Department of Pharmacology
Laboratory Manual
PHARMACOLOGY-II
Dr. Arunabha Mallik (M.Pharm, Ph.D)
Professor
Marri Laxman Reddy Institute Of
Pharmacy, Hyderabad
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
About MLRIP
To be an educational Institute of par excellence and produce competent pharmacy professionals
to serve the community through research and the ever-increasing needs of Industry.
1. Imparting quality education and innovative research for various career opportunities.
2. Creating conducive academic environment to produce competent pharmacy professionals.
3. Indoctrination of students adorned with high human values and make them aware of their
responsibility as health care professionals.
PEO 1: To produce graduates with sound theoretical knowledge and technical skills required
for their career opportunities in various domains.
Program PEO 2: To incite the students towards research and to address the challenges with their innovative
Educational
contributions for the benefit of the mankind.
Objectives
PEO 3: To instill the essence of professionalism, ethical commitment to become a health
care professional with sound integrity and adherence to the core human values in the service
of the society.
PROGRAM OUTCOMES
1. Pharmacy Knowledge: Possess knowledge and comprehension of the core and basic knowledge associated
with the profession of pharmacy, including biomedical sciences; pharmaceutical sciences; behavioral, social,
and administrative pharmacy sciences; and manufacturing practices.
2. Planning Abilities: Demonstrate effective planning abilities including time management, resource
management, delegation skills and organizational skills. Develop and implement plans and organize work to
meet deadlines.
3. Problem analysis: Utilize the principles of scientific enquiry, thinking analytically, clearly and critically,
while solving problems and making decisions during daily practice. Find, analyze, evaluate and apply
information systematically and shall make defensible decisions.
4. Modern tool usage: Learn, select, and apply appropriate methods and procedures, resources, and modern
pharmacy-related computing tools with an understanding of the limitations.
5. Leadership skills: Understand and consider the human reaction to change, motivation issues, leadership and
team-building when planning changes required for fulfillment of practice, professional and societal
responsibilities. Assume participatory roles as responsible citizens or leadership roles when appropriate to
facilitate improvement in health and well-being.
6. Professional Identity: Understand, analyze and communicate the value of their professional roles in society
(e.g. health care professionals, promoters of health, educators, managers, employers, employees).
7. Pharmaceutical Ethics: Honour personal values and apply ethical principles in professional and social
contexts. Demonstrate behavior that recognizes cultural and personal variability in values, communication
and lifestyles. Use ethical frameworks; apply ethical principles while making decisions and take
responsibility for the outcomes associated with the decisions.
8. Communication: Communicate effectively with the pharmacy community and with society at large, such
as, being able to comprehend and write effective reports, make effective presentations and documentation,
and give and receive clear instructions.
9. The Pharmacist and society: Apply reasoning informed by the contextual knowledge to assess societal,
health, safety and legal issues and the consequent responsibilities relevant to the professional pharmacy
practice.
10. Environment and sustainability: Understand the impact of the professional pharmacy solutions in societal
and environmental contexts, and demonstrate the knowledge of, and need for sustainable development.
11. Life-long learning: Recognize the need for and have the preparation and ability to engage in independent
and life-long learning in the broadest context of technological change. Self-assess and use feedback
effectively from others to identify learning needs and to satisfy these needs on an ongoing basis.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EX. NO. TABLE OF CONTENTS PAGE
Laboratory Orientation and General Objectives of the Course 3-4
1 Basic principle of Bioassay 5-7
To study the dose response curve of ach using rat
2 8-10
ileum
To perform and determine the strength of an unknown sample by three
3 11-12
point bioassay using isolated organ preparation.
4 To perform and determine the strength of an unknown sample of 13-14
histamine by four point bioassay using isolated organ preparation.
5 To record the concentration response curve of Oxytocin using rat uterus 15
preparation.
To study the anti-secretory and ulcer protective effect of cimetidine in
6 16-17
pylorus-ligated rats.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
LABORATORY ORIENTATION
The laboratory portion of this course is designed to study the different laboratory
animals, their applications and screening of different pharmacological agents
thoroughly than it is presented in lecture. Core learning will come from practical and
tissue studies. This method of ‘hands on’ learning should also enhance and strengthen
the knowledge you gain in lectures.
