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Unit | Pharmacoloq-2
ISaslc Rnolldo
Phamacoloq of drs ach on Cudocriul Sqstem

Andio qu ondAonlolic seroids

Sex hovmnmU
hovmons wic
pro duud

Fcboqn progasrona Ora Covracopthiu plaay

a
Cd Qud
aud eluased nunon becu
Sex hormonus
develbpmour f epoabuMm,

arm

arm
COxual
ImportautF
Drugs achng on UCWS. rol
Onadol Aeslds
ioassay
Sex eaids 0d ocodicojds,

Piiplus nd appliahans obioasscu

Typy Sex - Hormaus
Ty p a
Ph

Ph
of bioass

Bio ass0u of Lnsulin, Oxytocin, Vasopessin , ACTH

Aat d-tubocuraninl, dgualis, Histamia femada

nd S HT Mala
ll

ll
Atndvogun
mPu ESboguu oqoteron
we

we
a detouls not on Oral covtTaupti Testosjenone
Wrik hormonuy

nok DI drugs achng on (UeWE Dasialy elua fom femoe y

re

re
Elase pom
otqaud llps on
Oudho
Wrie a Ahortnolk on bosically tstes
developmusctofAxua) orgous
fAvabalic stsolds aul bu elps in
ud aluo lp
yAndhogtnd Ohd in (ovulolm
Ca

Ca
duelepmuF of Sexuc orqaus
Ecbogns Prqusteron
Cud
d up n elua o
OVUm
Dufin
(fsHfollida Atimulahin
Ond dasithy BioaAa wrik u bloavcan (SpOumeldgu
hrmmus
deuelopma of (oOvUm) seCrehion of eshauS
of dyet prodwchin also Support spamatoq auuis
Tnsulin d- tubo wvanina perm
(LH evtini 2inq hermos stimuat
Oxytoun Dugetceis - piggn ovvlahom, gulermu Stcnhon , etos teon
OChon

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wrie bhort noe on huulay glauds
O Requlabiou Aa
Andogun Anobolíc slvoids
oacdohopin homonaConk
relanq, hormanc
Atudvogns male sSux hormonus
HypotnalonU osteuio

Testostvou u andvdqn. Tt is Aei

a

Mauny studhusizic
main otural
1estitial cells Of -H testis, a
arm

arm
nd Amaler Qmount y OVaies d adkenal cortex)
AjcAGds
KiDnodo hopiw
/fu Pno
leutenizlvg homp/ 1ollick stimulaing
S4x

TLL Avbstan SCondary hormovu)

(auA developmN of
1
chaa ce in Min, inthdung focial aud bod 4
-8
Ph

Ph

hain qraw And vOtCA Choa

Testes (ONBles
qovedls inki bin
give hein achon b binding H Ovedvoge
Rept
Testortron hogen
ell

ell

|Progustevc
Auunogus MO Suuthh

dgans fAuhin
rew

rew

Sudhun'e
Anabeli q enten ino
Natua pvprl
MeHlealo tjerona ona
Hut ofF adulF mau produu
tlvoxy musJors Respond or al chaug fatosern Apoulsla
(S-12g of tapskm dady Ho t Polata
Ca

Ca

Apurt of q
14-0lkyl Auelfitvtd dadlvadjvts of
Adrenal cory producps Amalu
puloudt in boys
0udo q Amallu ex
testo tvmL (oaly ochve).
quovdtties of deluydroepiaudrostonu in gil
groun oF quutalu-pLnu
Rapad bou qouth, bsth in
f andro ShenadiMA Uotvm, Sewwnal eiclus,
weaR audvoguns prostale. uudknL well a n leath
youan pronmote muscl buulduug,
OvBAY
do produdsAlu qiouwm of hai Pubic bad,
oHeatpseonuy
. moUs tod body,
han Cud espaciallp 4 adid by exuerti

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of a pattn Appetie Improve an
male hegns
diubibuhon asuns T e- bLing
Geshegsn
giows voícL prevails
pumay emala Sx homon
cup C C

e e2 divelopmeiUano

a
po nsilblu fou Hu
. pcolciniy e sem Caud

arm

arm
Equlaion df u fenalt Hpioduch

. USually
a4ieDa e cte
SPCndoy Stxual chanaclerishs»
2nd win ptamoa Prettin globulin

playma V2 10-20 mUnURA Sex skuetd invlved InHu Hqulatin of

t
meuva an aL
Ph

Ph
Cycle changs expksucd Cycus
(Analboli'c ondhogunic sta4ds)
Anabolit stooids mpo vtaudin pegnonc

Thy mthuic Qndrogens with suppos e

S Sythuzid y OvO and placonda
ll

ll
gh anaoolic Qnd ower audvoqtnic) actiuw
smaly avmouvts
bu tis Oncadenalcorte
we

we
.Sexval qyowHh
Rape bon grouwHh
Th
promoe mucda he ajoendendogenw (exbogns
buildno
malu- esdone (oesbme) Eshadial (oesradisl)
re

re
hore Chuical Aubsan whih
T anR
Oud
qrow thu
uiol &esiol)
muscluy ono lps to bod
Ca

Ca
(Ethadiol is tu mos
Sonuim4 n llugal by ploy ports.
pincipo sboqun sece
ed
Naudrolon, Muhandaenons. ek.
Sqwtri
Anhiando GLNS Tnos arvs whih octagonds adhom df Cthiylehadiole, MUubandl
ek
0udrogens USac to deceaM CKCAsie
CReguloh
Saual desi e ulamida yprokona
e. abtody diuswa

