DR.

VISHWANATH KARAD MIT WORLD PEACE UNIVERSITY’S

SCHOOL OF HEALTH SCIENCES AND TECHNOLOGY, PUNE-411038

LABORATORY MANUAL
THIRD YEAR B.PHARM
SEMETER V [PCI PATTERN]
PHARMACOLOGY-II

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 Third Year B.Pharm (Semester V)

References books
1. A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019.
2. Ghosh M.N., Fundamentals Of Experimental Pharmacology, 3 rd Edition.
3. Kulkarni S.K., Practical Pharmacology And Clinical Pharmacy, Vallabh Publication,.
4. A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
Requirements for experiments
 Online Practical: Cap, Calculator, Red Pen, Scale, Pencil, Sharpener, Eraser, Rough
Note Book, Marker, Journal. Napkin, Hand sanitizer, butter paper, spatula, Plastic
box
 Offline practical: Clay, curve needle, sharp and blunt forceps and scissor, Syringe
(1 ml, 2 ml and 5 ml), black thread, cutter, graph papers, gum bottle.
Rules for students
1) Wear the apron before entering into Pharmacology laboratory
2) Carry all necessary requirements in laboratory as required in practical.
3) Do not touch any instrument without permission.
4) Do not roam, talk, laugh unnecessarily in laboratory and maintain decorum in
laboratory. The discipline of students will be constantly monitored and recorded.
5) Do not use mobile phones in laboratory. Keep it in bag in silent mode or switch off
mode. Emergency calls must be made or attended with permission and outside of
laboratory.
6) Always follow instructions given by laboratory attendant and concerned teacher.
7) Do not go outside during practical without permission.
8) Do not eat anything in laboratory.
9) Keep stools inside the platform before leaving the laboratory after practical.
10) Update and get checked journals regularly.

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PRACTICAL SYLLABUS
Experime Name of Experiment
nt No
1. Introduction to in-vitro pharmacology and physiological salt solutions.
2. Effect of drugs on isolated frog heart.
3. Effect of drugs on blood pressure and heart rate of dog.
4. Study of diuretic activity of drugs using rats/mice.
5. DRC of acetylcholine using frog rectus abdominis muscle.
6. Effect of Physostigmine and atropine on DRC of acetylcholine using frog rectus
abdominis muscle and rat ileum respectively.
7. Bioassay of histamine using guinea pig ileum by matching method.
8. Bioassay of oxytocin using rat uterine horn by interpolation method.
9. Bioassay of serotonin using rat fundus strip by three point bioassay.
10. Bioassay of acetylcholine using rat ileum/colon by four point bioassay

11. Determination of PA2 value of Prazosin using rat anococcygeus muscle (by
Schilds plot method).
12. Determination of PD2 value using guinea pig ileum.

13. Effect of spasmogens and spasmolytics using rabbit jejunum.

14. Anti-inflammatory activity of drugs using carrageenan induced paw-edema

model.
15. Analgesic activity of drug using central and peripheral methods

Note: All laboratory techniques and animal experiments are demonstrated by simulated
experiments by softwares and videos

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EXPERIMENT NO. 01
INTRODUCTION TO IN VITRO PHARMACOLOGY AND PHYSIOLOGICAL SALT
SOLUTIONS
References:
1) A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019.
2) Ghosh M.N., Fundamentals Of Experimental Pharmacology, 3 rd Edition, Page No.
21-28.
3) Kulkarni S.K., Practical Pharmacology And Clinical Pharmacy, Vallabh Publication,
Page No. 10-11.
4) A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
Introduction
1) Pharmacology is the science which deals with the study of drugs. The word
‘pharmacology’ is derived from the Greek words Pharmakon (A drug or poison)
and logos (discourse).
2) It broadly covers the information about the history, source, physiochemical
properties, physiological actions, mechanism of action, absorption, distribution,
metabolism, excretions and therapeutic uses of drugs.
3) Drugs are chemical substances used for the purpose of diagnosis, prevention,
relief or cure of the disease in man or animals.
4) Experimental pharmacology deals with effects of various test substances studied
on different animal species which is aimed at finding out safe therapeutic agent
suitable for public health as well as mechanism and site of action of a test
substance.
5) It is the basic step in the discovery of new drugs or studying the pharmacological
actions of already developed one using both preclinical and clinical study designs
in a stepwise phase of investigations.
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In Vitro Pharmacology
 In vitro pharmacology studies the biological effects of a drug in an isolated
environment, such as cell lines or tissues. This setup conveniently eliminates whole
organism physiological influences allowing for detailed analysis.
 In contrast, In vivo pharmacology is the study of biological effects of a drug in a
complex living organisms and is used to observe the complex physiological effects
of a drug.
 In vitro studies are conducted using component of an organism that have been
isolated from their usual biological surrounding such as microorganisms, cells, or
body.
 Microorganisms, cells, tissue, can be studied in artificial culture media therefore
these are also called ‘test-tube experiments’ because they are traditionally done in
test tubes, flasks, petri dishes.
 All the times, the results obtained from in vitro experiments cannot be considered
to predict same reaction of a n entire organism in vivo, basically in vitro
experiments provide better data to mathematical models.
Isolated tissue experiments.
In order to see the effect of drugs on tissues such as rectus abdominis muscles of frog,
guinea pig ileum, goat trachea, rat fundus, chicken ileum etc, the tissues must be
isolated from animal and kept in suitable physiological solution throughout the
experiment. This tissue is mounted to suitable instrument (e.g organ bath for chicken
ileum) and suitable physiological conditions such as temperature, salts and ions,
electrolytes, oxygen are provided to continue the contraction relaxation pattern as in
the body. The dose of suitable drug is given to the tissue under observation. The tissue
starts contracting. E.g. contraction of guinea pig ileum by addition of Acetylcholine.
Then with the help of rotating drum and lever, suitable response is produced on
kymograph paper. This procedure is repeated for maximum possible doses till ceiling
effect is observed. At last, the paper on drum is removed and responses of drugs are
analyzed.
Common isolated tissue experiments
Preparation Receptors commonly Bathing solution
studied
Frog rectus abdominis Ach (nicotinic, Muscarinic) Frog ringer

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Guinea pig colon 5 HT Kreb

Guinea pig ileum Ach (muscarinic Kreb or Tyrode
M3)histaminic H1,3,
Neurokinin NK 1, Bradykinin
B2
Guinea pig vas deferens EP3 Krebs
Mouse vas deferens Mu kappa and delta opiod, Kreb
dopamine D2
Rabbit jejunum Alpha2 adrenoceptors Tyrode
Rabbit heart Beta1 adrenoceptors Ringer-Locke
Rat or Rabbit aorta Alpha1, adrenocpetors, Kreb
5HT2, Neurokinin NK1,
Bradykinin, Angiotensin
Rat colon Neurokinin Kreb
Rat phrenic nerve Ach Kreb
Rat uterus Beta adrenoceptors, mu Kreb
opiod, bradykinin
Contact time
The time that is allowed for the drug to remain in contact with the tissue is called the
contact time. The contact time depends upon type of the tissue used.
Time cycle
A fixed time cycle is followed while recording any effect of the drug on isolated tissue
preparation. The fixed time cycle comprises of starting of recording from the base line.
Standard drugs
The commonly used agonists in isolated tissue preparation in Practical Pharmacology
are Ach, Tryptamine, histamine. Ach is neurotransmitter in body and acts on
muscarinic receptor. Smooth muscle such as intestine, bronchial muscle contract with
Ach or histamine.

Distance between writing point and

fulcrum
Magnification Value=
Distance between fulcrum and tissue
attachment point

Importance
1. Magnification value is one of essential concept for optimization of the tissue
experimentation conditions.S
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2. For high contracting tissue magnification value should be less and vice versa.
Load/Tension on the lever.
In bioassay, the load is applied to lever. The plasticin/clay is used to apply load. The
clay is rolled round over palm to make small circular ball of 500 mg and 10-12 cm
thread is tied over it in round manner. The other end of tread is tied to lever towards
organ bath direction but near to fulcrum. The load is applied when tissue is relaxing.
Other times load is kept at any elevated position on water bath. Initially lever is
adjusted keeping clay up.
The purpose of application of load is
 To relax tissue when tissue is not in presence of drug during washing.
 To provide proper tension to the tissue and thereby to provide some stretching
 To record contraction and relaxation pattern correctly, in successive way one after
other.
Note: the load is kept hanging when washing is done and kept elevated on water bath
corner when taking base line and injecting drug in all times till end of experiment. Do
not forget the procedure of it.
Ceiling dose:
The dose in which maximum response of tissue is obtained is called ceiling dose and
effect is called ceiling effect.
Dose response relationship
The relationship between dose of drug and response it shows is called dose response
relationship. Dose is directly proportional to height of response. The graph plotted
between percent response and dose, curve is obtained called as dose response curve.
It is also called as concentration response curve.
Temperature:
In order to get consistent effects it is important to maintain temperature of both
solutions at a specified level. The temperature of the tissue is maintained as that of in
the body. i.e. 370 C . e.g. when the bath temperature is decreased below 37 0 C the
tone of intestine is increased. The contraction and relaxation also increases.
Aeration:
Aeration oxygenates the PSS buffer and provides Brownian motion to distribute drugs
that will be introduced in the tissue bath during the experiment. Air with oxygen is
essential for survival of tissue, oxygen with 5 % CO 2 is needed for the proper
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functioning of the tissue. The aeration is provided experimentally by using aerator

which is connected to tube via IV set. The the tube solution ie. PSS is constantly
bubbled during experimentation (1-2 bubbles/sec) speed of bubbling affects
responses of tissues.
Note: Make sure aeration/bubbles does not cause tissue movement, which will disrupt
data recordings.
Ringer solution for mammalian isolated heart
Frog ringer----for heart and tissue
Tyrode ringer ----mammalian smooth muscle
Krebs solution---mammalian isolated organ nerve.
The Physiological Salt Solutions (PSS)
The physiological salt solutions are used to keep isolated tissue or an organ
preparation surviving as long as the experiment is over. It is important to choose the
particular type of solution in which tissue is known to survive. These salt solutions are
prepared carefully using analytical grade reagents and distilled water. The other
precautions to be taken are adjusting the proper pH of the final solution and aeration
with oxygen, mixture of oxygen and carbon dioxide or even bubbling with air. These
solutions should be clear and freshly prepared. There are various types of PSS
solutions prepared for various tissues. They are explained in preceding sections.
Purpose of each ingredient in PSS
1) Sodium chloride (NaCl): to maintain iso-osmolarity, contractility, isotonicity and
excitability
2) Potassium chloride (KCl): to maintain ionic balance
3) Calcium chloride (CaCl2): to maintain the contractility of preparation.
4) Sodium bicarbonate (NaHCO3): to provide alkaline pH.
5) Glucose: to provide energy
6) Sodium or potassium dihydrogen phosphate (NaH 2PO4 KH2PO4): acts as buffer.
7) Magnesium chloride or sulphate (MgCl 2, MgSo4): to stabilize the preparation and
hence to reduce spontaneous activity.
8) Distilled water: act as vehicle to dissolve various ingredients.
Compound Frog Ringer De Jalan Tyrode Kreb
(molecular Ringer Locke Hensle
weight) t

