'n .H 'S ',{8o1ouqco; Jo e}olr}su[ 'strlnrcEulJ3qd p rk LE.t tqi -N e
'/tltnrgrp slql Eullceralunoc ut sdleq
{1aEre1 ernleradtual aol Jo acueualuturu (t)
: ssessacord
Jaqlo Jo clleufzua raqla ,(e spunodutoc a,u1ce
,(tlucrEoloiq Jo uorle^rlceur alqrssod eql luarrardol pe^Jasqo eq 01 aA"q suorlnecard ursueJ
'salgedord l€crtuaqc-ocrs,(qd 1ue11ecxe qlr&\
lcnpord e senrt enssrl punorE eql Jo uoltezll-rqdofl 'secuelsqns e^rlJu eqt go uorllsodruoc
-ep prole ol elqtssod se ernleredurol € ,l\ol sB
l€ lno pauJec s 8ur,ftp ;o ssacord eql 'esaql
Jo uoll€ulquos € Jo Joqle puB loqoclE 'euo-lec" se qcns slue^los crueEro qtr,,t Eutlerp,{{aP
,(q paqstlduroof,B sr 3ur,(r6 'uollceJlxe JoJ
papoau senssll eqt ,(rp pu" lJelloc ol luetua^uoc
ueuo sI U anssll oql Jo {}r1;qeperre eql uodn
Surpuadep 'razremoll 'qse{ IIr}s are ,(eq} e11{n
sonssrl aql lJBJlxe ol elqEJrsep sI 1I suolllpuoc
lsoru Jopsll 'el€ls perJp Jo qse{ eql uI JeIIlIe
pasn oq ,(rur uotlrerlxe JoJ polcsles sonssll
'spue18 deeqs o1 pereduoc ueq,t
pelprll si ,{1ddns stl tnq }uanlt}suooeluos JoJ olqersep oJolu or€ spue18 esroq
'3'e slunoue e8rel ur atcads rulncrlred
lsql tuo{ sonss!} 3tl1 ulu}qo o1 e;qrssod
3q lou fUtU 1l 1nq ]uonlllsuoc e^!lc€
eql Jo eJotu uleluoc .{utu etcsds euo
q8noql 'enssrl poJlsop oqt Jo ,(llpqepe,re (c)
'urqdorlopeuo8 5o uotlceJlxc eql JoJ
pue13 eutloq JaAo pr)JJoJOrd e;tl spue18
,fuelrnlrd deeqs '3 'o onssll otu€s eql tuor3
q8noql ue^e Jeqlo sql uuql sJuelsqns
eql Jo eJOu ulsluoc ,{eu etcads eu6
'uoJlcBJlxs JoJ EtrB { c'l ! :nry. qlqclq^r tuo{ Ieurm? qr p rncc* :Cl (q)
:E 4qqo5
arrrlce r{11rcrEo1orsf,qd aqlJo rBJtKrr- s! (E)
: $ anssq rqnetued€ Jo uorlceles eql JoJ slseq egl tss}Jodult{ro,l sr seJuelsqns e,rrlce f11ec1Eo1op,{qd Jo son}-€Jlxo JoJ pesn oq ol senssll Jo uolltalas etLL
sl8lrel8tr/{
'uortecgtrnd Pue uotlceJlxe Jo
poqlau agl Jo eslor{r eql q enl8^ 1ear3 ;oaJE saJuqsqns eql Jo satgedo.ld IBsIuIeqrpue 1ucrs,(qd aq1 'spunoduoc eseql oipolBIeJ ,(1eso1o oJB Jo ploJels 'a1erp'(q
-oqJsc '1eg 'utalold Jo ad,$ etuos Jeqlls
eJ€ sasBestp snolJEA JO luaul€eJl Oql
ur pasn seJu€lsqns a,u1ce d11ec1Eo1ots,{q4
'pelnber sI uoll€l
-grrnd g: aar8ep qSlt{ B sasec lsour ut (c)
Pue 'letraleru lJaul Jo
,{1}uenb a8rel e qlt^\ po}€Isossu ,(11ensn
oJe saJuslsqns e^Ilr? f11ecl8o1olsf,qd (q)
'poolq
oql olul paEruqcsrp eJ€ Jo elull egl IIespunodtuoc raqlo olul uoIl€uJoJsiIsJl
leJrrroqr oSropun JeqlIO secuslsqns oql (e)
isnorroqel puu peleclldtuoc JeqluJ seouels
-qns o^rlJe ,(11ecr3o1ors.(qd qJns lo uollulosl
oqt e-IEril sJolcsJ osJgl 'flul€I I 'senssll lBlulue
ur sluno'rlle 3lnulu ur luaserd oIE SoJuslsqns
eArlJE ,{11ecr3o1ors,(qcI 3o requnu e8rel yIEreuaS
*d!I)NVHSIAVU'W
'977'atngol
'gg "cDtutot
'atnlop '(7'96€.
'(Soloutttop
'Lgz 'atntp
'gl ''!cs afi
''!cs atlZ
'€L ''ps 'l
cllocJ€N 11
uI '(916r)
gg "pg a1t
'tzgt-t7
'3 'A '0u
tuapnlswtDt
sPInlJ dPofl ry senssll
Ietrlruv urorJ slue8v leulf,IPsl\ Jo uolleredar6
sg
*i$
{{j
86
Upon removal the tissues are oftenfrozen using dry ice or liquid nitrogen.
(ii) minimum lapse of time between theslaughter of the animal and the freezingor extraction of the tissue; e.g. to pre-
vent the inactivation of insulin by pan-
creatic trypsin, the prncreas is ground
immediatelv and is either treated with
The Pharmstudent
acid-alcohol or is frozen.Some of the tissues used as raw-material for
the extraction of biologically active substances
is given in Table 1. There are certain general
methods and techniques of primary importancein the isolation and purification of many natural
substances, which are discussed first followed
by the description of certain specific methods'
Table 1 : Physiologically active Substances extracted
from animal tissues & bodv fluids.
Tissue/Body Fluid. Substance Extracted.x
Adrenal Cortex
Adrenal MedullaHeart MuscleAnterior Pituitary
Liver
Lungs
Ovary (Corpus luteum)([nterstitial tissue)
Pancreas
ParathyroidPituitary (Sheep)
Semen, Menstrual Fluid (Human)
Serum (Pregnant Mare) i
TestisThyroidUrine (Human Pregnancy)
Urine (Pregnant Mare)
Adrenal Cortex ExtractCorticosteroneDesoxycorticosteroneHyd roxycorticosteroneCortisoneDehydro corticosterone
AdrenalineCytochrome CSomatotrophin (Growth Hormone)Adrenocorticotrophin (ACTH).Thyrotrophin (Thyroid Stimulating Hormone).Follicle Stimulating Hormone (F. S. H.)Leutinizing Hormone (LH)Lactogenic Hormone (Prolactin)Liver ExtractHeparinHeparinProgesteroneEstroneEstradiolEstriolInsulinGlucagon
Parathyroid ExtractGonadotrophinProstaglandins
GonadotrophinTestosterone
ThyroxineGonadotrophinEstrone
Vol, XXI., 1l
General Metlfication of Tis
The finaltissues and b,
in chemical areven be differtfrom the natu.
ciples occur.effect of reage
and purificatioof minimuntreatment are l
substance is tcdone at log' tiwhich ar: ac
stirring. prr\effected this '*:
1. Isoelectric P
Proteins tttions, the stabifactors, namelyproteins show :
tion of theirpositive or negi
pH of the solur
definite chara
isoelectric poi,
electrically neupoles of an ele<
minimum solulat their isoelect
of the proteinr
and purificatiorgically active p:
Isoelectric p
adding acid orto adjust the plprotein. Tbe pr
centrifugationdiscarded or f.solution from
* Not necessarily on a comniercial basis.