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Man tmchom Maun n chy

pubu
pubpujal -fcmalus
cic Joy.
ChON.gtsn -T uhgen
Pon
,
Cha )
t baings abou

qrowh
qrouwlh of Ueus,alopion tubu aud vaaaMa, endoruhmm
CavsL cevixb bicmahik
cproductivê organ. Ahnnd Ou muCUS in
a

gcna epuhulum guts -hchenus

gain whih a
pevent Apwm pon paysng 1uh
arm

arm
- elponsiblu proluferahn peovulotoy T inducu pkgnanuy
of endomim in he
mamma glands
pha FaSL proliftuahionm h
eyogen Incea Coaquhl OF qonadobopins
Dlood nd Inteahuu allo Inhub StLehions of pituilay
t risk omboembolicm.
Ph

Ph

OF
wich pheven Ovulahom

linical U
wwrik a derails note on Oral (ontvacoptioe
Overt OVanlan (eaurnur's Cuhduaa)
fad Orad Conbhaaphioei
ell

ell

Secgmdantga ovclan fail for fiuskinq, Vaqjinal dyws and HO nouwn gir control Bl"
to pesevve bonu mass
Couta cupthion Thu hot dvvgs ohlch
in cobinahin pills
rew

rew

proae ad pven pkgnony Pesuphon mudicahions

baCoht.
udici Cotain hormons lR hogun
Proqarton
progsthin( sqpthuhic bu similm. 0s(pogusbsm)
Ca

Ca

serts lay Corpvs luttUm in th SeCom

po yc, and plocata during Rou oF tue hovmonu

of mnshv b
pregnont paanty Cnovmal munavnism
thoguu
Hestis
mal amounts aho SAcetto by ahd adena T OVones product tu egg
teus. Ovulahon) (fs)
ovum
cort Callnd (ova)tun

Tu ae AubAantag uhich Convent u (eAbogn paim

transpore antd falUopain tut. YLH
eluaj

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(UJcrus uleri Cauly Ora Cohacptives
Cmbaoptivej
Conbaceptivey
ypus of oral
Oral
wo maun
MAVMwY allopas
u )8 Combinahim pus

pill (mini pil)

a
Progustin- Only

Evdomublum

arm

arm
Covix va
Ceviol Combinahim pulls
Cana
and a iesi(progishin
Cousalns Q eshogen
Vaapna

MoA C phoqute).
Ph

Ph
Now
e fetilizd y ApAm in fallopian fubA
(Hpitholan
Now, tu moves UerUs Wh th Uteim unng
Cowbinahn ply
nP
ha ichinLd and pep t Hegele a
endombium
eL1/fSH (Liutove
glaud
ll

ll
featiua eg. Proouhin
(Esbegen (esrogen
1)
we

we
Ovonles

waintain Comsa
Phoqusterai
InbuF Secveion
lvele Ethogin
Rísing level of pragujevon tha CauSe glands in ining OF FSH ( ugahve
re

re
fudbadk o Prbguttu
edomalvm System)
to relea fluds 4that (teds) tu egg-feuilzad eg9).
SupeAA(Ovulahon) Jaei nubu Secrehn
Ca

Ca
Of LH
T also (ou ths tonnud ou mucus in t
peven pom pevent ovulalim)
Cevix to becm tuR which Spm
alle tu (udomuhm u
pasun hrough. Such as Way T tutken ng of ceviy muvS
discovrag implaahon.
on 99 hosn' bun fetiliud t level of both ehagin
which mwstuanm.
proguslerm bgn ofa (usR Ovvlahm pRgnont

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mosl popvlar avd efficaciou Cahacts

spnjans y)
most mulo ulein Codachom

tunlng mnsbualim.
Hhiny lespadiole Noguubol ee
duing.
(partutitim v DLh

fu Coodinatd
a

Adre gain Mausta Vomui hg ee

Classupohiom
a
arm

arm
wise pall tha Uleune
IOdhie
Coctain hormonus

) Mini pil (proquhin mly pal)

Ph

Ph

Uteun claxavds
Thy (ajain only proguhin |URani stimulaw J
Calcitonj
MOA cwical muwS Oxytocin
preuFovulahm 4 hickening of
Prostaglandins B2 adnoceptri
ell

ell

(adauvalina )/salbuan
pRven pHgnanty Erga dRal ids
frgamuhnnn)
tn ehicaty ower than Cwlolnahon pils
rew

rew

OOntocin hormm
Writ no On dug ah on ORS ptxkn secretd by u posejon
pihutany aong
Contracs vhuthmicaly ithVaopesunADH)
.Ueuinl mUSc both in uho anel
Ca

Ca

onginrin n th mutd itult. T 1notam h Cotrachm of ut mUIclly

wwo, Tu Coram
myomhiel cellb fundaa Ac 0) (poumoker)and
qive Cmducte Ochion potthal.
GytoinO Oxytocin C-PCR
Yeeuptor TP -DnG)
NOW
achu of paumgakar equlatd by Contra dchm
Drpalalsanion (by
(Sex homOnu 1a infux)

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.tko Thu
1 numbtr Of oxytocin eceptorsInCeaU mokea
mkea hostaglondint
during atr paF ofF PAcg nany IS-utuyt PGFu pole
PCE,PGLa ad
CudoonoUS Ukint Atimulats
CtrachionP Hel n child bity)

a
Usus Cahacin of ulavs
ch

arm

arm
relaxahm ofCewix/
th
USd oIndua Or auomin lalbour wn
Uterin muscll is not funchon tmq aduqvaclely Eigs Akalsud

AKo V sed
tohea postpartum hamorvhogi- Caouds u human
Ph

Ph
on (onrocted terus (
imudialely chuld biuth normal Atat fpllowina dlive),
oft
(exive blodd toss) Crqoman ha elaively luttlu etfcty.

