EVALUATION OF ANTI-DIABETIC ACTIVITY

OF CALYCOPHYLLUM SPRUCEANUM

Presented By,
U.SAILEELA
Regd.No: 17FJ1S0111
Under the guidance of
Mrs.G. KIRANMAI
Department of Pharmacology.
SSJ College Of Pharmacy

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 LIST OF CONTENTS

 Aim and objective

 Introduction
 Literature review
 Plant profile
 Experimental models
 Parameters

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 AIM AND OBJECTIVE

AIM:

 To study the evaluation of anti-diabetic activity of

Calycophyllumspruceanum in animal models.

OBJECTIVES:

 To evaluate the anti-diabetic activity of calycophyllum

spruceanum in streptozotocin induced diabetes mellitus.
 To estimate the blood glucose levels.
 To determine the body weight of the animal.
 To examine the histo-pathological section of pancreas.

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 INTRODUCTION

 Diabetes mellitus:
 Diabetes mellitus is achronic metabolic disorder caused
by lack of insulin or reduced insulin activity in the body
which results in hyperglycaemia.
 It also results in the abnormalities in the
carbohydrate,protein and fat metabolism.

 Insulin mainly regulates the blood glucose levels in the

body. Any defects in the insulin release or in the released
insulin activity causes diabetes mellitus.

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  ROLE OF PANCREAS

 Pancreas is a flattened elongated organ lying against the

posterior wall of the upper abdomen.
 Pancreas is the main organ which secretes the insulin from
its beta cells. The released insulin regulates the blood
glucose levels in the body.

 CLASSIFICATION OF DIABETES MELLITUS :

Clinically diabetes mellitus has been classified into three types

 Insulin dependent diabetes mellitus (IDDM)

 Non- insulin dependent diabetes mellitus (NIDDM)

 Gestational diabetes mellitus

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 ANTIDIABETIC-ACTIVITY
 The chemicals which are having the capacity to reduce the
increased blood glucose levels in the body are known to
posses the antidiabetic activity.

 These chemicals are screened by different methods for their

antidiabetic activity.

 LITERATURE -REVIEW
 CALYCOPHYLLUM SPRUCEANUM:

 Fabianos de vorgas (Feb 2016) Anti-oxidant activity

and peroxidase inhibition of Amazonian plants ,
extracts traditionally used as anti-inflammatory.

 dasilva APAB Lima Es (March 2018) Calycophyllum

spruceanum BENTH ameliorates acute inflammation
in mice.

 PLANT PROFILE
 Plant name - Calycophyllumspruceanum

 Common name - Capirone

 Kingdom - Plantae

 Phyllum - Angiosperms

 Order - Gentianales

 Family - Rubiaceae

 Subfamily - Cinchonoideae

 Genus - Calycophyllum

 Species - C. spruceanum

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 CALYCOPHYLLUM SPRUCEANUM :
 Description:
 Calycophyllumspruceanumis an evergreen tree with
a tall, narrow, open crown.
 It can grow up-to 20-30 meters.
 It is an Amazon native species.

 During the summer season it produces an abundant of

white, aromatic flowers, which are followed by
elongated seed pods with 3-5 seeds inside.
 Its bark is shed periodically to avoid lichens, fungi,
epiphytes and lianas.
 The plant grows extremely fast within eight years. The
wood is often cut for timber.

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 TRADITIONAL USES :
 Eye infections

 Anti-diabetic

 Anti-oxidant

 Cosmetic

 Skin infections

 Avoids lichens, fungi, epiphytes.

 Also used for age spots, cuts, wrinkles, scars, &

wounds.

 Also used to treat ovarian problems.

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 EXPERIMENTAL MODELS

 Alloxan induced diabetes mellitus.

 Streptozotocin induced diabetes mellitus.

 Fructose induced diabetes mellitus.

 Dehydroascorbic ascorbic acid induced diabetes.

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 INDUCTION OF DIABETES MELLITUS :
 Alloxan induced diabetes mellitus :

 All the groups of albino rats weighing 180-220 gm

except group 1 will be made diabetic by intra
peritoneal injection of alloxan monohydrate (150
mg/kg) which shows triphasic response in serum
glucose level.
 Alloxan will be weighed individually for each animal
according to the body weight and then solubilized with
0.2 ml saline 154mM Nacl just prior to the injection.
 Two days after alloxan injection, rats with plasma
glucose levels of above 140 mg/dl will be included in
the study,
 Treatment with plant extracts was started 48 h after
alloxan injection for 14 days.

