COLLEGE OF PHARMACY

MADRAS MEDICAL COLLEGE

CHENNAI - 03.

DEPARTMENT OF PHARMACOLOGY

PHARMACOLOGY III

PRACTICAL MANUAL

III YEAR B.PHARM. (6TH SEMESTER)

2025
 INDEX

PAGE
Ex. No DATE EXPERIMENTS
NO

1
DOSE CALCULATION IN EXPERIMENTAL
PHARMACOLOGY
2
EXPERIMENTS
EFFECT OF ANTI-ALLERGIC ACTIVITY OF TEST DRUG
BY MAST CELL STABILIZATION ASSAY

3
STUDY OF ANTI-ULCER ACTIVITY OF TEST DRUG IN
(CIMETIDINE IN PYLORUS LIGATED RAT) SHAY RAT et at.
METHOD.

4 STUDY OF DRUGS ON GASTROINTESTINAL MOTILITY

5 EFFECT OF AGONIST ON GUINEA PIG ILEUM

6 EFFECT OF ANTAGONIST ON GUINEA PIG ILEUM

7 EFFECT OF SALINE PURGATIVE ON FROG'S INTESTINE

8 EFFECT OF INSULIN HYPOGLYCEMIC IN RABBIT

9 RABBIT PYROGEN TEST

10 DETERMINATION OF ACUTE ORAL TOXICITY (LDso)

11 DETERMINATION OF ACUTE SKIN
IRRITATION/CORROSION OF TEST SUBSTANCE

12 DETERMINATION OF ACUTE EYE

IRRITATION/CORROSION OF TEST SUBSTANCE

13 CALCULATION OF PHARMACOKINETIC PARAMETERS

FROM A GIVEN DATA

14
BIOSTATISTICS METHODS IN EXPERIMENTAL
PHARMACOLOGY - student "T" test , Standard
Deviation, Standard Error of Mean (SEM)
15 BIOSTATISTICS METHODS IN EXPERIMENTAL
PHARMACOLOGY - chi square test

16 BIOSTATISTICS METHODS IN EXPERIMENTAL

PHARMACOLOGY - paired "T" test
 VISION, MISSION AND QUALITY POLICY

OUR VISION
 To Achieve Excellence in Pharmacy Education, Research and Services

OUR MISSION
 To produce Pharmacy Professionals of Global Standards by
 Providing Quality Education
 Pioneering Research
 Affording Pharmaceutical Services

QUALITY POLICY
The College is committed to achieve the Global Standards of "Health for All" by
producing Professionals par excellence in the field of Pharmacy through wholesome education
founded on high Ethical Standards.

We will continually improve the quality and achieve high standards in the delivery of
Education, Research and Pharmaceutical services.

 We will do this by
 Adopting a Structured Delivery of Education.
 Stimulate research by providing the necessary Infrastructure, Encouraging Publications in
Peer Reviewed Journals and Filing for Patents.
 Impart skill based training in areas of Hospital Services, Industrial Manufacturing
& Quality Assurance and Pharmacovigilance.
COURSE OUTCOME

 Ability to understand the mechanism of drug action and its relevance in the treatment of
Respiratory system.

 Ability to understand the therapeutic approach to management of gastrointestinal

diseases.

 Ability to understand the mechanism of drug action and its relevance in the treatment of
microbial infections.

 Analyses the problems associated with the drugs used for the treatment of various
disease including tuberculosis, malaria, viral disease, Fungal infection and cancer.

 Fundamental knowledge about the principles of toxicology and treatment of

poisoning.
 B.PIIARM SEMESTER -VI (PHARMACOLOGY III

PRACTICAL MANUAL

EXP. NO: I

CALCULATION OF DOSES OF DRUGS FOR ADMINISTRATION TO ANIMALS

Aim:

To calculate the amount of drug to be administered in animals.

Principle

The doses of drugs are generally expressed as mg/kg. Since the weight of the animals vary, it is necessary
to calculate doses of drugs for individual animals. The volume of drug administration should generally be
kept constant. In case of rats, the volume of injection should be ideally 0.5mg per 100g of the body weight by the
i.p route. In case of mice, the injection volume should be 1ml/100mg of the body weight. In order to
maintain these volumes, the preparation of the stock solution becomes important. It should be prepared in
such a way that the required amount of the drug is present in the permitted volume of the injection that can be
injected for that animal.

Dose Calculations

1. Calculate the amount of Diazepam required for a mouse weighing 30g. The dose
of diazepam is 2mg/kg by intraperitoneal route.

The weight of the mouse = 30g

The dose of Diazepam = 2mg/kg
Amount of Diazepam required = Animal weight /1000g x Animal Dose
=30x 2/1000 - 0.06mg

The dose to be injected is very small and it is converted to mcg = 0.06x 1000mcg = 60mcg
*Therefore the dose to be injected to the animal is 60mc*
2. Calculate the amount of Pentazocine required producing analgesia in a rat
weighing 220g. The dose of Pentazocine is 4mg/kg by intraperitoneal route

T h e w e i g h t o f t h e r a t = 2 20 g

The dose of Pentazocine = 4mg/kg

Amount of Pentazocine required = Animal weight /1000g x Animal Dose

= 220x4/1000 = 0.88mg
*Therefore the dose to he injected to the animal is 0.88mg to produce analgesia*

3. You are provided with a stock solution of Paracetamol which is 10mg/ml. The dose of Paracetamol
is 25mg/kg. Calculate the volume to be injected i.p. to a rat weighing 250g to demonstrate
analgesic activity.

The weight of the rat = 250g

The dose of Paracetamol =25mg/kg

Amount of Paracetamol required =Animal weight /1000g x Animal Dose
=250x25/1000 = 6.25 mg
The stock solution provided is 10mg/ml
10 mg of paracetamol = 1ml of solution
6.25 mg of paracetamol = 'X' ml of solution
X= 6.25 x 1/ 10 - 0.625ml.
*Therefore the dose to be injected to the animal is 0.625ml*

4. The antidepressant dose of Imipramine is 10mg/kg i.p. The stock solution of lmipramine
provided is 1mg/ml. Calculate the dose of Imipramine for a mouse weighing 25g and find out the
volume of stock solution to be injected.
The weight of the mouse = 25g

The dose of Imipramine =10mg/kg

Amount of Imipramine required =Animal weight /1000g x Animal Dose
=25x10/1000 = 0.25mg
The stock solution provided is 10mg/ml
1mg of imipramine = 1ml of solution

0.25 mg of imipramine = 'x' ml of solution

X = 0.25 x 1/ 1 = 0.25m1.
*The dose to be injected to the animal is 0.25m1*
5. The dose of Nimesulide is 1mg/kg i.p. Calculate the dose of Nimesulide required for a mouse
weighing 22g. The stock solution concentration of Nimesulide is 1mg/ml. Calculate the volume
of Nimesulide to be injected in the mouse.

The weight of the mouse = 22g

The dose of Nimesulide =1mg/kg
Amount of Nimesulide required =Animal weight /1000g x Animal Dose
=22x1/1000 = 0.022mg
The stock solution provided is 10mg/m1
1 mg of nimesulide = 1ml of solution
0.022 mg of nimesulide = 'x' ml of solution
X = 0.022 x 1/ 1= 0.022m1.
Dose to be injected is 0.022m1.
Since this volume is too small for injection. It is advisable to dilute the stock solution 10
times
1 mg of nimesulide =10m1 of solution (dilution 10 times)
0.022 mg of nimesulide = 'x' ml of solution
X = 0.022 x 10/ 1= 0.22m1.

*0.22 ml is the dose to he injected the animal*

6. The dose of Phentolamine in mouse is 4 mg/kg i.p. Calculate the dose of Phentolamine for a

mouse of body weight 25g.Thc stock solution provided is 2mg/ml. Calculate the volume to be injected.