UEWS plakly elaxed,

ll

ll
Brcast nttai u SeCrehim Of mulk from h
trgbmuhint
inappo

inuho stoen Cotracin ,tvs vedUun

we

we
mMammo qlond
bludg from plautal leid.

(Aavo etfilh)-
re

re
n huh yddn
CoUse Sussaind Cohadhon thaF inlnfm wih olodd
Ca

Ca
AO Miavgh pocuta Ond Cawse fctu dath Utun elaxants|
Seltdively
2- odsenocaptou agmsti, such as (vitadun)
P'oinaic or Salbutomo), inbut outocin-Inducud
ven (TV) o.M bu mosHy Irtvaveox nfvsim.
pHcgnovt
Cotrachion) o 4u
Inachvattd in UveL 4
d a Utrus.

USado peven pematu lobour )(dloy deluy upb

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Applicohiny
RIOASSAY

dekrmiu Thpolency) oF a dvu

(Bio+-(fA ssay protus procus of deleimins (doce (quanlily) of a drug equied to
cleler nunin -u (pohcncy) Of t
a

in

ti
liv Hes
SUitab blologial
pioduad a huaptulica Or tonit epons, e Colcoloim
arm

arm
dvug by usin (EDSDoso) cke.

Sysjem uke ,
avimols, iss , ioou ek..
effeche eha
dog doso
Outeunina relohive poitnty of acubttona (droq) by COrnpaing
Alo ps dvelopmaa ot nd drugs
àrs biological
attiul in th
Ph

Ph

With tho of a reftnce sJavndands

Thu maind ypus (Acc. to th klpn us

ell

ell

) Qvovtal ioossay

) Grndud oiosa
AlSo ULdto Sandovdyz Peparalion Ao that
rew

rew

ench pepurahon Cocpin Speeifiesl phoavmocolog4 Qctiuu-

Octiun-
1QUatal) bloassay

Pindiple
AlSO R10.0 rAaons (No)
Evd piut)
oL
*kespons Al or no", if. euhIr
Tu boMc principa Of
blosa to tnpa t tt Mximum esponse no esponsA w
Ca

Ca

Or

Substonu with inlernationga Adord pHpMahMs of uermudiole Hesponses

TIP,USP, BP ek
So ad find ou how mvch
eg. Digatals Codiac onus
Svbstanu Is vequied fo produ Sam bloloaical effrctr, n una pg /pouon

produca Sondord -fubocurarim hvabbir uad

by Dste
cbop umod

- compani slonm
of threshold esponsacompislon of

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td poi
(Anima
T ponss 0 ud n Hu Obsuwatioms u

Ihcl group
tha u CUn inueasomon In Hpmsu wih
Tk qoup
wuch Akou Inoeasemunt An u Conceurdim of dose

a
threshold dose
Thshold dou tve effrcs
Typus of

arm

arm
Spundavd Test
qrodid aay
ol so
a) Matclung
COmpore
b) 8tockehing

Tweshold clou oF sauolor

Conourahon of Conc. of sjaudovd Tnerpolatton
Ph

Ph
thHee poln
Unkvoun Thesnold dose of te
d) Multipu pointk bioa:sy Tur four poinr
i poin
i) Gradus bioassau
muans Aponsa proportionmal to igh 1gh poin
ll

ll
t do Ond Epons may e eetuwen no HEponse
we

we
tu moaimUm Hspon so

ceiling effects
re

re
Responsa
Ca

Ca
DOTR

T graduo dote espons Curve

dANd
plottiny qhaph wih dose X-O1S Ono

EApon&e On T-axis

Tt USually sig mold in Slapa

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Isulin 2o vnu+ Injulin
ioassoly OF inulin Hcoeuey wigu
Qnd dusole ormal Saun. (NOVmal Salin

Insulin Audihy wih H to pH S

HU ph 2)
a hormom mods by panRatic P-ulls OSphuol pRservaives
a

.T SyntaMzid 0 po-inulhin In endoplasmic etiiullum

Add 147. - 1-9 yuin a 0S
+
phno
arm

arm
final volu Ahould otan ou/m
prousd t bioloqially acthiv form insucda
Aetory hanuA Atou so1 in a Col plau
oU/m
fnchon wi thin AX mot 0-Smgm
Cappaov.)
lps to ontvol tha blood sugar 1vel by Cmveiv
Ph

Ph

Te bum 8tamdond
glutor itd glyogn and akso InCRaAe t glutoSt uptuR

Rabler mh
clls
Uptake Selechion of Robkeas (animal) - Ty Anould a haty4,
ell

ell

Eig about (23 Ka). A ttaia Ahauld

quca
glucogn -follos Otc.to Chovt. $VM ax (CPCsEA) ele-
rew

rew

for pefovn tost

Also we duf. OF byassay (Principl
Roblis mould bL maintoinud On Unifovm die bu
Fxpujmt model
fastcd for 19s efou 0may (tt)
Ralolai mumoc
Ca

Ca

mumad standond and Aanplu (dluhms)

prepanatn Camplus
use uy upan by dlutguHhnormial
Nau Aol
puR, d 0nd ostalinu insulin.
u -
SImilay,, tsF ampl
A0 a to Cotain (1/)
Sol
+(2U
vna 0.04092 mg spuified by nuaishy of uath
plutiou adso Apwdard
Govt: oF Indua f also eqinal
o iatemahon Uni) ppaucd.

