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1

SCREENING

OFANTIDIABETIC

PRESENTED BY:-

Salunkhe Pankaj ShrikantSemester 1st

M.Pharm {Pharmacolo!"

De#artment o$ Pharmacolo!

R.%.Patel collee o$ #harmac!&Shir#ur

SHRI

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2

Content :-

• History

• Definition & Classification of Diabetes

• Insulin

-Structural feature

-Mode Of Action

• Oral hypoglyceic agent -Classification & Mode of Action

• !lucose "stiation Method

• Screening techni#ues

-In $i%o

-In $itro

• eferences

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'

• DM (no)n since ages as *Madhumeha” i+e+s)eetness of disease urine in Ayur%eda by,Sushruta

• During first century !ree. physician ,Aeretausgi%es ter Diabetes as *to flo) through/

• In 01 by ,Dobson deonstrated Diabetes as bypresence of sugar in urine+

• In 0343 by ,De Mayer gi%es stateent aboutinsulin is ,the non-digesti%e part of pancreas & isletcell )as responsible for pancreatic diabetes+

• History :-

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5

-It is chronic etabolic disorder6 resulting froinsulin deficiency characteri7ed byhyperglyceia6 altered etabolis ofcarbohydrate6 protein 6 lipid & increases the ris.

of %ascular coplications+

-Diabetes is not a single disease6 it is

heterogeneous group of syndroe6 characteri7edby an e%aluation of 8lood !lucose caused by arelati%e deficiency of Insulin+

•Definition :- 16

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9

INSULIN LACK

HYPERGLYCEMIA KETOSIS

Glycosouria

Oso!ic "iur#sis

Loss o$ El#c!roly!#

Na%& K%& Ca%%

I'!rac#llular K% "#(l#!io'

Hy(o!#'sio'& S)oc*

Aci+osis

Loss o$ ,i#+

ca!io' i' Uri'#

"#)y+ra!io'

Hy(oosolari!y o$ .loo+

Loss o$ /a!#r

0oi!i'K#!o'ur#a

Hy(#r0#'!ila!io'

I(air#'!

o$ Glucos#

#'!ry i'

.rai'

I(air#'! o$

co'scious'#ss

I'!rac#llular

"#)y+ra!io'

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• Classification :-

0: T!#e-' ( )u*enile onset ( 'nsulinDe#en+ent Dia,etes Mellitus ;-

It occurs in nonbiased or before age '4+

Autoiune Disease of <-cell+ Autoiune

type 0A antibody destroy <-cell+ Circulating insulin is lo) or %ery lo) so

patient pro%e to .etosis+

=atient ust rely on ">ogenous insulin to

control the hyperglyceia+ Mostly Subcutaneous rout is preferred+

963663

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T!#e '' (Maturit! /nset (Non-'nsulin

+e#en+ent Dia,etes Mellitus :-

@sually occurs after age 54+ Obesity6 lac. of e>ercise

is aor ris. factor+

Moderate reduction of <-cell+

Insulin circulation is less B noral B high ay occur

hyperglyceia or hypoglyceia+

Here6 abnoralities in !lucoreceptor in <-cell so that

they respond at higher glucose concentration+

Oral hypoglyceic agent are as a part of treatent 6

e>ercise 6 dietary odification+

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0T!#e ''' ( M/DY Maturit! /nset Dia,etes in

Youn :-

In )hich dysregulation of glucose sensing or insulinsecretion is due to utation of particular gene+

Due to Horonal disorder+

reatent %aries often response to OHA

2T!#e '3 ( 4estational Dia,etes Mellitus :-!lucose intolerance associated )ith =regnancy+ It Is iportant to tight glyceic control close to

noral range during pregnancy 6because-

hyperglyceia can lead to congenital abnoralities

in fetus

reatent- controlled by diet6e>ersise

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Insulin:Structural $eature:-

High ol+ )t+ polypeptideha%ing )t+ about

?4?

