PHARMACOLOGICAL DIABETIC RATS

MASTER OF PHARMACY
IN
PHARMACOLOGY

By
Under the guidance of
ZAKARUL ISLAM
Dr.K B PRUSHTY
B.Pharm.
M. Pharm.,Ph.D.
(HT.NO. 21154P1015)
PROFESSOR & HOD
 CONTENTS

 INTRODUCTION
 REVIEW OF LITERATURE
 AIM AND SCOPE OF PRESENT WORK
 DRUG PROFILE
 INSTRUMENTS AND CHEMICALS
 METHOD DEVELOPMENT
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSION
 BIBLIOGRAPHY
INTRODUCTION

• Diabetes mellitus is a chronic metabolic disease caused by an absolute or relative

lack of insulin and or reduced insulin activity, which results in hyperglycemia and
abnormalities in carbohydrate, protein and fat metabolism. There are estimated
143 million people worldwide sufferings from diabetes, almost five times more
than the estimates ten years ago. This number may probably double by the 2030.
Therefore, the human population worldwide appears to be in the midst of an
epidemic of diabetes. Reports from the World Health Organization (WHO) indicate
that diabetes mellitus is one of the major killers of our time, with people in south-
east Asia and western Pacific being most at risk (Thivari et al.,2002).
Epidemiology

• Type-1 diabetes mellitus account for up to 10 % of all cases of diabetes mellitus

and results from an autoimmune destruction of the pancreatic β-cells. The
prevalence of β-cell autoimmunity appears proportional to the incidence of Type-1
diabetes mellitus in various populations. For instance, countries of Sweden,
Sardinia and Finland have the highest prevalence of islet cell antibody (3%-4.5%)
and are associated with the high incident of Type-1 diabetes mellitus, 22-35 per
100,000.
• Type-2 diabetes mellitus is heterogeneous disorder of glucose metabolism. Type-2
diabetes mellitus accounts for as much as 90% of all cases of diabetes mellitus and
usually results from defects in insulin sensitivity and a relative defect in insulin
secretion (Oki et al., 2002).
INSULIN

 Insulin, the hypoglycemic-anti-diabetic factor is a polypeptide hormone secreted by the β-cells of islets
of langerhans of pancreas. Insulin was the first hormone to be isolated from animal sources in pure
enough form to be administrated therapeutically and it was the first mammalian peptide hormone whose
biosynthesis by recombinant-DNA technology was achieved (Berne et al.,1993).
MECHANISM OF INSULIN ACTION
 Insulin is a major anabolic hormone. It catalyses the synthesis of various macromolecules and prevents
undue breakdown of proteins, carbohydrates and fat. Thus it promotes cell growth by deposition of
carbohydrates, lipids and proteins. The most clearly measurable action of insulin is to facilitate the
penetration of aminoacids and simple sugar through the cell membraneof skeletal and heart muscle which
otherwise have low permeability to these substances. Receptor sites on cell surface membrane bind
insulin and this reaction leads to changes both in cell permeability and the activity of enzyme system
within the cell.
Thus reduction of blood glucose is brought about by :

 Increasing glucogenesis

 Increasing entry of glucose into insulin sensitive cells such as myocytes, hepatocytes and adipocytes

 Inhibiting breakdown of lipids

 Increasing the rate of protein synthesis

 Stimulating some cell ion transport mechanisms such as Na+ / K+ / ATPase

 Decreasing gluconeogenesis

CLASSIFICATION OF DIABETES MELLITUS

 Clinically diabetes mellitus has been classified into three forms. They are :

 Insulin dependent diabetes mellitus (IDDM)

 Non insulin dependent diabetes mellitus (NIDDM)

 Maturity onset diabetes of youth (MODY)

 (Type I Vs Type II diabetes mellitus)

Type I (IDDM) Type II (NIDDM)

Onset < 20 years Onset > 30 years

Clinical Normal weight Obese
Decreased blood insulin Normal or increased
Anti-islet cell antibodies blood insulin
Ketoacidosis common No anti-islet cell
antibodies
Ketoacidosis rare

Genetics 50% concordance in twins 90% to 100% concordace

HLA-D linked in twins No. HLA
association

Pathogenesis Auto immunity Insulin resistance

Immunopathologic Relative insulin
mechanism deficiency
Severe insulin deficiency

Islet cells Insulitis early No insulitis

Marked atropy and Focal atropy and
Fibrosis amyloid deposits
β-cell depletion Mild β-cell depletion
 DIAGNOSIS OF DIABETES