'eJuBlsqnse^rlcsoqlJo UIEEB tl t@ t_ tF -'J llollnlos'
{1und unruix€ru ur€lqo ol ,(rgssacou sr s? sorurleqr Jo gd q1 Ed 5ry .r PaPJ?tsIP' fuuru sp palelrdgcuder pue llES eql eAorrer Jeqlle s[ ptr" oogl { n m'6ng:luoc
'o/ott,'a'e) uorlerluecuoJ ll€s ,(uy '{1lce.np tyes fq paqsrlduroco? $ uorlrtdFard +qaoq
ol pez,{l€.lp '.re1elr u1 pa^losslp ro pepuedsns
aru salelrdlcerd paluredes eq; 'uoueuorlcs{rog pe.{oldrua aq .{uru .(uo11ern1es o1og9 to o/o0g
pqos egl Eurppu fq perrarqce aJB tl€s Jo ssotl-sJluoJuoc qErq uorlelrdrcord I"ug eql ul :suoll-J"{ Joqlo elurudos ol JepJo ur uonrod elqnloseql ol pepps sr uorlnlos lJ€s eJotrN 'uousJllgro uorle8n;rrluec fq poleredas sr poruJoJ os el?l-tdtcard oql 'uorlce{ auo Jo uolleldrcard sesnec
13ql peqcseJ sJ sorl?.rlu$uoJ ? Illun ll"s eqt Jouollnlos pal€Jnt€s u Eurppe Jo slsrsuoc poqlaulereue8 eql .pesn seturlotuos oJ€ l?ql oslg sJer{loaJB eJoql qtnoql ,e1/{Jorlcala pasn fluomuoclsour eql sr eleqdlns tunruotutuv .suollJ"U
olut pelutedes sr lc?Jlxe Jo uoJlnlos uralord e
'l1ts clue?rour u? Jo suorl?JluoJuoc Surseorcur
Eursn ,(g 'se1,{1or1ca1a urElJeJ Jo suorl€Jlueruooredordgo ecuesard aql ur eleldrcerd surelor6
.s11ug crue8roul .{q uoltuuollJsrg .Z
'slsuel€ur
elllcu snourelord Jo uorl?JgrJnd pue uorlEJl-uecuoc JoJ poqleu llncgrp ? uorleuorlc€{ cIJl3
-eloosr eql sal€tu suollcB4 lrroJe.$p qlr,n ,(lt,rtlce
JO uorlBIsOSW SrqJ 'l?uol?ru Ueur Jo SlunoueSutf;err ur€luor srrorlJ€{ eseql uolllppe uI
pu? suor]3e{ crJlcoieosr eql Jo ouo u?q} eJow ulpqnoJ ueuo sr 3cu?lsqns e.tr1ou,{11ect3o1orsf,qd
eql 'sorxrl leJalas peleede; ,(pensn sr ssacord
srql 'sluroJ crrlcaleosr Jrerll le uorlulrdrcerda.r
pue uorlnlosor {q pagrrnd JaqlrnJ eJ€ etus}s-qns e^rlce eql ur€luoc leql saleldrcerd aq1'3uo1e3e ro loqoclE Jo sorunlo^ I€J3Aes Jo uou-rppu or{l ,{q ro eleqdlns runlucrurue qll^\ uor}
-eildr.lerd ,{q pe.louer eq uec 1r 'euulcu Alecr?-ololq sr prng luet€uredns yeug er{l uI sureluer
1nq1 uralord eril JI 'surilord alqnlosur JlJlJelaosr
Jeqlo st€lqo 01 osEq Jo prc" qll{{ pelsntpe
L8
,(q pa,rourer sr wanrd F*d *41 -matord
eruos Jo lurod culapc rp Gt HC qr rnfpe o1
sB os lceJlxe snosnbe ue ol ssq ro F{ 8rlppe
'surelord a.rq* .{11ect3
-o1ors{gd Jo roqunu e8rq e go uoqecglmd pue
uorleredas eql ur pellnseJ seq surelord aql 3o,(Uedord enbrun sq;. 'slurod crJlcoleos! Jrcql lsuollelrdloard unulxsur pue fltpqnlos tunw1ulllr
^\oqs surelord fueprl 'pleg clJlcole ue;o selod
eql 01 elerSru ol esBal pu8 IBJIn3II .{1ecu1ca1a
ere selcrped eql q3ig^t te 'ulod 44Jalaos!eql sB uaroIDI 'Hd cllsualcBJ?qc ollugep
€ st aJeql 'uralord ,(re,ra rog 'uoBnlos eqt 3o gdeql uodn Eurpuodap saEreqc e^I133eu ro eltllsod
arpbce seyctlred egl 'salilu"d Jleql Jo uoll-ap,(q eql uI secueJJJlp palJ?l,s moqs sutalord
I€n p l^Ip q' uot1e;p.(q puu eEreqc',(leru€u'sro1ce;
orrrl ,{q pau.re,roS sI qclq,\\ 3o ,{1qtqe1s oql 'suol}
-nlos l€pioiloc cr11,(qdorp,{q uro; sutoloJd
'uope1;dparl Jlrpeloosl'I
',(B^A srril palce.Uo
eq uac uollecgrrnd oqi pepl,rord '3utut1s
luslsuoJ qrl^A ,(11enperE popp€ oJ€ qclg&\
syuaSear e1n1rp Sutsn sernleradural d\ol le euop
oq ol sI {Jo&\ eql 'pelElosl eq o} sl scsBlsqns
epqul f la,rtleloJ € uaql\ alq€JnoAEJ eru luorul"oJl
lecrsiqd pue Iscttueqc sno:o8t.t ulnutultu Jo
sarnpeco.td,{1uo',(1tuabestro3'uo{}€cgr.rnd puu
uorlJuJlxe ut posn sluo^los pue sluo8eer -Io lcoJeeql 01 enp ,(1e3.re1 ,{1qeqo'rd sl slqJ, 'rncco seldtc
'urrd eltlce tqtr q3lq,4[ uI e]Bls IeJn]€u eql Luo{
,{lr,tlce 3o eer8ep pue pul{ ut lueJeglp 3q ue^e
,(eur ,(eq1 'setlreCord 1ecrs,{qd pue lecltuogc ul
luoJegrp eirnb eq ,(eru sptng ,{poq pue senssl}
JBr.urur? uIoU pol€losl slcnpord lsug oqlsplnljl fpog puu sanssll Jo uolluJg
-lrnd pu8 uollJuJlxg aql .roJ spoqlohtr trBrouac
t86r "IXX ',lol
'(auou.
'spoqle
psalolloJ
lernleu ,{r
ocuelrodtpreueE r
secuBlsqn
JoJ I€IJa11
JUaptustr't.
90
equent extractions may be required. The extract
thus obtained is fractionated by a specific pro-
cedure by which each active substance is effecti-
vely purified. Procedures for extracts of this
kind frequently consist of a combination ofisoelectric preciptation, the use of salts and
organic solvents and dialysis.
The natural occurrence of an active sub-
stance in a body fluid (blood, urine, bile) is in
a sense an extract. This obviates the preparation
of an extract, although difficulties may be en-
countered when working with body fluids that
do not arise when tissue extracts are fractionated.
Urine is a body fluid, sometimes in which,
depending upon the physiological state of the
animal, several products of biological impor-tance are found. Examples are the appearance
of chorionic gonadotrophin and estrogen in the
urine of pregnant women. Estrone is also
found in the urine of stallions. Both gonado-
trophin and estrogen have been concentrated
from urine by adsorption on to benzoic acid.
Water Insoluble Substances.
The compounds of this group are insoluble
in water in the purified form. In tissues and
body fluids they frequently occur in conjugated
forms which are water-soluble. The removal
of the tissue and the grinding and ertractionprocess are essentially the same as for water-
soluble suhstances, The extract is separated
from the tissue by centrifugation and the pro-
cess of extraction is repeated. The combined
extracts are fractionated. or are extracted withsome water-insoluble organic solvent; this latterprocess llertitions the active substance between
the extracting flurd and the immiscible solvent,
thereby giving a fairly concentrated solutionwhich is further purilicd by an appropliatemethod.
Body fiuids are usually extracted by refluxingthe acidified material to free the active sub-
stance from its conjugated
active compound is then
two immiscible solvents.
Specific Products.
l. Liver Extract.