The weight of the mouse = 25g

The dose of Pentolamine =4mg/kg

Amount of Pentolamine required =Animal weight /1000g x Animal Dose
=25 x4/1000 = 0.Img
The stock solution provided is 2ing/m1
2 mg of Pentolamine = 1ml of solution

0.1 me, of Pentolamine = 'x' ml of solution

X= 0.1 x 1/2 = 0.05ml

Dose to be injected in 0.05ml

Since this volume is too small for injection. It is advisable to dilute the stock solution 10 times

2 mg of nimesulide =10m1 of solution (dilution 10 times)

0.1 mg of nimesulide = 'x' ml of solution
X = 0.1 x 10/ 2= 0.5 ml.

* 0.5 nil is the dose to he injected the animal*

EX•NO•: 2

EFFECT OF ANTI-ALLERGIC ACTIVITY OF TEST DRUG BY MAST CELL

STABILIZATION ASSAY
Principle

In allergic disease mast cells plays an important role by defending the antigens. IgE antibodies
formed in response to antigen antibody complex attaches to the surface receptors of mast cells and rises
calcium influx leading to degranulation of mast cells which releases some pro-inflammatory mediators (also known
as local hormones) such as histamine and eicosanoids.

Aim

Aim of this experiment is to screen anti-allergic activity of the drugs.

Requirements : Cal software

Albino rats of (175-200g) of either sex

Drugs: Disodium Chromoglycate (50mg/kg i.p)

Reagents

Saline solution (0.9%), RPMI -1640, Buffer medium, (pH: 7.2 to 7.4), Egg albumin-(100mg/m1),
Toluidine Blue solution- (1%)

Instruments

Microscope with 10x magnificent lense.

Procedure

Mast cell stabilization activity.

Albino rats either sex are divided into two groups consisting of 3 animals in each Group-1
receives normal saline and Group -2 receives Di-sodium Chromoglycate 50mg/kg i.p for 3 days.

Inject 10m1/kg of 0.9% Saline into peritoneal cavity on fourth day to each animal. Massage the
peritoneal region of the animal gently for 5mins, and then collect the peritoneal fluid and transfer to
the test tube which is carrying 7-10m1 of RPMI buffer. Centrifuge the fluid for 400-500rpm.
Discard the supernatant and wash the pellets of mast cells twice with same butler
by centrifugation. Add egg albumin to the above cell suspension and incubate at 37°C for 10mins.
Later the suspension and has to stain with 1% toluidine blue solution and observe the slide under
Microscope for calculating the number of granulated and degranulated mast cell in each group.

(Total 100 cell are has to be counted from different visual areas)
Observationt

TOTAL NO . OF CELLS

GROUP GRANULATED DEGRANULATED

1. SALINE 12.55 + 1.80 87.44 + 4.62

2. DISODIUM GLYCOLATE 72.25 + 3.91 28.14 + 1.34

50MG/KG I.P

Conclusion

Pre-treatment of animals with Standard drugs stabilizes mast cell membrane and generates
nitric oxide as defensive mechanism that inhibits the release of Chemokine's. which are responsible for
vasoconstriction.

Report

On exposure to Histamine, Saline treated animal ( Albino mice )

shows allergic response where antihistamine ( Anti-allergic ) drug
treated animal ( Albino mice ) do not show allergic response.
Ex. NO: 3
STUDY OF ANTI-ULCER ACTIVITY OF EAR RAT DRUG IN
CIMETIDINE IN PYLORUS LIGATED RAT (S I AY RAT et al.) METHOD.

Principle
Peptic ulcers major disease that affect human g.i.t. The common clinical features of peptic ulcer
are hyper acid secretion and ulcer formation in the stomach and duodenal part of the intestine.
Peptic ulcer is a hole or open sore in the lining of stomach and duodenum it result probably due
to imbalance between the aggressive [acid, pepsin, bile and H.pylori] and the defensive
(gastric mucus and bicarbonate secretion, prostaglandins, nitric oxide and high mucosal blood flow,
innate resistance of the mucosal cells) factors.

Pyloric ligation procedure was described by shay et al. the basis for this model is that
accumulation of buffered gastric acid over a certain length of time leads to peptic ulceration
in rats whose pylorus has been ligated.

Neutralization of gastric acid by antacids or inhibition of acid secretion by drugs like H2 receptor
Mockers, proton pump [H+1C ATPase] inhibitors, cytoprotective prostaglandin analogs and
anticholinergics, ulcerprotective agents are the main modes of pharmacological treatment of
peptic ulcer.
Requirements: Cal software

Animal: Rats -150- 200 g overnight fasted.

Drugs: Anaesthetic ether, Cimetidine- 10mg/kg i.p, prepares a stock solution containing
2mg/kg of the drug and injects 0.5m1/ 100g b/w of animal. NaOH (0.01N), Topfer's reagent, Collodion.
Equipment: Dissecting microscope (100X magnificent), pH meter, burette, surgical instruments.

Procedure
Anaesthetise the overnight fasted rat with anaesthetic ether. Secure the rat on the operating
table give an incision of 1 cm long in the abdomen just below the sternum. Expose the stomach
Pass a thread around the pyloric sphincter and apply a tight knot. While putting the knot care
should be taken so that no blood vessel is tied along the knot. Close the abdomen wall by putting
the sutures. Clean the skin from any blood spots and bleeding. Apply the collodian over the
Wound. Keep the rat in a separate cage and allow it to recover. Another rat inject Cimetidine
(10mg/kg) i.p. After 15 minutes perform pyloric ligation. After 4 hour of pyloric ligation sacrifice
both animals by decapitation. Open the abdomen and tie the Oesophageal end (cardiac end) of the
stomach cut and remove the entire stomach from the body of the animal. Give a small cut to the
Pyloric region just above the knot and collect the contents of the stomach in a graduated
 centrifuge tube. Open the stomach along the greater curvature and wash it slowly under
the running tap water. Put it on the slide glass and observe under 10X for ulcers.

Score the ulcers as below

0= normal coloured stomach

0.5 = red ulcers
1.5 = haemorrhagic ulcers
2 = ulcer> 3 but < 5
3 = ulcers > 5
Mean ulcer score for each animal is expressed as ulcer index.
Centrifuge the gastric contents 1000 RPM for 10 minutes. Not the volume. Pipette out 1ml
of supernatant liquid and dilute it to 10 ml with distilled water. Titrate the solution against 0.0 IN
NaOH using Topfers reagent as indicator. (Dimethyl amino benzene with phenolphthalein and
used for detection and estimation of HCL and total acidity in gastric fluids). Titrate to the
endpoint. The percentage increase in the ulcer protection was also calculated,

P1= UI of control group- UI of treatment group/ UI of control group X 100

ULCER SEVERITY GRADE

S. No DRUG Score 0 Scorel Score2 Score 3

ADMINISTERED
I CONTROL 0.5 1 2 7

2 TEST 0 0.5 1 1

The ulcer index [ U1 ] is calculated by the following equation

U1 = UN +US + Up * 10 -1

Where, UN=average of number of ulcers /animals

US=average of severity scores

UP=percentage of animals with ulcers
ULCER INDE

S.NO DRUG UN US UP
ADMINISTERED

1 CONTROL 5.5 0.69 39.95

2 TEST 2.5 0.31 44.23

Inference
Anti- ulcer drug limited in processes antiulcer activity and possibly at via
multiple mechanisms including of the H2 histamine receptors/ H+K+ -ATPase, PG
modulation or anti-oxidation.

Report:
Higher number of ulcer count in vehicle treated group than drug treated and
reflects the antiulcer activity of drug ( Cimetidine )

Vechicle score 2 = ulcer > 3 and < 5.

Drug ( cimetidine ) score 1 ( spot ulcers )

EX.NO : 4
STUDY OF DRUGS ON GASTROINTESTINAL MOTILITY
EFFECT S OF DRU GS ON T HE CI LIARY MOTI LIT Y OF FROG
ESOPHAGUS
Introduction

cholinergic drugs cause contraction of cilia leading to increased movements.

Anti-cholinergic drug causes paralysis of ciliary leading to decreased movements

Aim : To study the effect of physostigmine and atropine on ciliary movement in frog
oesophagus (buccal cavity.)