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T potny esfinate amplt of bl6dd WIhda
wIhdrau
prinup OF a Het Campl is from tad rabeu, a J

by CompaA
ypogoN effe ct oF u upto
Shrs a nerval ot hr eauh,

Saumplu wm tha of ppav ahn of insulin.

td. Now Slood Sugan dijen mlnnd
blodd suqay level
Hi is allud "-inal

a
proedUR >
enc
Aymals diudsd Jnkoqusps of3 radbis

arm

arm
SeCnd pant
TM roldoets 0L T pu udo awa nolaay
noldar
A tL O eek Chodse Aamt aumals
Dhuy Ahoul4 ehdedaanel handle wth a avoa
agaln fusted fo 19 hrs Qud Inuhal blcod cuga
exutma
u deteminud.
pa - frs paat+ pa cKd qoub
Ph

Ph
Tpfoun tup ucond
NOu, May Chanaa

tMans- host animals which Hceived th Atamdad

O fin paa
OAL wen nd Hoived
-A sampla of bloc tapan fromt martna 0
ll

ll
now given L Audand
Co veln oF raho
Also
thos aimals
we

we
Ptsena of ecucing Sugar simatd pe lopmd of
Heceived
les
blooc by Sutabl chunal muthod
do ofF Atandad quen a hou doz o
tf Sampla Od viIa -VerA.
blood sugor
Thus Conc
alludnhia level").
re

re
Ths taF nown a Twin osS over tutP.
Thun four groups of rablils qiven
pary
ist |Sutnd pan
cutk sugan
Ca

Ca
subtwtauous (C) thjecim of Incoln a follou Level ^an
Sarpla Cop Sapla (u fin
Pabb Croup SapRe
pant.
Std-L Test-2
3 Std-1- I/
Std-2 Tet
Std -2 2U/ I L

Std-
Test-L Ju/ Tut IT
3
3 Test-2 - 2U/n
OfsL TV
Test- 2)T -L

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at
uan purconT of decease
.
blaod SLga o -firsF
m pu
IncoDajoL

halFhouv.
uud eLndpant u Calwlattd
fo M nd a
SSCand oburv
ovt of
MOUR muthod
which lnvulsa) o de
ua houd choractevisti convulsias atu (sc.echom
a

of insulin a elevufd emperctues

tu InwbalorY Qud
a obsevved
arm

arm
Th pirAnlag Comvul sloma proclucad and favtaaga Cmvulsis protut y tf Aampus

Aandard pparatioma COnpanL Compand of Btandna onpl

wth -hose
phocecu Aimalulana)
Convulcive mice b Aaved
munimum J00 m uegnig beuen 19-22gm of
Ph

Ph

O.S m of S dextroce sol"

sama srain USAd

ThoCA SUWIv may 2 Used aga4

animals
TuThuy ou ld bt maintain M (msant jek.

ThLy Ahould hsttd 18 hs Pio p H fo Ovnottn expumr aft Infeava of Onl

ell

ell

expeai munt wek .

Seunpl 1
rew

rew

PLpua
luhions AL pepaLd with ule Ao, o 0 to
Contan O.064 urr/m (std. ) ane 0.096 Unus/ (sta T),

SImalanly ts amplu o
Ca

Ca

diwdd four groups

Crp-1 SaumbL
Tnsulin ieckd sc.
4 fol low 11-
sd.2
Test L

T Test 2

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- TubOLUranin - Min. able anl USed T aAl duuidd
eoh Coutann
muscus
CaUSLA
t claxatian of skelulal

.Biocassay 8 rabbit
USIng Rabeil head- drop mutha

a
Principl
saax

arm

arm
Eis rablaus 4
ineced o manginal vein of
Vablbiks T
ea l t ralos uck mUs cis a Spucdand col
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ambUnf of He mangua
samplu Hauirtdto sieon appautv bragh tu in
produc u endpoint COmpoed wth tu total Spu
SpAA o.4 /min (Him aken abou lo min)
CumOUt of standand Aampla Kquidto DosL 0.o127. wfU Sali n
In
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produa siilan efecr.
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Selechon ofF Rabeiy
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houd Aaau CrOT-er tut Comied ou
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bíological
- Chsore pom a uathy Colony evorsduu to anmal Volahion,
hosA aubleits whl
Should be CUstonid uwith 4u teceivtdt standndamplu n
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expimrdal
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procedue do of expnl ma n MCR
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Samipl Compann
probuding ovtiud. Tha Aheuld laa paly with t nuan dbA C
ovabls auclend puponahion.