Aino acid lin.ages 0 aa

arranged in 2 chain+

A chain -acidic E 20 aa

8 chain -basic -- '4 aa

C Epeptide lin.age connectto A & 8 chain+

2 disulphide bond are

lin.ed in both aa+

6961

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# OA of Insulin:-

2 23 3

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/ral h!#ol!cemic aent Classification :- 1 'nsulin secretaous E they proote the insulin

release fro <-cell of pancreas+e+g++ Sulfonylurea ;- Tolbutamide,Acetohexamide

'nsulin sensiti5er E

Fi: it increases insulin upta.e & utili7ation by target

tissue+ e+g+hia7olidinediones;-osiglitazone,Pioglitazone

Fii: it reduces hepatic glucose output6 largely byinhibiting hepatic gluconeogenesis

e+g+ 8iguanides ; - Metfomin,Phetformin0 6-4l!cosi+ase inhi,itor E they act by delaying the

digestion of carbohydrate thereby decreasingglucose absorption

e+g+ Acarbose, Miglitol

163

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04 20

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Screenin! Tec"niue

In %i%o

- Cheical Induced Diabetes

i: Allo>an induced Diabetes

ii: Strepto7otocin induced Diabetes

iii: ?-SG Induced diabetes

- $iral Induced diabetes

- !enetically diabetic anial - Assay of Insulin

i: 8lood sugar lo)ering effect in abbit

ii: Hypoglyceic Sei7ures in ice

iii: 8lood Sugar deterination in ice

- 8lood !lucose lo)ering acti%ity of Antidiabetic Drugs

i: In abbits ii: In ats

- "uglyceic Clap techni#ue

- Inhibition of !lucose absorption in %i%o

04620

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In $itro

-Isolated at pancreas2

-Isolated Diaphrag of rat

' 5

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Glucos# Es!ia!io' M#!)o+

• 4lucose /7i+ase metho+-1

!lucose is o>idi7ed in !luconic Acid by glucose o>idi7e+

he hydrogen pero>ide liberated is reduced by pero>ides &O>ygen transferred to an accepter6 )hich is colour less in

the reduced for but coloured in o>idi7ed for+

• 4lucometer-

•$rine analysis-

Gualitati%e6Cheap6Con%enient+ 8enedict eagent are

used+ but Diagnosis cant based- ay sho) false %e6

false-%e

'6566

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• 8e7okinase en5!me metho+--9

he procedure is a fluoroetric rate ethod easuring

the foration of AD=H cataly7ed by iobili7ed glucose-9-

phosphate dehydrogenase and he>o.inase held )ithin a tinystirrer+ he en7ye stirrer is stable for at least t)o onths and

can be used o%er eight-hundred assays )ithout any loss of

acti%ity+

• 4l!cos!late+ 8aemolo,in 8,1c0

Measureent of blood glucose le%el in diabetic suffer

fro %ariation due to dietary inta.e of pre%ious day+ Jong ter

obecti%e assessent degree of diabetic control is better done

by easureent of glycosylated haeoglobin HbA0c6a inor

haeoglobin coponent present in noral person+ his isbecause non-en7yatic glycosylation of haeoglobin ta.es

place o%er 024 days+ his gi%es an estiated of diabetic

control for the =receding 9-04 days+

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0?

• 4lucose Tolerance Test 4TT- 0&2&9

Kasting 8lood glucose le%el is first deterined & then

blood glucose le%el are sapled inutes to hour after an

oral dose of glucoseF1g in '44 l )ater: + 8lood and

urine saple are collected at half hourly inter%al+

i i

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I' 4i0o Scr##'i'

F1 llo7an in+uce+ +ia,etes:-

=urpose E

Induction of diabetes using cheical allo>an+ In Most

species riphasic tie course is obser%ed+ FInitial rise of glucose is

follo)ed by decrease6due to depletion of islets fro insulin6again

follo)ed by sustain increase of blood glucose:+

nimal abbits ats 8eagle dogs

;eiht 2+4-'+ .g 04-244 g 0-24 .g

Route "ar %ein subcutaneously Intra%enously

Dose 04 gB.g 044-01 gB.g 94 gB.g

=rocedure- ' !roup are selected as FControl6 Standard6est :