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NaCl 110 (6.0)** 154 (9) 154(9) 137(8) 118(6.9)

(58.45)*
KCl (74.56) 1.9 (0.14) 5.6 5.6 (0.42) 2.7 (0.2) 4.7(0.35)
(0.42)
CaCl2 1.1 (0.12) 2.20.55 1.8 (0.2) 2.5
(110.99) (0.24)
(0.06) (0.28)
MgCl2 --- ------ 0.1 ---
(95.23)
MgSo4.7H2 ---- ---- ---- -- 1.2
0 (1.28)
NaHCo3 2.4 (0.2) 6.0 (0.5) 6 (0.5) 11.9 (1) 25 (2.1)
NaH2PO4 --- --- --- 0.4 (0.05) ---
(119.97)
KH2PO4 ---- ---- --- --- 1.2(0.16)
(136.08)
Glucose 11.1(2) 5.55(1) 2.28(0.5) 5.55 5.55
(180.16)
*Values in parenthesis against salt indicate molecular weight
** Values are in mM (g/l)
E.g. if 0.2 g of anhydrous CaCl 2 is required, a solution can be prepared from
CaCl2.2H2O
X= 0.2 (mol wt of CaCl2.2H2O)/ Mol wt of CaCl2
= 0.2 X147/111= 0.26 gm,
Where x is the amount of salt.
Preparation of PSS
 While preparing the solution absolute quantity of each ion and preparation among
each other with calcium and potassium must be maintained.
 Sodium ions are responsible for maintenance of excitability, contractility,
rhythmicity of muscle and nerves
 Potassium ions are responsible for increase of neuromuscular transmission and end
excitability of nerves
 Calcium ions increase force of contraction and tone of heart and decrease
excitability of nervous tissue
 Magnesium ions are responsible for contraction of smooth muscles.
Procedure for preparation of PSS
1) Weigh all chemicals accurately.
2) Use neat and clean bucket for preparation of PSS and make appropriate mark with
marker (10 liter) on bucket.

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3) Take some amount of distilled water (2 litres) in bucket.

4) Dissolve alkalies and sodium hydrogen phosphate, glucose, separately in a
distilled water in beaker.
5) Add the required amount of sodium bicarbonate by dissolving sufficient volume of
water. Add sodium bicarbonate at the time of setting ups the experiment since
calcium carbonate is liable to be precipitated if the calcium and bicarbonate are
kept long together
6) Dissolve other ingredients such as sodium chloride, magnesium chloride, glucose
in separate beaker and pour in main bucket.
7) Add calcium chloride separately in distilled water in beaker as it produces
precipitate.
8) Adjust the pH of solution and continuously stir the mixture.
9) Adjust final volume with distilled water
Different physiological salt solutions with their uses:

Physiological salt Tissue for which it is used

solution
Ringer lock’s solution Isolated rabbit heart
Frogs ringer’s solution Frog’s rectus abdominal muscle and leech
dorsalis muscle preparation.
Tyrode solution Rabbit intestine and guinea pig ileum
De-Jalon’s solution Rat uterus, duodenum and colon experiments
Kreb Henseleit Guinea Pig tracheal chain preparation and
solution: rabbit aortic strip preparation

Result
The in-vitro tissue experiment’s basic introduction and applications, types, uses of
Physiological salt solutions were studied
SHORT QUESTIONS
1) Define in vivo and invitro techniques.

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2) What are functions of each ingredient in PSS?

3) Which precautions are taken in isolated tissue experiments?

4) What are different PSS and for what purpose they are used?

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5) Define ceiling dose, Dose response relationship and Aeration.

EXPERIMENT NO. 02
EFFECT OF DRUGS ON ISOLATED FROG’S HEART
References
1) Practical’s in Pharmacology by Dr. R.K.Goyal, B.S.Shah Prakashan, Eighth edition,
Page 72-81
2) Hanbook of experimental pharmacology by S.K. Kulkarni, Page no. 155-164
3) Practical manual of pharmacology by Dinesh Badyal, Jaypee brothers publication,
First edition, 2008, Page 80.
4) X-cology software CD for demonstration.
5) A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019
6) A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
Requirement
Apparatus:
Starlings heart leaver, Marriott’s constant pressure bottles, symes venous cannula,
Sherrington’s recording drum, syringes and needles.
Drugs
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1. Acetylcholine 1 mg=1000 µg, Dissolve 1 mg of accurately weighed acetylcholine in

100 ml of distilled water. i.e. 1 mg in 100 ml water i.e. 1000 µg in 100 ml distilled
water (10 µg in 1 ml)
2. Adrenaline hydrochloride 10 and 100 µg/ml
3. Potassium chloride 10 mg/ml
4. Calcium chloride 10 mg/ml
5. Propranolol 10 µg/ml
6. Atropine sulphate 100 µg/ml
7. PSS: Frog ringer
Theory
Frog heart is red in color and conical muscular organ situated mid ventrally in the
front fraction of body in between two lungs. It is enclosed in two membranes inner
epicardium and outer pericardium having the fluid called pericardial fluid. The frog
heart is three chambered two auricles and one ventricle and valves to keep the blood
streaming one way.
Principle
Many drugs act on heart. Adrenergic and cholinergic drug produce opposite effect on
it by acting on different receptors. These drugs may influence the rate (chronotropy)
and force (inotropy) of contraction of heart. An increase in the heart rate is called
positive chronotropic response whereas decrease in heart rate is negative
chronotropic response. Similarly an increase in force of contraction is called a positive
inotroic effect and decrease in the force of contraction is called a negative inotropic
effect. Sympathomimetic amines such as adrenaline and noradrenaline produce
positive inotropic and positive chronotropic response whereas parasympathomimetics
such as acetylcholine produce negative chronotropic and negative inotropc response
on heart.
 Acetylcholine: Parasympathomimetics such as acetylcholine (in low dose)
produce negative chronotropic and negative inotropic response on heart. Ach
liberated at post ganglionic parasympathetic nerve fibers exerts inhibitory effects
on muscle cells due to its ability of increasing the permeability of the cell
membrane for potassium ions. The potassium ions under influence of Ach cause
quick repolarization of cell membrane causing rapid repolarization to shorten
action potential. Thus heart is inhibited. This is called as muscarinic action
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 Adrenaline/Epinephrine: Sympathomimetic amines such as adrenaline and

noradrenaline produce positive inotropic and positive chronotropic response. The
recording is characterized by increase in amplitude and beats. Adrenaline binds to
beta receptor on cell surface inducing conformational change which permits
interaction of G protein with stimulatory G protein. This activation causes binding
of GTP to alpha subunit causing dissociation of adenylate cyclase enzyme. This
causes conversion of ATP to c AMP and activation of protein kinase A. This PKa
causes increase in intracellular calcium ion concentration by increasing release
from store by releasing entry from extracellular fluid. This also causes increase
interaction of Troponin and with calcium and subsequent contractility of
myocardial cell. PKa also causes activation of phosphorylase enzyme which breaks
down glycogen and increase glucose concentration as energy source for
myocardial contraction so overall activity of heart increases (both rate and force)
 Noradrenaline/Norepinephrine: Norepinephrine is a sympathomimetic used in
the control of blood pressure during various hypotensive states and as an adjunct
treatment during cardiac arrest. It increases heart rate
 Isoprenaline: synthetic sympathomimetic with nonselective β adrenergic activity.
Stimulation of cardiac β1 receptors by isoproterenol increases heart rate, inotropy,
and resulting in an increase in cardiac output and systolic blood pressure.
 Calcium chloride (CaCl2): Calcium chloride is inorganic salt compound in lower
dose (less than 1%) increases heart rate and force of contraction but in high doses
decreases heart rate.
 Potassium chloride (KCL) is electrolyte produces same effect as that of Ach i.e.
negative chronotropic and negative inotropic effect and makes the heart to relax
completely.
 Atropine is parasympatholytic as such does not produce any effect except slight
increase in heart rate and force of contraction
 Propranolol is beta receptor antagonist on low doses sometimes increases heart
rate and contraction and in high doses it inhibits same
Observation table
Sr Drug Category Concentrat Dose Heart Amplitu
. ion Rate de
No

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1 Epinephrine Sympathomimetic 10 µg/ml 2 µg 65 75 Increase

2 Norepinephri Sympathomimetic 10 µg/ml 2 µg 64 68 Increased
ne
3 Isoprenaline Adrenergic cardiac 10 µg/ml 2 µg 66 91 Increased
stimulant
4 Calcium Inorganic 10 mg/ml 2000 66 45 Decrease
Chloride compound salt µg d
5 Propranolol Beta blocker 1 mg/ml 200 67 48 Decrease
µg d
6 Acetylcholine Parasympathomim 10 mg/ml 2 µg 66 39 Decrease
etic d
7 Potassium Electrolyte 10 mg/ml 2000 66 05 Decrease
Chloride supplement µg d
8 Atropine Parasympatholytic 100 mg/ml 20 µg 67 70 No
Sulphate change

Langendorff Assembly set up of heart experiment

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Fig. Mounting of heart (first) and response of various drugs on heart

(Second)

Result

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Following effects are observed on heart after administration of drugs

Sr Drug Category Concentrat Dose Heart Amplitu
. ion Rate de
No
1 Epinephrine Sympathomimetic 10 µg/ml 2 µg 65 75 Increase
2 Norepinephri Sympathomimetic 10 µg/ml 2 µg 64 68 Increased
ne
3 Isoprenaline Adrenergic cardiac 10 µg/ml 2 µg 66 91 Increased
stimulant
4 Calcium Inorganic 10 mg/ml 2000 66 45 Decrease
Chloride compound salt µg d
5 Propranolol Beta blocker 1 mg/ml 200 67 48 Decrease
µg d
6 Acetylcholine Parasympathomim 10 mg/ml 2 µg 66 39 Decrease
etic d
7 Potassium Electrolyte 10 mg/ml 2000 66 05 Decrease
Chloride supplement µg d
8 Atropine Parasympatholytic 100 mg/ml 20 µg 67 70 No
Sulphate change

Assignments

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 Third Year B.Pharm (Semester V)

1. List cardiac stimulants and depressants.

2. Why should I.V. Potassium and Calcium injections be given slowly?

3. Why is the dopamine preferred over adrenaline in cardiogenic shock?
4. List the uses and adverse effect of (i) Propranolol (ii) Digoxin (iii) Adrenaline?
5. List cardiac depressants
6. Explain the effects of following drugs on frog’s heart Acetylcholine, Calcium
chloride Potassium chloride, Adrenaline and propranolol