Tlte Pharmstudent
soluble form. Thepartitioned between
The importance of liver in the treatment ofanemias was established by Minot and Murphy
in as early as 1926. I-ater the haematopoietic
principle in the liver was identified as cy4noco-
balamine. Although today, pure crystalline cya-
nocobalamine is exclusively obtained by the ferm-
entation process, the methods developed for liverproducts have established so well that they are
still popular. Here briefly the preparation ofthe lever fractions has been dealt, (flow chart
is given in the Appendix).
Liver, collected from healthy oxen or sheep
are ireeze stored. Liquid extract of Iiver is
made by extracting the minced liver with a
reserved maceration process. The alcoholicextract is concentrated under reduced pressure,
the active constituent is precipitated by dehy-
drated alcohol, and the precipitate redissolved
in alcohol (509/"). Finally a solution of the
purified liver extract is made in alcohol-glycerin-
vrater tnixture.
A more purified liver extract is obtained bydigesting the minced liver with proteolytiuenzyme (papain at pH of 5.0 to 5.5 at 603C),
to remove a major unwantcd constituent pre-
sent in the liver extract i. e. prolein. The pro-teinaceous substances cause severe immunereactions on injecting. After filtering off theinsoluble residue the aqueous extract is concen,trated under reduced pressure and low tempe-rature or subjected to freeze drying. This pro-duct is suitable for oral administration. Theaqueous extract is treated further with 70o/o
alcohol, the precipitate of unwanted proteinousfraction removed and the filtrate evaporated.Still further purification is effected by passing
Vol. XX!.,l9
through an iduct so obta
process chart
appendix.
2. Heparin.
This anticc
buted in the bstitial mucosr
etc. Heparin r
tissue of slaug
quant!ties froris extracted rr;
ration for a l':
brings abLlut I
minirrrising i=
Further e:
of 2o7o sodiunsulphate at ,<O
70oC proteins
filtered out.to the filterEheparin to,eet
proteinous inu'ith trlpsin i
is reprecipitat
a;:lcne. lear j
1- ->-
-aF
in aac:c -;comp::r+ l:remorai b-"
sodiura ca:ltrseparat* our I
and other ne
The heparin is
it as heparin-lplex and thus I
The insolubie
with ammon::
heparin is eff
barium salt,
;
,l
#s
€g
{a3a
"$
.i-sqns JrloJ jo tunutixPtu € oldu puE Sut8nJulual [ic'-us e ur F]rr-rsxf s :lFm{.r--:-: 3lr11nser
,(q pere,rorer sr ile:t-\:. oql 'pJlcgrlxJ ocr ol oul pue uortecr;!:rE_:-tlr uq ::-.-.:trrrd st
cJuelsqns J,\riJt? 3r{i uodn spuadap pesil iueAios oJuE}Gqns a^l1J€:g: :€::|: --*:: -: ---;:..a 3r-11 Joeql Jo od,r'1 eq1 'uorlJ?Jl):o 3AI]tJ_Ue JoI ourll olunlo^ otll JI paaiqEi:r :ia $t:=-:.-' aql IIe3o potred e JoJ uelEqs {llEilsll sr ojn}xr'rr Jql psu .lr.ro lseel le p:1--f.irF}:j ---- :rplsarpue'(euclroe fuc loqoJie J4ti siualios rtueS:c 3iil 'luuf,liliu l.;eut 1go{ i-18}tr:;f: ::---r--!s olJO SIJ€S "siseq 'SplJE .Jo su.-,ilnlos snolri L'i: paSnJlllual Sl oJnlxltg agl q--,t 1" :;:--r 'lqfluJo 'Je,ij''i) lusllos rr]1 Jo eurnloA olrr:U:p B -ii,\o Jo sJllou lE.ralos .toJ plo3 ;:--t :: FuBlsqllA\ peYIru si rep,ncd (rp r.1 1 'ensti] qstr-l .r'ril .'t pr$oiie ,(;:rranbe.r3 sr onssti Jo uotsu3dsnsse {?&. 3ql3s 3ql LlJiltu ur il.lo pir.rlu:, sr rn-(slt 1-.- 'iilss.ro sasgq .spif,B
Jo suorlnlos snoanbgpalrp Jo ttcijluJlxo a{il '.:ruolols till.'r1 ur1]le,lr .lniln lo .iole^\ r.:r Sol$?JSnS alqnios-releu Sutl-fqei., uei{l cils€Jp sgal si Doqi€tr rrql 'p?,\i)i-iio.i --iejlro Jo.l p:s,i ,i;irnsn s}u.)AIoS ',{llpeer e.rotrr
sI J01e,'r, iielii JOI{10 slt13n1rlS{JOf, 3nSSrl Cr ',:.-, rri:,:ni :1:-.,ja ot s-'.\l3s ll ir:rx,!, e lil€JlsoprunnseA JapLlil l)oleJp,{uap pire uezolj sr at'issr1 Si :'.i-::r.i:r! si -,-.-'j j}.r .r,ri.:-t E.jo i!rtruii?ell ,.iiSBJp
ouno;3 Oql .il 'o3€;S rroliJeJlil cql 1e ,-ilaq rlrr:: t:: i'*euj j-r ijoni\lt-.!!i pri€ uotleJnl
Jo sI slql islLte'tJos liueS.rt-, 3ur,{oldile poiite,-i1 -rrol l:.r..? .i .:ic?lr3?ld t.ie saJnpoJoJd plltua^oqe.lql Sutsn dq pa,\oLuar ere sprdil oriSSrf riin,''.:r11' .rrrr\los ploJ q]r11 ilq3noroql paxrui
'Jc?Btissop B ur t{e3rss;p l-:r '.i;uu Pnnc:? 'socuelsqns eAIltE otll Jo
3 JeAo 8une1d .{q pol-lp JorilJnJ eq f€w enssrt uollE.\Iljeul lusltJtl 01 suollnef,ord ,(;essaoau
oql 'JI" nr {lp o1 pe,\\oiJ€ sr ensst} eqt pup uorl Japun i?{.ulu? aql olo{ psAolueJ sI enssll eqJ-?Jllg uorlcns .,(q penoruer sr lue^los eqt l-doru acu'lsqns alqnlos Jaf'd\-1"aJ1 i?url pu€ pJrql oql rouv ']ua\los qso{,(q pecelder pue Buruoqdrs ,{q pe,lo{ual sr ruar 'seuouJoq
{os oql 3o i.red :aieerS eq1 .srnoq lz Surrnp xas lPplolsls pc? ieJIlJoJ 'sulluslta alqnlos
seull [rJe,\es poJJus puB arnte.radua] uoor -leJ Jo lsISuoJ dno:3 puocos eql Jo spunod
1e ido>1 ,{ltuanbar3 sr arnixrru aql pu€ anssrl -tuoJ 'se1erp,{qoqrec pue seplloelcnu osutelold
Jo eunlo^ 3ql salull xts ol rnoJ sr pasn lue^los lsotll spnlJut dnorE lsJg eql 3o spunoduro3
egl Jo erunlo^ eql 'ouotoc? ,{11ensn sr gctq^^ 'pegrJnd pu€uollsJpfqrp JoJ pesn lue^los oql otur punor8 palelo$ i(11eug ere ,{eql uaq^\ 'Ja}?^\pue l"ulue eql u(-iry IB^orusJ tage ,(lolerpeurrur ur elqnlosut aJu sacqelsqns eql JaqleqlA. (ll)polooo sr 1r 'perrp eq ol sr ons$l e uor{i11 Jo .spuDl snotJBA Jo
'uolls^Ilc€ul lue^aJd ol lue^Ios redord e qlr^{ srrorlnlos snoonbs ur Jo JJ13,A[ uI elqnlosanssll qse{ oql tesJl ol luulrodul sr 1r snql e]B peur"lqo sscuulsqns 3qt JaI{l3tIl[ (l)'rrrJoJ pegrJnd ur ecuelsqns eql 3o ,{:ezrocar eq} isplngJoJ solllnlglp sesod seulloluos slqJ 'utelord ,{poq pue sonssrl ruorJ peure}qo secue}sqnse eq lou feur scuelsqns s^Ilc? aql qanoql uo^e eql 3o ,{trlrqnlos Jo srs€q eql uo peJaprsuoo oqens$r qse{ € Jo lcerrxe snornbu u€ ur pamulqo III^ spoqieu cglceds oqr Jo uorldl.'sep y,i11ensn sr ureloJd elqeJaprsuo3 .uollucgrrnd s1r
ro3 pordopu sI rellrsa peluaserd spogle111 IBr splnlg
^pofl puB
-eua8 eql Jo uoD€trJgluos € Jo .uo ,rq1," p"1n 1 sanssrJ. Jo uollcBJlxfi oql roJ spoqlol^l cgleed5
-ueJuoJ sI loEJlxe oql tou Jo Joqlorll[ .llEs oql .uollu8ruJrluec f,q
oAoIueJ o1 pez,(1erp pu€ luollos aql Jo errrnlol po^oureJ oq ,{psue uec pu" seleltdtcard IJtelord
T86T,'IXX 'IOA
€ql 'JaJnq I3ql Jo luro(
Jo JoJnq e I
,{q s1 1cu.ttx
ernpacord r
-c3{-11€s upoqlouI slq
oq s?c su(
'eSreqc cut:
Joqlre uo u
egl 'aJnleJi-IweS eql -Il
srs,{1erp go
Jraql sa{Eu
-tuoJ se 'ensuol cruB3,
uorsnJrp Jo
ornleredura
elenbape ur
urnurldo 3
lC€JlXe u€ i'lcellxe ,(re
utdo.rlopuu,
eql sr eldrenssrl oluor
pol€Jluacuc
-qns eAr13€
'paglr
pculqluoJ :paleeder ,(1
qlla\ potnl'luoqJosp"
lou IIIA lslB qlla\ p3r
'uoll€Jiig J(
sr lr qclgll\paJJrls puE
eql lereueS
luapnlsutJp 68
90
equent extractions may be required. Tbe extract
thus obtained is fractionated by a specific pro-
cedure by which each active substance is effecti-
vely purified. Procedures for extracts of this
kind frequently consist of a combination ofisoelectric preciptation, the use of salts and
organic solvents and dialysis.