• Urine test
• Fasting blood glucose level
• Acetone breath
• Insulin assay
• Oral glucose tolerance test :
• C-peptide assay :
• Glycated hemoglobin (HbA1c) test
 NEED OF WORK
• The treatment of diabetes mellitus is based on oral hypoglycemic agents and
• insulin. However, in the indigenous Indian system of medicine good number of
• plants were mentioned for the cure of diabetes and some of them have been
• Experimentally evaluated and principal were isolated (Grover et al., 2002). WHO
• (1980) has also recommended the evaluation of the effective of plants in
• Conditions where there are no safe modern dr
• AIMS AND OBJECTIVES
• To evaluate anti diabetic activity of Lupinus albus leaves exrtact in alloxan induced diabetic rats.
•
• To estimate the insulin levels in various groups of animals
•
• To estimate the lipid profiles levels in various groups animals
•
• To estimate the liver glycogen levels in various groups animals
•
 LUPINUS ALBUS LEAF DESCRIPTION:

Scientific classification
Lupinus
Kingdom: Plantae

(unranked): Angiosperms sundial lupine (Lupinus perennis)

(unranked): Eudicots

(unranked): Rosids

Order: Fabales

Family: Fabaceae

Subfamily: Faboideae

Tribe: Genisteae[1]

Subtribe: Lupininae

Genus: Lupinus
L.

Type species

Lupinus albus L.

Subgenera
 Lupinus
 Platycarpos
 MATERIAL AND METHODS

• Glucose estimation kit : Span Diagnostics Ltd. Surat

• Cholesterol estimation kit : Span Diagnostics Ltd. Surat
• Triglycerides estimation kit : Span Diagnostics Ltd. Surat
• HDL estimation kit : Span Diagnostics Ltd. Surat
• Serum creatinine reagent : Pinnacle Marketing Pvt. Ltd. Mumbai

• The dose of lupines albus leaves was selected taking into consideration that an normal
• adult man weighing approximately 1kg consumes 10 g to be taken an hour before the
• principal meal, twice a day.
According to the conversion table M.N.Ghosh 1971 the human adult dose multiplied by

• 0.018 gives the rat dose (for a rat weighing 200g)55

• Therefore rat dose : 10 g x 0.18 / 200g of a rat

• Or

• 180 mg / 200g of rat

•
• The diavedic powder was dissolved in 2% w/v acacia suspension and this suspension
•
• administered twice a day.
•
 Programme: Procedure:
Mode End point

• Wavelength
Temperature
Optical path length
505 nm (490 – 550 nm)
37 o C or R.T.
1 cm
Pipette
tubes marked
into
Blank Standard Test
Blanking Reagent blank Serum or - - 10 μl
Incubation time ( minutes) 10 at 37 o C or 30 at R.T. Plasma
Sample volume 10 μl Glucose - 10μl -
Working reagent volume 1000 μl standard
Concentration of standard 100 mg/dL Working 1000 μl 1000μl 1000μl
Linearity Upto 5oo mg/dL glucose reagent
Stability of color 1 hour
Maximum absorbance 2.000
limit
Units mg/dL

The above were mixed well , incubated at 37 o C for 10 min or R.T. for 30 min and the

absorbance of sample (As) and of standard (Astd) was read against blank .
Calculation :
Glucose (mg/dL) = (As / Astd) x concentration of the standard
• Estimation of serum cholesterol:
•
•
• Span diagnostics kit was used for the estimation of serum cholesterol .
•
• Method:
•
• Enzymatic , ( Cholesterol Oxidase – Peroxidase ) , Endpoint colorimetry , (www.ncbi.nlm.nih.gov/pubmed/10692346)
•
• Single Reagent Chemistry , with LCF ( Lipid clearing factor )
•
• Principle:
•
• The estimation of cholesterol involves the following enzymatic reactions
•
• Cholesterol esters CE Cholesterol + Fstty acids
•
• Cholesterol + 02 CO Cholesten – 3 – one + H2o
•
• H2o2 + Phenol + 4-APP POD Quinoneimine dye + H2o
•
•
• CE = Cholesterol esterase
•
• CO = Cholesterol oxidase
•
• POD = Peroxidase
•
• 4APP=4-Aminoantipyrine
•
• Absorbance of quinoneimine was measured at 505nm which proportional to cholesterol
•
• concentration in the specimen
 Programme: The basic assay parameters
Mode End point
Wavelength 505 nm (490 – 550 nm)
Temperature 37 o C
Optical path Length 1 cm
Blanking Reagent Blank
Incubation time ( minutes) 10 at 37 o C
Sample volume 10 μl
Working reagent volume 1000μl
Concentration of standard 200 mg/dL
Linearity 750 mg/dL
Stability of color 1 hour
Maximum absorbance limit 2.000
Units mg/dL