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EXPERIMENT NO. 03
EFFECT OF DRUGS ON BLOOD PRESSURE AND HEART RATE OF DOG
References
 X-cology software CD for demonstration.
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
Requirement
Apparatus:
Research Kymograph, operation table, U shaped mercury manometer tuberculine
syringe. Burette arterial venous and tracheal cannula, connecting rubber tubing,
dissection box, cotton and thread.
Drugs
 Sodium citrate: 8.5 % (anticoagulant)
 Heparin: 500 IU (Anticoagulant)
 Normal saline: 0.9 NaCl
 Morphine: 1 µg/kg
 Urethane: 1.5 gm/kg
 Adrenaline: 100 µg/ml
 Acetylcholine: 100 µg/ml
 Histamine: 100 µg/ml
 Noradrenaline: 100 µg/ml
 Isoprenaline: 100 µg/ml
 Tolazoline: 1 mg/ml
 Atropine: 1 mg/ml
 Propranolol: 1 µg/ml
 Mepyramine: 1 mg/ml
Animal: Dog, Sex male, Weight 10 kg
Anesthesia used: Morphine + urethane (1.5 gm/kg)

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Theory
Dogs especially beagles (puppy) are widely used in biomedical research, testing,
education because they are gently and easy to handle. They are used as models for
human and veterinary diseases in cardiology, endocrinology, bone and joint studies
that tend to be highly invasive. They are used for the safety assessment of new drugs
for human or veterinary use after using drugs in rodents as per regulations. The
normal values in dogs are
Blood Pressure:
Systolic: 110-160 mm of Hg, Diastolic 60-90 mm of Hg, Normal BP: 85-120 mm of Hg
Heart rate: 60-100 beats. Young doggies ordinarily have higher pulses: upto 180
beats for every moment.
Respiratory rate: standard respiratory rate for 10-34 breaths for each moment.
Body temperature: canine normal body temperature is between 100.5-102.5 F (38-
39.20C)
Principle
• Drug-induced effects on the cardiovascular system remain a major cause of drug
attrition.
• While hemodynamic (blood pressure (BP) and heart rate (HR) and
electrophysiological methods have been used in testing drug safety for years,
animal models for assessing myocardial contractility are used less frequently and
their translation to humans has not been established clearly.
• The goal of these studies is to determine assessment of contractility and
hemodynamics of heart, by administering drugs on heart to detect clinically
relevant positive and negative effects on myocardial contractility, blood pressure
and rate of heart.
Observation:
 Adrenaline (Adr): It is sympathomimetic catecholamne which produces effect
through alpha, beta 1 and beta 2 receptors. As a result of beta 1 receptor
stimulation there is increase in heart rate, force of contraction and thereby
increase in BP. Immediately due to reflex inhibition there is slight decrease in BP
but as the drug reaches to the periphery where there is alpha 1 receptor
stimulation which produces vasoconstriction hence further rise in BP is observed.
Its action is slowly terminated by monoamine oxidase (MAO) and catechol o methyl
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 Third Year B.Pharm (Semester V)

transferase (COMT). When concentration of adrenaline is reduced beta 2 receptor

action is seen in which there is slight fall in BP (secondary fall).
 Noradrenaline (NA): it is sympahomimetic catecholamine having alpha receptor
action. HR is slightly decreased with NA. Rise in BP due to vasoconstriction.
 Isoprenaline (ISP): it is sympathomimetic amine and produces specific beta 1
and beta 2 receptor actions. Beta1 receptor stimulation produces increase in BP
and HR. As drug reaches periphery, there is vasodilation and decrease of BP.
 Acetylcholine (Ach): It is parasympathomimetic amine which stimulates both
muscarinic and nicotinic receptors. In low doses, it produces only muscarinic
receptor action and hence there is fall in BP due to dilatation of blood vessels. HR
no change. In high doses, Ach acts on nicotinic receptors present on skeletal
muscle and autonomic ganglia blocking muscarinic receptors. Autonomic ganglia
is stimulated due to which there is rise of BP
 Histamine: it is autocoid which causes vasodilatation through H 1 and H2 receptors.
When histamine is administered to dog, there is fall in BP
 Propranolol: The response of isoprenaline is blocked by Ppl.
 Phentolamine: the response of noradrenaline is blocked by Phentolamine i.e
alpha blocker
 Mepyramine: The response of histamine is blocked by mepyramine.
Effect of various drugs on heart
Observation table
Drug name (Dose in Pharmacological Actions
mg/kg)
Epinephrine It stimulates the alpha and beta adrenergic
(Adrenaline) receptors. Conventional doses will increase
Dose Range : 1 – 3 Mg/kg the BP followed by a short fall before
Body Weight reaching the basal level (biphasic response
due to alpha and beta receptor responses).
The heart rate decreases due to vagal reflex.
Norepinephrine It stimulates mainly the alpha and beta1
(Noradrenaline) Dose receptors. The heart rate is generally reduced
Range: 2 – 5 Mg/kg Body due to vagal reflex in response to increased
Weight BP.
Isoprenaline Dose Range: 2 It is a potent, non- selective beta adrenergic
-5 stimulant. It increases the systolic BP, but
Mg/kg Body Weight decreases the diastolic BP. Because the
decrease is more pronounced than the
increase, the mean arterial pressure typically
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 Third Year B.Pharm (Semester V)

falls.
Acetylcholine Dose Range Acetylcholine (ACh) leads to a sharp a fall in
2-5 mg/kg BP which returns to basal level quickly.
Histamine Dose Range: Acts on H1 and H2 receptors to produce a fall
2-5 in BP Stimulation of H1 produces a rapid
Mg/kg Body Weight onset short lived decrease in BP whereas H2
stimulation leads to a fall characterized by
slower onset and longer duration.
Ephedrine Dose It acts on both alpha and beta receptors and
Range :100-200 Mg/kg in addition enhances the release of
Body Weight norepinephrine from sympathetic neurons. It
increases the BP and heart rate.
Phentolamine Dose : This drug, an alpha blocker, reduces BP
1000Mg/kg Body Weight
Propranolol Dose : 1000 It is a beta blocker which reduces BP and
Mg/kg Body Weight heart rate.
Atropine Dose: 750 This drug is a muscarinic cholinergic
Range : 500-1000 Mg/kg Body antagonist. It competitively antagonizes Ach.
Weight
Cimetidine It is a H2 blocker, which also partially blocks
Dose : 5000 Mg/kg Body the effect of histamine on BP.
Weight

SHORT QUESTIONS
1) What is normal blood pressure in dogs?

2) Explain action of adrenaline on dog heart.

3) Explain effect of isoprenaline on dog heart.

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 Third Year B.Pharm (Semester V)

EXPERIMENT NO. 04
STUDY OF DIURETIC ACTIVITY OF DRUG USING RAT/MICE
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
Requirement: Metabolic cages, graduated measuring cylinders.
Drugs: Normal saline (0.9%), urea (900 mg/kg oral) hydroflumethiazide (1mg/kg,
oral) Frusemide (5 mg/kg oral)
Animal: Rats
Theory
Diuretics are the compounds which increase the flow of urine. They are used in
various diseases such as cardiac failure, liver cirrhosis, hypertension, water poisoning
and certain kidney diseases.

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 Third Year B.Pharm (Semester V)

Fig. Classification of diuretics

Adverse effects
Hypovolemia, hypokalemia, hyperkalemia, hyponatremia, metabolic
alkalosis, metabolic acidosis, and hyperuricemia
Principle
Normal urine output in rats is 1-2 ml/rat/day. Hence to get measurable quantity of
urine, the rats are first hydrated. The urine output is increased after administration of
diuretics like urea, hydroflumethiazide and frusemide. This increase in urine output is
measured in measuring cylinder and compared with normal urine output.

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 Third Year B.Pharm (Semester V)

Fig. Metabolic cage

Metabolic cage
These well-engineered Metabolic & Diuresis Cages supply uncontaminated samples of
effectively separate urine and feces of the rodent. Metabolic cages give an ideal
separation of water through a special style of the funnel and of the separation cone.
Animal waste is directed through a collection funnel and onto a linear diffuser. The
diffuser allows solid matter to travel down the top ridges of a 50% incline, passing
over a urine port, and into a fecal collection vessel. Liquid waste flows along grooves
down the inclined diffuser, passing through the urine port, and into a urine collection
vessel. A urine deflector prevents excess urine from splashing and contaminating
fecal specimen. Separate urine and feces samples are collected in two standard 50
ml. centrifuge vessels.
Groups
Group 1= Normal saline
Group 2=Saline + Urea (900 mg/kg, oral)
Group 3=Saline + hydroflumethaizide (1 mg/kg, oral)
Group 4= Saline+ furosemid (5 mg/kg oral)

Observation table
Amount Groups 1 2 3 4
of urine After 15 2 3.5 3 4.2
collecte min
d in ml After 30 3 4.5 4.2 4.0
min
After 1 2.5 3.5 3.0 3.8
hour
Total 7.5 11.5 13.2 16
volume
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 Third Year B.Pharm (Semester V)

Average 2.5 3.83 4.4 5.33

Interpretation and Calculations:

Result: The diuretic activity of drugs after 1 hour are found as under
1) Normal Saline = 2.5 ml
2) Saline + urea (900 mg/kg, oral) =3.5 ml
3) Saline + hydroflumethaizide (1 mg/kg, oral) = 3.0 ml
4) Saline+ furosemid (5 mg/kg oral) = 3.8 ml

SHORT QUESTIONS
 Define diuretics and write its uses.

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 Third Year B.Pharm (Semester V)

 Classify diuretics with examples.

 Write adverse effect of diuretics

 Write mode of action of loop diuretics

EXPERIMENT NO. 05
TO STUDY THE DOSE RESPONSE CURVE OF ACETYLCHOLINE USING FROG
RECTUS ABDOMINIS MUSCLE.
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
 Handbook of Experimental Pharmacology by S. K. Kulkarni, Vallabh Publication,
Third Edition, 2009, Page No. 85.