At times you will be working individually, in pairs or in groups of three or four. Each
lab period is loosely structured to begin with a short introduction to the exercise that
highlights the activities of the day, what materials are available for use and any
changes in procedures. After that you will work independently to learn the material.
There is never enough time in lab to go over each and every item that you are assigned.
The lab is a designated a time when you have access to materials that you will not have
available during home study time. Some of the information assigned in lab you can
learn at home, particularly animals, instruments, method, procedure, application,
mechanism of action of drugs etc.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
GENERAL OBJECTIVES OF THE COURSE
At the end of the practical training in general, and experimental pharmacology
the learner shall be able to:
1. List the various dosage forms and enumerate their advantages and disadvantages.
2. Advise patients about the proper use of medication devices, storage of medicines
etc.
3. Retrieve drug information from appropriate sources.
4. Appreciate the role of good laboratory practice in promotion of rational
diagnostics, therapy, and experimentation.
5. Realize the cardinal role of ethics in experimentation.
6. Order monitoring of drug levels where indicated and take appropriate remedial
measures.
7. Prescribe rationally and in an individualized pattern.
8. Plan and carry out experiments to demonstrate the effect of drugs in experimental
animals and isolated tissues.
9. Critically appraise drug advertisements.
10. Apply fundamental statistical tests to experimental data and interpret results.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EXPERIMENTAL NO- 01
Object: Basic principle of Bioassay.
Introduction:-
Bioassay is defined as estimation or determination of concentration or potency of
physical, chemical or biological agents by means of measuring and comparing the
magnitude of the response of the test with that of standard over a suitable biological
system under standard set of conditions. An assay is a form of biological experiment; but
the interest lies in comparing the potencies of treatments on an agreed scale, instead of in
comparing the magnitude of effects of different treatments. Biological assays or
biological standardizations or simply bioassays are methods used for estimation of the
potency of substances by observing their pharmacological effects on living animals (in
vivo) or isolated tissues (in vitro) and comparing the effect of these substances of
unknown potency to the effect of a standard.
Principle of Bioassay:-
Active principle to be assayed should show the same measured response in all animal
species. Bioassay involves the comparison of the main pharmacological response of the
unknown preparation with that of the standard. The method selected should be reliable,
sensitive, and reproducible and should minimize errors due to biological variation and
methodology. The degree of pharmacological response produced should be reproducible
under identical conditions. The reference standard and test sample should have same
pharmacological effect and mode of action, so that their DRC curve run parallel and their
potency ratio can be calculated. Activity assayed should be the activity of interest.
Individual variations must be minimized/accounted for. Bioassay might measure a diff
aspect of the same substance compared to chemical assay. The test solution and standard
should be compared for their established pharmacological effect using a specified
pharmacological technique.
Types of Bioassays
There are three main types of bioassays (other than qualitative assays):-
1. Direct Assays
2. Indirect Assays based upon quantitative responses
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
3. Indirect Assays based upon Quantal responses (“all or none”)
• Direct Assay:- Doses of the standard and test preparations are sufficient to
produce a specified response, and can be directly measured.
• Indirect Assay:- In indirect bio-assays the relationship between the dose and
response of each preparation is first ascertained. Then the dose corresponding to a
given response is obtained from the relation for each preparation separately.
• Quantal Assay:- This response is in the form of “all or none” means no response
or maximum response. These can be biossayed by end point method.
Predetermined response is measured which is produced by threshold effect.
Quantal Responses are population response based on an all-or-nothing (0 or 1 –
presence or absence) response such as death.
Concentration of unknown = Dose of the standard × Concentration of standard/
Dose of test
• Graded Assay:- It is proportional to the dose and response may lie between no
response and maximum response. Graded Responses can be any type of measured
responses in isolated tissues in particular, but also in whole animals. Such
responses are infinitely graded and there are a large number of them.
Examples – contractions of muscle, blood pressure, blood sugar concentrations, etc.
• Matching Method:- In this type of assay the test substance and the standard are
applied and the responses obtained are matched by a trial and error process until
they produce equal effects.
• Bracketing Method:- Bracketing bioassay is performed by selecting two
standard doses, which will give a close bracket on either side of the response
produced by the unknown. The working dose of standard is first determined in the
sensitive part of dose-response curve, that is, a dose that will approximately
produce 50% of the maximal concentration.
