabetic rats receive the same medications, all of which are
given orally.
Evaluation:-
Blood glucose change was measured and evaluated with a standard.
7)Adrenaline induced acute hyperglycemia PG -18

Principle:-
• Adrenaline functions as an anti-insulin regulating hormone. It increases the rate
of glycogenolysis and increases the blood glucose.
Procedure:-
•A dose of 0.1 mg/kg of adrenaline is administered intravenously to adult albino
rats.
• The dose takes up to four hours to reach its maximal hyperglycemic effects.
• Appropriate route is used to provide drug samples for screening, and blood
glucose level is measured.
• The blood glucose level is determined.
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Non obese diabetic mouse [NOD MOUSE] -genetic model for induction of type-I
diabetes mellitus
Principle:
• NOD mouse is model for IDDM.
• When autoantibodies are produced in conjunction with the autoimmune death of pancreatic beta
cells, hypoinsulinemia develops.

Procedure:
For more than 20 generations mice are bread in laboratories by slibing and mating.

The spontaneous development of IIDM in mice is achieved after 20 generations of slib mating.

Between the ages of 100-200 days, diabetes develops suddenly (Denoted by severe glycosuria,
polyurea & weight loss).
The drug sample for screening is administered to the animals.
BB RAT( BIO- BREEDING RAT) PG -20

•An important rodent model of human type 1 diabetes (T1D) is the BB

rat, which has been used to examine the pathophysiology of diabetes
and look into possible intervention therapy for use in clinical trials.
•After 50 to 90 days of age, the Diabetes-Prone BB (BBDP) rat develops
autoimmune Type 1 Diabetes mellitus on its own.
•The Diabetes-Resistant BB (BBDR) rat has similar diabetes-susceptible genes
as the BBDP, but does not become diabetic in viral antibody-free conditions.
• In well-managed conditions and during a brief, consistent time span,
these diabetes-inducible rats experience hyperglycemia (14–21 days).
• If insulin treatment is not started, diabetic animals become severely
hypoglycemic, hyperglycemic & ketotic within a few days.
WBN/KOB RAT PG -21

• Aged males of an Inbred wistar strain known as the WBN/kob rat have
been shown to exhibit spontaneous hyperglycemia, glucosuria &
glucose intolerance.
•At 21 weeks of age, these animals show signs of reduced glucose
tolerance and glucosuria.
•Even at 12 weeks of age, there are noticeable reductions in the quantity
and size of islets.
•In the exocrine portion, fibrinous exudation and pancreatic tissue
degradation are seen, particularly in the vicinity of the pancreatic ducts
and deteriorated islets in men 16 weeks old.
•In addition to axonal alterations, these rats experience demyelinating
neuropathy, primarily affecting the motor system.
 GOTO-KAKIZAKI RAT PG -22

• The Goto-Kakizaki (GK) rat is a non-obese, non-hypertensive rodent model of type

2 diabetes mellitus (T2DM).
• Multiple mechanism:-
o Impaired glucose-stimulated insulin secretion (GSIS), Decreased beta cell mass,
increased leptin hormone, Changes in insulin action, Increased plasma GOT and
GPT, and Higher content of basal phosphorylation of GSK-3β.
• ZUCKER-FATTY RAT
• The Zucker-fatty rat is a classic model of hyperinsulinemic obesity.
• A basic autosomal recessive (fa) gene causes obesity, which manifests at a young
age.
• Similar to human NIDDM, obese Zucker rats have peripheral insulin resistance,
hyperinsulinemia, and moderate glucose intolerance.
• However, they often have normal blood sugar levels throughout their lives.
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GOTO-KAKIZAKI RAT
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