27
 Third Year B.Pharm (Semester V)

Requirement: Student kymograph, student organ bath, aeration tube, aerator,

aeration tube holder, frontal writing lever, lever holder, screw clip, forceps, rubber
tube, tuberculin syringe, pithing needle, scissor, forceps
Drugs: Acetylcholine stock solution (1 mg/ml), PSS-frog ringer solution bubbled with
air
Animal: Frog
Physiological and experimental conditions:
 Temperature of organ bath: 37 ± 0.050 C
 Tension on lever: 500 mg
 Aeration: Air bubbled with oxygen and carbon dioxide
 Magnification value: (should be near to 5)
 Base line: 30 Sec, Response: 60 Sec
 Speed of Drum: 0.25 rpm
 Concentration of standard Ach = 10 µg/ ml
 Stabilization of tissue time: 30 min
Theory
Dose response relationship demonstrates hierarchical responses to medication or
agonist wherever a rise of response is recorded with increase of the dose or
concentration of drug. This relationship is well explained by dose response curve
(DRC). The DRC is S shaped curve. The first part of the curve (25 % graph) has poor
discrimination between doses and response whereas central portion shows bigger
sensitivity to concentration and therefore increasing responses [see figure]. The last
part of curve shows the ceiling result where no any additional increase in response is
seen even more increase in dose or concentration.
Response

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 Third Year B.Pharm (Semester V)

Fig. Responses obtained on kymograph drum

Fig. Dose response curve

Advantages and applications
1. The method is easy to carry and study.
2. The technique is simple and less time-consuming. It is employed in those
preparations where the tissue is slowly contracting and slowly relaxing.
3. Large dose range can also be plotted and errors are distributed all through the
graph with the help of logarithmic transformation of doses in DRC.
4. The study of antagonist effect and agonist comparison is easy.
5. Dose-response, which involves the principles of pharmacokinetics and
pharmacodynamics, determines the required dose and frequency as well as the
therapeutic index for a drug in a population.
6. The therapeutic index (ratio of the minimum toxic concentration to the median
effective concentration) helps determine the efficacy and safety of a drug.
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 Third Year B.Pharm (Semester V)

7. Increasing the dose of a drug with a small therapeutic index increases the
probability of toxicity or ineffectiveness of the drug
8. The pharmacological profiles of individual drugs can be compared based on DRC
Disadvantages
1. Dose–response relationships generally depend on the exposure time and exposure
route (e.g., inhalation, dietary intake); quantifying the response after a different
exposure time or for a different route leads to a different relationship and possibly
different conclusions on the effects of the stressor under consideration
2. The errors in the method cannot be denied.
Principle
Frog rectus abdominis muscle contains nicotinic N2 receptors and acetylcholine work
as an agonist. Frog rectus abdominis muscle is voluntary muscle. At the
neuromuscular junction, nerve impulses liberate Ach from nerve ending into the cleft
between nerve fiber and muscle. This acetylcholine causes a depolarization of the
muscle fiber which in turns sets off a muscle action potential and contraction of the
muscle fiber.
Acetylcholine is cholinergic drug which act on cholinergic receptors namely muscarinic
receptors. Its effect is contraction of smooth muscle so it is employed in
pharmacology practical to assess its effect on smooth muscle (rectus abdominis
muscle).
Acetylcholine is an organic molecule that acts as a neurotransmitter in many
organisms, including humans. It is an ester of acetic acid and choline, There are two
main classes of acetylcholine receptor (AChR), nicotinic acetylcholine receptors
(nAChR) and muscarinic acetylcholine receptors (mAChR). Nicotinic AChRs are
ionotropic receptors permeable to sodium, potassium, and calcium ions. They are
stimulated by nicotine and acetylcholine. They are stimulated by Muscatine and
acetylcholine. Muscarinic receptors are found in both the central nervous system and
in the peripheral nervous system of the heart, lungs, upper GI tract, and sweat glands.
ACh is sometimes used during cataract surgery to produce rapid constriction of the
pupil.
Effect of Ach on muscle- Contraction of smooth muscle by acetylcholine is
mediated by activation of Muscarinic receptors of which M2 and M3 subtypes are
present in longitudinal muscle of guinea pig intestine. In single cells, Muscarinic
30
 Third Year B.Pharm (Semester V)

receptor activation evokes calcium release from stores which raises the internal free
calcium concentration and causes opening of calcium-activated potassium channels.
The rise in internal calcium suppresses the voltage-dependent inward calcium current.
A third important effect is the opening of channels which cause depolarization of the
membrane and so increase action potential discharge and contraction in the whole
muscle.
Precaution for good responses
1. Clean the assembly before experiment
2. Balance lever horizontally with load.
3. Maintain required temperature of water bath.
4. Maintain required and constant speed of drum.
5. Maintain slow and smooth aeration to tissue.
6. Prepared stock solution, working solution, tyrode solution in distilled water.
7. Make PSS for experiment with exact quantities and add calcium chloride at the
end in PSS to avoid turbidity and precipitation. PSS should be clear
8. Try to minimize handling of tissue, remove extra tissue cautiously. This is to
prevent damage of receptors.
9. Keep less quantity of Tyrode solution in internal tube in such a way that tissue is
just dipped into Tyrode. This will not dilute the drug much and response will be
obtained.
10. Maintain constant dose response cycle (time cycle) to maintain tissue sensitivity
Donts
1. Do not start button of water bath in absence of water in organ bath
2. Do not rotate drum manually when knob is closed.
Observation table
Sr. Dose of Log dose Height of % response
No. Acetylcholine of Ach contraction
(response)
ml µg in mm
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2

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 Third Year B.Pharm (Semester V)

7 6.4
8 12.8
9 25.6

Calculations and Graph:

A) Dose in µg calculations
Concentration of Ach standard: 10 µg /ml, i.e. 10 µg for 1 ml so for 0.1 ml

B) % response calculations: (maximum response’s dose is considered as 100%)

C) Graph:
Plot the graph is between percent response on Y axis and log dose on X axis
respectively

Interpretation & Result: The dose response curve of Acetylcholine using frog rectus
abdominis muscle was observed and ceiling effect was observed at dose of
______________ml Ach and height of response is observed as ___________ (100%)

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 Third Year B.Pharm (Semester V)

SHORT QUESTIONS
Define Dose response curve.

Write advantages and disadvantages of DRC.

Write mechanism of Ach action on contraction of smooth muscle.

Write principle of determination of dose response relationship of Ach using frog’s

rectus abdominis muscle

EXPERIMENT NO. 06

33
 Third Year B.Pharm (Semester V)

TO STUDY THE EFFECT OF PHYSOSTIGMINE AND ATROPINE ON DRC OF

ACETYLCHOLINE USING FROG RECTUS ABDOMINIS MUSLCE AND RAT ILEUM
RESPECTIVELY.
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
 Handbook of Experimental Pharmacology by S. K. Kulkarni, Vallabh Publication,
Third Edition, 2009, Page No. 88.
A) TO STUDY EFFECT OF PHYSOSTIGMINE ON THE DOSE RESPONSE CURVE
OF ACH USING FROG’S RECTUS ABDOMINIS MUSCLE.
Requirement: rotating drum, student organ bath, aerator, frontal writing lever,
forceps, tuberculine syringe, beaker, spatula, cotton, pipette with rubber bulb, scissor,
forceps, Petri plate, curved needle and thread.
Drugs: Ach stock solution (1 mg/ml) Ach standard solution (10 µg/ml), Physostigmine
stock solution (2 µg/ml) Physostigmine standard solution 10 µg/ml.
PSS-Frog ringer solution bubbled with air
Animal: Frog
Physiological and experimental conditions:
 Temperature of organ bath: 37 ± 0.050 C
 Tension on lever: 500 mg
 Aeration: Air bubbled with oxygen and carbon dioxide
 Magnification value: (should be near to 5)
 Base line: 30 Sec, Response: 60 Sec
 Speed of Drum: 0.25 rpm
 Concentration of standard Ach = 10 µg/ ml
 Stabilization of tissue time: 30 min

Principle

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 Third Year B.Pharm (Semester V)

Physostigmine (also known as eserine) is a parasympathomimetic alkaloid,

specifically, a reversible cholinesterase inhibitor. Neostigmine acts by interfering with
the metabolism of acetylcholine. It is a covalent (reversible - bond hydrolyzed and
released) inhibitor of acetylcholinesterase, the enzyme responsible for the breakdown
of acetylcholine in the synaptic cleft of the neuromuscular junction. When
physostigmine is present more acetylcholine is available for response and the length
of contraction of Ach is increased. It indirectly stimulates both nicotinic and
muscarinic acetylcholine receptors. As a result, the action of Acethyl choline on
smooth muscle potentiated, and concentration response curve is shifted to left in
presence of Neostigmine. (Agonistic effect)
Calculation of Physostigmine (Neostigmine)
Preparation of Neostigmine solution (10 µg /ml)
1ml = 0.5 mg = 500 µg
5 ml Neostigmine Ampoule contains
5 ml = 500 X 5= 2500 µg
5 ml is dissolved in 250 ml water
2500 µg = 250 ml water
So 1 ml contains= 2500 X 1 ÷ 250 = 10 µg /ml
Response:

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 Third Year B.Pharm (Semester V)

Observation table
A. For Ach solution
Sr. Dose of Acetylcholine Response in % Response
No. ml µg mm/cm
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2
7 6.4
8 12.8
B. For Neostigmine + Ach solution
Sr. Dose in ml (Neo + Ach) Response in % Response
No. ml of Neo µg of Ach mm/cm
and Ach
1 0.1+ 0.1
2 0.1+ 0.2
3 0.1+ 0.4
4 0.1+ 0.8
5 0.1+ 1.6
6 0.1+ 3.2
C. Percent Potentiation
Sr. Dose of Ach % % Potentiati %Potentiat
No. ml (A) µg Respon Respons on ion
se e (D)= (C) (E)=[(D)-
Ach (B) Neostigmi X 100/(B) 100]
ne
+ Ach (C)
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2
7 6.4
8 12.8
9 Mean
Calculation and Graph
A) Dose of Ach in µg calculation
Concentration of Ach standard: 10 µg /ml, i.e. 10 µg for 1 ml so for 0.1 ml

36
 Third Year B.Pharm (Semester V)

B) % Response calculation (maximum response’s dose is considered as 100%)

Graph: Plot the graph is between percent response on Y axis and log dose on X axis
respectively
Interpretation and result
There is observable Potentiation of responses of Ach after adding Physostigmine and
the concentration response curve of Ach is shifted to left in direction, so
Physostigmine confirms the agonistic effect on smooth muscle of frog. The percent
Potentiation was found to be______________%
B) TO STUDY EFFECT OF ATROPIN ON THE DOSE RESPONSE CURVE OF ACH
USING RAT ILEUM.
Requirements
Animal: Rat ileum
Drugs: Ach stock solution (1 mg/ml) Ach standard solution (10 µg/ml), Atropin
standard solution (10 µg/ml).
Physiological solution: Tyrode solution
Equipment: Kymograph, IV set
General requirements: beaker, spatula, cotton, pipette with rubber bulb, scissor,
Petri plate, curved needle, thread, anesthetic chamber or bottle etc.
Physiological and experimental conditions:
 Temperature of organ bath: 37 ± 0.050 C
 Tension on lever: 500 mg
 Aeration: Air bubbled with oxygen and carbon dioxide
 Magnification value: (should be near to 5)
37
 Third Year B.Pharm (Semester V)