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Dwlna t RXptalma n
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Cgem muthwd (tu amoVn of shad kqujnd to produu huis

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effceF Is aeen -u Lethal dos of h

exud)

hot HA San
SA OF
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Sp.rdes DCed expeumo aud

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BIOASSAY OF OXYTOCIN:
(but hichud)
Oxytocin is a peptide hormones and Neuropeptide. Oxytocin is normally produced by
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the paraventricular nucleus of the hypothalamus and released by the
and
finally potenty Cakolaltd in clehntd tnd
posteriorpituitary.
dividinq aveuge ehal do of thu Oxytocin is a natural hormone that causes the uterus to contract. Oxytocin is used to

qum induce labor or strengthen labor contractions during childbirth, and to control
Sanp of test CLd expHsu nik pu
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bleeding after childbirth.
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Oxytocin is also used to stimulate uterine contractions in a woman with an incomplete
or threatenedmiscarriage.
Different bioassay methods used for oxytocin are
o By contraction of the rat uterus
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o By depression of the blood pressure in chicken Record the contractions of the muscles produced by the addition to the bath of two
o By measurement of milk ejection pressure in a lactating rat etc. doses of the Standard Preparation suitably diluted with the above solution. The doses
Principle: should be such as to produce clearly discriminated, submaximal contractions.The
>The poteney of oxytocin injection is determined by comparing its activity with that of required doses normally lie between 10 and 50 micro Unitsper ml of bath liquid.
the standard preparation of oxytocin under the conditions of the followingmethod of When maximal contraction has reached replace the bath liquidby a fresh solution.
assay The doses should be added at regular intervals of 3 to Sminutes depending upon the
a

Standard preparation: rate of recovery of the muscle. a

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The standard preparation is consisting of a freeze dried preparation of oxytocin with Dilute the preparation being examined so as to obtain the responses on the addition of
human albumin and citric acid (supplied in ampoules containing 12.5 units) or any two doses similar to thoseobtained with the Standardpreparation.
other suitable preparation, the potency of which had been determined in relation to the The ratio between the two doses of the preparation being examined should be the
International standard. same as that between the twodoses of the Standard Preparation and this ratio should
The Unit is the specific oxytocin activity corresponding to that yielded by 0.0005 g of keptconstant throughout the assay.
the Standard preparation. The two doses of Standard preparation and the two doses of the test preparation
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The standard preparation is as per the 4h international standard for Oxytocin, should be given according to a randomized order or Latin square design and at least
established in 1978. six responses toeach should be recorded.
Experimental Methods: Measure all the responses and calculate the result of theassay by statistical methods.
1) By contraction of the rat uterus: 2) By depression of the blood pressure in chicken:
Use female rats weighingbetween 120 and 200 g. Immediately before the assay
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Anaesthetize a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anesthetic
confirm the rat is in oestrous or preoestrus by vaginal smear. that will maintain aprolonged and constant high blood pressure.
Inject 100 microgram of oestradiol benzoate intramuscularly 18-24 hours before the Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the
assay.Kill the rat and suspend one horm of the uterus in a bath containing a solution of
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popliteal artery and cruralvein.

the following composition:
Cannulate the popliteal artery and record the bloodpressure.Cannulate the crural or
Composition (% w/v) brachial vein.
Sodium chloride 0.662
Prepare standard solution with saline so that the volume to be injected is between 0.1-
Potassium chloride 0.045
0.51ml.
Calcium chloride 0.007
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Inject 2 doses of standard solution into cannulated vein and record blood pressure.
Sodium bicarbonate 0.256
The doses should be such that as to produce clearly discriminated, precipitious,
Disodium hydrogen phosphate 0.029
submaximal decreases in B.P.
Sodium dihydrogen phosphate 0.003
The required doses normally lie between 20 and 100 milli units.
Magnesium chloride 0.010
The interval between injections should be constant and lie between 3-10 minutes
Dextrose 0.050
depending on the rate at which B.P. returns tonormal.
Maintain the bath at a temperature of 32° C so that spontaneous contraction of the Dilute test sample before use with saline solution so as to obtain responses similar to
uterus are abolished and preparation maintain its sensitivity.
those obtained with the standard preparation.
Oxygenate the solution with a mixture of 95% of oxygen and 5% of carbon dioxide.

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The ratio between the two doses of the test preparation being examined should be Clamp the strain gauge so that a slight tension is applied to the teat andits natural
same as that between the two doses of the standard preparation and the ratio should be alignment is preserved and connect the gauge to a potentiometric recorderadjusted to
kept constant throughout the assay. give full-scale deflection for an increase in milk-ejection pressure of about 5.3kPa.

two doses of Standard preparation and the two doses Inject all solutions through the venous cannula using a 1-ml syringe graduated in0.01
The of the test preparation
should be given according to a randomized order or Latin square design and at least ml and wash them in with 0.2 ml of saline solution.
>Prepare a solution of the Standard Preparation and a solution of the preparation

a
six responses toeach should be recorded.
beingexamined in saline so that the volume to be injected is between 0.1 ml and
Ifanimal rapidly become insensitive due to repeated injections of the solutions

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0.4ml.
another animal must be used. Measure all responses and calculate the result by
standard statistical methods. Choose two doses of the Standard Preparation such that the increase in milk-

