Objective Principle Requirements Experimental specifications (conditions) Preparation of ACh and Physostigmine stock and std. solutions Preparation of frog ringer solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation of magnification value (Mf) Graphical presentation of CRC/ DRC Result and interpretation

1. Experiment No. 8
Effect of physostigmine on DRC of acetylcholine
using frog rectus abdominis muscle
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
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2. Overview of Discussion
• Objective
• Principle
• Requirements
• Experimental specifications (conditions)
• Preparation of ACh and Physostigmine stock and std. solutions
• Preparation of frog ringer solution (PSS)
• Procedure
• Kymograph recording of contractions
• Observation table
• Calculation of magnification value (Mf)
• Graphical presentation of CRC/ DRC
• Result and interpretation
2

3. Objective
To study synergistic effect of physostigmine on the CRC/ DRC
of acetyl choline (ACh) using isolated frog rectus abdominis
muscle preparation.
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4. Combined effect of drugs
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When two or more drugs are given simultaneously or in quick succession they
may be either indifferent to each other or exhibit synergism or
antagonism. The interaction may take place at pharmacokinetic level or at
pharmacodynamic level.
Synergism (Greek: Syn- together; ergon- work)
When the action of one drug is facilitated or increased by the other, they are
said to be synergistic. In a synergistic pair, both the drugs can have action
in the same direction or given alone one may be inactive but still enhance
the action of the other when given together.
Synergism can be of two types-
(a) Additive The effect of the two drugs is in the same direction and simply
adds up-
Effect of drugs A + B = Effect of drug A + Effect of drug B
Side effects of the components of an additive pair may be different-do not add
up. Thus, the combination is better tolerated than higher dose of one
component.
Principle

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(b) Supraadditive (potentiation) The effect of combination is greater than
the individual effects of the components-
Effect of drug A + B > Effect of drug A + Effect of drug B
This is always the case when one component is inactive as such.

6. The rectus abdominis muscle, also known as the abdominal muscle, is a
paired muscle running vertically on each side of the anterior wall of
the human abdomen, as well as that of some other mammals.
There are two parallel muscles, separated by a midline band
of connective tissue called the linea alba.
It extends from the pubic symphysis, pubic crest and pubic
tubercle inferiorly, to the xiphoid process and costal cartilages of
ribs V to VII superiorly.
The proximal attachments are the pubic crest and the pubic symphysis.
It attaches distally at the costal cartilages of ribs 5-7 and the xiphoid
process of the sternum.
Functions It is an important postural muscle. It helps to flexing lumbar
spine. It plays an important role in breathing and helps to keep
internal organ intact
Rectus abdominis muscle
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7. Rectus abdominis muscle of the frog is the voluntary skeletal muscle
that produces a slower contraction in response to the acetylcholine
(ACh).
Isolated rectus abdominis preparation is an isolated preparation of
tissue which is widely used for study of ACh as it operates on
nicotinic ACh mechanism at neuromuscular junctions (interaction
occur at Nm subtype of receptor present on neuromuscular
junction).
Cholinergic drug like Ach stimulate nicotinic receptors and produces
skeletal muscle contraction due to opening of Na+ channel.
Physostigmine is reversible anti-cholinesterase (carbamate) which
inhibits the metabolic breakdown of ACh by forming a reversible
complex with an enzyme acetyl cholinesterase (AChE) by interacting
with anionic (-) site of AChE. The step II (Splitting) and step III
(Regeneration of enzyme) analogous to ACh hydrolysis are occurs
slowly and hydrolysis of ACh delay by 1-8 hours. As a result, the
action of ACh is potentiated.
The CRC of ACh will be shifted to the left without suppression of
maxima in the presence of physostigmine. 7