 Base line: 30 Sec, Response: 60 Sec

 Speed of Drum: 0.25 rpm
 Concentration of standard Ach = 10 µg/ ml
 Stabilization of tissue time: 30 min
Principle:
Rat colon is an intestinal smooth muscle. Ach causes the contraction of smooth
muscle by acting on muscarinic receptors. Atropin blocks muscarinic receptors in the
smooth muscle. Therefore atropine blocks Ach induced contractions by inhibiting
Muscarinic receptors in rat (chicken) ileum. The DRC of Ach will be shifted to right in
the presence of atropine. The nature of antagonism is of competitive type and so
surmountable.
Calculation of dose of atropine
Preparation of Atropine solution (10 µg /ml)
1 ml Atropine Ampoule contains
1ml = 0.64 mg = 640 µg
1 ml Ampule is dissolved in 64 ml water
640 µg = 64 ml water
So 1 ml contains= 640 X 1 ÷ 64 = 10 µg /ml
Response:

Observation Table
For Ach solution

38
 Third Year B.Pharm (Semester V)

Sr. Dose of Acetylcholine Response in % Response

No. ml µg mm/cm
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2
7 6.4
8 12.8

For Atropine + Ach solution

Sr. Dose in ml (Atro + Ach) Response in % Response
No. ml of Atro µg of Ach mm/cm
and Ach
1 0.1+ 0.1
2 0.1+ 0.2
3 0.1+ 0.4
4 0.1+ 0.8
5 0.1+ 1.6
6 0.1+ 3.2
7 0.1+ 6.4

Percent Inhibition
Sr. Dose of Ach % % Inhibitio %
No. ml (A) µg Respon Respons n Inhibiti
se e (D)=(C)X on
Ach (B) Atropinis 100/(B) (E)=[100-
ed Ach (D)]
(C)
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2
7 6.4
8 12.8
9 Mean

Calculation and Graph

A) Dose of Ach in µg calculation

39
 Third Year B.Pharm (Semester V)

Concentration of Ach standard: 10 µg /ml, i.e. 10 µg for 1 ml so for 0.1 ml

B) % Response calculation (maximum response’s dose is considered as

100%)

C) Graph: Plot the graph is between percent response on Y axis and log dose on X
axis respectively

Interpretation and result

There is observable suppression of responses of Ach after adding atropine and since
the concentration response curve of Ach is shifted to right in parallel fashion, the
antagonism is confirmed and nature of antagonism is competitive. The percent
inhibition was found to be
___________%

40
 Third Year B.Pharm (Semester V)

SHORT QUESTIONS
 What is agonism and antagonism?

 Discuss mechanism of Physostigmine and Atropine on Ach induced contraction of

smooth muscle.

 Describe principle involved in agonistic effect of Physostigmine and antagonistic

effect of Atropin on DRC of Ach using suitable tissue preparations

 Name four examples of agonistic drug and antagonistic drugs in clinical

Pharmacology.

EXPERIMENT NO. 07
41
 Third Year B.Pharm (Semester V)

BIOASSAY OF HISTAMINE USING GUINEA PIG ILEUM BY MATCHING METHOD.

References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and company (medical
publishers) Jalandhar, 2019
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020.
 Essentials Of Pharmacotherapeutics By FSK Barar, Page No. 49-52
Theory:
Biological standardization or bioassay is defined as the assessment of activity of
preparation by measuring the effect on living organism or tissues. Bioassay is the
procedure to determine the quantitative relationship between the dose or
concentration of drug and the magnitude of response it produces. Such procedure
utilizes intact animals, isolated living tissues, animal preparations or micro-organisms.
Basic Principles for conduction of bioassay procedures
There are certain principles which must be followed to obtain valid results.
Principle-1: All bioassays (laboratory studies, toxicity studies, clinical trials) must be
comparative against a standard drug or preparation.
Principle-2: The standard and the new drug should be as far as possible identical to
each other. If it is so, their dose-response curves will have the same slope and would
be parallel; i.e., the potency ration would be constant all along the response curve.
Principle-3: The method for comparing the unknown and the standard drug should
preferably (but not essentially) test the therapeutic property of the drug. Ideally, an
analgesic should be tested for analgesic properties, and an anticonvulsant for
anticonvulsant activity. However, it is not always possible to do so, e.g., for insulin
lowering of blood glucose would be the ideal potency index, but a commonly used
method is the mouse convulsion method, in which the percentage of mice developing
convulsions with the standard and new insulin is compared.
Principle-4: The method should estimate, as far as possible, and allow an estimate of
the error due to biological variation in different animals/persons at any time, and in
the same animal/person at different times
Methods of Bioassay:
1) Quantal bioassay:
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 Third Year B.Pharm (Semester V)

 Quantal assay is an ‘all or none’ phenomenon. For example, Insulin-induced

hypoglycemic convulsive reaction or the cardiac arrest caused by digitalis.
 In both these cases the end point is an all or none response i.e. either convulsions
or no convulsions, similarly the cardiac arrest.
 This method is also called as End point method. In this type of assay the threshold
dose is measured for obtaining a specific biological end point in each animal.
 In these assays the drug (standard or test) is administered slowly to the animal
until some sharply observable effect (endpoint) is produced.
 The amount of the drug or the time for reaching the end point is then noted. Then
the comparison between the average results of two groups of animals (one
receiving standard and other the test) is done.
 Example. Bioassay of digitalis in cats. Here the cat is anaesthetized with
chloralose and its blood pressure is recorded.
 The drug is then slowly infused into the animal through vein at the rate of 1ml per
minute till the heart stops beating and blood pressure falls to zero.
 The volume of the fluid passed into the vein (threshold dose) is note in each
animal. This is the minimum lethal dose of digitalis in cats.
 Two series of such experiments one using standard digitalis and other using test
preparation of digitalis is done and then potency is calculated as follows

Dose of std.
Concentration Of Unknown = ----------------- × Concentration
of Standard
Dose of test

 Direct end-point assay are applicable when an end – point can be reached in each
animal; the drug effect appears rapidly; and the drug effect is directly proportional
to the dose.
 In case it is not possible to measure individual effective dose fixed doses are
injected into groups of animals and the percentage of mortality at each dose are
injected.

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 Third Year B.Pharm (Semester V)

 In this percentage of positive effect measured. A quantal response is obtained and

the percentage of positive effect at each dose is computed. The unknown is then
compared with a standard with respect to potency is causing the quantal effects.
Most familiar example of this method is in toxicity testing. E.g. LD 50
determinations.
2) Graded Bioassay:
 Graded response bioassay is based on the observation that there is a proportionate
increase in the observed response with a subsequent increase in the concentration
or dose. Graded response may be assessed by using either the whole animal or a
part of the animal. The parameters employed in such bioassays are based on the
nature of the effect the substance is expected to produce.
 Example: Contraction of smooth muscle preparation for assaying histamine or the
study of blood pressure response in case of adrenaline.
 The graded bioassays can be performed by employing any of the following
techniques.
Matching Bioassay
 It is the simplest form of all methods of graded bioassays. In this type of bioassay
the response of the test substance is taken first and the observed response is tried
to match with the response that is obtained with the standard drugs.
 Several responses of standard drug are recorded till a dose matching response to
that of test substance is observed. A corresponding concentration is thus
calculated.
 Advantages: This assay is employed when the sample size is very small.
 The assay does not depend on assumption of Dose response relationship
 Disadvantages: Since the assay does not involve the recording of concentration
response curve, the sensitivity of the preparation is not taken into consideration.
Therefore, the precision and reliability are not very good by this method.
 Purely subjective method and have experimental errors. The potency of drug is not
exact because this is a crude method.
Bracketing method:
 This method is made use of when the test sample is small. It is also a simple assay
procedure. In this method a constant dose of the test is bracketed by varying doses

44
 Third Year B.Pharm (Semester V)

of standard till the exact match is obtained between the test dose and the
standard dose.
 Initially, two responses of the standard are taken. The doses are adjusted such
that one is giving response of approximately 20 % and other 70 % of the
maximum.
 The response of unknown which in between two responses of standard dose, is
taken. The panel is repeated by increasing or decreasing the doses of standard till
three equals responses are obtained.
 The dose of test sample is kept constant. In the end responses of the double dose
of the standard and test are taken. They should give equal responses.
 Concentration of the test sample can be determined as follows:
Dose of Standard
Concentration Of Unknown solution = ------------------------ ×
Conc. of Standard
Dose of test

This method has following limitations:

1. This method is time consuming and records of response occupy a larger area of the
drum as far as tracings are concerned.
2. The match is purely subjective, so there are chances of error and one cannot
determine such errors.
3. This method does not give any idea of dose-response relationship.
However, this method is particularly useful if the sensitivity of the preparation is not
stable. Bioassay of histamine, on guinea pig ileum is preferably carried out by this
method.
Interpolation method:
 This method is based on the assumption of dose – response relationship. At first,
the concentration response curve due to graded doses of a standard substance
followed by the dose response curve of the test substance is recorded.
 Then two standard doses and one test dose are selected from the respective DRCs
such that they lie on the linear portion of the DRC.
 The test dose is selected in such a way that its response is greater than that of
smaller dose of standard and is lesser than that of larger dose of the standard.
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 Third Year B.Pharm (Semester V)

 Bioassay is then carried out with the selected standard and test doses in 2 – 3
cycles. The height of contraction of all the standard and test doses in the bioassay
is measured.
 Then, a log DRCs is plotted with the mean values of the standard responses and
the dose of the standard producing the same response as produced by the test
sample is directly read from the graph and the concentration of the test sample is
determined.
 It is simple method. The precision and reliability of the assay is much better as
compared to the earlier method as the sensitivity of the preparation is assessed
prior to testing the unknown sample.
Application of bioassay methods:
The various application of bioassay methods are follows:
1. Screening of new compounds for biological activity. Even synthetic products are
subjected to these methods, as chemical structure does not truly predict
pharmacological activity.
2. A bioassay method is used to ascertain the potency of drug and serves as the
quantitative part of any screening procedure.
3. Standardization of drugs of natural origin.
4. Estimation of biologically active substances like acetylcholine, adrenaline, nor-
adrenaline and serotonin in body fluids or tissue extracts.
5. Diagnosis and research: Bioassay may help in diagnosis of various clinical
conditions e.g. gonadotropins for pregnancy. The concentration of gonadotropins in
the blood or urine may be estimated by injecting these fluids in animals.
6. Sometimes stability studies are also conducted by bioassay.
7. Bioassay also measures any toxicity.
Requirements:
1) Animal and tissue: Chicken ileum
2) Drugs: Histamine standard solution (10 µg/ml), Stock solution 1 mg/ml &
Histamine test solution of unknown concentration
3) Physiological solution: Tyrode solution
4) Equipment: Organ bath and rotating drum

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 Third Year B.Pharm (Semester V)