Principle

Cilia in the buccal cavity and in the oesophagus help in the movement of food
particles. Similarly, the importance of mucociliary function has been established, in
respiratory tract and of pulmonary diseases such as chronic bronchitis, asthma and in
cystic fibrosis. The integrity of mucociliary function is very important in these air- way
diseases. Cilia exhibit a great degree of autonomy in that they are capable of functioning in
the absence of nervous innervations.

It has also been demonstrated that the acetylcholine present in the mucous
membranes of trachea and buccal cavity helps in the ciliary movement. Acetycholine
serves as a local hormone and the presence of choline acetylase support the fact that
acetycholine is locally synthesized in the mucous membranes.

Requirements: Cal Software

 Animal- frog
 Drugs- acetylcholine stock solution (1µg/ml) physostigmine stock
solution (1µg/ml). Artropine stock solution (1µg/ml)
 Physiological solution -- Normal saline
 Equipment- Frog board, poppy seeds or tiny pieces of cork.

Procedure
I. Decapitate the frog and pin the frog to the frog board on its back
2. Pin the lower jaw to the abdomen cutting sufficiently the buccal cavity and
exposing the oesophagus. Keep the buccal cavity and the opening of the
oesophagus wet by irrigating it with normal saline.
3. Fix two parts i.e. from a point in the lower jaw to the beginning of the
oesophagus.
Keep this distance constant to measure the time taken by the particle to move
from a point in the lower jaw to the beginning of the oesophagus.
4. place a poppy seed or a small piece of cork at the premarked spot in the jaw. Turn on
the stop-watch and note the time taken by the object to reach the beginning of the
ocsophagus. Repeat this several times,
5. Put a few drops of acetylcholine on the buccal cavity and after 10 min repeat step 4.
Note the time

6. Put a few drops of physostigmine on the buccal cavity and after 10 min repeat step 4.
Note the time
7. Wash the buccal cavity. With normal saline. Put a few drops of atropine on the buccal
cavity. After 10 min repeat the step 4. Note the time.
8. Find out the difference in the time taken by the object to move between the pre-marked
distance in the buccal cavity in presence of saline, physostigmine and atropine.

Inference - Physostigmine reduces, and atropine enhances the time taken by the object to
move from the pre marked point in the lower jaw to reach the esophagus respectively.

S.no Drug Reading Reading Reading Mean

1 2 3
1 Salaine (control) 34 s 29 s 32 s 32 s

2 Acetylcholine 22 s 18 s 17 s 19 s

3 Physostigmine 14 s 14 s 14 s 14 s

4 Atropine 43 s 46 s 51 s 46 s

Interpretation of the results

The Most effective drug is Physostigmine,

Because the drug takes short time to increase the movement.

The order of the drugs depending on the shortest time as following

1. Physostigmine
2. Acetylcholine
3. Atropine

Report :

The drug ( acetylcholine shown increased motility

The drug ( atropine ) show decreased motility ( movement ) .
The drug (physostigmine ) show increased motility ( movement)
5
0
4
5
4
0
3
5
3
0
2
Salaine (control) Acetylcholine Physostigmine Atropine

Materials Picture
Materials pictures

Frog wooden board Poppy seeds

In Situ experiment
 PHYSIOLOGICAL SALT SOLUTIONS USED IN
ISOLATED TISSUE EX PERI MEWS

Aim : To study about the physiological salt solntion used in isolated tissue

experiments.

Need for physiological salt solutions (PSS)

Once a tissue or a muscle is isolated from the animal it can he maintained

and kept alive only by the use of physiological salt solution (PSS).

General composition

PSS contains various ions similar to the amounts in blood, plasma and
extracellular fluids. They help in maintaining the functions of different tissues. The
salt solution contains a mixture of cations, anions and glucose in distilled water. Some
of the anions and cations present in the PSS and their functions arc given below:

Sodium ions

It maintains the isotonicity of the fluid. In the absence of sodium ions, the
cells are incapable of producing action potential. Sodium ions affect the
contraction of the muscle indirectly by influencing calcium uptake.

Potassium ions
It is also necessary to produce action potential. The lack of potassium ions
increases the uptake of calcium. Excess of potassium has depolarizing action.

Calcium ions
It plays a role in maintaining the excitability of the membrane. Excess of ions
facilitate coupling and contractility.

Magnesium ions
Magnesium relaxes the tissue following contraction of tissue by drugs. Calcium
ions and magnesium ions are antagonists.

Glucose

It serves as a source of energy for the isolated tissue.

Chloride ions
It plays a part in maintaining the osmotic pressure of the salt solution.
 Bicarbonate and Phosphate ions

It serves as a buffer to maintain the pH of the PSS.

Some of the examples of Physiological salt solutions are Frog ringer, Ringer
Locke, Modified Kreb's, Tyrode, Kerb's Hensleit, DeJalon.

Composition of some physiological salt solutions

compoun Frog Ringer Ringer Locke De Jalon Tyrode Kerbs-Hensleit

NaCl 6 9 9 8 6.9

KCl 0.14 0.42 0.42 0.2 0.35

CaCl2 0.12 0.24 0.06 0.2 0.28

MgCl2 - - - 0.01-0.1 -

MgSo4.7H2o - - - - 1.28

NaHCO3 0.2 0.5 0.5 1 2.1

NaH2PO4 - - - 0.05 -

KH2PO4 - - - - 0.16

Gluose 2 1 0.5 1 1
 DOSE RESPONSE CURVE

Normally an increase in concentration or dose of drug shows an increase in

response to the drug. When the doses are plotted against the response, the curve
obtained is referred to as the dose response curve.
To plot the dose response curve, graded effects are obtained by
aministering varying concentration (or doses) of the drug. For an isolated tissue
experiment, the proceeding dose has to be washed before adding the next dose. At
least 4-5 doses are used to cover the range between the smallest and the largest
(maximal) response. Maximal response is that which does not increase with further
increase in dose.

When doses are plotted against the response directly, the DRC is a
hyperbola. But when the log dose are plotted against the response, the MC is
sigmoid or "S" shaped with a linear portion in the middle portion. The lower and
upper parts of the curve arc relatively flat while the middle portion is steep. This
linear portion is the most sensitive part of the curve and working doses are
calculated from this region.

How to draw n log dose response curve?

The log of doses is plotted on the X axis or the doses can he directly
plotted on a log paper. The response for each dose is measured and this is plotted
on the y axis against the corresponding doses.
The log transformation has the following advantages:

 Results can be plotted even when the doses vary over a 1000 IOW range.
 It helps to compare effect of 2 dose response curves. When the lines are

parallel, the distance between the 2 lines is a measure of potency ratio of the 2

drugs.

The DRC is characterized by 3 parameters:

1. Maximal or ceiling effect: Referred to as the E max or efficiency.

2. Position of the curve in relation to the abscissa: When the curve is more to the left, it
is more potent and vice versa.
3. Slope of the curve: When the slope is steeper it indicates that the discrimination
between doses is good which makes the assay more precise.
 GENKRAL PROCEDURE FOR MOUNTING OF GUINEA PIG ILEUM PREPARATION

 prepare sufficient quantity of physiological salt solution. Fill up the reservoir and the tissue bath with this
solution.
 Attach the frontal Writing lever onto the fulcrum and balance it by adding plasticine at one encl.

 Adjust the fulcrum in such a manner that a magnification of 7-8 is maintained.

 0,5 g tension is applied to the lever on the arm between fulcrum and writing point at a distance equal to
that of the distance between fulcrum and point of tissue attachment to provide the required tension to
the tissue.
 Sacrifice the animal by cervical dislocation or stunning method.

 Cut open the abdomen and identify the colon. The right flexure, i.e., the sub-hepatic region of the
colon where the ascending colon turns to become the transverse colon (ilea-caecal junction) or
the ileum is cut out and placed in the shallow dish containing Tyrode solution which is aerated.

 The mesenteric layer of the ileum / colon is removed and a tissue measuring about 2-3 cm is cut
from this. The preparation must not be pulled at any time and should be handled gently. The lumen
is gently cleaned.

 One end of the tissue is tied with the tissue holder (J-tube) which is mounted inside the organ
bath containing Tyrode solution maintained at 37°C and the other end of the tissue is connected to the
lever. The tissue may get damaged if it is attached to a lever which is not properly balanced. The
aeration speed should be adjusted to an optimal level.