Procedure:

Pipette into tubes Blank Standard Test

marked
Serum - - 10μl
Standard - - -
Cholesterol Reagent 1000μl 1000μl 1000μl

The above were mixed well , incubated at 37 o C for 10 minutes, the absorbance of
standard and sample were read against reagent blank at 505 nm within 60 minutes .
• Calculation:
•
• Cholesterol concentration (mg/dL) = Absorbance of test x 200
• Absorbance of standard
• Cholesterol concentration (mmol/L) = concentration (mg/dl) x 0.0259
• Estimation of serum triglycerides:
•
• Span diagnostics kit was used for the estimation of serum triglycerides.
•
• Method:
•
• Enzymatic (GPO/Trinder) , Endpoint Colorimetry , Single Reagent Chemistry with LCF (Lipid Clearing Factor)
•
• PRINCIPLE:
•
• The estimation of triglycerides involves the following enzymatic reaction :
•
• Glycerol + LPL Glycerol + FFA
•
• Glycerol + ATP GK Glycerol – 3 – Phosphate + ADP
•
• Glycerol – 3 – Phosphate + o2 GPO DHAP + H2O2
•
• 2H2O2 + 4 APP POD Quinoneimine dye + 4 H2o
•
•
RESULTS
Effect of oral administration of lupine albus leaves( 100 mg/kg , p.o) , luine albus
leaves(200mg/kg , p.o) and glibenclamide ( 5 mg/kg , p.o) on glucose tolerance test in normal rats
•
Serum glucose levels (mg/dl) / % change
GROUP
0 min 30 min 60 min 120 min

Group 1 71.71± 0.63 97.98± 0.74 123.81± 0.94 100.98±0.74

36.56± 2.13% 72.70 ± 1.90% 40.85± 2.14%

Group 2 81.85± 0.49 90.08± 0.34 78.1± 0.28 88.65± 0.3191

10.06± 0.41%*** -4.56± 0.85%*** 8.33± 0.802%***

Group 3 77.81± 0.77 86.66±0.33 76.28± 0.20 81.38± 0.33

11.43± 1.37%*** -1.88± 0.85%*** 4.64± 1.36%***

Group 4 76.58± 0.63 108.2± 0.51 88.35± 0.47 100.6± 0.182

41.30± 1.40 ***
17.96± 2.80% ***
31.38±1.10%***

Group 1: Control (2% w/v acacia suspension p.o)

Group 2: lupine albus leaves(100 mg/kg, p.o)
Group 3: lupine albus leaves(200 mg/kg, p.o)
Group 4: Glibenclamide (5 mg/kg, p.o)
Values are expressed in mean ± SEM of serum glucose mg/dl , n = 6
Values are expressed in mean ± SEM of percentage change in serum glucose , n = 6
P < 0.001 when compared to the control group 1
In normal control rats, the serum glucose levels increased by 89.06%,
98.86% and 19.58% at 30, 60 and 120min after feeding glucose (10gm/kg, p.o).
 Effect of administration of lupine albus leaves (100mg/kg,p.o, 200mg/kg,p.o), and
glibenclamide (5mg/kg, p.o) for 14 days on serum HDL-cholesterol levels in fructose fed rats.

SERUM
GROUP TREATMENT HDL
(mg/dl)

Control (Normal control) 50.06 ± 0.16

Group – 1

Diabetic control 21.1 ±0.15 **

Group – 2

Lupine albus leaf extract

38.3 ± 0.17**
Group – 3 ( 100 mg/kg , p.o)

Lupine albus leaf extract

45.83 ± 0.15**
Group - 4 (200 mg/kg , p.o)

Glibenclamide ( 5 mg/kg , p.o)

49.3 ± 0.20*
Group - 5 (reference standard)
 Effect of administration of lupine albus leaves(100mg/kg,p.o, 200mg/kg,p.o) and glibenclamide
(5mg/kg, p.o) for 14 days on serum creatinine levels in fructose fed rats.

Administration of lupine albus leaves (100mg/kg,p.o, 200mg/kg,p.o), for 14 days showed serum
creatinine levels of 57.82±1.49µmol/lt and 50.79±1.10µmol/lt respectively. This was 30.21% and
38.70% significantly lower as compared to that of fructose fed rats 82.86±1.26µmol/lt.