The natural occurrence of an active sub-
stance in a body fluid (blood, urine, bile) is in
a sense an extract. This obviates the preparation
of an extract, although difficulties may be en-
countered when working with body fluids that
do not arise when tissue extracts are fractionated.
Urine is a body fluid, sometimes in which,
depending upon the physiological srate of the
animal, several products of biological impor-tance are found. Examples are the appearance
ofchorionic gonadotrophin and estrogen in the
urine of pregnant women. Estrone is also
found in the urine of stallions. Both gonado-
trophin and estrogen have been concentrated
from urine by adsorption on to benzoic acid.
Water Insoluble Substances.
The compounds of this group are insoluble
in water in the purified form. In tissues and
body fluids they frequently occur in conjugated
forms which are water-soluble. The removal
of the tissue and the grinding and extractionprocess are essentially the same as for water-
soluble suhstances, The extract is separated
from the tissue by centrifugation and the pro-cess of extraction is repeated. The combined
extracts are fractionated, or are extracted withsome water-insolublc organic solvent; this latterprocess prrtitions the active substance between
the extracting fluld and the immiscible solvent,
thereby giving a fairly concentrated solutionwhich is further purified by an appropriatemethod.
Body fiuids are usually extracted by refluxingthe acidified material to free the active sub-
stance from its conjugated
active compound is then
two immiscible solvents.
Specific Products.
l. Liver Extract.
The Pharmstudent
soluble form. Thepartitioned between
The importance of liver in the treatment ofanemias was established by Minot and Murphy
in as early as 1926. Later the haematopoietic
principle in the liver was identified as cya-noco-
balamine. Although today, pure crystalline cya-
nocobalamine isexclusively obtained by the ferm-
entation process, the methods developed for liverproducts have established so well that they are
still popular. Here briefly the preparation ofthe lever fractions has been dealt, (flow chart
is given in the Appendix).
Liver, collected from healthy oxen or sheep
are ireeze stored. Liquid extract oI liver is
made by extracting the minced liver witb. a
reserved maceration proccss. The alcoholicextract is concentrated under reduced pressure,
the active constituent is precipitated by dehy-
drated alcohol, and the precipitate redissolved
in alcohol (50ob. Finally a solution of the
purified liver extract is made in alcohol-glycerin-
vzater tnixture.
A more purified liver extract is obtained bydigesting the minced liver with proteolytiL;
enzyme (papain at pH of 5.0 to 5.5 at 60cC),
to remove a major unwantcd constituent pre-
sent in the liver extract i. e. protein. The pro-teinaceous substances cause severe immunereactions on injecting. After filtering off theinsoluble residue the aqueous extract is concen-trated under reduced pressure and low tempe-rature or subjected to freeze drying. This pro-duct is suitable for oral administration. Theaqueous extract is treated further with 70o/o
alcohol, the precipitate of unwanted proteinousfraction removed and the filtrate evaporated.Still Further purification is effected by passing
Vol. XXL,19
through an i
duct so obta
process chart
appendix.
2. Heparin.
This anticr
buted in the tstitial mucos:
etc. Heparin ;
tissue of slau3
quantities froris extracted uration for a 1-r
brings abour r
minimising irFurther e:
of 2o7'o sodiurr
sulphate at 5O
70oC proteins
filtered out.to the filtere,heparin toget.
proteinous inwith trypsin ir
is reprecipitatacetone, leavi
tion. Almostcan be effected
in acidic me
complexes are
removed bysodium carbon
separates out I
and other ne
The heparin is
it as heparin-bplex and thus fThe insolublervith ammonia
heparin is effi
barium salt, r
sopllded io Jaqunu
aql se u,\\oul oslB
E seleJcos'..Pue16 J3]seur,,
'pue13 , r4m1rd aq1
st)npo.Id {tDt!ruld ','tJorlET
-r11e1s,(:c suols€q ullnsul 3o 1ets,{-rc e qltlt Sutpaas
ro 3ul1pq3 'eplJolgc culz Jo '{1r1uenb 11erus
e Surureruoc ragnq ileqdsoqd Jo o1?Jll3 Jo
el€le-le 3urppu pua lale,^ uI ullnsul Eut'tlosstpar
,(q paurelqo sI i'lnsul aur11e1s,{r3 'euolocs qlt^\
'pelqldlceJd pue loqoJlE ctptre qll'\\ pasodtuocep
sl 1I 'peutsiqo ueeq seq atelrtd j1 'ullnstrt
pegrrnd ,(1q8rq sa.'rri '(S S-g'S i1d) turod ctrtr
-eleosl sll le uollnlos snoanbe wo:1 uoteltdtlrd
I€urd 'sopndedflod rtru,(zua uo:i uotlerrdas
alelduoc ? oJnsue ol paleadar ssecord eql pue
paAlosstpoJ st ernpecord lno-pallss luor3 ullnsul
epnJ) 'elercrd ol uolsJoluoc dq ro sPlJolqc
unrpos Jo uolllpp" eql ,{Q uollnlos Eutllnsar aql
uror; paletrdtlard st ullllsul epnJJ 'l€tJoleu '{tleJ
elsredes 01 pel[qc uaq] sl l€lJol€ur pal€JluoJ
-um eql '3ogg EurpeaJxe lou arnleredual e 1e
runncu\ ut pelJ€JluecuoJ pu€ poglpIJB 'lceJlxe
J€elJ B ulelqo ol poJellg utu8e 'EtuotutDs I{lL'r\
pazrl€Jlneu 'suqap anssll eAOUIsr o1 paSnJtrluac
sr rr€rlxe eql '(s'e-o's Hd te )i,99-99 aulaq
uo!l€Jluacuos Ioqocl€ [Eus eql) ,{lpaleader
loqoJl€ poglptce tlll^\ pelJ"Jlxe pue alnleJedrual
^\ol ls peJuil.u sI Isuel€tll uazo4 eq1 '.{1ate
-rpe{uul uazo4 pue s8rd Jo uexo tuo{ polJalloc
sr urlnsul Jo uolloeJlxe egl JoJ SseJCu€d
: sdl\olloJ s€ peull
-1no eq feur uotlecgtrnd 3o poqleu leraua8 y',(dereqt Jo uoll3npoJd raqlre ut eceid ,(ue puno3
JeAou suoll?Jedald roa.r1 Jo csec oql uI s€ 'slJellxa
€pnJc 1e 1 prder os ssAA uIInsuI Jo uollecg
-trnd o I JoJ spogleu 3o luerudole,\op eqlullnrul '€
'PezuezrPd
{1;rug pue p.1llp 'rar1la pue lor{ocl€ t{11^