5) General requirements: beaker, spatula, pipette, scissor, Petri plate, curved

needle, thread, lever, kymograph paper, sketch pen tip, clay, pencil, scale, blade
etc
Physiological conditions:
 Temperature: 37 ± 0.050 C
 Tension: 500 mg
 Aeration: Air bubbled with oxygen and carbon dioxide
Principle
In matching bioassay method, first standard dose response curve is taken till
ceiling effect followed by 0.1 ml, 0.2 ml, 0.4 ml doses of test are taken till
any of test solution dose is matched (by height) with any dose of standard
solution. The concentration of test drug can be easily revealed. One of the main
advantages of this method is that sensitivity of the tissue is first determined by prior
plotting of a concentration-response curve with the known agonist as is the case with
acetylcholine.
Histamine: Histamine is an organic nitrogenous compound involved in local immune
responses, as well as regulating physiological function in the gut and acting as
a neurotransmitter for the brain, spinal cord, and uterus. Histamine is involved in
the inflammatory response and has a central role as a mediator of itching. As part of
an immune response to foreign pathogens, histamine is produced by basophils and
by mast cells found in nearby connective tissues. Mechanism of action- Histamine
exerts its effects through H1, H2, H3 and H4 receptors. Histamine H1 receptor are
reported to be expressed on enterocytes, connective tissue cells, muscle layer, blood
vessels, immune cells and ganglion cells of the myenteric plexus in the human
intestine. Contraction of smooth muscle by Histamine is mediated by activation of H1
histamine receptors
Response:

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 Third Year B.Pharm (Semester V)

Observation Table:
For standard (Concentration of standard=10 µg/ml)
Sr. No. Dose in ml Dose in Response
μg in mm/cm
1 0.1 1
2 0.2 2
3 0.4 4
4 0.8 8
5 1.6 16
6 3.2 32
7 6.4 64
8 12.8 128
For Test:
Sr. No. Dose in ml Dose in Response
μg in mm/cm
1 0.1 1
2 0.2 2
3 0.4 4
4 0.8 8
5 1.6 16
6 3.2 32
7 6.4 64
8 12.8 128

Interpretation of matching doses:

______ml of test Histamine solution is matching with________ml of standard Histamine
solution.
Calculations
Formula:
Dose of Standard

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 Third Year B.Pharm (Semester V)

Concentration of Unknown solution = ------------------------ × Conc. of Standard

Dose of test

Result
The concentration of test solution of Ach by matching bioassay method using isolated
chicken ileum preparation was found to be…………. µg/ml

SHORT QUESTIONS
1) Define bioassay. Write any four applications of bioassay.

2) What are graded and quantal bioassays?

3) Write principle involved in bioassay of histamine by matching method.

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 Third Year B.Pharm (Semester V)

4) Discuss the mechanism of action of Histamine on smooth muscle.

EXPERIMENT NO. 08
BIOASSAY OF OXYTOCIN USING RAT UTERINE HORN BY INTERPOLATION
METHOD.
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 54.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020. Page 37
 Handbook of Experimental Pharmacology by S. K. Kulkarni, Vallabh Publication,
Third Edition, 2009, Page No. 97.
Requirements
 Animal: Female rat (120-150 g, overnight fasted)
 Tissue: Uterine horn
 Drugs: Oxytocin stock solution, 10 µg/ml, stilboestrol
 Apparatus: student organ bath, aeration tube, tissue holder, frontal writing lever,
fulcrum, Sherrington’s rotating drum, reservoir, screw clip, rubber tubing etc.
 PSS: De Jalon
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 Third Year B.Pharm (Semester V)

 Expeimental conditions:
 Temperature: 30-32 o C
 Tension on lever: 500 mg
 Magnification value: nearly 5
 Aeration: atmospheric air.
Theory
Oxytocin is peptide hormone and neuropeptide. Oxytocin is usually made by
hypothalamus and released by posterior pituitary. It is responsible for sexual
reproduction, childbirth. It is secreted in blood in response to stretching of the cervix
and uterus during labor. This helps with birth and production of milk. The sensitivity of
uterus to oxytocin depends on estrus cycles. Various stages of estrus cycle can be
identified by preparing the vaginal smear and observing under microscope. An adult
2-3-month old female rat has an oestrus cycle of 5 days. The estrus cycle can be
divided into different stages A) Dioestrus, characterized by the presence of
leukocytes in vaginal smear, B) Proestrous, Oestrus characterized by large no of
nucleated epithelial cells C) frank estrus cornified epithelial cells D) Metaestrous,
mixture of nucleated, cornified epithelial cells and leukocytes (If the rat is not in
estrous, it can be induced by the administration of oestrogen preparation (stilboestrol
(0.1 mg/kg, sc; 24 hr before).
Frank oestrus uterus is highly sensitive to oxytocin and hence preferred for the
bioassay.
Principle
This method is based on the assumption of a dose-response relationship. At first, the
concentration-response curve due to graded doses of a standard substance followed
by the dose response curve of the test substance is recorded. In this method, dose
response curve of standard solution is plotted on graph paper by taking log dose on X
axis and % response on Y axis. The percent response of the test drug is traced on a
standard drug curve and interpolated by dotted line to the corresponding log dose
scale on X axis. This is done for all % response of test dose. The antilog of log dose of
test solution is taken, and corresponding concentration for 1 ml solution is calculated.
The average of all concentrations gives actual concentration of test solution.
Response:

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 Third Year B.Pharm (Semester V)

Observation Table:
Standard solution
Sr. Dose in Dose in Log dose Respons %
No. ml μg e in response
mm/cm
1 0.1 1 0
2 0.2 2 0.3010
3 0.4 4 0.6020
4 0.8 8 0.9030
5 1.6 16 1.2091
6 3.2 32 1.5051
7 6.4 64 1.8061
8 12.8 128 2.1072
Test solution
Sr. Dose in Height of % response Log The
No. ml response (Consider dose antilog
(mm/cm) Standard From of log
dose as the dose
100%) graph (μg)
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2
7 6.4
8 12.8

C] Graph: % responses on Y axis and log doses of Ach on X axis

Calculations:
From Test solution observation table,
1) If 0.1 ml contains ______μg, then 1 ml will contains= ______μg

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 Third Year B.Pharm (Semester V)

2) If 0.2 ml contains _______ μg then 1 ml will contain= _______ μg

3) If 0.4 ml contains ______ μg then 1 ml will contain= ______ μg
4) If 0.8 ml contains ______ μg then 1 ml will contain= _______ μg
5) If 1.6 ml contains ______μg then 1 ml will contain= _______ μg

Concentration of unknown test solution = Average of all above doses.

Result: The concentration of unknown solution of Oxytocin by interpolation bioassay

method using isolated rat uterus horn preparation was found to be …..…. µg/ml

SHORT QUESTIONS
1. What is oxytocin? Write its clinical applications.

2. Write phases of oestrus cycle in rat.

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 Third Year B.Pharm (Semester V)

3. Discuss principle involved in interpolation bioassay method

EXPERIMENT NO. 09
BIOASSAY OF SEROTONIN USING RAT FUNDUS STRIP BY THREE POINT
BIOASSAY METHOD.
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 57.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020., Page 41.
Requirements
Equipment: kymograph, student organ bath, frontal writing lever, scissor, forceps,
thread, plasticine, pithing needle.
Animal: Rat (150-200 g, overnight fasted)
Tissue: Fundus strip.
Drug: 5 HT (Stock solution 10 µg/ml)
PSS: Kreb solution
Tension: 500 mg)
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 Third Year B.Pharm (Semester V)

Theory
In this bioassay, two responses of standard drug and one response due to test sample
are taken into consideration. At first, the concentration-response curve due to graded
doses of standard substance followed by the dose-response curve of the test
substance is recorded. Then, two standard doses and one test dose are selected from
the respective DRCs such that they lie on the straightest and steepest part of the
DRC. Two standard doses are selected so that they have 20% and 80 % of
maximum reaction and assigned S1 and S2. The test T dose is selected in
such a way that its response is greater than that of smaller dose of standard
and is lesser than that of larger dose of standard. If the selected doses of both
the standard and test are randomized several times and response calculated as mean,
this will minimize the error resulting from sensitivity changes. Bioassay is then carried
out with the chosen standard and the test doses in 3 successive cycles as per the
Latin-square design. The responses are recorded in order of
S1, S2, T.
S2, T, S1.
T, S1, S2.
The height of contraction of all the standard and test doses in the bioassay is
measured. Then, a log DRCs is plotted with the mean values of the standard
responses in the bioassay and the dose of standard producing the same
response as produced by the test sample is directly read from the graph and
the concentration of the test sample is determined. The concentration of the
unknown can also be determined mathematically as follows:
n1 T – S1 n2
Concentration of Unknown = ---- × antilog {---------- × log
------} Cs
t S2 – S1 n1

n1 = Lower standard dose.

n2 = Higher standard dose.
t = Test dose.
S1 = Response of n1.

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 Third Year B.Pharm (Semester V)

S2 = Response of n2.
T = Response of test (t)
Cs = Concentration of standard.
Dose pattern to be followed:
S1 S2 T
S2 T S1
T S1 S2
Serotonin is also known as 5 hydroxytryptamine or 5 HT. It is essentially fond in the
brain, guts and blood platelets. Serotonin is utilized to transmit messages between
nerve cells, it is believed to be dynamic in tightening smooth muscles. It also controls
body’s rest wake cycles and inner clock. Low serotonin levels are related to
depression.
Principle:
Rat fundus is extremely sensitive tissue for examination of substances like 5 HT,
histamine, acetylcholine and bradykinins. Unlike the intestinal smooth musle, this
preparation is slow contracting and slow relaxing type. Rat fundus is usually used for
bioassay of serotonin. The fundus is grey in color and thus easily identified from
pyloric part. A zig zag preparation of the fundus strip is prepared in an attempt to
expose maximum part of tissue to the drug.
Response

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 Third Year B.Pharm (Semester V)

Doses panel:
S1= 0.1 ml (response of lower standard dose)
S2 = 0.8 ml (response of higher standard dose)
T = 0.8 ml (middle test dose of S1 and S2)
Observation table
Sr. Different Height of different responses (mm)
No. Responses 1 2 3 Mean
1 S1 07 09 10 09
2 S2 15 15 15 15
3 T 12 12 12 12

Calculations
n1 T – S1 n2
Concentration of Unknown = ---- × antilog {---------- × log ------} Cs
t S2 – S1 n1

Result: The concentration of unknown solution of serotonin by three-point bioassay

method using rat fundus was found to be_________________
SHORT QUESTIONS
1) Write principle of three-point bioassay method.

2) What is serotonin? Explain its physiological role in body.

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 Third Year B.Pharm (Semester V)

3) Write formula for calculating unknown drug concentration by three-point bioassay

method and write meaning of each term used in it.

