Experiment- 9

Bioassay of serotonin using rat fundus strip by three

Point bioassay
AIM- To perform bioassay of serotonin by three Point bioassay
method using rat fundus strip preparation.
Requirements-
Animal- Rats [150-200 gms]
Physiological salt solution- Kreb’s solution
Tissue- fundus
Drug- serotonin stock solution (10 ug/ml)
Instruments – organ bath, dissecting board, aerator,
kymograph.
Principle-
Rat fundus is a very sensitive tissue used for the study of
several naturally occurring substances like 5-
hydroxytryptamine, histamine, acetyl choline and bradykinin.
Unlike the intestinal smooth muscle (ileum) this preparation is
slow contracting and slow relaxing type.
Rat fundus is generally employed for the bioassay of serotonin.
The fundus in upper part of stomach is grey in colour and easily
identified from pyloric part which is pink in colour. A zig- zag
preparation of fundus strip is prepared so as to expose
maximum portion of the tissue to drug. The tissue is sensitive
to serotonin [1ng/ml], histamine [0.05-1 ng/ml] and acetyl
choline [ 0.2-0.5 ng/ml] respectively.
Principle of three Point bioassay-
Three Point bioassay method is based on assumption dose
response relationship. Log dose response curve is Plotted and
dose of the standard producing the same response as produced
by the test sample is directly read from the graph so as to
estimate the potency of the test sample.
In three Point bioassay the DRC [dose response curve] of the
standard, test sample is first obtained from the responses due
to graded doses. From the DRC of the standard two standard
doses are selected in such a way that they produce 20% and
80% of maximum response respectively and are designated as
S1 and S2. The responses of these doses lie on the steepest and
straightest [linear] part of the curve.
After selecting the standard and test doses the bioassay is
performed by recording the standard and test responses in a
randomized fashion.
Procedure-
1.sacrifice the rat by a blow on head and bleed to death
2. cut open the abdomen and expose the stomach. Identify the
fundus of stomach incise it from the junction of pyloric part and
put it in the dish containing kreb’s solution.
3. Incise the fundus from the lesser curvature and open it
longitudinally. Give alternate Zig-zag cuts to make a fundus
strip preparation and tie both ends with the thread and mount
the preparation in the organ bath containing kreb’s solution at
370c and aerate the tissue.
4. apply 1 gm load and allow the preparation to equilibrate for
30 min. using frontal writing lever with 10-12 time
magnification record the contractions due to increase in
concentration of serotonin.
5. record a DRC dose response curve of standard serotonin
solution using atleast four doses and record DRC of test solution
using atleast four doses
6. select two concentrations of standard drug electing
submaximal responses and designate them as S1 and S2 having
a dose ratio of 1:2
7. select one dose of test lie between two standard responses
and designated it as T.
8. Record three sets of responses adding them to organ bath in
random fashion by using latin square design. Calculate the
mean of each response.
9. plot the graph with log dose on x-axis and % response on y-
axis and interpolate the Test [T] response on to the DRC of
standard in between S1 and S2 to find the standard doses that
gives equivalent response of that of test.
10. calculate the potency of the test drug by converting the log
dose of the standard that has produced an equivalent response
as that of test in to anti-log and report the potency.
Report-

Experiment - 10
Effect of physostigmine on DRC of acetylcholine using
frog rectus abdominis muscle.
Aim – To study the effect of physostigmine on the dose-
response curve of acetylcholine using frog rectus abdominis
muscle
Requirements-
Animal-frog
Drugs-1. Acetylcholine stock solution [1mg/ml]
2. physostigmine stock solution [1mg/ml]
Physiological salt solution- frog ringer solution
Principle
Frog rectus abdominis muscle is an intestinal smooth muscle.
Ach cause the contraction of the muscle by acting on
muscarinic receptors. Physostigmine is an anticholinesterase
substance and it inhibits the metabolic breakdown of
acetylcholine. As a result acetylcholine levels increase at the
synapse, physostigmine indirectly stimulates both nicotinic and
muscarinic receptors due to increase in acetyl choline at the
synapse. Physostigmine is used in diagnosis and treatment of
myasthenia gravis. It is antidote used for atropine poisoning.
The concentration response curve of acetylcholine will be
shifted to the left in the presence of physostigmine.
Procedure
1. pith the frog and lay it on its back on the frog dissecting
board. Pin the four limbs, remove the skin on the abdomen
and expose the rectus abdominis muscle.
2. Cut and prepare two rectus muscle preparations and tie a
thread to the top and bottom of each muscle preparation
before detaching the muscle from the body of the frog.
3. Mount the preparation in up-right position in the organ
bath containing frog ringer solution under a tension of
1gm. there is no need of maintaining the organ bath
temperature since it is an amphibian tissue preparation.
Bubble the organ bath with air.
4. Relax the tissue for 45 minutes during which wash the
tissue with fresh frog ringer solution for at least four times.
5. Record a dose response curve of acetylcholine using
frontal writing lever 90 sec contact time and a total of 5
min time cycle may be used for proper recording of the
response using atleast four doses.
6. Add physostigmine (2ug/ml) to the reservoir containing
frog ringer and irrigate the tissue for 30 min
7. Repeat the concentration response curves of acetylcholine
in presence of physostigmine. label and fix both
concentration response curves.
8. Measure the height of the response [mm] and plot a dose-
response curve one in the absence and other in the
presence of physostigmine. Note the potency in response
of acetylcholine.
Report-
 Experiment-11
Determination of PD2 value using guinea pig ileum
Aim- To determine PD2 value of histamine in the presence and
absence of mepyramine by using isolated guinea pig ileum
Requirements
Animal- guinea pig (400-600 g, fasted overnight)
Instruments- student organ bath, Aeration tube, kymograph,
frontal writing lever, syringe, petri plate.
Drugs- 1. Histamine stock solution (10ug/ml)
2. mepyramine stock solution (10ug/ml)
Physiological salt solution- Tyrode solution
Temperature- 370c
Basal tension-0.5g
Magnification -10 times
Aeration- 0xygen [ 02] supplied through aerator
Principle-
The PD2 value is defined as the negative log of molar
concentration of the drug producing 50 percent of maximal
response or the negative logarithm to base 10 of EC50.PD2
value also known as EC50 describes the potency of the drug.
Higher the PD2 value, lower EC50 value- reflects higher
potency.
PD2 = -log 10 [EC50]
Histamine is an autacoid and have profound physiological effect
in the body. Histamine besides producing triple response it also
has spasmogenic response on intestinal smooth muscle. By
acting on H1 receptors it causes the contraction of intestinal
smooth muscle (ileal preparation). Mepyramine is a selective
H1 receptor antagonist it antagonizes the contraction of
smooth muscle.