paqs"^l st pauroJ snql ur.ledaq tunlpos '01€u
-oqJrc urirlpos qtl.{\ luoulluart fq 11es unlpos
I6
eql ol psrieanc GqE. r 1tPn -sqs :nlJ€qsll sB uorltgllEls;F '{ }Eqst s :uedoq
5o uorlecgrrnd llqt-rnJ g:S -E&cE:: qll,t.r
posodtuocap st xalduoc qitd=q *r::= a{I'sapr.leqctes,(1ocl prlneu u:o{ F€3 s; =;
t:1d
-ruoJ oulplzueq-utredaq Jo 3urJru(1.cjE:; :? 1l
Surleltdtcerd ,{q pagund JeqlJnJ sr uueJ3q :qI'uorlnlos uI seplrBqJcus,(1od IeJlnJu Jaqlc puP
urredaq clplce eql gulqaq Eur,real lno salzredas
slBuOql?J tunIpEJ olqnlosuJ 'aleuoqJ?3 IlnIPo!qlr.!\ polPoJl ucql pu€ uollBJllu ,(q pairouer
sr xaldutoc aq1 'pateltdrcerd eru saxelduroc
uralotd-untupe-t uaq.\\ 'runtpau ctptce uI
eFucltlJ urnIIEpEf, Jo uolllPpe '(q patcaga 3q u€J
uraro:d -lo uolleulutla aleldruoc lsouIIV 'troll
-nlos ur sutalord palsa8rp aql Sutleel 'ouolace
Jo loqorle Jo uoqrppe ,(q paleltdtcerder st
urredell 'tnnlperu eulle{1" ue ut utsd'ft1 q1t"tl
Surtsa8rp .(q panourer st ,{ltrndurt snoutelo;d
eql 'urelord ollltl P qll&\ raqleEol utredaq
3o ateildrcard e sp1et,{ lcsJlxe poJellg aql o}
Iogosl€ ro suolec€ Jo uolllppv 'lno parallg
uaql are ,{aql 'pa1e1n8eor e:e sulelord 3691
ot lr€lxe srqr Sulteeq ,{[ 'c 009 1e aleqdlns
runruorurue o/og'g pu" aprxorp,(q unlpos o/oz lo
uorlnlcs e qllrd e[Iop sI uollcBJlxo JsqlJnc
'setltlndtut Sutsrrrttuttu
sngl 'sensstl Jo uollcnJlsop 1er1.red lnoqe s8upq
srs,{1o1ne qcrqtr 8u1rnp s,(ep ^reJ
€ JoJ uolluJ
-areu ,(q autles lectEolots,(qd glIA\ pal3€Jlxe sI
enssrl lu€Aalal oql 'snssll JaAII Uror; satliluenb
11eus ,{1uo pu€ sl€ruluE esnoq'.re1qFn€ls Jo Onssll
3un1 ruo.t3 peulelqo ,{1e8re1 aou sl uuudell 'cle
'ensstl a,ttlceuuol JEIncse.l-tred'€soontu lelllls-,relur's8unl 'ra,rt1 Surpnlcut ',{poq eq} uI pa}nq
-ulsrp flepl,tr st eldtcutrd lutln8eoctlut slql'ultodag 'Z
'xtPuadde
eql trr ua,rt8 sl slo€Jlxs JOAII Jo 1'reqc ssecord
V 'uotlcafut toJ olqullns sI peulslqo os lcnP
-ord aq1 'rulnloc e8ueqcxe-uor uu qEnorql
t86t "ixx'loA
Surssed ,{q p
'palerodeae
snoutelord p:o/o1L qpn' toqJ 'uorlPJ:
-o:d srql ',
-:dure1 mo1
-uacuoc sI lc1
eqt go Ewre
euntu{Ijr oJ3
-ord aq1 'r-erd luanltls'(Cc09 te s'trrfloelordfq paut4qo
-ulrec,{1E1oq
aql Jo uotlpe^losslpsJ
-,(qep fq p:
'alnsserd pe:
crlor{o3le e
3 qlllt\ JeAl
sr JaAII IO
deeqs ro ua
lreqc arog)
3o uollerudere faql lerre, r1 ro3 ped
-ureJ eqt ,{q
-e,(c eutl1els
-ocou-P,(c se
cgtrelodoleuKqdrn;41 pu
Jo luatulEeJ
ueealeq p(
eql 'luroJ
luapnlswrL
92
which exert physiological action. Frotn the
anterior lobe of the pituitary at least the follow-ing six hormones have been definitely establi-
shed and corimercially made available.
(I) Adreno-ccrticotropichormone (ACTH),also known as Corticoiropin;
(II) Thyrotropin or Thyrotropic hormone
(TH);(III) Growth Stimulating or Somatotropic
hormone (STH);
(IV) Lutenizing (LH) or Interstitial cell
stimulating horirione (ICSH) :
(V) Follicle stimulating hormone (FSH) :
(VI) Luteotropic or Lactogenic hormone
(LtH) or Prolactin.
The last three named are the hypophyseal
gonadotropins. Sonte of these hormones when
obtained from different animals show differing
activities from species to species. It is not
possible to obtain sufficient amounts of human
The Pharmstudent
Growth Stimulating Hormone (StH) to satisfyany therapeutic demand. Also, human pregn-
ancy urine and pregnant mare serum are richsources of chorionic gonadotropins, with simi-lar activities to those exhibited by the hypho-physeal gonadotropins. The thyrotropic hor-mone is easily replaced by the directly acting
thyroxin. Besides these reasons, the anterior
lobe hormones being all proteinous in nature
are liable to cause immune reactions' Therefore,
ACTH remains the only major hormone ofwide clinrcal importance. However, some
preparations of the other horomones mentioned
above are marketed by a few leading pharma-
ceutical manufactu rers.
The anterior pituitary gland, obtained from-
sheep, oxen, pigs or horses are collected from
lieshly slaughtered animels and either deep'
frozen or dehydrated till further treatment' The
following general scheme is then followed for
the extraction of dilferent hormone fractions.
Vol. XXL, l9
chromatograpcurrent extr:lc
4b. Posterior
The preser
pituitary was
in 1895. Honprincipie war
pituitary. Poendocrine glaroxytocin and t
The poste
obtained fronrthe slaughter
frozen or tiirepeatedl-r i:finally oL-'.1 ce
containing aiiposterior iot'ethe smooth n
prepared sim;defatted postr
aqueous aceticthe aid of hear
lated and a'-rtr
elevated tempe
be used forfiltration or aur
of the rwo cc
ressin) of the p<
may be utilizer
(i) One hund:dned glar
litres of 0
is h€ated
950 C dur
quickly ar
luble resi<
litre of the
(ii) The combi
small voluThe conce
Extraction with ammonia atI
Fractionation with 0.25 M amtnoniuinI
pH 8.
sulphate at pH 4.
I
Precipitate alpH 5.4 : LtH
I
Precipitate atpl{ 4.8;ACTH
I
Filtrite :
adjust pFl to 7. lncrease arnmoniumsulphate concentration progressively.
a. 6 M... ......Ppt. of St. Hb. 4 N4..........,.Ppt. of LH & TH
Only the extraction of ACTH has been dealt
here in further detail. l{onrcgenised fresh glands
or dried poivdered glands are treated with acidic
acetone (10o/") or alcohol (709b), in rvhich
ACTH is soluble but other proteins are in-
soluble. Raising the acctonc concentration to
90o/o causes tire precipitation of ACTH, rvhich
can be redissolved a;id the fractionation repeatcd'
Luteotropic horirtone still remains associated
with ACTH. It can be separated by adjusting
c. 8l\'L .........Ppt. of ESH.
the pH suitably and precipitating, while ACTHremains in solution. The ACIH is then precipi-
tated by the addition of alcobol or acetone.