• Interpolation Method:- This is a simplest form of graded response assay and
involves no statistical data and many calculations. In this assay the dose response
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
curve is fist obtained from different doses of standard ach solution. The
concentration of unknown is then read from the standard graph. Interpolation
method of bioassay is less time consuming and yet reliable compare to matching
type of bioassay.
• Three Point Bioassay:- In three point bioassay, the DRC of standard & test
samples is first obtained from the responses due to graded doses. From the DRC
of standard, two standard doses are selected in such a way that they have
produced 25% & 50% of the maximal response respectively & are designated as
S1 & S2. The responses of these doses lie on the steepest & straightest part
(linear) of the curve. From the DRC of test sample one test is selected such that it
gives a response which lies in between the two standard responses that is it gives
a greater response than S1 & a smaller response than S2 & is designated as T.
After selecting the standard & test doses, the bioassay is performed by recording
the standard & test responses in randomized fashion as per Latin square design.
The pattern of addition of doses is S1, S2, T; S2, T, S1 & T, S1, S2 in 3
successive cycles. The mean values of height of contraction for all the 3 doses are
calculated and are used in plotting the graph so as to estimate the potency of the
test sample. The precision and reliability of this method is much better than
matching and bracketing methods of bioassay & the sensitivity of the isolated
tissue preparation is assessed prior to testing the unknown sample.
• Four Point Bioassay:- The classic 2X2 parallel assay involves being able to
measure parallelism where drugs acting through the same mechanism are
expected to produce parallel dose-response curves.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EXPERIMENTAL NO- 02
Object: To study the dose response curve of ach using rati leum
REQUIREMENTS:
Animal: Rat (of either sex weighing between 200-250g.)
Drugs: Acetylcholine (1 µg/mL, 10 µg/mL, 100 µg/mL)
Apparatus: Reservoir, Tubing, isolated organ bath, organ tube
heating coil, Thermostat, Isotonic frontal writhing lever, Recording
drum, Aeration tube cum tissue holder, Haemostatic forceps, sketch
pen tip, ink etc.
EXPERIMENTAL CONDITION:
Physiological Salt Solution : Tyrode
Temperature : 37 ± 1o C
Basal Tension on Lever : 500 mg
Contact time : 30 sec.
Aeration : Carbogen
(95% O2 and 5% CO2) Magnification of the
response : 10 times
Drug : Acetylcholine Chloride (1, 10 or 100 μg/mL)
Molecular weight of drug : 181.78
PRINCIPLE:
Acetylcholine produces a dose dependent concentration of rat ileum smooth muscle. First
taken the two equipotent response of same dose and then taken the graded response.
THEORY:
Graded Dose Response Relationship Curve of Acetylcholine on Frog Rectus
Muscle:
➢ Single biological unit, either a single animal or an isolated tissue is used.
➢ It depends upon an observation that graded increase (in geometric
proportion) in the dose of drug gives proportional rise in the magnitude of
biological response.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
➢ Actually, beyond a specific dose level, biological response increases in
proportion to the increase in dose. This dose level is known as ‘Threshold
dose’.
➢ Such proportional rise in biological response occurs only up to a dose level
known as ‘Ceiling dose’, beyond which a steady biological response is
achieved even after increasing the doses.
➢ Shape of Graded DRC, when plotted as ‘dose Vs Response’ is a ‘Parabola’
➢ Shape of ‘Log Dose Vs Response’ curve is a ‘Sigmoid’ line
or is having ‘S’ like shape.
PROCEDURE:
➢ The assembly is set up and the arrangements are made for the above
mentioned condition.
➢ A rat fasted over night was anaesthetized by chloroform and sacrificed by as
per CPCSEA recommended guidelines.
➢ The abdominal cavity was quickly opened through a midline incision, leum
is separated and mounted in the organ bath.
➢ One end of the ileum is tied to the aeration tube and the other is connected to
the isolated frontal writing lever.
➢ The ileum is allowed to stabilize for half an hour. During this period the PSS
is changed after every ten min. Once the tissue is stabilize, raded doses of
Ach are added to at defined time period of interval for obtain contractile
responses.
➢ 00 sec: Start the drum and record a base line for 30 sec.
➢ 30 sec: Add the first dose of drug in organ bath and take the response for
another 30sec.