EXPERIMENT NO. 10
BIOASSAY OF ACETYLCHOLINE USING RAT ILEUM BY FOUR-POINT BIOASSAY
METHOD.
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 57.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020., Page 41.
 Handbook of Experimental Pharmacology by S. K. Kulkarni, Vallabh Publication,
Third Edition, 2009, Page No. 99.
Requirements:
Animal: Rat ileum
Drugs: Ach stock solution (10 µg/ml), Ach test solution of unknown concentration
Physiological solution: Tyrode
Equipment: Kymograph, IV set
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 Third Year B.Pharm (Semester V)

General requirements: beaker, spatula, cotton, pipette, scissor, Petri plate, curved
needle, thread, clay, kymograph papers etc
Physiological conditions:
Temperature: 37 ± 0.050 C
Tension: 500 mg
Aeration: Air bubbled with oxygen and carbon dioxide
Principle
4-point bioassay: This method is almost similar to the 3-point bioassay, but in this
method 2 doses of the standard (S1, S2,) and 2 doses of the test (T1, T2.) are used. The
selection of the standard doses and test doses should be such that they all should be
submaximal (i.e. the response is directly proportional to the dose.) they also lie on the
linear portion of the CRC and the ratio between the smaller to greater dose should be
1:2. i.d. S2 response is double than S 1 and the response of T 1 (dose) should lie in
between S1 and S2. T2 dose should be double than that of T 1. The selection of the test
doses is done in such a way that volume of T 1 is selected to give response approx
equal to that of S1. The volume of T2 is mathematically calculated by employing the
Latin-square design. If the selected doses of both the standard and test are
randomized several times and response calculated as mean, this will minimize the
error resulting from sensitivity changes. The responses of the chosen standard and
the test doses are recorded in 4 successive cycles in the order of following
randomization.
The height of contraction of the entire standard and the test doses in the bioassay is
measured and their mean values are calculated. The precision, reliability and
reproducibility of this method is high. The concentration of the test sample can be
determined by graphical method as well as mathematically by employing the
following formula:
n1 (S1+ S2) –
(T1 + T2) n2
Concentration of Unknown = ---- × antilog
{-------------------------- × log ------}
t1 (S 2 + T2) – (S1+
T1) n1

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 Third Year B.Pharm (Semester V)

 n1 = Lower standard dose.

 n2 = Higher standard dose.
 t1 = Low dose of test
 t2 = High dose of test.
 T1 = Response of t1.
 T2 = Response of t2.
 S1 = Response of n1.
 S2 = Response of n2.
Dose pattern to be followed:
S1, S2, T1, T2.
S2, T1, T2, S1.
T1, T2, S1 S2.
T2, S1, S2, T1.
Acetylcholine effect and information, refer experiment no. 05 page no. 25

Observation
Sr. Different Height of different responses (mm)
No. Response 1 2 3 4 Mean
s
1 S1
2 S2
3 T1
4 T2

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 Third Year B.Pharm (Semester V)

Calculation
n1 (S1+ S2) – (T1 + T2) n2
Concentration of Unknown = ---- × antilog {-------------------------- × log ------}
t1 (S2 + T2) – (S1+ T1) n1

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 Third Year B.Pharm (Semester V)

Result: The concentration of unknown solution of Acetylcholine by four-point

bioassay method using rat ileum was found to be________________

SHORT QUESTIONS
1) What is the four-point bioassay method

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 Third Year B.Pharm (Semester V)

2) Write pharmacological actions of Acetylcholine and give its effect on GIT.

EXPERIMENT NO. 11
DETERMINATION OF PA2 VALUE OF PRAZOSIN USING RAT ANOCOCCYGEUS
MUSCLE (BY SCHILDS PLOT METHOD).
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 66.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020., Page 50.
 Handbook of Experimental Pharmacology by S. K. Kulkarni, Vallabh Publication,
Third Edition, 2009, Page No. 95.
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 Third Year B.Pharm (Semester V)

Requirements:
Animal: Rat anococcygeus muscle
Drugs: Prazosin ( 1 µg/ml) and Norepinephrine (10 µg/ml)
Physiological solution: Kreb’s Solution
Equipment: Student organ bath, aerator, frontal writing lever, forceps, rubber tubes,
scissor and forceps, beaker, spatula, cotton, pipette, scissor, Petri plate, curved
needle, thread, clay, kymograph papers etc
Physiological conditions:
Temperature: 37 ± 0.050 C
Tension: 500 mg
Aeration: Air bubbled with oxygen and carbon dioxide
Theory
The pAx value is calculated as the negative logarithm of molar concentration of the
antagonist required to reduce the effect of multiple dose (x) of the agonist to that of
single dose in the absence of antagonist. The use of pA x value is convenient method
for evaluating competitive antagonism. Higher the pA x value, more potent is the
antagonist.
The value of pA2 is a negative logarithm of the molar concentration of competitive
antagonist, which requires a doubling of the concentration of agonist to compensate
for the action of the antagonist. The meaning of pA2 is the affinity of the antagonist
to the receptor
The pA2 value indicates the concentration of antagonist when double the agonist is
required to have the same effect on the receptor as when no antagonist is present.
Rat anococcygeus muscle is smooth muscle which is attached to the bone. This
muscle arises independently from one or more, usually two upper coccygeal vertebrae
in the middle of pelvic cavity. The muscle at their origin lies near to one another and
at the back of the terminal colon. The muscle can be easily and quickly dissected out
and are about 3 cm long, 150-300 µg thick and about 0.5 cm broad. The muscle in
male rate is stronger, heavier and broader than those in females. They have thick
adrenergic innervations and contracts to NA, Ach, and Serotonin but not to histamine.
Prazocine: Prazosin is sympatholytic medication used to treat hypertension, anxiety
and PTSD. Prazosin is alpha 1 blocker that acts as an inverse agonist at alpha

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 Third Year B.Pharm (Semester V)

adrenergic receptors. These receptors are found on smooth muscle where they are
responsible for vasocontrictive action of NA. They are found in CNS.
Norepinephrine (NE) Norepinephrine is a organic chemical called catecholamines
which is hormone and neurotransmitters.
Procedure:
 Record concentration dependent concentration response curve till peak effect.
 Select two doses bearing 1:2 dose ratio and eliciting submaximal responses (A and
2A) for PA2 determination
 Standardize the tissue with the selected doses of acetylcholine. A tissue is said to
be standardized when it responds identically to the same dose of an agonist when
repeated.
 Record the concentration due to the double dose of Norepinephrine (2A) in the
presence of varying concentrations of Prazocine (B 1, B2, B3)
 Consider the response due to double the dose of Nor epinephrine (2A) before
adding prazocine as 100% response. Determine the corresponding percent (%)
response to this dose of Norepinephrine (2A) in the presence of varying
concentrations of Prazocine
 Plot the graph representing negative log of molar concentration of atropine
employed along X-axis and % response along Y axis.
 pA2 value is defined as the negative log of molar concentration of prazocine
required to reduce the effect of dose 2A to A respectively
 Read out the pA2 value for prazocine from the graph directly. It will correspond to
the % response obtained with half the dose of Norepinephrine (A).

Response:

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 Third Year B.Pharm (Semester V)

Calculations
Sr. Molar Molar Response % response
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 Third Year B.Pharm (Semester V)

No. concentration of concentrati (mm)

Noradrenalin on of
Prazosin
1 A
2 2A
3 2A B1
4 2A B2
5 2A B3

Result:

SHORT QUESTIONS
1) Explain pA2 value

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 Third Year B.Pharm (Semester V)

2) Explain rat anococcygeus muscle

3) Write note on Prazosin and nor adrenaline

EXPERIMENT NO. 12
DETERMINATION OF PD2 USING GUINEA PIG ILEUM
References

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 Third Year B.Pharm (Semester V)

 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.

Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 70.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020., Page 55.
Requirements:
Animal: Guinea Pig
Drugs: Acetylcholine (Stock solution 1 mg/ml)
Physiological solution: Tyrode Solution
Equipment: Student organ bath, aerator, frontal writing lever, forceps, rubber tubes,
scissor and forceps, beaker, spatula, cotton, pipette, scissor, Petri plate, curved
needle, thread, clay, kymograph papers etc
Physiological conditions:
Temperature: 37 ± 0.050 C
Tension: 500 mg
Aeration: Air bubbled with oxygen and carbon dioxide
Principle
The EC50 value describes relative potency of the drugs. Lower the EC50 value, more
potent is the drug. Sometimes the same is indicated as pD 2 value which is defined as
negative log of molar concentration of the drug producing 50% of maximal response.
If pD2 value is high then potency of the drug also high. In calculating the pD 2 value,
the % of the maximum response produced by each does is plotted against log molar
concentration of the drug and the concentration producing 50% response is read as
pD2 Value. The EC50 or pD2 values are used to express the affinity of a drug. Both the
values can be interconvertible employing a simple equation i.e. if EC50 is expressed
as (m X 10-n) then pD2=n-log m. Dose ration is calculated by comparing the doses of
Ach, needed to produce 50% response in presence and absence of drug.
Response

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 Third Year B.Pharm (Semester V)

Observation
Sr. Dose of Ach Log dose of Height of %
No. ml µg Ach contraction in response
mm
1 0.1
2 0.2
3 0.4
4 0.8
5 1.6
6 3.2

Graph
 Plot a graph showing dose of Ach on X-axis and response on Y-axis
 Plot a DRC on semilog graph paper taking log dose of agonist on X-axis and %
response on Y-axis
 Plot a third graph showing log molar conc. Of Ach on X axis and % response on Y
axis.

Result:
The PD2 value of Acetylcholine using guinea pig ileum was found to be_____

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 Third Year B.Pharm (Semester V)

SHORT QUESTIONS
1) Explain pD2 value. What is importance of pD2 value?

2) Discuss mechanism of Acetylcholine on guinea pig ileum

EXPERIMENT NO. 13
EFFECT OF SPASMOGENS AN SPASMOLYTICS USING RABBIT JEJUNUM
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 Third Year B.Pharm (Semester V)

References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 77.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020. Page 59.
Requirements:
Animal: Rabbit jejunum
Drugs: Acetylcholine (Stock solution 1 mg/ml), Physostigmine (10 µg/ml), Atropine (4
µg/ml) Adrenaline (20 µg/ml) propranolol (100 µg/ml)
Physiological solution: Tyrode Solution
Equipment: Student organ bath, aerator, frontal writing lever, forceps, rubber tubes,
scissor and forceps, beaker, spatula, cotton, pipette, scissor, Petri plate, curved
needle, thread, clay, kymograph papers etc
Physiological conditions:
Temperature: 37 ± 0.050 C
Tension: 500 mg
Aeration: Air bubbled with oxygen and carbon dioxide
Theory:
Spasmogens is a substance which induces spasms or substance which can produce
contraction of smooth muscle such as histamine, serotonin, and bradykinins,
acetylcholine.
Smasmolytics: They are also called antispasmodics. They are suppressing muscle
spasms.
Principle:
Rabbit intestine is smooth muscle which shows regular pendular movement having
nonstop contraction and relaxation. So, for studying effect of drugs on intestinal
movement, rabbit intestine is an ideal preparation. It is supplied by ANS. Rabbit
intestine contains muscarinic and adrenergic receptors. Ach acts on muscarinic
receptors and Physostigmine increases the spasm and pendular movements. This
action is blocked by muscarinic blockers. Similarly, the adrenaline acts on alpha and
beta receptors and exhibits inhibitory effect on pendular movements. The adrenaline

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 Third Year B.Pharm (Semester V)

action is inhibited by adrenoreceptor blockers like alpha receptor blockers and beta
blockers.
Response:

Note: Correlate the agonism and antagonism from experiment number 2.