The ACTH can then be redissolved suitably
and purified by chromatography on oxycellulose.
Weakii' acidic solutio;r ot' corticotropin is
ailsorbed on to columns of oxycellulose and
eluted bi' dilute hydrochioric acici' Extremeiy
pure preparations of corticotropill is obtained by
i'ecourse to several techiliques like ion-exchange
:,
t
€
$
g
A
crozLroq qllA\ palesJl s! oulJn 'uotlcuJlxa aq1 JoJ \tr!r FIa. s ::EF :€F-=:a- r ;qI',{cueu8a.rd 3o ,(ep ql08 pue q109 otll Euunp '?Jnler}iG;i :!c{ :s :Jr_ - r ic'- : '" .;rlrspJJ-aJox; Suraq lunorue >1ead eqt 'auun eql € ol pel?jls--tlra*a s[:E: --a-,- :] ::J.I (Il)ui paloJJxe f1lzug pue poolq eql olur rile,to eqi :=,{'_. F;- _-_- ::t:lJO euBlqruau cruo[oli3 eq1 ,(q palsJces sI i qlr.\\ palJsJr:\:-el -sr q-aq? :-:..i.:, : :.:ll(ClU) trrdorlopeuo8 ctitor.roqr ueutnq 3I{I -osur erlt e.\orua] 01 per3'g si ::; . 1::nb'urnros e.ruur lueu3old ro:utrn L!€Lunq luuttda,rd pal()or 'polraC Jnoq JIEq e 6uu:: 3,-c6Jaqile sI ss[Ielsqns Slql Jo uotlJeJlxo 3tl1 JoJ Jo a:nle:acjL'.t;l c ol .(linnperf P:]E?q s!
seornos ul€tu eql 'seuoutlcq crdo:lopetto8 sacnp OJttlxlu o'-if 'Pl3e JIlaJu ;9o;i'O Jo slllll-o:d pue13 ,{:e;rn1d Jouo}u€ aqt qSnoqily 0l qlls pJlr€.jlxe sr l?rJolsru puel8 p;trp
strtdottopouog 't -auolecg l€lcJ3uliuoc 3o su2.1fi poJpunq euo (l)
: peztllln eq ,(eur
8ur,uo11o5 oql 'lcuJlxe eqol JolJolsod eqt3o (utsser
-dose,r pue ulJol.{xo) s}uouoduroJ o,^Al erll Jo
uolleJe(I3s JoJ (JeAaA\oH '8ul,re1co1ne Jo uolleJllg
fq uor1ez11qra1s JetJ" Eurlcafut JoJ pasn eq
qJns ss uec uoltrnlos sIqI 'seJnleJedural pel€Aela
13 pal€AIIJEuI eJe salu{zue ct1,{1o1iiu pue peluJ
-n8eoc oJB suteloJd polu?.laun ']€ag Jo ple eql
qlt.t Jo ploJ oql ul Jaqlla plc€ cllaJ€ snoonireoogZ0 gtlt etrSSIl eqol JolJelsod pelluJep
pus pel-*rssap 3uricu.tlxe ,{q i(1durs paredard
iq ufJ 'lraga Sutlelnulls alrsnrJr gloous eql
'JrlalnrpltuB'ctco1.(ro'arnsserd) eqol rotralsod
aqr jo selll^u3e [JEtuud eq] Jo 11e Eututeluoc
g-.eJl\a Ireltnltd Jor-Ielsod'pe.lep'rnod,{11nug
sr 1r pije puu13 aql e1erp,{qap ol ,{lpeleedar
euoleJ€ q'.111 ,{ioletpeiuurt pe}€eJ} Jo uezou
deap .leqire pue sl€ulu€ eql .;o re1q8n"ls eqt
raqe dleletpeuul pelcelloo ouexo tuorJ peut€lqo
,{11ensn sI pueJB ,(rellnltd rotrelsod eql'utssatdosott pau ulcol,ko
(1aueu 'seuourtoq o,t1 p1er,( o1 puu13 euIJcopuo
lsJg eql selr ,fuelrnlrd rotrelso4 'frzltnltdrorralsod eql uL{yuo punoJ sulrr eldtcutrd
stql icql psuoqs ,{1}uenbasqns '11eznog '9681 u}
reJaeqJs pue Jo^IIO fq palelocstp sun freltnltdaql ur eldrcurrd ernsserd ? Jo acueserd eql
sauoutlog ,{to7y114 'tolra$od 'qt
'rIo!lJEJlxe lueJrnt-Jolurlur3 ro ss.(1urpo'|1ceJe 'fqdaEoltuorqc
l86t ''IXX 'loA
'enbruqcat uorlnq-IJlsrp lueJJnJ-Jalsnoo ro,(qderSolcruoJr{c(elruoluoq uo) uorld.lcspe ro eEceqJyo-uor
1o dlaq eqt ,(q euop sr uorlecgrrnd JaqlJnJ'urco1fxo pue urssaldose.t e;nd lsomle sp1ar,(
suorlcs4 ls€l pu€ qug eql Jo uoflEuoll-c€UeU 'suorlce{ qloq Jo ,(1nr1cc parrur
Surssessod sleqio 'suorlcu.l3 crroi.irrr pup
rossord ur.raqorJ oJu suorlJ€{ is€l aqi ptrE
qgg eql '(uor1cer3 crcol,(xo au: -1o isour)Jarllo runolorlad puu Jeqlo IW 000+ 'r uorl
-J€r; o...r1ce-rossa:d eqt 3o rsou) reul! 'lur
0S, 'Joqte 1u g1 'auotaJu ltu 997 'aucrl;re
[n gZ(, '(uotirzr3 rr:ur) pr-ae ctlare : Jaqia
wnelorled puz 'J3qle 'auolef,E lo suourFpe
e,ussaccns fq par€uortJeJj pue J a0? leprce crlecE ?;io "l pe^lossrp
^\ou sr seuoul
-Joq Jo oJnlxrrlr paleildrretd aq1 'stuur8
91 o1 9 r.uor-l seue.\ p1at,( eq1 'pelrp pue
Jaqle qll^\ pogsu,tr sr eleltdtrarcl aq1 '{lat
-alduroc euolul oll eql selelldlcerd luerulearl
slqJ 'reqla utnalorled Jo ltu 00SZ tilll'ruorll pue raqta 1{qte Jo Itu OszI qll/rl lsJU
pelBaJl SI 13€Jlx3 plcE c!103€ eql 'pJSn
Suaq luaalos Jo lu 00t Jo eunlo^ I"lole'prcu crtace iercul8 3o suotlrod ellssoJtns
qtla palJerlxa sI lrnpord lno'pelles aql (lll)'(surerS OV-02lnoqe p1al,{) Psr
-ap.rod pae p?up go paralyg s! aleltdtca.rd
orII 'atuqdlns untuoultu? 3o suutzr8 699
€6
eBuuqcxa-uo
,(q peurulqo
,{louraJlxapus osolllll:sr uidorlo:'eso1n 11ar,(x<
,(1qe1tns Pa
'euolac€ JO
-rdrcerd ueq
HJCV EI!q.
'HS:I.
HITl11 'lS J
'd1aa,tssa.
lunluou
'suollsE
JoJ poa\oll
eqI 'lseu-d:ep reqt
ruo{ pelc
rl,o{ psul
-eu.reqd ip0uorlusru
euos 'Je,,
JO AUOIuJ(
'eJoJoJoqIoJnl€u uI
JOTJOIU€ 3l
Eurlcu ,(1t:
-roq cldo:-oqd,(q aq
-lurs qlIAqcIJ eJ? 1
-u8ard ue
,(gstlus o1
luapnlswt
94
acid when the hormone and other proteins are
adsorbed. After filtration the adsorbate is
extracted with acetone. Many small molecularweight components are removed by this process
leaving the hormone as insoluble residue (about
500 mg per litre of urine). The residue is norv
extracted with 50o/o alcohol (pH 6.0;, when the
hormone goes into solution, leaving behindimpurities. Raising the alcohol concentrationof the extract to 90o/o and lowering the pHto 5.0 precipitates the active material. Thisis again dissolved in water, pH adjusted to5.0 and treated with chloroform to givean
emulsion. Tl-re emulsion is then repeatedly
extracted with water, from rvhich the
hormone can be obtained in a pure form by
precipitation with alcohol or by dialysis.