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• "%aluation

@se any suitable ethod for collection of blood+ and

estiation of blood glucose le%el+

Copare the result of test )ith standard and control+

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Stre#to5otacin in+uce+ +ia,etes:-

=urpose E

Induction of diabetogenic acti%ity of antibioticstrepto7otacin+ he copound turned out cytoto>ic to <-cellof pancreas+

Initially blood glucose increases up to 04-244 g L6 9-?hour after insulin increase up to 5 ties Fhypoglyceicphase: after 25-5? hyperglyceia up to ?44 gL+

=rocedure-

Male istar at E 04-244 g

!roup E est6 Standard6 Control

SN+ Dose E 94 gB.g intra%enously

"%aluation

@se any suitable ethod for collection of blood+ andestiation of blood glucose le%el+


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0 /ther Dia,etoenic com#oun+ in+uce+ +ia,etes:-

=urpose E

Se%eral other copound to induce syptos of diabetesandBor obesity+ hese copound also sho)s triphasicglyceic reaction+

=rocedure-

Anial E Cats 6abbits 6!olden Hasters 6Mice

!roup E est6 Standard6 Control

Cheical E Dithi7one ?-SG F?-p-toluene-sulfonylaino-#uinoline:

?-8SG F?-ben7ene-sulfanylaino-#uinoline:

Dose E 54-044 gB.g intra%enously

"%aluation @se any suitable ethod for collection of blood+ and

estiation of blood glucose le%el+


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2 3irus in+uce+ Dia,etes ;-

=rinciple-

-It causes type-I diabetes ellitus+

-he D-%ariant of Encephalomyocarditis %irus F"MC-D:selecti%ely infects and destroys pancreatic <-cell +

-Islet cell destruction )as associated )ith chronic isletcell inflaation6 ele%ation of islet cell antibody6 andprolonged presence of %iral A in the pancreas+

9 4eneticall! +ia,etic animal :-

88 at-

8io 8reeding rat is a odel of spontaneous diabetesassociated )ith insulin deficiency due to autoiune

destruction of pancreatic beta cell+

"8B(O8 at-

Spontaneous hyperglyceia6 glycocosourea ha%e beenobser%ed in aged ale of istar Strain+

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DK BA-KA at-

Coonly referred to as the fatty rat6 is a genetically obese6hyperglyceic rat established by transfer of fatty gene froNuc.er rat to )istar (yoto rat+

OD Mice-

OD ice is a odel of ype-I diabetes

OD ice De%elop diabetes bet)een 044 and 244 days of

age+ As )ell as rapid )eight loss6 polyuria6 se%ere !lucosuria+

OD ice caused by specific -cell ediated6 destruction ofinsulin producing beta cell+

Chinese Haster E FCrecetulus griseus) he no+ of pancreatic islet )as decreased 6 and the cell of the

reaining islet )as decreased and other cell )ere abnoral+

8SJ of diabetic haster )ere ele%ated fro noral of004gL up to 944 gL+

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2

OD Mice 8io 8reeding rat

Chinese Haster E FCrecetulus griseous)

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ssa! o$ 'nsulin

8lood sugar lo)ering effect in abbits-

=urpose- A biological assay of insulin preparation in coparison

)ith a suitable standard using the blood sugar lo)ering

effect in rabbits+

=rocedure- 9 rabbit in each group+ )t+at least 0+?.g 25 h before test rabbit is pro%ided )ith food ad libidum.

During test food !ater !ithheld until blood sam"le ta#en.