SERUM
GROUP TREATMENT LDL
(mg/dl)

Control (Normal control) 29.08 ± 0.14

Group – 1

Diabetic control 84.13 ± 0.13**

Group – 2

Lupine albus leaf extract

41.71 ± 0.14**
Group – 3 ( 100 mg/kg , p.o)

Lupine albus leaf extract

35.2 ± 0.18**
Group - 4 (200 mg/kg , p.o)

Glibenclamide ( 5 mg/kg , p.o)

30.76 ± 0.18**
Group - 5 (reference standard)
 DISCUSSION

• Oral glucose tolerance test was done in normal rats by giving glucose 10g/kg , p.o) post half
an hour after administration Lupinus albus leaves(100mg/ kg , p.o) Lupinus albus leaves
powder (200mg/kg , p.o) and glibenclamide ( 5mg/kg , p.o) so as to confirm whether the
formulation under evaluation has any extrapancreatic ( apart from those involving insulin )
actions like the up regulation of the insulin receptors or the inhibition of gluconeogenesis.
•
• Lupinus albus leaves and contains Gymnema sylvestris and Momordica charantia. These have
been individually shown to possess antihyperglycaemic activity with varied mechanisms of
action (Yoshikava et al.,1991). Gymnema contains gymnemic acid which is reported to inhibit
the adrenohypophyseal stress response and hyperglycaemic response to adrenaline and
growth factor. Momordica charantia has been shown to increase peripheral utilization of
glucose. Hypercholesterolemia and hypertriglyceridemia have been reported to occur in
diabetic rats. Under normal circumstances insulin activates enzyme lipoprotein lipase and
hydrolyzes triglycerides. However in insulin deficient subjects it fails to activate the enzyme
and causes hypertriglyceridemia. Oral feeding of Lupinus albus leaves prevented
hyperlipidemia which could possibly be due to activation of enzyme lipoprotein lipase.
• Liver glycogen level may be considered as the best marker for assessing
hypoglycemic activity of any drug. A decrease in liver glycogen in diabetic animal
is reported to be due to less availability of the active form of enzyme glycogen
synthetase which in turn has been reported to be responsible for the
incorporation of glucose moieties into the pre-existing glycogen chain and due to
the decrease in the rate of glycogenesis.
• In diabetes, the decrease in body weight is associated with decreased rate of
glucose utilization and impaired carbohydrate metabolism . lupine albus leaves
treatment seems to have regulated these disturbances at the cellular levels. The
ability of Lupinus albus powder to protect the body weight loss seems to be due
to its antidiabetic activity.
•
• Treatment with Lupinus albus leaves has shown an increase in body weight when
compared with control group in both models.
•
• From the above observations, it is seen that Lupinus albus leaves lowers serum
glucose, serum cholesterol, serum triglycerides, serum creatinine and increases
liver glycogen and body weight in type II diabetes model in rats.
 CONCLUSION

• Lupinus albus leaves also significantly reduced the

hyperglycemia in glucose fed normal rats .Lupinus albus leaves,
hyperglycemia in Type II diabetes mellitus model and alloxan
induced insulin resistance model as noticed from animal
experiments Lupinus albus leaves could also be of benefit in
overcoming accompanying cardiovascular complications of
diabetes mellitus. Further studies will be necessary to establish
the probable mechanisms of action of Lupinus albus leaves.
These could include measurement of serum insulin levels and
liver enzyme levels involved in glucose metabolism.
 BIBLIOGRAPHY
•
• 1. Tiwari Ak, Rao JM. Diabetes mellitus and multiple therapeutics approaches of
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• 3. Kamtchouing P, Kahpui SM, Djomeni Dzeufiet PD, Tedong EA, Dimo T. Anti-
• diabetic activity of methanol/methylene chloride stem bark extracts of Terminalia
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• 2006 Apr; 104: 306-9
• 4. Hongmei chem., Sachin Brahmabhatt, Akansksha Guptha & Avadesh. Sharma C.
• Duration of streotozocin-induced diabetes differiantely afeect p38- mitogen-activating
• Protein kinase (MAPK) phosporylation in renal and vascular dysfunction.
• Cardiovascular diabetology 2005 jul; 4(3):1-18
• 5. World Health Organization. Defination, diagnosis and classification of diabetes
• mellitus and its complications-Report of a WHO Conclusion. Geneva: World Health
• Organisation; 1999;2
• 6. Maureen HI. Definition and classification of diabetes mellitus and the criteria for
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• fundamental and clinical text. 3rd ed. Philadelphia: Lippincott Williams & Wilkins
• 2004; 457-67