Approximately 6mg of HCG per litre of urine
will be obtained with an activity of about 6000
unit per mg.
The pregnant mare serum gonadotropin
(PHSG), resembles in activity to the two ante-
rior pituitary hormones LH and FSH. The
extraction of this hormone is best exemplified
by the flow sheet given in the Appendix.
Products obtained from Human Blood
Although blood and some blood products are
included in most of the Pharmacopoeia as a
therapeutic substance, it is still in some ways
different to the other drugs listed in the officiai
monographs. Blood is a unique tissue containing
living cells and a variety of complex consti-tuents. Biood in its entirety or tne several
parts separated from it is used extensively intherapy.
The present century has increasingly used
blood and blood derivatives in medicine and
surgery. We have come a long way from
'bloodJetting' (bleeding and use of leeches),
to that of 'blood transfusion'. The differentcomponents of blood have their own thera-
The Pharmsudent
peutic functions and one means of economising
in the use of human blood consists of separating
the components and using each individually.Mechanical separation of plasma from cellularcomponents is easy but the protein constituents
are separable only by methodical fractionation.The various blood products, official in thePharma-copoeia is given in the table below.
Table 2: List of some blood products officialin British Pharmacopoeia (BP) & United States
Pharmacopoeia (USP).
Product Official in
Anti-A Blood Grouping Serum
Anti-B Blood Grouping SerumAntihemophilic Human Plasma
BIood Platelets (Human)Dried Human FibrinogenDried Human Plasma
Dried Human Serum
Dried Human ThrombinHuman Antihemophilic FactorHuman Normal Immunoslobulin
Inj.Iodinated ( 1 2'r J)HumanAlbumin Inj.Iodinated (1'1I) HumanAlbumin Inj.Immune Huntan Serum
GlobulinNormal Human Serum AlbuminPlasma Protein Faction (Human)
(Hunran Albumin Faction).Red Huqian Blood Cells
Rho (D) Immune Globulin Inj.Whole Human Blood
USP
USPUSP
USPBP, USP
BP
BP
BP, USP
USP
BP
BP
BP
USP
USPBP, USP
BP, USP
USP
BP, USP
Vol. XXI.,l9i
centrifuging is
and cells. The
6 days after ttted human RIhours of prel
bacterial contrplasma.
The R. B.
fragile in natu
side the body'
lation of bloorare kept inact
temperature.
exchange dec
glycolysis pers
Iactic acid re.i,
level. The RIsodium ions ar
tance of the nSince low
cons;derabl,v
easily presen
made to keep
but the tbax-
cells. Howeverol have prov,illL - i.1 rrlr ln
RBC t'r.38of - l*-C. Ipracticable- R
months at - liand a lars :their use. Ge ,
luouil1 '*r<:.solutions c-l gd
CSSS tnking ur*
Ieucoc"rtes art
Though tlcollection, pr,
made it possib
f,or therapy' t
Red Human Blood Cells (Erythrocytes)
Red human blood cells may be prepared at
any time during the dating period of the whole
blood from which it is derived, except that if
'slass0^ poolq
eql ulo{ lB^ouIaJ uo pefoJlsep '{actpaururt ere
s1e1e1e1d leql EopI eql potnJeJ seq '(sreuteluoc
euoorlis ur s1e1a1e1d Jo JolcsJ ,{ltsuep /(ol otl} Jo
eSeluu,rpe 3ur>1e1) Suritgtrluoc qll^\ p0ulqutoJ
ssocord stql 'el€1e3? Iutllclsc Jo unlpos Jo
uorlnios u 3o ,(yluenb 11utus ro/uot;n1os eu11es
ounlo^ eErel e ,{q pelnle uogl aq ,{uru s1e1ete1d
eql, 'poolq oql {uo{ suol "u1€l
e^otueJ ol posn
surser e8ueqcxa-uoll€c oql fq pexg ?J€ os pue
urnrcl€J qyA\ pel?lcossB eJu stalaleld 3ql lEqr
sreedde trI lessel egl tuc{ poolq Jo lE^otuor uO
'paqsrlq€isa 11en elnb s! '(slolc Jo uollceJllroc
pue utlsuldoqruoJgl Jo uoll€turoJ) pootq Jo uoll-ulnEroc eql ur elor Jleql lnq ulslloq4ou Jloql
lnoqe u&\ou{ sl ellllT 'poolq eql Jo luenll}suoc
relnllec 1se1q8r1 pu€ lsellsuls eql eJE s10la1€ld
'Eun1 eq1 uI Pexg rtoddee; Pu€ Jnoq u?
u"ql ssel ut reedslp ,{eql uorsuadsns uI Jo IUPaJO
€ s€ polceiul e.re selfcccnel ueq,t$ lnq p3'\ll-lJoqs
suorlezrreserd eql sr ,(1uo lou 'suoune3aid asaql
go elrdsul 'Joi sl eJnl€JodtDal runullCo agl pue
Ierluesse sI sseuIJEC 'ulalcnucqlJ.ixoep .;o saoe:t
puB olslaJE ulnlpos 3o .(lrtuenb IIsuIs € 'ptle ctq
-Jocse Jo saJell 'ut1sl33 SutweluoJ lnq urumqie
lnoqll.rt sptollol pu" splonEls.(rc luale'roooutllltrt ', Jo an[€A gd e qlrs erpau pr*ds ut
pe,rreserd are iaqr 'auelquaur aql Sutualstotu 3o
,gredord aql scq qJrga\ aurqf,su 1e6n3u1uec uqo3
letceds aql ,{q paleredas are sal,(locnol uag41
'etrBJqluslrr eAtsoqpe u€ ultill/r\
ruszldolord parerp,(q Jlstl] Jo anpr,r .{q apEer;
peiopuoJ are ,{aql esn€ceq qloq e^Josard puu
eleredes 01 llnJglp ere ,(aql 'Je,letto11 'lsaJalul
relncrlred Jo tuagl se>leu salPogllus Jo uollsl
-:odsueJl Pu€ uotlstuJoJ ?ql ut ueql Jo ewos '{q
pe.{z1d tred aqt osle 'setlredord cry(co8uqd rleql
pue seu.izua ul qclJ are sal,(cocnel 'e3e1s 1e1
-uarurradra 3ql Irl [lrls sr s1e1e1e1d pue sa1'(cocnel
Jo uolsnJsuert 391 lsql plBs aq ,{eu lI suosseJ
,{ueu ro1 'sutalord eurseld uI€lJeJ puu s1e1e1e1d
's1iar poolq allq,{ s€ qcns 'uoJlerotralep ptder
96
oSrapun [gaqr F rqil El; ;:-i'-q] roJ
suollcelJ Pcan IEJa!$ g,:l, il,R-c :i epsu
e^€q uorleales3Jd por krc-c -:.nlD3[loc
pooiq roJ spoqlau FlGq: :q- =c.q1 sqqd F =tioorl
'sJnoq o,nl otdn tulrl ssac
-o'td aruosra.qulnJ € sI slql :loJeJ{p 5o suounlos
petn[p ,{lSutsuercur q CgU ogl Sulqs?'n {pnonu
-rluoc ,(q pe^o[ual sq fettr 1orec,(13 egl 'osn naql
01 rolJd '1orecf,18 3o uotgodord e8rel e puz
atrBJlIc Sututeluoc tunlpolu 3 ul 'C, 0Z- w sqluoul
g oldn rog pauesard usoq s€q 3gg 'elqecrlce'rd
.{re,r lou oJE suoulpsoo q3ns q8noql 'Jo6L - Jo
eJnluladuol e a^rlJns ol punoJ ueeq eA€q Jg'l{aql Jo ]dg1 lnoqe 1orec,(13 Jo uollnlos %09B ul 'Sutsttuo.rd elrnb aq o1 pe,rord eABq IoJ
-ac,{13 Sursn slueuuedxe etuos '''e^e,l\oH 'sllec
eqt s,{oJlsep ,(1pnsn ssecord Sullrreg} eq} lnqseJnleredual Ja,^Aol ua^e l€ uroql deel ol epetu
Suraq are sldualle I€JeAeS 'perrrsserd f11see
eje slleJ egl 'uslloqelelu sJe,{ol ,{lqereplsuoc
(3{r9 pue ;' uae.r"laq) sernlereduel ^\ol
eculs
'seseaJJap ,tllunperE allsJqtueu eql Jo ecuel
-srsar aql f,lluant'asuoc J3l?lA PIIB SUOI tuupos