3) By measurement of milk-ejection pressure in a lactating rat: ejectionpressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher
dose.
Select alactating rat, in the third to twenty-first day after parturition and weighing
As aninitial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an
about 300 gseparate it from the litter and 30 to 60 minutes later anaesthetise (for
upper dose ofl.5 to 2 times this amount may be tried.
example, by theintraperitoneal injection of a solution of Pentobarbitone Sodium).
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Tie the rat to anoperating table, maintained at 37, by its hind legs leaving the front
Choose two doses of the preparation beingexamined with the same inter-dose ratio,
matching the effects of the doses of theStandard Preparation as closely as possible.
legs free. Cannulatethe trachea with a short polyethylene tube of internal diameter
about 2.5 mm in such amanner so as to ensure a free airway; apply artificial
Inject the four doses (two doses of theeStandard Preparation and two doses of the
preparation being examined) at intervals of 3to 5 minutes.
respiration only if necessary.
The two doses of Standard Preparation and the two doses of the preparationbeing
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Cannulate an external jugular or femoral vein with a polyethylene tube of
examined should be given according to a randomised block or a Latin squaredesign
internaldiameter about 0.4 mm which is filled with saline solution and closed with a
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and at least four responses to each should be recorded. Measure all the responsesand
pin.
calculate the result of the assay by standard statistical methods.
Shave the skin surrounding the inguinal and abdominal teats and excise the tip of
BIOASSAY OF VASOPRESSIN:
oneteat, preferably the lower inguinal teat. Insert a polyethylene tube of internal
Biological Assay for Vasopressor Activity
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diameterabout 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to
Principle:
obtainappropriate measurement of pressure (3 to 10 mm depth), into the primary teat
The vasopressor activity is estimated by comparing the activity of thepreparation
ductwhich opens onto the cut surface and tie firmly in place with a ligature.
being examined with that of the Standard Preparation of arginine vasopressinunder
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Connect thiscannula with a suitable strain gauge transducer (such as that used for
the conditions of a suitable method of assay.
recording arterialblood pressure in the rat) and fill the whole system with a 3.8% w/v
Standard Preparation:
solution of sodiumcitrate or saline solution containing 50 Units of heparin sodium per
The Standard Preparation is the Ist International Standard forArginine
ml to prevent clottingof milk.
vasopressin,established in 1978,consisting of freeze-dried synthetic
After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into theteat
argininevasopressin peptide acetate with human albumin and citric acid(supplied in
duct through the transducer to clear the milk from the tip of the cannula.
ampoulescontaining 8.20 Units),or another suitable preparation the potency of which
(Thisprocedure may be repeated durin the assay should obstruction arise from milk has beendetermined in relation to that of the International Standard.
ejectedinto the cannula). Suggested Method:

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Inject slowly into the tail vein of a male albino rat weighing about300 g a solution of pressureisabout 4 kPa for the lower dose and about 7 kPa but always submaximal for
a suitable alpha-adrenoceptor blocking agent.for example 10 ml perkg of body weight the higherdose, the ratio of low to high dose being determined by the response and
of a solution prepared by dissolving 5 mg of phenoxybenzaminehydrochloride in 0.1 usually being 3to 5.
ml of ethanol(95%),adding 0.05 ml of IM hydrochloride acid anddiluting to 5 ml with As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose
saline solution. twodoses of the preparation being examined with the same inter-dose ratio, matching
a

After 18 hours,anaesthetise the rat with ananaesthetic that will maintain a prolonged
and uniform blood pressure.After 45 to 60minutes,tie the rat on its back to the Inject a
theeffccts of the dose of the Standard Preparation as closely as possible.
doses atintervals of 10 to 15 minutes. The two doses of the Standard
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operating table by its hind legs. Preparation and the twodoses of the preparation being examined should be given in a
Cannulate thetrachea with a short polyethylene tube of external diameter about 2.5 randomised block or aLatin square design and four to five responses to each should be
mm and dissect acarotid artery ready for cannulation.Then cannulate the femoral vein recorded.
close to the inguinalligament. Measure allthe responses and calculate the result of the assay by standard statistical
Retract the abdominal muscles to expose the inguinal ligament.Retract thesuperficial methods.
pudendal vein to one side and dissect the femoral vein towards the inguinalligament BIOASSAY OF ACTH:
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rom the corresponding artery. ACTH (Adrenocorticotropic hormone, corticotropin) is polypeptide tropic hormone
>When dissecting,a deep branch reaching thefemoral vein must be found and tied off to (39 amino acids) secreted by the anterior pituitary gland.

prevent bleeding during cannulation.Tie ashort polyethylene cannula of external ACTH stimulates the production of cortisol, a steroid hormone important for
diameter about mm into the femoral vein by twoligatures and join by a short piece regulating gucose, protein and lipid metabolism, suppressing the immune system
response, and helping to maintainblood pressure.
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of flexible tubing to a 1-ml burette with an attachedthistle funnel containing saline

solution at about 37. Official Preparations
Firmly fix a wet absorbent cottonswab to the thigh so as to cover the incision and Corticotropin injection: Is a sterile solution , in a suitable diluent, of the polypeptide
from the pituitary glands of mammals. Potency range should be 80.0- 120.0 % of
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cannula.
At this stage inject through thevenous cannula 200 Units of heparin,dissolved in USP cartiotropinunits.
saline solution,per 100g of bodyweight. Corticotropin for injection, antimicrobial agent. Repository corticotropin injection is