!roup Inection

0 Standard sol+ 2

2 Standard sol+ 0

' Assay sol+ 2

5 Assay sol+ 0

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"%aluation E

Calculate the response of each rabbit to each inection+

Kro the su of t)o 8SJ %alues substrate its response

to standard solution 0 & fro standard solution 2+

!roup Differences Indi%idualresponse

otalresponse

0 Std+sol+2-assay sol+0 P0 0

2 Assay sol+2-std+sol+0 P2 2

' Assay sol+2-std+sol+0 P' '

5 Std+sol+2-assay sol+0 P5 5

@S=

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2?

< 8!#ol!cemic sei5ure in mice1=&-0

=urpose-

)in crosso%er ethod for bioassay of Insulin usingblood glucose le%el in ice+

=rocedure-

Anial - S)iss albino F)t+ 24 Q 2 g:

!roup - 5 !roup Feach R04 anial :

Solution - Standard F 2 diluting )ith 4+3L acl6 =H-2+5:

est F 2 diluting )ith 4+3L acl6 =H-2+5:

on fasting ice are used+ F s+c+rout:

"%aluation-

he ice are obser%ed for 0+ hour+

he no+ of ice are recorded that are dead6 con%ulse+

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< Bloo+ Suar +etermination in mice

=urpose-

8iological assay of Insulin using hypoglyceic sei7ures inice+

=rocedure-

Anial - 39 ice of either se> F)t+ 24 Q g:

!roup - 5 !roup

Solution - Standard F diluting )ith 4+3L acl6 =H-2+5:

est F diluting )ith 4+3L acl6 =H-2+5:

Mice depri%ed food 2-24 hour iediately preceding test

"ach of prepared sol+4+0 lB04g inected s+c+

ot less than 2+ h later6 each sol+ Adinistered to secondgroup+

"%aluation- ">actly 2+ h later6 a saple of 4 l of blood is ta.en

fro orbital %enous sinus of each ice+

8SJ is deterine+ =otency is calculated by t)in-cross-o%er

assay

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'4

Bloo+ lucose lo>erin acti*it! o$ anti+ia,etic +ru

$ Bloo+ lucose lo>erin acti*it! in Ra,,it

=urpose-

abbit has been used since any year for standardi7ation ofinsulin+

=rocedure-

!roups Fstd+6 test6 control: rabbits of either se> F'+4-5+ .g:

Anial .ept on oral diet+ Oral glucose lo)ering agent are

applied by ga%ages in 0 lB.g of 4+5L starch suspension or i+%+in solution+ se%eral dose are gi%en to different groups+ controlgroup recei%e %ehicle only+

"%aluation-

8y puncturing of ear %ein6 blood 04 l is )ithdra)niediately and 06 26 '6 56 6 256 5?6 & 12 hours+

8SJ is deterine+

A%erage blood sugar %alues are plotted %erse tie for eachdosage

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'0

$ Bloo+ lucose lo>erin acti*it! in Rats%

=urpose-

ats are used for screening as )ell as for #uantitati%e

e%aluation of blood glucose lo)ering agent+

=rocedure-

Male istar rats F0?4-254 g: are .ept on standard diet +

!roups of 5-1 non-fasted anial are treated orally or

intraperitoneally )ith %arious doses+

Control group recei%e only %ehicle+

"%aluation-

8lood is )ithdra)n 04 l fro tip of tail iediately beforeand 06 26 '6 6 256 5? hours+

8SJ is deterine+

A%erage blood sugar %alues are plotted %erse tie for each

dosage

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'2

Eul!cemic %lam# Techni?ue;-

=urpose-

@seful ethod of #uantifying in &i&o insulin sensiti%ity inhuan+ In this techni#ue a %ariable glucose is deli%ered toaintain euglyceia during insulin infusion+

=rocedure- -Male istar rats F04-244 g : are fasted o%ernight+

-Anaestheti7ed )ith =entobarbital F 54 gB.g6 i+p+ :

-Catheters are inserted into ugular %ein for blood collectionand a feoral %ein for insulin and glucose infusion+

-Insulin infusion rates6 9 and '4 @B.gBin+ -!lucose infusion rate is adusted so as to aintain 8SJ at

basal le%el

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''