 The preparation is allowed to stabilize for 30 min

 For recording the response

The drum speed is adjusted in such a way that it rotates at a speed of 0.12mm/s.

A 4 minutes time cycle is maintained for proper recording of the responses for this tissue. A
contact time of 30 sec is followed as rat ileum/colon is a slow contracting tissue

 0min : start the kymorgraph

 30 sec : Add the Ach

 1 min : stop the kymograph and wash the tissue 3 times at time interval

 3mins : start the kymograph

( if the tissue has not fully recovered, it ,may be necessary to wash the tissue again before
proceeding for the next dose.)
 EX NO: 05
EFFECT OF AGONIST ON DODE RESPONSE CURVVE OF
ACETYLCHOLINE USING GUINEA PIG ILEUM
Aim
To study the potentiating effect of Neostigmine on acetylcholine response using Guinea pig ileum.
Principle
Guinea pig ileum is an intestinal smooth muscle.

,Acetylcholine acts by holding with the cholinergic receptors present in the intestine and produces
contractions. Potentiation is a phenomenon in which two drugs when combined together. Produce an
effect which is greater than the sum of the effects of the individual drugs.

Neostigmine is a reversible anti-cholinesterase drug which prevents the metabolism of acetylcholine by

inhibiting the enzyme acetyl-cholinesterase and potentiates the action of acetylcholine. This is seen as
potentiation of response of acetylcholine in the presence of neostigmine.

Requirement : Cal software

Animal: Guinea pig
Tissue used: Ileum/colon
Physiological Salt Solution: Tyrode solution
Lever: Frontal writing lever
Drug : Acetylcholine stock solution ( 1 mg/ml). Neostigmine (1 mg/ 1 ml )

Procedure

 Set up the tissue as described in" General procedure for mounting Guinea pig ileum_
 From the stock solutions of Ach and Neostigmine, dilutions are suitably made to get 100µg/ml and
10µg/ml solutions.
 After the tissue stabilizes for 30 mins, the response of increasing doses of Ach are recorded i.e
1,2,4,8,16µg.
 Add neostigmine to the inner bath at a concentration of 2µg/ml and allow the tissue toirrigate in this
solution for 20 mins. (e.g lithe bath volume is 20 ml the amount of neostigmine required is 2x20=40
µg.)
 From the concentrations of Ach tried before, choose any one concentration which shows a good
contraction and add this to the inner bath without washing the neostigmine and record its
contraction.
 Wash the tissue with the tyrode and continue to record the contractions of the same concentration
of Ach till the response of Ach shows the original response

Report
EX NO: 6

EFFECT OF ANTAGONIST ON DRC OF ACETYLCHOLINE

USING GUINEA PIG ILEUM

Aim

To study the effect of atropine on acetylcholine response using guinea pig ileum.

Principle

Guinea pig ileum is an intestinal smooth muscle and is rich in M3 and H1 receptors acetylcholine the
contractions 01 the smooth muscle by acting on muscarinic receptors (M3).

Atropine is a muscarinic receptors blocker in the smooth muscle. It competitively blocks the
action of Acetylcholine. This is seen as inhibition or total block of the contractions produced by
acetylcholine on rat ileum/colon.

Requirements: CAL software

Animal: Guinea pig
Tissue Used: Ileum/colon
Drug : Acetylcholine stock solution ( 1 mg/m1), Atropine stock 1mg/m1
Physiological Salt Solution: Tyrode solution
Lever: Frontal writing lever

Procedure

 Set up the tissue as described in "General procedure or mounting Guinea pig ileum.
 From the stock solutions of Ach and Atropine, dilutions are suitably made to get 100µg/m1 and
10µg/m1 solutions.

 After the tissue stabilizes for 30mins, the response of increasing doses of Ach are recorded i.e 1, 2,
4,8,16µg.

 Add Atropine to the inner bath at a concentration of 21.1g/m1 and allow the tissue to irrigate in this solution
for 20mins. (e.g if the bath volume is 20 ml , the amount of Atropine required is 2x20=40 µg.)

 From the concentrations of Ach tried before, choose anyone concentration which shows a good
contraction and add this to the inner bath without washing the Atropine and record its contraction.
 Wash the tissue with the Tyrode and continue to record the contractions of the same concentration of Ach
till the response of Ach shows the original response.

Report
EX NO : 7

EFFECT OF SALINE PURGATIVE ON FROG'S

INTESTINE
Aim
Effect of saline purgative on Frog's intestine.

principle

Increase in the volume of content of the bowel stretch the colon and produce normal stimulus and contraction
of muscles. Salt comprising of highly charged ions. It does not cross cell membranes freely. So they remain inside
the lumen and retain water through osmotic forces. Increase the volume of content of bowel. Stretch the colon
and produce normal stimulation and contraction of muscle leads to defecation.

Requirements: CAL Software

Animal : Frog
Hypotonic solution: 0.9-0.45% saline
Hypertonic solution: 27% (Magnesium sulphate)
Isotonic solution —Frog Ringer solution
Instruments: Frog board, Pithing needle, dissecting board, Tuberculin syringe with needle.

Procedure
Pith the Frog and place it on dissecting board. Expose the abdominal cavity and carefully trace the
small intestine. Make the small intestine 3-compartment by tying threads of different colour. Such

a way that no fluid can move to other compartment.

Inject the 0.2 ml of hypotonic solution on compartment-I (saline)

Inject the 0.2 ml of hypertonic solution on compartment-II(Magnesiumsulphate)

Inject the 0.2 ml of isotonic solution on compartment-III (Frog Ringer solution) wait for
20mins after injection and observed are to be recorded.
 Observation

DRUG COMPARTMENT EFFECT

Normal saline (0.9%) I SHRUNKEN

Magnesium Sulphate (27%) II SWOLLEN

Frog ringer solution III NO CHANGE

Conclusion
HYPOTONIC - It’s causes the fluid to move from lumen in to circulation by process osmosis there by SHRINKS
the tissue.
HYPERTONIC- Its moves the fluid from cell in to the lumen and SWELLS the tissue
ISOTONIC - It doesn't shows any fluid movement across the Intestinal membrane

Report:
EX NO : 8

EFFECT OF INSULIN HYPOGLYCENIIC IN RABBIT

Bioassay: Biological assays or "bio-assays - are a set of techniques for estimating the potency or strength of an "agent"
or "stimulus " by utilizing the "response- or "effect- on biological system OR experimental living subjects.
Principle of Bioassay: The basic principle of bioassay is to compare the test substance with the standard preparation
of the same and to find out how much test substance is required to produce the same biological effect, as produced by
the standard.
Bioassay experimental Models/Methods for Insulin
Rabbit Model: Observation of hypoglycemia effect
Albino Rats/Mice model -Observation of hypoglycemic effect and hypoglycemic convulsion.
Isolated rat diaphragm model- observation of glycogen contents
Rat's Epididymal Fat model- observation of glucose metabolism.
Standard solution
Standard solution is prepared by using pure, dry, crystalline insulin and One unit contains 0.04082mg or 40.82
microgram. This unit is specified by Ministry of Health, Government of India and is equivalent to international unit (IU).

Standard and Test sample dilution: I U/m1 and 2 U/ml solution is freshly prepared from stock solution (20U/m1) of
standard and test solutions.

Principle: The potency of test sample is estimated by comparing the hypoglycemic effect of the sample with that of the
std. preparation of Insulin.

Animal selection
Experimental protocols should be approved prior to the Experiment by IAEC *(Institution animal Ethics committee)

Thereafter, healthy rabbits (2-3kg) are selected and habituated for the experiment in standard laboratory environment
condition as per the norms of CPCSEA* (Committee for the purpose of Control and Supervision of Experiments on
Animals). Govt. of India.
After that animal are kept in fasting for 18hr before performing the assay. Water is withdrawn during the experiment.

*IAEC and CPCSEA are the committee constitute in India

.
Preparation of standard solution for Bioassay
20unit (40.82 x 20= 816.4 microgram) pure insulin is dissolved in small volume (<1m1) normal saline and acidify it with HCL to pH
2.5. 0,5% phenol is added as preservative.
Add 1.4% to 1.8% glycerin. Final volume should contain 20units/ml. Store the solution in a cool place and use it within six months.