Then tie in a carotid cannula of external diameter about 1

mm and connect by corticotropin in a sterile solution of partially hydrolyzedgelatin and is intended for
acolumn of saline solution containing heparin with a suitable pressure measuring subcutaneous and intramuscular use. This solution has beenadopted as the reference

devicesuch as a mercury manometer of internal diameter about 2 to 3 mm. standard for the bioassay.
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The central and peripheral nervous system including both vagus and o Packing: Preserve in single-dose or multiple-dose containers of Type-1glass.
associatedsympathetic nerves is left intact. No artificial respiration is necessary. o Storage:Store in cold place.
Taking care that noair is injected, inject all solutions through the venous cannula by o Labeling:njection recommends intravenous administration

means of a 1-ml syringegraduated in 0.01 ml and wash in with 0.2 ml of saline Purpose and rationale
solution fronm the burette. This is a historical assay method. Administration of pituitary ACTH decrease the
ascorbic acid present in the adrenals. The depletion of adrenal ascorbic acid is a
Dilute the extract of the Standard Preparation and the preparation being examined
withsaline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. function of the dose of ACTH administered. This relationship has been used for a

Choosetwo doses of the Standard Preparation such that the elevation of the blood quantitative assay ofACTH.

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Solutions:
Solution A: Five units of test or standard dissolved in 0.25 ml of0.5% phenol solution
and diluted with 8.1 ml of 15% gelatin solution (Now 0.5ml contain 300 mU ACTH).
solution B: Three ml of solution A diluted with 6 ml gelatin solution.
Nowconcentration reduced to 100 mU ACTH/ 0.5 ml. 1
ml of solution B diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.02%
solution C: Again 3 ml of solution B diluted with 6 ml of gelatin solution, theresulting

a
ascorbic acid solution (solution C)
solution contains 33 mU ACTH/ 0.5 ml.

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Procedure Preparation of other solutions
Male Wistar rat (100-200 g) are hypophysectomized (pituitary gland removed by Sulfuric acid (85%) is obtained by adding 900 ml concentrated sulfuric acid to 100 ml
surgery) one day prior to the test. distilled water.
For one test with 3 dose of test preparation and standard solution used for the study. Two g dinitrophenolhydrazine are dissolved in 100 ml 9 N H2S04 (75 ml distilled
Number of hypophysectomized rats required: at least 36(preferably 60).
water and 25 ml concentrated sulfuric acid).
The hypophysectomized rats are randomly distributed in to sixgroups. Each rat
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Six g thiourea are dissolved in 100 ml distilled water.
receives subcutaneous 0.5 ml of the variousconcentrations of test or standard.
Three hours after injection, the animals are anesthetized andboth adrenals removed, Calibration
freed from extraneous tissue andweighed. The rats are sacrificed and the scull opened > Trichloroacetic acid (4%) is added to 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 ml of the
to verifycompleteness of hypophysectomy. 0.02% ascorbic acid solution (solution C) and 1.0, 1,5 and 2.0 ml of the 0.2% ascorbic
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The adrenals are homogenized in glass tubes contains 200 mgpure sand and 8.0 ml of acid solution to reach a final volumeof 8.0 ml (Solution B).
4% trichloroacetic acid and the ascorbicacid determined. (Roe and Kuether 1943).
>100 mg charcoal is added to cach sample and thoroughly mixed byshaking for 1 min.
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The potency ratio including confidence limits is calculated withthe 3+3 point assay.
After 5 min the solutions are filtered.
Ascorbic acid determination:
An aliquot of 0.1 ml of the 6% thiourea solution is added to 2.0 mlof the filtrate
Reagents
followed by 0.5 ml dinitrophenylhydrazine solution.
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0.02% ascorbic acid solution
85 % sulfuric acid (9N H2S04) > The mixture is shaken and heated for 45 min at 57°C in a waterbath.
.0.02 g/ml of dinitrophenolhydrazine in 9N H2S04 The solutions are placed in an ice-cold water bath and withfurther cooling 2.5 ml of
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0.06 g/ml of thiourea are dissolved in distilled water the 85% sulfuric acid are added.
Charcoal The calibration curve is established at a wave length of 540 mmusing the solutions
Preparation of 0.02% ascorbic acid solution
without ascorbic acid as blank.
100 mg L-ascorbic acid are dissolved in 100 ml of 4% trichloroacetic acid (lmg/ml
solution)(Solution A=1% solution)
2 ml of Solution A diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.2%
ascorbic acid solution (solution B)

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produces effects by acting on the histamine receptors (H1, H2, H3 and H4) present on
target cells.
Bioassay of histamine on isolated guinea pig ileum can be determined by graded
bioassay procedure i.e. i) Matching bioassay, i) Interpolation bioassay, ii) Bracketing
assay, iv) Multiple point assays.
Bioassay of histamine in biological samples can be studied by using different bioassay
a

a
methods. Depending upon pharmacological action, histamine can be assayed by:
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o Contractile effect of isolated ileum of guinea pig,
o Contractile effect of uterusof guinea pigand
o Fall in blood pressure of anaesthetized and atropinized cat. Guinea pig's uterus
Bioassay of histamine using guinea pig ileum:
Principle:
Guinea pig ileum is very useful preparation for the bioassay methods. It is more
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BIOASSAY OF HISTAMINE: sensitive to histamine. Contractile response of histamine to ileum is due to presence of

Histamine is present in almost all the animal tissues mostly within mast cell and H1 receptor.