➢ 60: Stop the drum and give wash until the tip of lever rich to baseline.
➢ Continue above procedure for next doses.
➢ Measure the height of concentration at different doses of Ach.
➢ Tabulate the observations into three columns as Dose of Ach, Height of
concentration (in mm) and % response.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
OBSERVATION TABLE:
Sr. Drug Conc. Dose of drug in mL Response in % Response
No Name of drug mm
1. 0.1 2 20
2. 1 0.2 5 25
3. µg/mL 0.4 7 35
4. 0.8 9 45
5. 10 0.16 12 60
6. µg/mL 0.32 14 70
7. Ach 0.64 16 80
8. 100 0.128 17 85
9. µg/mL 0.256 20 100
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EXPERIMENTAL NO- 03
Object– To perform and determine the strength of an unknown sample by three
point bioassay using isolated organ preparation.
References – Kulkarni S.K., Handbook of experimental pharmacology, Vallabh
prakashan, first edition 2009, page no.92-94
Requirements –
Drug - Histamine stock solution
Physiological solution – Tyrod solution
Procedure –
1. The guinea pig is scarified by a blow on the head and carotid blending.
2. Cut open the abdomen and lift the caeccum to trace the ileo-caecum
junction cut and remove a few centimeters long of ileum portion and
immediately place it in the watch glass containing tyrode solution.
3. Take one piece of ileum of 2-3 cm long and tie the thread to top and
bottom ends without closing the lumen ,mount the tissue in the organ bath
containing Tyrode solution maintained at 32-350c and bubbled with 0.2 or
air.A tension of 0.5gis applied and the tissue is allowed to equilibrate for
30 min. before adding drug to the organ bath.
4. Record concentration dependent response due to histamine using frontal
writing lever.\contact time of 30 sec. and 5 min. proper recording of the
response.
5. Record at least 4 concentration dependent response curve due to
histamine.
6. Select two doses standard say S1 and S2 which produce sub maximal
response.
7. Select a dose of the test sample t, which is likely to produce a response
less than the response produced by S2 dose of the standard as shown
below.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
8. Measure the of concentrated produced by S1 dose as S 1 .
9. Measure the height of concentration produced by S2 dose.
10. Measure the height of concentration produced by S2 dose.
11. Measure the height of concentration produced by t1 dose as t1..
12. Calculate the potency of the test solution.
Potency and test solution:
S1 T1 – S1 S2
………… X antilog ……………. X log ………..
T1 S2 –S1 S1
Results:-
The method for finding the strength of an unknown sample of Histamine by three
point bioassay using Guinea pig ileum preparation had been demonstrated.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EXPERIMENT NO. – 04
Object – To perform and determine the strength of an unknown sample of
histamine by four point bioassay using isolated organ preparation.
Reference – Kulkarni S.K. Handbook of experimental pharmacology, vallabh
prakashan first edition 1987 reprint 2009, page no.92 to 94
Requierment –
Animal – Guinea pig (400-600gm overnight fasted)
Drugs – Histamine stock solution
Physiological solution –Tyrode
Procedure –
1. The guinea pig is sacrifieced by a blow on the head and carodid bleeding.
2. Cut open the abdomen and till the caeccum to trace the ilium caecal
junction. Cut and remove a few centimeter long of the real portion and
immediately place it in the watch glass containing tyrode solution trim the
nesentry and with gentle care olean the contents of the ileum by pushing
the tyrode solution into the lumen of the ileum. Cut the ileum into small
segments of 2-3 c.m. longs.
3. Take one piece of ileum of 2-3 c.m. long and tie the thread to top and the
bottom ends without closing the lumen and mount the tissue is allowed to
equilibrate for 30 min. before adding drugs to the organ bath.
4. Record concentration dependent response due to histamine using frontal
writing lever, contact time of 30sec. and 5 min. time cycle are kept for
proper recording of the responses.
5. Record at least 4 concentration dependent response due to histamine.
6. Record graded response with the standard solution histamine until peak
effect is observed.
7. Select two concentration (A,B) of the standard drug, elicting sub-maximal
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
drug responses(S1,S2) andbearing a dose ratio 1:2 preferentially.