Procedure-
1. Sacrifice the guinea pig by a blow on the head and lay it
on the dissecting board and pin the limbs to the dissecting
board.
2. Cut open the abdomen by a midline incision and lift caecum
to trace ileum. Cut and remove a traced few cm of ileal portion and
immediately transfer it in to a petri dish containing Tyrode
solution.
3. Cut ileum small segment 2-3 cm long and Tie a thread to the top
and bottom and mount the preparation in up-right position
in the organ bath containing Tyrode solution under a
tension of 0.5g. maintaining organ bath temperature at
370c and bubble the organ bath with air[oxygen]
4. Record at least four [4] DRC (Dose response curve) due to
histamine by using frontal writing lever and allow contact time of
30 seconds and 5 minutes time cycle are kept for proper
recording of the responses.
5. Add mepyramine 1-2ug/ml to the reservoir containing
Tyrode solution and irrigate the tissue with mepyramine
containing Tyrode for 30 min.
6. Repeat the DRC of histamine in the presence of
mepyramine
7. Label and fix the tracing and plot the graph and calculate
the EC 50 (PD2) value and note the nature of antagonism

REPORT-
Experiment-12
Anti-inflammatory activity of drug using carrageenan
induced paw-edema model
Aim-
To study the anti-inflammatory activity of the drug using
carrageenan induced paw-edema model.
Requirements:-
Animal- Rats (150-200g)
Drugs – 1. Carrageenan – 1% w/v solution
2. Indomethacin stock solution- 4mg/ml [dose 20mg/kg
S.C]
3. normal saline -0.9%
Apparatus- digital plethysmometer ,syringes, oral feeding
tube.
Principle:-
Inflammation is the tissue response to infection, injury, irritation
or foreign substance. It is a part of defence mechanism but
when it abnormally increases it causes damage. The
inflammation reaction is readily produced in rats in the form of
paw edema with the help of irritant substances like
carrageenan, formalin, bradykinin, histamine, 5-
hydroxytryptamine, mustard or egg white.
Carrageenan is a sulphated polysaccharide obtained from sea
weed. It causes release of inflammatory mediators such as
histamine, 5-hydroxytryptamine, bradykinin and
prostaglandins. When injected it produces inflammation and
edema too within a few minutes of injection.
Indomethacine is a powerful anti-inflammatory agent with
analgesic and anti-pyretic properties. It inhibits the production
of eicosanoids by inhibiting Cox pathway and reduce the
edema.
Procedure:-
1. Weigh the animals [rats] and number them.
2. Make the mark on both the hind paw (right and left) just
beyond the Tibio-tarsal junction.
3. Note the initial paw volume of both right and left paws of
each rat by mercury displacement method.
4. Divide the animals in to two[2] groups , each group
containing six[6] rats.

Group-I -CONTROL GROUP- Inject normal saline

Group-II- TEST GROUP- Inject indomethacin drug
subcutaneously (s.c)

5. After 30 minutes inject 0.1 ml (1 % w/v) carrageenan in

plantar region of left paw of control group as well as
indomethacin treated group.
6. Right paw of each rat will serve as reference non-inflamed
paw for comparison.
7. Note the paw volume of both the legs of control group
treated with normal saline and indomethacin treated rats
at 15min, 30 min, 60 min and 120 min after carrageenan
challenge.
8. Calculate the % difference in right and left paw volume of
each rat of control group and indomethacin treated group.
9. Compare the mean % change in paw volume in control
group and indomethacin treated group.
10. Express the % edema inhibited by anti-inflammatory
drug indomethacin.

Report-
Anti-inflammatory activity of indomethacin drug was
studied and observed by using carrageenan induced paw
edema model.