Interpretation and result

Acetylcholine produced contraction of the rabbit jejunum. Atropine sulphate is a
muscarinic receptor blocker; therefore, it reduced the spasmogenic effect caused by
acetylcholine. Physostigmine also produced mild spasmogenic effect and increased
the peristaltic movement of the jejunum. Atropine sulphate blocks the effect of
Physostigmine.

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 Third Year B.Pharm (Semester V)

SHORT QUESTIONS
1) Define spasmogenic and spasmolytics with example. Write principle involved in
this experiment.

2) Explain mode of action of Physostigmine.

3) Write clinical uses of propranolol and atropine.

EXPERIMENT NO. 14

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 Third Year B.Pharm (Semester V)

TO STUDY ANTI INFLAMMATORY ACTIVITY OF DRUGS USING CARAGEENAN

INDUCED PAW EDEMA MODEL
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 80.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020. Page 63.
Requirements:
Equipment and apparatus: Plethysmograph, verneir caliper, syringes, oral feeding
tube etc
Animal: Rat (150-200 g)
Drugs and solution:
Carrageenan (prepare 1% w/v solution and injects 0.1 ml into planter region)
Indomethacin (20 mg/kg,s.c., stock solution 4 mg/ml of the drug and inject 0.5 ml/100
g of the body weight of the rat
Normal saline solution
Theory:
Inflammation is reply of immune system to foreign organism or antigenic substance
and characterized by edema, erythema, swelling, pain, tenderness and burning
sensation.
Signs of inflammation: the five classical signs of inflammation are heat, pain,
redness, swelling, and loss of function. Inflammation may be acute and chronic.
Mediators of inflammation:
Bradykinins, leukocytes, lysosome granules, histamine, T cells, leukotriene,
interleukins, PG
Antiinflammatory drugs:
Corticosteroids, Acetyl salicylic acid, Indomethacin, ibuprofen
Indomethacin is NSAID used to reduce fever, pain, stiffness and swelling. It acts by
preventing the production of PG molecules responsible for above symptoms. it is
powerful anti-inflammatory drug with analgesic and antipyretic properties. If inhibits
production of eicosanoids like prostacyclin, thromboxane and PGs by inhibition of
cyclo oxygenase and thereby reduces edema in this model.
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Plethysmograph is used to measure the paw volume. Paw edema is the method
used for testing acute inflammation. Among other methods of screening, this is
simplest and most commonly used technique. It is basic assembly containing
mercury. The mercury uprooting due to plunging of the paw can be straightforwardly
from scale appended to the mercury section or modifying the mercury level in the arm
B to the first level by moving B up-down and taking note of the volume required to get
the level of both arms equivalent.
Carrageenan are a family of linear sulfated polysaccharides that are extracted
from red edible seaweeds. They are widely used in the food industry, for their gelling,
thickening, and stabilizing properties
The development of edema induced by carrageenan injection causes an acute and
local inflammatory response. In the early phase (0–1 h), histamine, serotonin,
and bradykinin are the first mediators involved, whereas prostaglandins and various
cytokines such as IL-1β, IL-6, IL-10, and TNF-α are implicated in the second
phase. intraplantar injection of carrageenan led to time-dependent development of
peripheral inflammation, which resulted in a significant increase in the levels of tumor
necrosis factor α (TNF-α) and interleukin 1 (IL-1) β, nitric oxide (NO) and prostaglandin
E2 (PGE2) and also iNOS and COX-2 protein expression in inflamed paw.

Fig. Plethysmometry
Principle:
The method is based on the ability of anti-inflammatory agents to inhibit the edema
produced in the hind paw of the rat after injection of carrageenan. The carrageenan is
sulphated polysaccharide acquired from ocean weed and by causing the arrival of

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histamine, 5 HT, bradykinins and PG which produces aggravation and edema. The
volume of the injected paw is measured before and after the application of irritants.
The paw volume of treated animals is compared with control.
Procedure:
 Weight the animals and number them
 Make the mark on both the hind paws (right and left) just beyond the tibio tarsal
junction, so that every time the paw is dipped in the mercury column up to the
fixed mark to ensure constant paw volume.
 Not the initial paw volume of both the paws of each rat by mercury displacement
method
 Divide the animals into two groups each comprising of at least four rats. To one
group inject saline and to the second group inject indomethacin subcutaneously.
 After 30 min inject 0.1 ml of 1% (w/v) carrageenan in the plantar region of the left
paw of control as well as indomethacin treatment group.
 The right paw will serve as reference non-inflammatory paw for comparison.
 Note the paw volume of both legs of control and indomethacin treated rats at 15,
30, 60, 120 min after carrageenan challenge.
 Calculate the percent difference in the right and left paw volumes of each animal
of control and indomethacin-treated group. Compare the mean percent change in
paw volume in control and drug treated animal and express as percent edema
inhibition by the drug.
Observation table

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Interpretation and result:

Indomethacin showed anti-inflammatory action against carrageenan induced paw
edema.

SHORT QUESTIONS
1. Define inflammation and write its signs.

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2. Enlist mediators of inflammation

3. Give the principle in anti-inflammatory activity of drugs using carrageenan induced

paw edema model.

EXPERIMENT NO. 15
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TO STUDY ANALGESIC ACTIVITY OF DRUG USING CENTRAL AND PERIPHERAL

METHODS
References
 A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T.
Baviskar and Dr. Prakash Patil, PV books, S Vikas and Company (Medical
Publishers) Jalandhar, 2019, Page no. 86.
 A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and
Sunil Pawar, Nirali Prakashan, Second edition, January 2020. Page 68.
 Handbook of Experimental Pharmacology by SK Kulkarni, Vallabh Prakashan,
2009, Page no. 125.
 S.B.Kasture, Handbook of Experiments In Preclinical Pharmacology, Career
Publication, First Edition, 2007, Page. 72-73
CENTRAL METHOD
Requirements:
Apparatus: Volumetric flask, syringe (1 ml), beaker etc
Drugs: Pentazocine injection OR morpine injection 5 mg/ kg
Instrument: Hot plate analgesiometer, (Eddys hot plate) animal weighing balance
Animals: Swiss albino mice (3 mice) 20-25 gm
For demonstration on computer only: X cology software CD
Principle:
Analgesia is defined as a state of reduce awareness to pain, an analgesic are
substance which decrease pain sensation by increase threshold to painful stimuli.
Painful stimuli in experimental animals can be produced by applying noxious stimuli
such as theral radiant heat, chemical irritant such as acetic acid, or any acid or
physical pressure such as tail compression.
Pentazocine/Morphine is a synthetically-prepared prototypical mixed agonist–
antagonist narcotic (opioid analgesic) drug of the benzomorphan class of opioids used
to treat moderate to moderately severe pain. Onset of action is 15-30 min on
oral/im/sc use and duration of action is 2—3 hours. Binds to opiate receptors in the
CNS, causing inhibition of ascending pain pathways, altering the perception of and
response to pain by minimizing action of enkephalin
Hot plate method: In this method heat is used as a source of pain. Animals are
individually placed on a hot plate maintained at constant temperature (55 0 C) and the
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reaction of animals, such as paw licking or jump is noted as end point. Analgesics
increase the reaction-time. The method was described by Eddy and Leimbach

Fig. Hot plate analgesomer

Tail flick method: The tail flick test is a test of the pain response in animals, similar
to the hot plate test. It is used in basic pain research and to measure the
effectiveness of analgesics, by observing the reaction to heat. Most commonly, an
intense light beam is focused on the animal's tail and a timer starts. When the animal
flicks its tail, the timer stops and the recorded time (latency) is a measure of the pain
threshold. Alternate methods can be used to apply heat, such as immersion in hot
water.

Fig. Tail flick analgesometer

Dose calculations:
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For dose in mg
Pentazocine/Morphine dose 10 mg/kg
10 mg for 1000 gm, so 50.3 gm= 0.50 mg
52.6 gm=0.52 mg and 54.1 gm=0.54 mg
For dose in ml
Pentazocine ampule contains 30 mg/ ml pentazocine
So 30 mg is dissolved in 10 ml water, so 1 ml contains 3 mg/ml (stock
solution)
Now, for 0.50 mg drug how many ml?
3 mg for 1 ml, so 0.50 mg requires 0.16 ml drug.
Similarly, 0.52 mg requires 0.17 ml drug and
0.54 mg will require 0.18 ml drug.
Observations (as in Software CD)
A) Tail flick method
Sr. Time taken to flick the tail Time taken to flick the tail
No before morphine after morphine
administration (sec) administration (sec)

B) Hot plate method

Sr. Basal reaction time (Sec) Reaction time (Sec) after
No. before morphine morphine administration
administration
Paw licking Jump Paw licking Jump
response response
1. 3 5.6 4.6 7.1
2. 4.3 2.8 5.9 3.9
3. 6.3 3.2 8.2 5.2
4. 2.9 4.2 4.7 5.7
5. 1.6 5.2 2.6 6
6. 2.3 3.8 4.6 4.6
Mea 3.4 4.13 5.1 5.41

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Result: Pentazocine/morphine was found to increase reaction time on mice on tail

flick and hot plate analgesometer. Hence it has analgesic activity. The average
maximum percent increase in reaction time was found to be 8.66 sec at 60 min as
per X cology software CD experiment (demonstration) by tail flick method and 5.1
Sec for paw licking and 5.41 sec for jump response in hot plate method.
PERIPHERAL METHODS
TO STUDY THE ANALGESIC EFFECT OF DRUGS ON MICE BY USING ACETIC
ACID INDUCED WRITHING TEST IN MICE
Requirement
Animals: Mice (25-30 g)
Drugs: Morphine Sulphate (Dose 5 mg/kg, s.c, stock solution 0.5 mg/ml and
administer 1 ml/100 g of body weight of animal
Acetic acid 1% v/v (inject 1 ml/100 g of body weight of the animals
Normal saline (0.9 %)
Principle
Writhing response: it is a response or painful reaction shown by an animal following
administration of certain chemicals. Writhing response includes abdomen constriction,
twisting of trunk and expansion of hind legs.
Painful reactions in animals may be produced by chemical also. Intraperitonial
injection of pheynylquinone, bradykinins or acetic acid produces pain reaction which is
characterized as writhing response. Abdominal constriction, twisting trunk and
extension of hind legs (stretching) responses by the animal are taken as reaction to
chemically induced pain. Analgesics inhibit this writhing response.
Observation:

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Result:
The writhing response of mice was reduced by morphine administration. Hence
morphine has analgesic activity.
SHORT QUESTIONS
1) Define analgesia

2) Classify analgesic drugs

3) What is writhing response? How it is induced by chemicals? Which chemicals are

used?

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4) Write mode of action of morphine.

5) Discuss principle involved in analgesic activity measurement by hot plate, tail flick
and writhing response.

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