8. Frog’s Rectus
Abdominis Muscle
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9. Requirements
Animal: Frog.
Physiological solution: Frog ringer solution.
Chemicals: Acetylcholine (stock solution- 1 mg/ml), Physostigmine
(stock solution- 1 mg/ml), Fixing solution.
Instruments: Sherrington recording drum , Student organ bath
Aerator, Insulin or tuberculin syringe to inject drugs in small
fractions, Dissecting board and various dissecting instruments.
Frontal writing lever and stand, Pipette, Stop watch etc.
Miscellaneous: Kymograph paper, plasticin, clips, and thread.
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10. Experimental specifications (conditions)
 Isolated tissue- Isolated Frog’s rectus abdominis preparation
 Drug- Acetylcholine (ACh) (Stock solution- 1 mg/ml, Std. conc.- 1,
10 and 100 μg/ml), Physostigmine (stock solution- 1 mg/ml, Std.
conc. 2 μg/ml)
 Physiological salt solution (PSS)- Frog ringer solution
 Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact
time- 90 seconds, Washing period- 3 minutes
 Applied load/ tension- 1 g
 Bath capacity- 40 ml
 Bath temperature- Doesn’t matter
 Speed of rotation of drum- 0.25 mm/ second
 Magnification value (Mf) = d (F-W)/ d (F-T)
 Aeration- Normal air (1- 2 bubbles/ second)
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11. Preparation of ACh stock and standard solutions
Stock solution- 1 mg/ml or 1000 μg/ml
 Dissolve 1 mg of ACh in 1 ml of distilled water → 1000 μg/ml
Standard solutions
 Dilute 1 ml of stock solution up to 10 ml with distilled water → 100 μg/ml
 Dilute 1 ml of 100 μg/ml solution up to 10 ml with distilled water → 10 μg/ml
 Dilute 1 ml of 10 μg/ml solution up to 10 ml with distilled water → 1 μg/ml
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Preparation of Physostigmine stock and standard solutions
Stock solution- 1 mg/ml or 1000 μg/ml
 Physostigmine salicylate injection contain 1 mg/ ml of physostigmine →
1000 μg/ml.
Standard solutions
 Dilute 0.1 ml of stock solution up to 10 ml with PSS → 10 μg/ml
 Dilute 2 ml of 10 μg/ml solution up to 10 ml with PSS → 2 μg/ml

12. Preparation of frog ringer solution (PSS)
 Prepare 1 litre of frog ringer solution by dissolving NaCl (6.0 g),
KCl (0.14 g), NaHCO3 (0.2 g), and glucose (2 g) in distilled
water.
 CaCl2 (0.12 g) should be dissolved separately in distilled water
to avoid chances of precipitation of salt.
 Mix CaCl2 solution to the higher volume of PSS.
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13. Procedure
 Pith or stun the frog and lay it on its back on the frog-dissecting board.
Pin or tape the four limbs to the frog board.
 Remove the skin on the abdomen and expose the rectus abdominis
muscle
 Cut and prepare two rectus muscle preparations from each frog. Tie a
thread to the top and bottom of each muscle preparation before
detaching the muscle from the body of the frog.
 Mount the preparation in up-right position in the organ bath containing
frog ringer solution under a tension of 1 g. There is no need of
maintaining bath temperature since it is an amphibian tissue
preparation. Bubble the organ bath with air.
 Relax the tissue for 45 minutes during which wash the tissue with fresh
volume of frog ringer for at least four times.
 Record the contraction due to ACh using frontal writing lever. Contact
time of 90 seconds and 5 minutes of total time cycle may be followed
for recording of response of each dose of standard concentration.
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14.  Record the CRC/ DRC of ACh std. using at least five gradual doses.
 Add physostigmine (2 μg/ml) to the reservoir containing frog ringer
solution and irrigate the tissue with eserinised ringer for 30 minutes.
 Repeat the CRC/ DRC of ACh std. (same conc.) in presence of
physostigmine.
 Properly label and fix both the CRCs/ DRCs of ACh std. in absence and in
presence of physostigmine.
 Measure the height of the response (cm/ mm) and plot both the CRCs/
DRCs graphically.
 Note the potentiation in the response of ACh. Read the relative EC50/
ED50 values and calculate the dose ratio using the formula-
 Describe the shift of CRC/ DRC of ACh std. in presence of physostigmine
and interpret the result.
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15. Kymograph recording of contractions
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Fig. Dose Response Curve (DRC) of ACh Std. in absence and
in presence of physostigmine.

16. Observation table
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17. Graphical presentation
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18. Calculation of magnification value (Mf)
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Magnification value (Mf) = d (F-W)/ d (F-T)
Where-
d (F-W) → distance between fulcrum and stylus (writing tip)
d (F-T) → distance between fulcrum and point of attachment of
tissue
Dose Ratio (DR)
DR= Antilog (A)/ Antilog (B)

19. Result and interpretation
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The synergistic effect of physostigmine on CRC/ DRC of ACh std.
using isolated frog rectus abdominis muscle preparation was
interpreted as the shift of CRC/ DRC of ACh std. towards the left side
in the presence of physostigmine without suppression of maxima,
which is an indication of potentiation type of synergistic effect.
The dose ratio by the graph % Response Vs log (dose) was found to
be-...............
The magnification value was found to be- ...............

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Thank You...