Preparation of test solution for bioassay : same as standard solution.

Rabbit Model: Experimental protocol/ procedure

Group No Group Name Treatment

I Std-1 Std-1 dilution, s.c
II Std-2 Std-2 dilution, s.c
III Test-1 Test-1 dilution, s.c
IV Test-2 Test-2 dilution, s.c

Animals are divided into 4 groups and each containing 3-5 animals.
1 .First Part of test: A. "Initial Blood sugar Level"
A sample of blood is withdrawn from marginal ear vein of each rabbit and examine the reducing blood sugar ( mg/ml) before insulin
treatment.

B. "Final Blood sugar Level"

After standard and test insulin injection from each rabbit , a sample of blood is withdrawn up to 5 hrs. at the interval of 1 hr. each blood
sugar is determined again. This is known as " Final Blood Sugar Level".
2.Second Part of Test: Cross Over Test
The same animals are used for the second part after one week. Again animals arc fasted and initial blood sugar is determined. The
grouping is reversed, means those animals which received the standard are given the test and those which received the test are now
given the standard. Those animal which received the less dose of standard are given the higher dose of the test sample and vice-
versa

I Std-1 Std-1
II Std-2 Std-2
III Test-1 Test-1
IV Test-2 Test-2
Observation:
"Hypoglycemic effect" rabbit are put in an air incubator at 30"C and observed for one and half hr. The rabbit which produce hypoglycaemic effect are
taken out the incubator and observed. The percentage (%) hypoglycaemic effect of test is compared with the standard.

Report
EX NO : 9
RABBIT PYROGEN TEST

Aim: The aim is to test the rabbit pyrogen test.

Principle:
The pyrogen refer to all the substance that cause as increase in fever, also known as pyroxia.
Upon entering into contact with pyrogens, rabbit's have an increase in their temperature, just humans.
The pyrogen test is aim to check the existence of pyrogen by using rabbits. Based on measurement of the
increase in the rabbit's temperature upon being injected with a product t might contain contaminant of
the pyrogen type.

Requirement: CAL Software

Drugs: Quantity of injection: Unless otherwise specified, 10 ml of the sample per kg of bo mass of
the animals is used /injected. Rabbit must be healthy and mature. New Zealand or Belgi white's rabbit
breads are used for the test. Testing rabbits must be individually housed between 20°C to 23°C
temperature. The temperature should not increase or decrease more than ± 3°C.
 Selection of Animal: Healthy, adult rabbit need to be selected for the test.
and the minimum weight of the rabbit should not be less than 1.5kg.
 Housing of Animal: Testing rabbits should not be kept in the places like
environmental problems, noise areas, and presence of strangers (unknown places).
Equipment and material used in testing: Glassware, Syringes, Needles.

 Retaining Boxes: The box should be comfortable for the rabbits.

 Thermometers : Standardized position in rectum should be precision of 0.1°C

PROCEDURE:

Perform the test at an environmental temperature similar to that of the room where in the
animal are housed. Withhold food from the test animals before and during the period of
experimentation.
Trake 3 rabbits and fixed them in rabbit holder. Insert the rectal thermometer into the rectum
of the test animal to a constant depth in the Range of 60 to 90 mm, and read the temperature after a
sufficient period of time. Determine the temperature of the Test animals 3 times at 1 hours intervals
before the injection of sample.
When the 2nd and 3rd temperatures showed little difference, the latter is taken as the control temperature. Do
not use animals whose 2nd and 3rd temperatures exceed 39.80C even if these two values are similar.
When the sample to 37°C before injection, and administer intravenously through an ear vein
hour 15 minutes after the third temperature recording. Read the temperature 3 times at 1 hour
intervals after injection. Difference between the control temperature and the highest temperature
is taken to be the rise in body temperature.
 Sr. O b se r v at i o n Inference
NO
1 112 or 3 rabbits show an individual Positive
rise of> 0.6 °C than respective control
temperature
If only I animal shows a temperature Repeat the test on a group of
2 rise of> 0.6 C . if the sum of the
0 five other rabbits
temperature rises of the 3 animals >
1.40C
If 2 or more of the 5 rabbits show an Positive
3 individual temperature rise of> 0,6 ° C

Conclusion: When the pyrogen test is positive, the sample is considered to be

rejected.

Disadvantages/ Limitations:

It is a long test. It is an inadequate method to determine pyrogen in medicines,

such as steroids etc. It is qualitative test only not quantitative.

Preliminary test (sham test)

ntravenous injection of sterile pyrogen-free saline solution to exclude any

animal showing an unusual response to the trauma (shock) of injection. Any

animal showing a temperature variation greater than 0.6°C is not used in them

an in test. All glassware, syringes and needles must be pyrogen free by heating

at 250°C for not less than 30min_ warm the pyrogen free solution up to 38.5°C.

Interpretation of result

No of Rabbits Individual Temperature Test Result

Temperature rise Rise in group

3 Rabbits 0.60C 1.8 Pass

If above 3 not pass
3+5 = 8 rabbits 0.610C 4.88 Not Pass

If above test not passes the sample is said to Pyrogenic

Report :
Ex. No: 10

DETERMINATION OF ACUTE ORAL TOXICITY (LD50)

Aim

To determine the acute oral toxicity ( LD50 ) of a given drug

Principle

The determination of ED50 (the dose effective in producing certain expected

response in 50% of the animal group) values helps in ascertaining the potency of a
drug in terms of a reference standard. The calculation of ED 50 value is done when
a drug is showing graded response. But when the response is quantal or all-or-done,
the ED50 value becomes LD50 (the dose lethal to 50% of the animal group). Both these
values i.e., ED 50 and LD 50 arc important for knowing the safety of a drug. The ratio
between LD50 and ED50 (LD 50 /ED 50 ) represents therapeutic index. Greater the
therapeutic index, safer is the drug. The therapeutic index of most of the drugs which
have a low margin of safety is generally close to unity. Ideally one would like to
determine a dose that is effective in most of the animals (ED99) and least toxic to most
of the animals of the group (LD50). These values can, however be calculated from the
graph.

There are various methods used to calculate LD 50 values viz, the graphical
method, arithmetical method and the statistical approach. For research purpose, the
most widely used method is that of Litchfield and Wilcoxon (1949). For the routine
practical Glasswork, Read and Muench. Miller and Tainter or the Karher's method can
be used. For calculating LD50 by either method find out the least tolerated (smallest)
dose (100% mortality) and most tolerated (highest) dose (0% mortality) by hit and trail
method. Once these two doses are determined, select at least 5 doses in between the
least tolerated doses, and observe the mortality due to theses doses. Generally. mice
are used for this purpose and each dose group should consist of 10 animals. One can
determine the LD 50 value by different Routes of administration. The LD 50 value of a
new drug is determined by oral as well as by one of the parenteral routes (ip, iv
or im) of administration.
OBSERVATION

Procaine hydrochloride toxicity data in mice

GROUP DOSE LOG DOSE DEAD/TOTAL %DEAD CORREDTED PROBIT

1. 68 1.83 0/10 0 2.5 2.04

2. 75 1.87 1/10 10 10 3.72

3. 91 1.96 2/10 20 20 4.16

4. 110 2.04 6/10 60 60 5.25

5. 146 2.16 9/10 90 90 6.28

6. 161 2.21 10/10 100 97.5 6.96

Apply correction for 0% and 100% dead.

(observe the mice for 2 hour for death or recovery)

0% dead = 100(0.25/n); 100% dead = 100(n-0.25/n)

n= total no of animals

Requirements
 Animal mice (20- 25g)
 Drug procaine hydrochloride.