 Fructose induced diabetes mellitus :

 Male albino rats weighing between 180-220 gm will be

used for this study.
 The animals will be divided and treated as follows for 3
weeks.
(1 kg fructose diet consists of casein, high protein207
gm, DL methionine-03 gm, fructose -600 gm, lard 50
gm, cellulose-79.81 gm, mineral mix 50 gm, zinc
carbonate 0.04 gm, vitamin mix 10 gm, foodcolour
green-0.15 gm.
 After treatment with fructose diet and drugs for 3
weeks the blood sample will be withdrawn by retro
orbital plexus puncture and biochemical parameters
such as glucose was estimated.
 During the treatment the body weight will be recorded
and will be compared among all groups.

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 STREPTOZOTOCIN INDUCED DIABETES
MELLITUS :

 Male Wistar rats weighing 150–220 g fed with a standard

diet are injected with 60 mg/kg streptozotocin
(Calbiochem) intravenously.
 As with alloxan, three phases of blood glucose changes
are observed. Initially, blood glucose is increased,
reaching values of 150–200 mg% after 3 hours.
 Six–eight hours after streptozotocin administration, the
serum insulin values are increased up to 4 times,
resulting in a hypoglycemic phase which is followed by
persistent hyperglycemia.
 Severity and onset of diabetic symptoms depend on the
dose of streptozotocin.
 After the dose of 60 mg/kg i.v., symptoms occur already
after 24–48 hours with hyperglycemia up to 800 mg%,
glucosuria and ketonemia.
 Histologically, the beta-cells are degranulated or even
necrotic. A steady state is reached after 10–14 days
allowing the use the animals for pharmacological tests.

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  GROUPING OF ANIMALS :

GROUPS TREATMENT

Group 1 Normal control (vehicle)

Group 2 Diabetic control (STZ + Vehicle)

Group 3 Reference standard ( STZ +

Glibenclamide)

Group 4 STZ + Low dose of extract

Group 5 STZ + Intermediate dose of extact

Group 6 STZ + High dose of extract

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 Parameters

 Blood glucose levels.

 Body weight.

 Histo-pathological section of pancreas.

 Serum lipid profile.

 DETERMINATION OF BLODD GLUCOSE LEVELS :

 Blood samples were withdrawn from tail tip of animal.

 Blood glucose estimation can be done by one touch
electronic glucometer using glucose test strips.
 Test was repeated for 7 and 14th day of the study.

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 DETERMINATION OF BODY WEIGHT:

 The body weight of the animal was measured regularly

during the study by using the balance.

 HISTOPATHOLOGICAL SECTION Of PANCREAS :

 Sacrifice the animal by anesthesia.

 Pancreas was removed from each animal and
collected in 10% formalin solution.
 Sections were cut and stained with hematoxylin
and eosin for histological examinations.

 ESTIMATION OF SERUM LIPID PROFILE :

1) ESTIMATION OF TOTAL CHOLESTEROL

REAGENTS:

 Sodium cholate
 Phenol cholesterol
 Cholesterol oxidase
 Peroxidase
 4-aminoantipyrine
 Cholesterol standard

METHOD:

 1.0ml of reagent is mixed with 10µL of standard

and 10µl of serum sample in different test tubes.
 The test tubes are incubated for 10min at room
temperature (16-25ºC) or for 5min at 37ºC.

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  Absorbance of standard and sample is measured
against blank, 1.0ml of reagent at 500nm using
spectrophotometer.

FORMULA:

Cholesterol = absorbance of test x concentration of standard

absorbance of standard

The amount of total cholesterol is estimated as mg/dl.

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 2) ESTIMATION OF TRIGLYCERIDES(GPO-PAP
method)

REAGENTS:
 Triglyceride standard
 Sodium phosphate buffer
 Hydrogen peroxide
 Amino antipyrine
 Triglyceride enzyme mixture
METHOD:

 GPO-PAP method is based on enzymatic

colorimetric method.
 1ml reagent is added to 10µl of sample and 1ml
reagent is added to 10µl of standard in different
test tubes.
 Test tubes are now incubated for 5min at 37ºC
or 10min at room temperature.
 Absorbance of standard and the sample is read
against blank at 505nm.

FORMULA:

Triglycerides = absorbance of test x concentration of standard

absorbance of standard
The amount of triglycerides is estimated as mg/dl.

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