Preparation of Standard and other solution:

basophil granules. Tissues rich in histamine are skin, gastric and intestinal mucosa,
Prepare the stock of Tyrode solution. Also prepare the standard stock solution of
lungs, liver and placenta.
histamine (l mg/ml) and then different concentrations of histamine by serial dilution
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Non-mast cell histamine is present in brain, epidermis, gastric mucosa and growing method.
regions. Histamine is also present in blood, most body secretions, venoms and Procedure:
pathological fluids. It is now known to play important physiological roles. Histamine Sacrifice the 24 hr fasted guinea pig by stunning on the head and carotid bleeding. Fix
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the animal on the dissecting board by tying its legs. Open the abdominal cavity by a
small horizontal cut followed by vertical midline incision and expose the abdominal
organs.
Trace the ileocaecal junction by lifting the caecum, then go upwards up to 8 cm from
the junction and cut the 2-3 cm long segment of ileum muscle (excluding the terminal
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5-8 cm, contains excess of excitatory a-adrenergic receptor, which may interfere in
the study).
Immediately place it in a petri dish containing aerated warm Tyrode solution. Slowly
remove the mesentery attached to the muscle and then gently clean the lumen of ileum
by passing luke warm Tyrode solution through it with a pipette or syringe.
Mount the preparation in the inner organ bath containing Tyrode solution (20 ml)
maintained at 37°C. Tie the bottom end of the muscle to the hook of aeration tube and
the upper end to the isotonic frontal lever by a thread without closing the lumen.

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Adjust the magnification of response to 7-10 fold. Aerate the tissue with 02 or Preparation of Standard and other solution:
carbogen slowly (40-60 bubbles per minutes).
Prepare the stock of Krebs solution. Prepare the standard stock solution of serotonin
Stabilize the tissue for 30 min by applying a tension of 0.5 g weight attached to the
lever, during which wash the tissue with fresh Tyrode solution once in every 10 min.
(1 mg/ml) and then different concentrations of serotonin by serial dilution method.
Use 5 min time cycle with contact time of 30s for recording the contraction Procedure:
dependent responses of tissue due to histamine on the smoked drums.

a
Sacrifice the 24 hr fasted rat by stunning on the head and carotid bleeding. Fix the
Record the responses of standard and test compounds.
animal on the dissecting board by tying its legs. Open the abdominal cavity by a small

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Record the responses of test compound i.e. unknown concentration of histamine with
gradually increasing volume, till obtaining a response (T) which would lie on the horizontal cut followed by vertical midline incision and expose the abdominal organs.
linear portion (30-70%) of the CRC. Fix the response obtained due to volume of T. Identify the stomach and separate it from the abdomen by gently cutting its cardiac
Record the graded responses of standard solution and test solutionof histamine. and pyloric end. Place it in a petri dish containing aerated warm Krebs solution.
The potency of the test sample is calculated in relation to that of the std. preparation
> Incise the fundus of the stomach (upper grey part) from pyloric part (pink and thick
by dividing the average lethal dose of the sample to the test and expressed as units per
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gram. part). Cut the fundus from the lesser curvature and open it longitudinally. Then cut the
BIOASSAY OF 5-HT: fundus at the midline into two equal parts. Give alternate transverse cuts (zig-zag cut)
5-Hydroxytryptamine (5HT) is the biologically active local hormone, low molecular
on opposite sides of the muscle to make a fundal strip preparation (as shown in fig.
weight, and also an important neurotransmitter in the brain and periphery.
9.1).
It was first detected in serum (serotonin) and enterochromaffin cells of gut mucosa
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(enteramine), and later both were identified to be SHT. Today the terms 5HT and Mount the preparation in the inner organ bath containing Krebs solution (20 ml)
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serotonin are used interchangeably. maintained at 37°C. Tie the bottom end of the muscle to the hook of aeration tube and
About 90% of body's content of 5-HT is localized in the intestines; most of the rest is
the upper end to the isotonic frontal lever by thread. Adjust the magnification of
in platelets and brain. It is also found in wasp and scorpion sting, and is widely
response to 10-15 fold. Aerate the tissue with 02 or carbogen slowly (40-60 bubbles
distributed in invertebrates and plants (banana, pear, pineapple, tomato etc.).
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The concentration of 5-HT in biological samples can be assayed by:lsolated fundus per minutes).
strip of rat stomach, Isolated terminal colon of rat,Isolated rat uterus,Perfused rabbit > Apply 1g load and stabilize the tissue for 30 min during which wash the tissue with
ear.
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fresh Krebs solution once in every 10 min.
Isolated fundus strip of rat stomach
Use a 5 min time cycle with contact time of 90s (since the fundus strip muscle
Principle:
Rat fundus preparation is a very sensitive tissue for the bioassay methods and is useful contracts slowly and relaxes slowly) for recording the contraction due to serotonin.
to study the action of several substances like 5-HT, ACh, PGE2 and bradykinin. Record the graded responses of standard solution and test solutionofserotonin.
Fundus strip preparation is slow contracting muscle. Fundus part of stomach can be
The potency of the test sample is calculated in relation to that of the std. preparation
easily identified by its grey colour and situated above the pink coloured thick pyloric
by dividing the average lethal dose of the sample to the test and expressed as units per
part. A zig-zag preparation of the fundal strip is prepared so as to expose maximum
portion of the tissue to drug. gram.

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