Procedure
1. Use overnight fasted mice. Each dose group should consist of 10 mice.
2. Inject the drug by intraperitoneal route and observe the animal for 2 hours for death due to
acute toxicity
3. The table below gives the results of a LD50 study done with procaine in mice using Miller
and Tainter (1944) method. Note that a per cent mortality values are converted to probit
values by reading the corresponding probit units from the probit table.
4. Plot the probit values against log does and read LD50 values as the dose that corresponds
to probit value
Report
 EX.NO: 11

DETRMINATION OF ACUTE SKIN IRRITATION

Principle

 The test chemical to be tested is applied in a single dose to the skin of an experimental animal, untreated skin
areas of test animal serve as the control.
 The degree of irritation / corrosion is read & scored at specified intervals.
 Animal showing continuing signs of severe distress and / or pain at any stage of the test should of the
humanely killed and the test chemical assessed accordingly.

Requirements: CAL Software

 Animals should be individually housed.

 Rabbit at least 12 weeks old and body weight 1.5kg to 3.0kg.
 The temperature should be 20°C for rabbit.

Preparation of animals

 Approximately 24 h before the test, fur should be removed by closely clipping the dorsal area of the trunk
of the animal.
 Care should be taken to avoid abrading the skin, and only animals with healthy, intact skin should be used.
 Some strains of rabbit have dense patches of hair that are more prominent at certain times of the year. Such
areas of dense hair growth should not be used as test sites.

Procedure

 The test chemical should be applied to a dorsal / flank region (approximately 6cm2) of skin
and covered with gauze patch.
 The test chemical should be applied to the gauze patch, which is then applied to the skin.
 The patch should be loosely held in contact with the skin by the means of a suitable semi-occlusive dressing for
the duration of the exposure period.

Dose level: A dose of 0.5 ml of liquid or 0.5g of solid or paste is applied to the test cycle
Initial test (using one animal)

 Up to three test patches are applied sequentially to the animal.

 The first patch is removed after 3 minut
  If no skin reaction is observed, a second patch is applied at a different site and removed after
one hour.
 If the observation at this stage humanely he allowed to extend to 4 hours, a third patch is
applied and removed after 4 hours and the response is graded.
 If a corrosive effect is observed after the 3 Sequential exposures, the test is
immediately terminated.
 If a corrosive effect is not observed after the last patches removed, the animal is observed for
14 days, unless corrosion develops at an earlier time point.
 In those cases in which the test chemical is not expected to produce corrosion but
may be irritating, a single patch should be applied to one animal for 4 hours.
Confirmatory test (with additional animals)
 If a corrosive effect is not observed in the initial test, the irritant response should be
confirmed using up to two additional animals each with one patch, for an exposure period of
four hours.
 If an irritant effect is observed in the initial test, the confirmatory test is may be conducted
by exposing two additional animals simultaneously.

Clinical observations
All animals should be examined for signs of erythema and oedema and responses scored at 60
mins and then 24, 48 & 72 hours after patch removal.

GRADING OF SKIN REACTIONS

ERYTHEMA AND ESCHAR FORMATION

ERYTHEMA SCORE
No erythema 0
Very slight erythema 1
Well defined to severe erythema 2
Moderate to severe erythema 3
Severe erythema to Escher formation 4

OEDEMA FORMATION
OEDEMA SCORE
No oedema 0
Very slight oedema 1
Slight oedema 2
Moderate oedema 3
Severe oedema 4

Report :
EN.NO: 12

DETERMINATION OF ACUTE EYE IRRITATION

Aim
To determine the acute eye irritation using albino rabbits.

Preparation of animals

Albino rabbits are preferentially used for acute eye irritation test. Both eyes
of each experimental animal selected for testing should be examined within 24 hours
before testing starts. Animals showing eye irritation, ocular defects, or pre-existing
corneal injury should not be used.
Housing and feeding conditions
Animals should be individually housed. The temperature of the experimental
animal room should be 20°C (± 3°C) for rabbits. Although the relative humidity should
be at least 30% and preferably not exceed 70%, other than during room cleaning, the
aim should be 50-60%. Lighting should be artificial, the sequence being 12 hours light,
12 hours dark. Excessive light intensity should be avoided. For feeding, conventional
laboratory diets may be used with an unrestricted supply of drinking water.

Test procedure

Use of topical anesthetics and systemic analgesics

The following procedures arc recommended to avoid or minimize pain and

distress in ocular safety testing procedures. Alternate procedures that have been
determined to provide as good or better avoidance or relief of pain and distress may
be substituted. Sixty minutes prior to test substance application (TSA), buprenorphine
0.01 mg/kg is administered by subcutaneous injection (SC) to provide a therapeutic
level of systemic analgesia. Five minutes prior to TSA, one or two drops of a topical
ocular anesthetic (e.g. 0.5% proparacaine hydrochloride or 0.5% tetracaine
hydrochloride) are applied to each eye. In order to avoid possible interference with the
study, a topical anesthetic that does not contain preservatives is recommended. The
eye of each animal that is not treated with a test article, but which is treated with
topical anesthetics, serves as a control. Users should be aware that multiple
applications of topical anesthetics could potentially cause a slight increase in the
severity and/or time required for chemically-induced lesions to clear.

Application of the test substance

The test substance should be placed in the conjunctival sac of one eye of each
animal after gently pulling the lower lid away from the eyeball. The lids are then gently
held together for about One second in order to prevent loss of the material. The other
eye, which remains untreated, serves as a control.
Irrigation
The eyes of the test animals should not be washed for at least 24 hours
following instillation of the test substance, except for solid, and in case of
immediate corrosive or irritating Acts. Cond it ions of was hing shou ld be
carefu lly document ed, e.g., time of washin g; composition and temperature of'
wash solution; duration, volume, and velocity of application.
(I) Testing of liquids

For testing liquids, a dose of 0. I ml is used. Pump sprays should not he

used for instilling t he substance directly into the eye. The liquid spray should
he expelled and collected in a container prior to instilling 0.1 ml into the eye.
(2) Testing of solids

When testing solids, pastes, and particulate substances, the amount used
should have a volume of 0.1 ml or a weight of not more than 100 mg. The test
material should be ground to a tine dust. The volume of solid material should be
measured after gently compacting it, e.g. by tapping the measuring container. If
the solid test substance has not been removed from the eye of the test animal by
physiological mechanisms at the first observation time point of 1 hour after
treatment, the eye may be rinsed with saline or distilled water.

Initial test (in vivo eye irritation/corrosion test using one animal)

It is strongly recommended that the in vivo test be performed initially using

one animal. Observations should allow for determination of severity and reversibility
before proceeding to a confirmatory test in a second animal. If the results of this
test indicate the substance to be corrosive or a severe irritant to the eye using the
procedure described, further testing for ocular irritancy should not be performed.

Confirmatory test (in vivo eye irritation test with additional animals)
If a corrosive or severe irritant effect is not observed in the initial test, the
irritant or negative response should he confirmed using up to two additional
animals. If an irritant effect is observed in the initial test, it is recommended that
the confirmatory test be conducted in a sequential manner in one animal at a
time, rather than exposing the two additional animals simultaneously. If the
second animal reveals corrosive or severe irritant effects, the test is not continued.
If results from the second animal are sufficient to allow for a hazard classification
detemiination, then no further testing should be conducted.

Observation period
The duration of the observation period should be sufficient to evaluate
fully the magnitude and reversibility of the effects observed. However, the
experiment should be terminated at any time that the animal shows signs of
severe pain or distress. To determine r eversibility of effects, the animals should be
observed normally for 21 days post administration of the test substance. If
reversibility is seen before 2ldays, the experiment should be terminated at that
time.
Evaluation of acute eye irritation

The ocular irritation scores should be evaluated in conjunction with the nature and severity
of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an
absolute standard for the irritant properties of a material, as other effects of the test material arc
also evaluated. Instead, individual scores should be viewed as reference values and are only
meaningful when supported by a full description and evaluation of all observations.

Observation
OPACITY OF EYE Score

No ulceration / opacity 0

Scattered or diffused opacity l

Translucent cornea, iris obscured 2

Opalescent cornea, iris not visible, pupil size is 3

not discernible

Opaque cornea, iris not discernible 4

Report
EX PT : 13

PHARMACOKINETIC PARAMETERS CALCULATION

1. NSAID- Aceclofenac sodium was administered at the dose of 100mg to the patient. The
plasma drug concentration at various time periods as follows: Calculate Cmax , tmax , & AUC..