"%aluation-

8lood glucose conc+ are deterined fro saple collection at-in inter%al during the 34 in clap techni#ue+

he glucose etabolic clearance rate is obtained by di%idingglucose infusing rate by steady state blood glucoseinfusion rate

"uglyceic clap echni#ue

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'5

< 'nhi,ition o$ 4lucose a,sor#tion in 3i*o-

=urpose-

he inhibition of glucose absorption deterined by blood

glucose after adinistration of starch or disaccharides+

=rocedure-

- Male istar rat F04-244g: .ept on standard diet6 free

access to tap )ater+

-09 hour prior to e>perient food is )ithdra)n but not )ater

-2+ gB.g Starch recei%e by stoach tube for control group &

)ith %arious dose α-aylase inhibitor in test group

-After6 046 246 '46 946 024 & 254 in6 blood is )ithdra)n and

deterined blood glucose+

-he anial are sacrificed after these inter%al & residual

starch in stoach 6 intestine is deterined+

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'

"%aluation-

he %alues of starch content in stoach & intestine as

)ell as blood glucose 6 seru insulin %alues are

copared bet control and treated anial+

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'9

< 'solate+ Rat Pancreas:- -

=urpose-

@sed for studying the effect of drug on Insulin6 glucagons6

soatostatin secretion+

=rocedure-

Adult istar rat F04-244 g: are fed ad libitum .

=ancreas are reo%ed under =entobarbital F4 gB.g i+p+:

hrough a portal %ein canula (rebs-ringer bicarbonate buffer

)ith 2L bo%ine albuin & + ol+Bl glucose is perfuse at

rate of 0+1 lBin+ at pressure 044Hg+

=erfusate is collected e%ery in+ for '4 in+ after first in+

test drug added till 0 in+ ne>t 09-'4 in glucose is perfuse

I' 4i!ro Scr##'i'

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'1

"%aluation

Insulin6 !lycogen6 Soatostatin are estiated usingadioiunoassay+

he effect of test drug on horone secretion of pancreasin response to ele%ated glucose le%el is copared )iththe control+

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'?

'solate+ +ia#hram o$ rat-

=urpose-

Deterination of Insulin based on the stiulation of glucose

upta.e by the isolated diaphrag fro rat +

=rocedure E

-Anial - Male Sprague Da)ley F14-044 g:

-Anial sacrificed during anesthesia and diaphrag arecarefully reo%ed6 spred out and di%ided into t)o e#ualpieces

-Hei diaphrag are incubated in (rebs buffer solution )ithcarbogen )ith M glucose6 insulin or copound to betested+

-After '4 in+ heidiphrag are blotted on tissue6 grounded onporcelin ortar pestle chilled )ith li#uid nitrogen

-After 5 hour at -244C Saple centrifuged 2444g for 04 in+

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'3

23=reparation of Diaphrag

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"%aluation-

he concentration dependent of T @-05 CU glucose upta.e

and con%ersion into glycogen and concentration by insulin orinsulin ietic copound are deterined+

•R#$#r#'c#s:

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52

•R#$#r#'c#s:-

0: Dr +Shi%ai =+ !a)ade+ Ho) and hats Of

=haracology+ irali =ublication6033 0st ;2'0-2'2

2: Dr+Mohd+ Asla6Dr+Surender Singh+ esearchMethodology and Statistics in =haracology+

Acadee publication624490st ;09?-012

': Cortan6(uar6collins+obbins =athology 8asic of

Disease +Harcourt Asia Jtd+ 24449th

;305: Harsh Mohan+ e>tboo. of =athology + Vaypee

8rothers 62449th ?5'6?'