8. Select two suitable volume of the test solution by trail and error method in
such a waythat the response(T1) due to the lower dose of the test (C) lies
preferentially between S1 and S2 . the higher volume of the test solution
selected would be D such that the dose ratio B/A = D/C all the four
responses (S1,S2 , T1, T2) due to the doses thus selected (A,B,C,D) must lie
on the linear part of standard curve.
9. Standardise the tissue with the concentration A.
10. Record four sets of responses due to A,B,C,and D,adding them to organ
bath in a random fusion.
11. Label and fix the tracing.
12. Measure various responses to calculate the mean of each (S1,S2 , T1, T2 )
13. Calculate the potency ratio(M) using following formula:-
Potency ratio = x1/ y1 X antilog
𝑇2−𝑆2+𝑇1−𝑆1 𝑋2
𝑋 𝑙𝑜𝑔 𝑋1 = M
𝑇2−𝑇1+𝑆2−𝑆1
Where, X1 = Lower volume of the standard drug (A)
X2 = Higher volume of the standard drug (B)
Y1 = Lower volume of test solution (C)
Mean S and T = the mean responces
14. Determine the strngth of the unknown solution of histamine using the
concenteration of the standard drug and potency ratio (M).
Report – the method for finding the strength of an unknown sample of
histamine by four point bioassay using guinea pig ileum preparation has
been demonstrated.
Results:-
The method for finding the strength of an unknown sample of Histamine by four
point bioassay using Guinea pig ileum preparation had been demonstrated.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EXPERIMENT NO.-5
Object: - To record the concentration response curve of Oxytocin using rat uterus
preparation.
Requirement:-
Animal- Female rat (120-150gm)
Drugs Oxytocin, Stilbesterol,
Dejalon solution.
Procedure:-
1. Examine the vaginal snear under microscope to know about the proper stage of
oestrus cycle, if the rat is not in Frank oestrus inject 0.1 mg/kg of stilbesterol and
wait for 24 hours.(Vaginal smear is prepared by taking a drop of the slide glass)
2. Cut open the pelvic region and expose both the horn and uterus separate them
gently from the surrounding fatty material and transfer them to a dish containing
DeJalon solution. When the rat is in oestrus generally the uterus is fleshy and pink
in colour.
3. Two separate pieces (2-3cm long) of uterine preparation can be made.
4. Mount the uterine preparation in the organ bath containing DeJalon solution at 30-
320C.Apply a tension of 0.5gm and allow the tissue to equilibrate for 30 minutes.
5. Record contraction due to different coenteration of Oxytocin using frontal writing
lever.Contact time of 30 seconds and 3 minutes time cycle is used for proper
recording of the responses.
6. Label and fix the tracing and draw the concenteration-response curve.
Results:-
The CRC of Oxytocin using rat uterus has been recorded.
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
EXPERIMENT NO.-6
Object: - To study the anti-secretory and ulcer protective effect of cimetidine in pylorus-
ligated rats.
Reference: - (1) Gupta S.K., Drugs screening methods, first edition,2004, Jaypee
Brothers publication, Page no.- 301-307.
(2) Indian Drugs, Volume 48, issue no.- 03, march 2011,page no.- 31-32.
Requirement:-
Animal- Wistar albino rat (150-200gm)
Drugs Cimetidine (Dose 10 mg/kg, ip)
Topfer’s Reagent
Equipment:
Dissecting microscope, surgical equipment’s
Procedure:
1. Anaesthetize the overnight fasted animal.
2. Give an incision of 1 cm long in the abdomen just ellow the sternum.
3. Expose the stomach. Pass a thread around the pyloric sphincter and apply a tight
knot.
4. To another rat inject cimetidine. After 15 min perform pyloric ligation as
described.
5. Give a small cut to the pyloric region just above the knot and collect the contents
of the stomach in a centrifuge tube.
6. Open the stomach along the greater curvature and wash it slowly under the
running tap water.
Score the ulcer as bellow:
0= Normal colored stomach
0.5= Red colouration
1= Spot ulcers
1.5= Haemorrhagic sreaks
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
Department of Pharmacology
2= ulcer ≥ 3 but ≤ 5
3= ulcers > 5
Mean ulcer score for each animal is expressed as ulcer index.
OBSRVATION TABLE
Sr. No Treatment Dose (mg/kg) Ulcer Index
Report:
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Marri Laxman Reddy Institute of Pharmacy, Hyderabad.