Time Time (Hrs) Plasma Drug Conc.(µg/m1)

t0 I 16.50 Co
t1 3 56.84 C1
t2 5 18.10 C2
t3 7 15.05 C3

t4 9 3.5 C4
t5 12 0.0 C5

Calculation
1. Cmax = 56.84µg/m1
2. Tmax = 3hrs
3. AUC =

AUC calculation by Trapezoid rule; AUC between l and 3 hours, 3&5 hours, 5&7hours, 7&9 hours,
9&12hours calculated by following equation

[ AUG ]tn tn – 1 = Cn-1 + Cn /2 ( tn – tn-1)

Result: AUC = 205.23 µg / ml / hour

2. An IV injection of antibiotic of dose 100mg/k body weight administered to patient.
A blood sample withdrawn at different time intervals and drug content is
estimated.

Time Time ( Hrs) Plasma Drug Conc. ( µg/ ml )

t0 0x0 0 y0C0
t1 1x1 21 . 2 y 1C 1
t2 3x2 10.1 y 2C 2
t3 6x3 7.4 y 3C 3
t4 8 x4 3.9 y4C 4
t5 10 x 5 2.1 y5C 5

Find out the slope, Half- life, cmzx , tmax ., rate constant K of IV inj of antibiotic;

Calculation
Slope : log y2-log y1/x2-x1
Rate Constant (k) = -slope x 2.303
Half Life= t1/2 = 0.693/K

Result
Slope 1 = -0.161
K = 0.3707
T1/2 =1.86hrs
C max =21.2
Tmax = 1 hour
3. The following data obtained after oral administration of anti-viral drug to
the patient having common cold

Time Time(Hrs) PlasmaDrug Conc. (µg/ml)

T1 1 2.5c 0
T2 2 6.6c1
T3 3 4.2c2
T4 4 2.9c3
T5 5 1.5c4
Find out Cmax ,t max & AUC.

Calculation
Cmax= 6.6 µg/m1
tmax, = 2hours
AUC =

AUC calculation by Trapezoid rule; AUC between land 2 hours, 2&3 hours, 3&4hours, 4&5
hours, calculated by following equation

[ AUG ]tn tn – 1 = Cn-1 + Cn /2 ( tn – tn-1)

Report: AUC = 15.7 pg/ml/hour

•
4. At a dose of 10mg/kg bodyweight, IV injection of ceftriaxone administered to the
patient. The following data obtained us follows:

Time (firs) Plasma Drug

Conc.(µg/ml)

0x0 0 yo
4 xl 25.5 y1
8x2 12.0 y2
12x3 6.5 y3
16x4 3.15 y4
20x5 1.5y5

Calculate Cmax, tmax Slope, t1/2, Rate constant (K)

Calculation
Slope: log y2-log y1/x2-xi

Rate Constant (k) = -slope x 2.303

Half Life= t 1/2= 0.693/K

Result
Slope= -0.0817
K =0.188
T1/2= 3.68hrs
Cmax= 25.5 µg/ml
Tmax= 4 hours
 BIOSTATSTICS METHODS

STANDARD DEVIAION
Definition: In statistics, standard deviation is a measure of how spread out a set of data is around
its mean (average). It indicates how much individual data points deviate from the mean. It’s often
denoted by the Greek letter sigma (σ),

σ = (√ð Ex2—(Ex)2 / n) ÷ n-1

Find the standard deviation of ESR Erythrocyte sedimentation rate, found to be 3,4,
5.2,4,5.3 and 4 in 8 normal individuals.
SD = (√ð Ex2—(Ex)2 / n) ÷ n-1

EX = 3+4+5+2+4+5+3+4 = 30

EX -2 = 9+16+25+4+16+25+9+16 =120

SD = √120-(30)2/8
8-1

= √120-112.5/7 = √7.5/7 = √1.07 = 1.03

Result: Standard deviation= 1.03

Find the standard deviation of ESR Erythrocyte sedimentation rate, found to bet,
2, 3.4,2,3 and 1 in 7 normal individuals

SD = √EX2--(EX)2 / n
n-1

EX= 1+2+3+4+2+3+1 = 16

EX2= 1+4+9+ 16+4+9+1 = 44

SD =√44-(16) 2 /7 ÷ 7-1

= 0.4/6 = 1.1126

Result: Standard deviation = 1.1126

Standard Error of Mean

Definition: Standard error of the mean (SE) In a small sample size the arithmetic mean would be an
approximation of the "true mean" of the whole popualtion, and therefore, subjected to error. In such
cases the error of the observed mean is calculated. The SE allows finding out the range in which the true
mean would lie. The extent of the error is inversely proportional to the square root of the number of
observations (n) and is directly related to the extent of the standard deviation. The value of Standard
decreases as the sample size increases.

SEM = SD / √n

Where SD is the standard deviation of the sample and n is the sample size.

I. Standard error of the Mean Calculation:

Systolic blood pressure of 566 males was taken mean BP was found to be

128.8mmhg and SD 13.05mm. Find 95% confidence limits of BP within the population mean
would lie. Calculate the SEM.

SEM = SD/ √n 13.05 / √566 = 0.55

95% confidence limits

Mean BP + 2SE = 128.8+2 x0.55 = 129.9

Mean BP — 2SE= 128.8-2x0.55=127.7

II. Systolic blood pressure of 266males was taken mean BP was found to be
124.4mmHg and SD 15.05mm. Find 95% confidence limits of BP within the
population mean would lie. Calculate the SEM.

SEM = SD/ √n 15.05 / √266 = 15.05/16.31 = 0.922

95% confidence limits

Mean BP + 2SE = 124.4 + 2 x 0.92 = 126.24

Mean BP — 2SE= 124.4-2x0.92= 122.56

ES.N0: 14
STUDENT "T" TEST

Definition : Student's t-test is a statistical test used to test whether the difference between the response of two groups is statistically
significant or not. The Student's T test can only he used when the data in each sample can Said to distributed normally around the Mean.
This means the frequency of data at different values would form a bell-shaped curve when drawn as a graph.

1. Calculation:

Two hatches of capsules A and B are prepared by two processes. Samples of 6 capsules from each batch are subjected to
disintegration time of the Indian Pharmacopoeia. Does a significant difference exist between the means of the two batches?
The data is given in table. Disintegration time in minutes of 6 capsules of the batches A & B
S.No Batch A
Batch B
1 10.5 6.5
2 11.2 7.1
3 12.6 6.8
4 14.0 7.5
5 14.7 7.1
6 12.8 6.8

t = XmA-XmB
ʃ SA2/nA + ʃSB 2/nB
Mean XmA= 12.63
XmB= 6.967
Standard Deviation= (√Ex2—(Ex)2/n)÷n-1
Standard Deviation of Batch A
EXA2 = 110.25 + 125.44+158.76 + 196+ 216.09+163 = 970.38
(
EXA2) =10.5+11.2+12.6+14.0+14.7 +12.8 = (75.8)2 = 5745.64
S.D A= Route of 970.38 — 5745.64/6
n- 1
Route of 970.38-957.61/5
= Route of 12.77/5 Route of 2.554 = 1.598
S.D A = 1.598
Standard Error of mean A = S.D /In = 1.598/16 = 0.6525

Standard Deviation of Batch B

EX B2 = 42.25+50.41+46.24+56.25+50.41+46.24 = 291.8
 (EX)2 = 6.5 + 7.1+6.8+7.5+7.1+6.8 =(41.8)2 = 1747.24

SD B = Route of .291.8-1747.24/6 =√29I.8- 291.2/5

5
=ʃ0.12 = 0.3464

Standard Error of meanB = 0.3464/ /6 =, 0.1414

XmA -XmB
t=
ʃSA/nA+ SB/nB

XmA = 12.63 XmB = 6.967

SA=1.598 SB = 0.3464

t= 12.63-6.967
ʃ (1.598)2 /6 + (0.3464)2/6

5.663
ʃ0.4455

t= 8.485

Degrees of freedom (DF) = ni+n2-2 = 6+6-2=10

REPORT:
The hypothetical t value from the t —table at 5% significance level and 10 degrees of freedom = 2.228

Calculated "t" value =8.485

The calculated value of "i" is more than the hypothetical "t" value given in the table. The difference is
significant. Null hypothesis is not applicable.