: (+D+ripathi+ "ssential of edical =haracology+

Vaypee 8rothers +6244'th

;'?4-'?19: H+=+ ang6 M+M+Dale6 V+M+itter6 =+(+Moore

+=haracology+ Churchill Ji%ingstone+244'th;2'9-

20+

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5'

1: K+S+(+8arar+ "ssential of =haracotherapeutics+S+Chand62443th ;'54-'53

?: Arthur C+ !uyton6ohn "+hall6 e>tboo. of Medical=hysiology +Harcourt 8race & Copany6033?3th ;3?2

3: ichard D+Ho)land6 Mary V+ Myce.+ JippincottsIllustrated re%ie)s6 =haracology6 Jippincot )illias &il.ins62442'rd edition;2?0-2?3

04: H+ !erhard $ogel + Drug Disco%ery and "%aluation=haracological assay+springer+24422nd;Chapter-.+;351-0422

00: he @nited states =haracopeias 6 Asian edition6 @S=-2?6244F020:;22?1

02: +aghuraulu6 (+Madha%an air & S+(alynasundara + A Manual of Jaboratory echni#ues6national institute of utrition6ICM6Hydrabad6244'2nd 04'-045

0': Anne augh6 Allison !rant6 oss & illisons6Anatoy And physiology in Health & Illness+ Churchill6 2440 3th;22 6 2'62'1

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55

05: S+8hard)a et+al6 Antihyperglyceic acti%ity of a#+">tract of lea%es of cocculus hirsutus' J+: in allo>aninduced diabetic ice6 Indian ournal of =haracology6

feb+2449'?;53-'+0: =+D +Doeni et+al+6Hypoglyceic effect of methylene

chloride root e>tract of ceiba "entandra in noral andDiabetic rat6 Indian ournal of =haracology6 Vune 2449 '? ; 035-031

09: Sh)eta !uptaet+al6 Antidiabetic 6antihypercholesterolae-

-ic and antio>idant effect of occium santum FJ: seedoil6 Indian ournal of ">periental 8iology6april-244955;'42-'45+

01: $enugopal =+ Menon et+al+ Antidiabetic & Antihyperlipidaeic effect of alcoholic syzium cumini seed inallo>an induced diabetic rat 6 Vournal of "thnopharacology 62445630243-20'

0?: = Orti7 Andrade et al Antidiabetic effect on

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5

0?: =++Orti7 Andrade et+al+ Antidiabetic effect on

allo>ianis7ed and noroglyceic rat of Tournefortia

harrt!egiana.Vournal of "thno pharacology 6 2446

040 ;'1-52

03: S+Helata et+al+ "ffect of a#+e>tract of fruit of ithania

caagulens on glucose utili7ation by rat

Heidiaphrag+Indian Vournal of atural

=roduct20F2:; 24-22

24: aanarayana (6 et+al+6Antidiabetic6antihyperlipideic

and Kree radical sca%enging acti%ities of Ayur%edic

Medicine6 Indian Drug6 March 2442'1F':;0'4-0'0

20: Alan !+ 8a>terW6 o)ena C+ Duc.)orth6 Models of

type0 Fautoiune: diabetes Coparati%e !enoics

Centre6 Vaes Coo. @ni%ersity6 Australia Drug

Disco%ery oday; Disease Models $ol+ 06 o+ 5

244550-5'+

22 l S 0 Xi i K 2 !

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59

22+ oel So)ar06 Xian ping Kang26 !ary

S)eeney26WDrug Disco%ery oday; Disease Models

$ol+ 26 o+ ' 244;0?0-0??

2'+ Dr+ Jibor $elise. +6Hypoglyceic Sei7uresInstitution; Albert "instein College of Medicine at

Peshi%a @ni%ersity Mentor ; 8y (a Jai =oon

24. www.diabetescare.com

25. www.americanassociationofdiabetis.com26. www.hopkinsmedicine.org

27. www.diabetis.diabetiso!rnal.com"

2#. www.t!b!les.com"..."mice$#nod$sept.2%%4.pg

2&. '.(.).*erry. *harmacological E+periment,s on

solated preparation E./.0. (i1ingstone &7%32 nd 3

2

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51

‘?’

ANY QUESTIONS

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