WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Daude et al. World Journal of Pharmacy and Pharmaceutical Sciences

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Volume 5, Issue 1, 1221-1225 Short Communication ISSN 2278 – 4357

BIOASSAY OF HISTAMINE BY USING ISOLATED CHICKEN ILEUM

Rakesh B. Daude*, Soniya V. Mishra, Raosaheb Y. Ghegade and Sunil V. Amrutkar

*Assistant Professor, Department of Pharmacology, Gokhale Education Society’s

Sir Dr. M. S. Gosavi College of Pharmaceutical Education and Research, Prin.T.A.Kulkarni
Vidyanagar, Nashik-422005, Maharashtra, India.

ABSTRACT
Article Received on
14 Nov 2015, Study of isolated organ/tissue preparation study has to sacrifice

Revised on 05 Dec 2015, laboratory animals just for teaching purpose. The objective of present
Accepted on 27 Dec 2015 investigation was to study effect of histamine on dose response curve
(DRC) and to find out unknown concentration of histamine by

*Correspondence for interpolation and three-point bioassay using isolated chicken ileum.
Author The results suggested that histamine produced a dose dependent
Rakesh B. Daude increase in response of contraction using tyrode physiological salt
Assistant Professor,
solution (PSS). Unknown concentration matches nearby with actual
Department of
concentration by interpolation and three-point bioassay. The result
Pharmacology, Gokhale
Education Society's Sir shows that the chicken ileum is suitable for performing bioassay of
Dr. M. S. Gosavi College histamine, an alternative to isolated ileum preparation from laboratory
of Pharmaceutical animal (guinea pig) without sacrificing the experimental animals.
Education and Research,
Prin.T.A.Kulkarni KEYWORDS: chicken ileum, histamine, interpolation, three-point
Vidyanagar, Nashik-
bioassay.
422005, Maharashtra,
India.
INTRODUCTION
The usually accepted methods for bioassay of histamine are cat's blood
pressure, guinea pigs uterus and the guinea pigs ileum. The most widely used tissue for the
bioassay of histamine is guinea pigs terminal portion of the ileum. (Chowdhury et.al.,
1956).Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA), a statutory body framed under the Prevention of Cruelty to Animals Act, 1960,
which is duty bound to take all such measures as may be necessary to ensure that animals are
not subjected to unnecessary pain or suffering before, during or after the performance of
experiments on them. CPCSEA has instructed Universities and College to avoid experiments

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Daude et al. World Journal of Pharmacy and Pharmaceutical Sciences

on animals wherever it is possible to do so for teaching purpose and introduce use of

alternative to animal experimentation like CDs, computer simulations, models, in-vitro
methods etc. Substitute to in vivo animal experiments are in vitro cell culture technique and in
silico computer simulation. Still there is need to develop alternative to animal testing.
(Undale et.al., 2013). Also restriction to experiments using laboratory animals for bioassay
has provoked researcher to search for alternate tissues. (Parthasarathy, 2014). Guinea pig
ileum has been used to study various receptors present in ileum. It responds to most of the
common spasmogen and particularly suitable for study of histaminergic receptors. For these
study laboratory animals has to be sacrificed. Chicken ileum is simply obtainable from the
slaughter house and animal need not to be killed just for teaching and demonstration purpose.
(Bhutada et.al., 2006). Use of chicken ileum might reduce, refine and replace the number of
animals for laboratory experiment just for teaching purpose. No attempt has been made to
study effect of histamine on isolated chicken ileum. The objective of present investigation
was to study effect of histamine on isolated chicken ileum is easily available from slaughter
houses as an alternative for dose response curve (DRC) and estimation of unknown
concentration of histamine by interpolation and three-point bioassay. Histamine (stock
solution: 1mg/ml) used in the study was purchased from S.D.Fine Chem. Mumbai. Initially
different physiological salt solutions were used. But tissue responses to histamine were not
uniform and taking long time for relaxation. Therefore tyrode solution was used for
experimentation. Composition of tyrode physiological solution were (in g/liter) NaCl 8.0;
KCl 0.2; CaCl2 0.18; MgCl2 0.1; NaHCO3 1.0; NaH2PO4 0.05 and glucose 2.0 weighed
accurately and dissolved in distilled water maintained at 32-35˚C.

Fresh intestine of Chicken were obtained from a government registered slaughter house. The
tissue was immediately transported to the laboratory under ice and kept for aeration. The
intestinal content were removed by washing with PSS. With gentle care, the mesentery and
adhering tissues were removed. The ileum was cut into small segments of 2-3 cm long and
mounted the tissue in the organ bath containing PSS maintained at 32-35˚C ,bubbled with air
and allowed to equilibrate for 30 min. Mounting was similar to guinea pig ileal preparation
mounting.(Kulkarni, 2009) Tension adjustments were made and tissue were washed after
every 15 min. The histamine induces the contraction were recorded on kymograph by using
frontal writing lever. Contact time of 30 sec, and 5 min time cycle was kept constant
throughout the experiment which was followed by 3 times washing with PSS at an interval of
1 min. Concentration of histamine were added in geometric manners. DRC was recorded till

