In this slide contains introduction, role, mechanism, and assay of oxytocin. Presented by: P.PAVAN KALYAN (Department of pharmaceutical analysis ). RIPER, anantapur

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
BIOLOGICAL TESTS AND
ASSAY OF OXYTOCIN
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
P.Pavan kalyan
(Reg. No. 20L81S0705)
Under the guidance/Mentorship of
shakir basha
Associate Professor
Dept. of Pharmaceutical Analysis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Introduction to Oxytocin
• Role of Oxytocin
• Oxytocin - mechanismm of action
• Biological assay of Oxytocin
Contents

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
Introduction
• Oxy -Rapid
Tocos –Labor
• Oxytocin synthesized in both sexes ( male and female ),well
recognised physiological effects only in women.
• Pituitary gland consist posterior lobe which produce Oxytocin
and diuretic hormone.

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
• It stimulates the contraction of the
uterine smooth muscle and memory
gland.
• Oestrogen progesterone and prolactin –
responsible for production of milk by
mammary gland but ejection require
Oxytocin.
• It facilitates the contraction of uterus.
Role of Oxytocin

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• Neuropeptide made in hypothalamus that stimulates
contractions , that expel the infant from uterus.
• Responsible for milk letdown and triggered by the nipple
stimulation of suckling.
• Oxytocin called love and binding hormone. It has a very
special affect on mothering .
• Psychologically , Oxytocin promotes a feeling of well being
and tranquility.
• It enables the growing sense of love and attachment to the
infant. The more the infant suck the more Oxytocin is
produced.
Oxytocin – Mechanism of action

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Principle
potency is determined by comparing it’s activity
- depression of BP
- Contraction of uterus
- Milk ejection of pressure
- Vasopressor activity
With standard preparation of Oxytocin.
Standard Preparation
It consisting free dried synthetic Oxytocin peptide with
human albumin , citric acid ( 12.5 units ).
Biological assay of Oxytocin

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Method – A
. Depression of the BP in the chiken
Test animals : cockerel ( young male chiken )
Weight : 1.2 – 2.3 kg
Method – B
By contraction of the rat uterus
Test animals : female rat
Weight : 120- 200 g

8. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Method – C
. Milk ejection pressure in lactating rat
Test animals : lactating rat ,3 – 21 days after parturition
Weight : 300 g
Method – D
Vasopressor activity
Test animals : male rat
Weight : 300 g

9. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
Depression of the B.P in chiken
Test animals : cockerel (young male chiken) 1.2- 2.3 kg
• Anesthetized cock- prolonged and remove popliteral
artary and crural vein.
• Cannulate the popliteral artery and record B.P response .
• Cannulate the crural vein or branchial vein.
• Prepare standard solution with saline injection 0.1 -0.5 ml.
• Inject 2 doses of standard solution in to cannulate vein and record
B.P response.
• Dose should be cause decrease in B.P.
Method – A

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• Interval between 2 injections, between 3-10 min depends on the rate
at which B.P return normal.
• Dil.test preparation with saline so as to get same response as
standard.
• The ratio between standard and test should be equal.
• If animal rapidly becomes insensitive to repeated injection the
solution another must used .
• Measure all responses and calculated results of the assay by standard
statistical methods.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Contraction of the rat uterus
Test animals : female rat ,120 – 200 g
• Inject 100 micro gram of oestradiol benzoate IM in to female rat
before the assay.
• Kill rat and suspend one horn of uterus in organ bath containing a
solution of following Nacl, Kcl ,Cacl 2,Na2HCo3 ,NaHPo4,
NaH2Po4 ,Mgcl2 , dextrose. Temp at 32’C.
• Bath liquid required dose between 10-50 units / ml .
• Oxygenate solution with mix of 95% of O2 and 5% of CO2 record
contraction of muscle.
Method – B