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 2. Student "t" Test Calculation
The table shows gain In weight of two lots of young rat ( 28.80 day old )
maintained on two different diets ( high and low protein). Calculate whether the
change in the body weight observed is due to diet or not.
Gain in actual body weight (g)
SI.No Group-1 Group-11
I 134 70
2 146 118
3 104 101
4 119 85
5 124 107
6 161 132
7 107 94
8 83
9 113
10 129
11 97
12 113

n=12 EX1=1440 n = 7 EX2 =707

Mean X1=120 X2 = 101

Standard deviation of Group I

= (ʃEx2 — (Ex)2/n) ÷n-1-ʃ177832-(1440)12/11

= ʃ177832-2073600/12 =ʃ177832- 172800

11 11

= ʃ5032/11 = √4457.45

Standard Deviationi=21.38

SEI=SD/ʃn = 21.38 = 21.38/3.4

√12

Standard Error of mean1= 6.172

EX No.15

ANALYSIS OF VARIANCE (ANOVA)

Definition: Analysis of Variance (ANOVA) is a statistical test used to compare the means of two or
more groups. It helps determine if there's a statistically significant difference between the means
of those groups. Essentially, ANOVA analyzes the variability within each group compared to the
variability b etween the groups to see if the differences are likely due to chance or a real
effect.
Treatment of animals with four different drugs (Drug A, B, C & D) causes alteration in the brain tissue
concentration of acetylcholine (ng/mg). Is there any significant difference observed between the treatment groups?
The control group (E) received only the vehicle. The data is computed in the table.
Control E Drug A Dug B Dug C Dug D Total
17 19 18 20 24
21 22 16 23 28
19 25 17 25 29
11 18 13 20 25
Observation (n) 4 4 4 4 4 20 (n)
17+21+19+11 19+22+25+18 18+16+17+13 20+23+25+20 24+28+29+25 410
∑X = 68 = 84 = 64 =88 = 106 (total)

68 ÷4 =17 84 ÷4 =21 64 ÷4 =16 88 ÷4 = 22 106÷ 4 = 26..5

X ( mean)
172 + 212+ 192+222+252+182 182+162+172+132 202+232+252+202 242+282+292+252 8824
∑X2 192+112= 1212 = 1794 = 1088 = 1954 = 2826 (total)

(68)2 ÷ 4 = (84)2 ÷ 4 = (64)2 ÷ 4 = (88)2 ÷ 4 = (106)2 ÷ 4 = 8689

(∑X)2 ÷ n 1158 1764 1024 1936 2809 (total)

One-Way ANOVA Analysis

Correction Factor (C.F.)
C.F. = T2 / n (410)2 / 20 — 8405
Total Sum of Squares
Total Sum of Squares = (∑x 2 ) - C.F. 8824 — 8405 = 419

Between Groups Sum of Squares

Between Groups Sum of Squares = (∑x2) / n - C.F. 8689 — 8405 = 284

Error Sum of Squares

Error Sum of Squares = Total Sum of Squares - Between Groups Sum of Squares
= 419 — 284 = 135
 Degrees of Freedom (D.F.)
Between Groups (D.F.) = No. of Groups — I 5_1=4
Total (D.F.) = Total Observations —1 20 - 1 = 19
Error (D.F.) = Total D.F - Between Groups D.F
19 _ 4 = 15

Mean Squares
Mean Square (Between Groups) =
= Between Groups Sum of Squares / Between Groups D.F. 284 / 4 = 71
Mean Square (Error) = Error Sum of Squares / Error D.F. 135 / 15 9

F-Ratio
F = Between Groups Mean Square / Error Mean Square 71 / 9= 7.89
Thus, there is a significant difference between groups.

Dunnett's t-test to determine effect of Drug against control group (E):

t =[X1- X2 ]/ √(2S2/n)
Where, S2 = Mean Square (Error) = 9.
X 1 = Mean of test group (A, B, C, D)
X 2 = Mean of standard group (E)

Calculations for Each Group:

For Group A: t =121 - 171/ √(2x9/4) = 4 / √4.5 ≈ 1.886

For Group B: t = [16 – 17]/ √(2x9/4)= 1 /√4.5 ≈ 0.472

For Group C: t = [22 – 17]/ √(2x9/4) = 5 / √4.5 ≈ 2.358

For Group D: t =[26.5 – 17]/ √(2x9/4) = 9.5 / 44.5 ≈ 4.481

REPORT:

Drugs C and D differ significantly from Control (E). Drug D is highly effective.
EX.NO: 16

CHI SQUARE TEST

Principle

The Chi square test is generally applied to the analysis of quantal or all or none responses
Pearson's Chi Square Test

 Chi-Square test is method of testing the significance of difference between two proportions.
 It has the advantage that it can also be used when more than two groups are to be
compared.
 Commonly applied in biomedical research

Chi-Square test calculation

To find out the protective dose of diazepam in relation survival against a challenge of a
convulsive dose of pentylenetetrazole (PTZ). Diazepam (1 and 4mg/kg) showed anti-convulsant effect
against full-blown convulsions due to pentylenetetrazole (80mg/kg) in mice. Each group consisted of 10
animals. The result are tabulated as below to find out the degree of significance.

Formula
N ((ad — bc] — N/2)2
2
Xy =
(a+c) (b+d) (a+b) (c+d)

Calculation

Number of animals showing

Treatment Groups
Convulsions (+) No Convulsions ( - )

A Control ( PTZ ) 10 0

B Diazepam ( 1mg/kg) + PTZ 6 4

C Diazepam ( 4mg/kg) + PTZ 3 7

1. Effect of ling/kg dose of diazepam
Treatment Number of animals showing Total
Groups Convulsions (+) No Convulsions (-)

A a 10 b0 10

B c6 d4 10

Total 16 4 20

20( [10x4) – (0x6)] – 20/2)2

Xy2 =
(10+0) (6+) (10+6) (0+4)

18,000
=
6400

= 2.81 ( Not significant)

2. Effect of 4mg/kg dose of diazepam

Number of animals showing
Total
Treatment Groups Convulsions (+) NO Convulsions
(—)
A 10 0 10
C 3 7 10
Total 13 7 20

20 ([(10x7) — (0x3)] —20/2)2

Xy2 =
(10+0) (3+7) (10+3) (0+7)

72,000

9100

= 7.91

From the x2 table the anticonvulsant effect of diazepam at 4mg/kg dose is significant (P<0.01) as
compared to 1mg/kg dose (P>0.05).
EX NO : 17

WILCOXONS SIGNED RANK TEST

DEFINITION: The Wilcoxon signed-rank test is a non _ parametric rank test for statistical hypothesis testing
used either to test the location of a population based on a sample Of data. or to compare the locations of two populations
using two matched samples.
Two different strains of mice were raised at eleven different breeding centers. Co-variation between paired
groups was noted in respect of their body weights. The observations are tabulated in the following table:

Centers Strain Strain di Rank Ranks with less

1 2 of di frequent sign
1 21.4 20.2 +1.2 4
_
2 13.4 12.1 +1.3 5

3 19.8 16 2 +3.6 7
4 36.4 31.5 +4.9 8.5
5 22.1 22.4 -0.3 1.5 1.5
6 14.9 14.9 0.0 - -
7 37.7 34.5 +3.2 6
10.2 11.0 -0.8 3.0 3.0
8
9 26.1 21.2 +4.9 8.5
10 41.5 35.5 +6.0 10.0
11 14.3 14.0 +0.3 1.5

OBSERVATION:
 Calculation di = ( Strain 1 – Strain 2 ) for each pair.
 Ignore cases where di = 0 ( center 6 )
 Rank the absolute values of di, ignoring signs.
 Identify ranks with the less frequent sign to compute T . T= 4.5.
Since n = 10, referring to Wilcoxon tables, T = 4.5 corresponds to p < 0.02( for two tailed test).
 Therefore there is a significant difference between the groups.