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Daude et al. World Journal of Pharmacy and Pharmaceutical Sciences

maximum response (E max or ceiling effect) to histamine was obtained and height of
response were measured in terms of mms. Interpolation bioassay was carried out in six set of
experiments with standard concentration 5,10,15,20 & 30ug/ml. Record the contraction due
to graded doses of test histamine. Measure various responses to calculate the mean of each
test response. Plot the graph with log dose on X-axis and % response on Y-axis and
interpolate the response of test on to the DRC so as to find the standard dose that gives an
equivalent response of that of the test. Calculate the potency of the test by converting the log
of the standard dose that has produced an equivalent response as that of test in to antilog and
determine potency of test (ug/ml). To measure the potency of the test solution, four different
set of experiment were carried out by three point bioassay method. Two doses of standard (S1
and S2) from the DRC of standard drug and one dose of test (T) which lies between S1 and
S2 were selected. Three sets of responses using S1, S2, and T were recorded for histamine in
randomized Latin square fashion and mean heights of S1, S2, and T responses were
measured.

Potency= n1/t x antilog {T-S1/S2-S1 x log n2/n1 }x C

Where n1 = lower standard dose; n2 = higher standard dose; t = test dose; S1 = response of n1 ;
S2 = response of n2; T = response of test (t) and C=concentration of standard. (Vrushabendra
et.al., 2014)

Interpolation bioassay, Histamine (1 ug to 30 ug) produced a concentration dependent

increase in concentration of isolated chicken ileum preparation. These results show that
histamine can induce contractions in isolated chicken ileum. Concentration of histamine used
were 5, 10, 15, 20 & 30 ug/ml and concentration obtained after performing bioassay nearly
match with the actual concentrations. Table no. 1 show the concentration obtained after
performing bioassay.

Table 1. Concentration of unknown sample of histamine by interpolation bioassay

Concentration of Concentration of
histamine used (ug/ml) histamine obtained (ug/ml)
05 5.37 ± 0.1739
10 9.275 ± 0.2056
15 14.08 ± 0.2501
20 19.12 ± 0.2394
30 26.39 ± 0.6397
(*Values are expressed as Mean ±SEM from 6 preparations for individual test samples of
Histamine.)

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Daude et al. World Journal of Pharmacy and Pharmaceutical Sciences

Concentration of the test was determined using S1 (2 ug) and S2 (4 ug) by three point
bioassay (Table 2). The potency of unknown sample of histamine was found to be 14.03 ±
0.1707. The actual value of T was 15ug and by bioassay, it was 14.00ug that is 93.34% of the
actual strength.

Table 2. Three point bioassay of histamine using isolated chicken ileum preparation
Height of response in mm
S1 (2 ug) S2 (4 ug) T
26 50 38
26 51 39
26 51 38
25 51 38
(* Three point bioassay with Histamine was carried out in four experiments)

Experiment was design to study effect of histamine on DRC and estimate unknown
concentration using isolated chicken ileum which will mimic the actual laboratory condition
without sacrificing the experimental animals. Histamine produced a dose dependent increase
in response of contraction using tyrode physiological salt solution (PSS). Unknown
concentration matches nearby with actual concentration by interpolation and three-point
bioassay. By interpolation bioassay, potency of test concentration was found nearly match
with actual concentration. Three point bioassay results have given good response for about 4
to 6 hrs. The result shows that chicken ileum is suitable for performing bioassay of histamine.
Ileum preparation is most widely used for the study of drugs on isolated muscle preparation.
It consists receptors like muscarinic, histaminic, GABAergic, serotonergic, and
adrenoreceptors. (Ghosh, 2005 and Jain et.al., 2012) The advantages of using chicken ileum
preparation are that it is cheap, easy to mount in organ bath, stable and reproducible for about
4- to 6- hours and no need to sacrificed the experimental animals.(Jain et.al.,2012).
Furthermore this is needed to perform antagonistic study. However, the model has certain
limitation, i.e., ileum has to be brought from slaughter house for study and the characteristic
and nature of receptor present are not fully understood. They use to be studied by radioligand
binding assay. (Bhutada et.al., 2006). Bioassay is used to identify the nature of receptors and
to find out the unknown substance. M3 muscarinic receptors were reported in chick ileum.
Chick ileum gives significant stability and reproducibility. (Parthasarathy et.al., 2013)

Chick ileum can be used as an alternative for tissues from guinea pigs for bioassay of
histamine. Chicken ileum preparation can be used to demonstrate anti-Histaminic and anti-

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Daude et al. World Journal of Pharmacy and Pharmaceutical Sciences

spasmogenic activity. It is also suitable for carrying out different experiments like pA2 value;
multiple point bioassay, drug antagonism and comparison of different agonists.

ACKNOWLEDGEMENT
We are grateful to the Laboratory Assistant and Attendant of GES’s Sir Dr. M.S.Gosavi
College of Pharmaceutical Education & Research, Nashik for successful completion of these
experiments.

REFERENCES
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for pharmacological experimentation on isolated tissue preparation. Journal of Advanced
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isolated chicken ileum: Alternative to laboratory animals for isolated tissue
experimentation. IOSR Journal of Pharmacy, 2012; 2(5): 39-45.
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8. Ghosh MN. Fundamentals of Experimental Pharmacology. 2005; 2nd Edition. Calcutta:
Scientific book agency.

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