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• Record contraction produces by addition of two dose of
std.preparation.
• Dose should be added at regular interval (3-5 minutes).
• Similarly record the contraction of test preparation as a standard.
• Ratio between two doses of test and two doses of standard should be
equal.this ratio kept constant through out the assay.
• Measure all response and calculate results of assay by standard
statistical methods.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Milk Ejection pressure in lactating rat
test animals : lactating rat,3-21 day after parturition,300 g.
• Separate from litter and 30-60 minutes later anesthetized (IP
pentobarbitone Na ).
• Tie rat to an operation table,at 37 degrees ,by its hind legs leaving
front legs free.
• Cannulate trachea with a short PE tube of i.d 2.5 mm in such a
manner so as to ensure a free airway.apply artificial respiration only
if necessary.
• Shave the skin surrounding the inguinal and abdominal teats and
excise the tip of one teat,preferally the lower inguinal teat.
Method – C

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• Insert a P.E tube i.d 0.3 mm and e.d 0.6 mm,to a deapth sufficient to
obtain appropriate measurement of pressure ( 3-10mm depth),in to
the primary teat duct.
• Connect this cannula with a suitable strain gauge transducer.
• Prepare 3.8 % w/v of Na citrate/ saline , contain 50 units of heparin
Na/ml to prevent the clotting of milk.
• Then inject 0.005-0.2 ml of this solution in to teat duct through
transducer.
• Then slight tension is applied to the teat and connect the gauge to a
potentiometric recorder for an increase in milk ejection pressure of
5.3 kpa.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• Prepare a solution of standard and test preparation in saline solution,
so that the volume to be injected is between 0.1-0.4 ml .
• Choose two doses of standard preparation such that the increase in
the milk ejection pressure is about 1.35 kpa for lower dose and about
2.7 kpa for high dose.
• Choose two doses of the test preparation with matching effects of
doses of the standard preparation as closely as possible.
• Inject four doses (2 doses of standard and 2 doses of test ) at intervals
of 3-5 minutes.

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
• 2 doses of standard and 2 doses of test , should be given according to
randamized block or a Latin square design and at least four responses
to each recorded.
• Measure all responses and calculate results of the assay by standard
statistical methods.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Vasopressor activity
Test animals : male rat ,300 g
• Vasopresser activity of test with standard preparation of arginine vasopresin
(Freeze dried syn.arginine vasopressin peptide acetate with human albumin
and citric acid).
• Inject slowly in to the tail vein of male albino rat weight 300 g – solution of
a suitable adrenoreceptors blocking agent,(10 ml/kg body weight ), solution
prepared by 5 mg of phenoxy benzamine HCl in 0.1 ml of ethanol (95% ),
adding 0.05ml of 1 M HCl and dil to 5 ml with saline.
• After 18 hr ,anaesthetize rat- that will maintain prolonged and uniform B.P.
• After 45-60 min ,tie the rat on its back to the operating table by its hind legs
.
Method – D

18. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
• Cannulate trachea with short P.E of E.D 2.5 mm and dissect carotid
artery ready for cannulation.
• Then cannulate the femoral vein close to the inguinal ligament.
• Dissect femoral vein towards inguinal ligament from corresponding
artery.
• Tie a short P.E cannula of E.D about 1 mm in to femoral vein by two
ligatures.
• At this stage inject through venous cannula 200 units of heparin,
dissolved in saline for 100 gm of body weight.
• Then tie in a carotid cannula of E.D about 1 mm and connect to saline
contain heparin with a Hg manometer of I.D about 2- 3 mm.
• No artificial respiration is necessary.

19. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• Dil.extract of standard and test preparation with saline,that volume to
be injected is between 0.1 to 0.5 ml.
• Choose 2 doses of the standard preparation such that elivation of the
B.P is about 4 kpa for lower dose And 7 kpa for higher dose.
• Choose 2 doses of test preparation with same inter dose ratio
matching effects of dose of standard preparation.
• Inject doses at intervals of 10 – 15 minutes.
• 2 doses of std and 2 doses of test Preparation should given in
randamized block / latin square design and 4- 5 responses to each
record.
• Measure all responses and calculate results of the assay by standard
statistical methods.

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20