urr8 pue suol {unISS?1od esol SglI otll 'la^ot
1eor8o1ors,(qd ,troleq gd aql secnpoJ pIJB cllcel
E-urllnser pIIB eulll atuos JoJ slstsrad srs,{1oe'(1E
1nq 'sarnleraduel no1 l€ sasueJcap aEueqcxa
snoase8 pu€ elPJ clloq€telu eq1 'ernleradutet
^dol B tc poolq Eutlresard ,{q s,rtlcuut 1de1 ere
f,gd aql puu papp€ sr alBrllc 'poolq Jo uollsl
-nieoc aql luererd oI 'lFcgIIp st dpoq eql epls
-lno uoll€AJesercl slr 'oJoJeJaql 'eJnlsu ur ep8er;
eru '(selcsndro3 poolg peg) 'C 'g 'U aql'euseld
yo uorleredes aqt Eurrnp uoll€quetuoJ IslJelc€q
Jc {slr at{l Jo esn€coq uotleredard Jo sJnoq
lz o1 zl urqtr,\^ pasn eq lsnur c8l{ u€unq pai
-EJluecsoc oql 'u^!BJp sI poolq egtr Jeue s'(up 9
ueql rel?l lou ap€Iu s1 uotlrederd eqJ,'sll3c pu"
euseld go uotlerudas eql JoJ pesn sl Sut8n3trluac
t86l ''IXX'loA
JI l3{} tdarxlaloq,t\ sgl Jo
1e paredard,
(sa:
dso'dgdsn
dsn'dg
dsn'dgdsn
dsn
ds
dg
dsdsn
dsn'dgdsdiI
dsn'dsdsndsndsndso
ut l€trlgo
sel?ls pallu
I€IcIUo s]31
'a\olaq
eql ur l€l:'uorlBuorlcE
sluenulsuo.J€lnller ruol'[llenptirtpoSurleredas g
Sutstuouocr
lUapnsutloql
96
Plasma anil Plasma Fractions
Vlhole Dried Plasn'r,t
Plasma proteins are more stable than the
blood ccll and so plasma is aspirated from the
sedimented red cells of time expired whole
blood. The plasma from some donors contain
haemagglunating antibodies of the ABO system
which upon transfusion leads to intravascularagglutination and haemolysis. This risk can be
avoided by neutralising the haemagglutins withthe soluble blood group substances present in
the plasma of other donors of appropriate bloodgroups.
The plasnra fronr time expired human
c;trated blood is collected into l0 donor pools,
constituted so that to each of the 9 bottles ofGroup A, I bottle of Group B or AB is included
to remove isoantibodies. It is then siphonedinto transfusion bottles in 400m1 quantities.
These minute bottles are stored at 2aC and thenfrozen by spinning them on a vertical aris atabout 1,000 rpm at -25tlc in a current of airfrom a fan, In this way a cone is forced dowathrorigh the liquid spreading the pla.sma evenlyaround the inside periphery' of the bcttle where
it is supercooled and thus causing ti.e lieezrngof piasma. It is then drieci irom lhe liczet statefirstiy bv vacuunl and linaliy over phosphoiiispeutoxiclc. f iie plasma v,,ilicir *'culd othcrrvrseh:rve beeit il;,iii for use rnay be pr.eserved irr ihedri:d stiic anii i"rsid afrcr recolstitutiug witirpyr()gen-iree distil led rvatc-''.
Litltrid Pissnrii
The preservatici of liquid plasma and serunrhas presented manv difficulties. largely Cue icrtlie fact that these liquids are iiorntally ratheropaque and tend to form a sedinient on standilgthus making it iinpossible to distinguish sierrieand non-sterile material. Two methods of trear_nlent a!'c available : Firstiy, the plasma may bedried by lyophilization soon after collection; the
' The Pharmstudent
second method is bi' filtration through anaseptic filter unit. The latter pro!-ess suffers from,the disadvantage cf activation of plasma pro-thrombin by the magnesium of the asbestos.filter pad and the subsequent conversion offibrinogen to fibrin, causing clots and shreds toappearin the filtered plasma. Prior alkalinisationof the plasma and later adjusting the pH withCO, after filtration was not satisfactory as
reported by Bushlel and Whitley.
Several otber methods are available forremoving fibrinogen and prothrombin fromplasma :
(a) Clegg and Dible prepared a transfusionmaterial by recalcifying citrated plasma.
The clots were broken by shaking withglass beads and the plasma sterilised bypassing through a seitz filter.
(b) Reid and Bick prepared serum by therecalcification of centrifuged plasma.
(c) Maizels de:cribed elaborately a process forremoving fibrinogen by adsorption onKaolin, the process being quite lengthyand also because the process lowers theprotein content it is not very much in use.
(d) Gardiner anci McFarlane independentiyshonc.l that alcohol and ether when used
ili lc\r.' tempelatures are one of the bestagents te.r obtaining clear plasu:a andssiuni l:.iviog good keepin_e qualities.
Fracf ionation of PlasmaInciividuai protein components of the piesma
can be rr-'parated from each other and also pre-parcd in concentrated lorm by means of preci-
pitatic'n. Precipitation is brought about brin-girlg iirto use the tbllowing properties ofploteins :
(i.1 miuimuin solubility at the isoelcctric poii:t(ii) selective precipitation without der:aturation
by the acldition of organic solvents likediethyiether and ethanol.
Vol. XXI,798l
(iii) alterationvariation ilmethod.
The chief 1
namely, albumirated from the I
using concentriThe salting-outelective and inaration of bloo,poses. It rvas I
dated by Cohnfractions u'ere
narnely, albumi:cess methodirxiiassociates at H;the plasma x'hirthe electrophc'lToday a methoby Cohn et al is
centres in the *The develol
methods of pla
his co-n'orkenneed for a procrtrated human a
-iu:tilg the iveLLJlSg_:!!Lr
ccEcest=I€E;ioss froe trr:given in Tabxcjected tc' i--,*:the compciticre
l3
€I
'alJlu€ slql Jo uollsJ?deJd eql uI ocu€plnE
pue dleg slq JoJ ',{ltsrallun npulH sereueg
'fEolouqcal Jo elnll]strI 'sJllneceuJeqd Jo
luewpedeq 'rarn1ce1 'l1pue6 ') 't 'rI I {u€qlol seqsr.t Joqtnu eql : luawa8paluoutlcy
'xtpuoddY
eql uf poluesard sI ssecord oq] Jo oullno uB
EurrrlS eueqcs V 'uolleuollJ?{ euseld JoJ Joqle
Eursn 3o ,{lrpqrssod eq1 eleEtlse^ul ol sra{Jo^l
-oc s;q pus {cl,rua) pal zulsqd ptnbg 3o
uoqurederd eql JoJ sa:nleradual A\ol ls Jegle
Eulsn go eIIelJedcI l ,(q uotlerlsuouap aql
't olq"I ut pel?Jaunuo s€ suolllsoduroc eql
poa\oqs uoll€uttuexe cllaroqdorlcala ol pelcof
-qns uega\ strollJeU oseql 'g s1qe1 ut uszr€
se 'euseld uEIrInq pelood I€IrrJou {uo{ suol
-lc?{ urBtu er.g parederd uqo3 'UOtleJlueJuoc
uralord'ernle:odurel'en1el 11d'uotlzlluacuocIorreqla 'ql3uer1s JIuoI-selqBIJe^ e^g eqt Sultsnf
-p€,{g 'uoIsnJSIIsJ} JoJ ultunqls ueung po}"Jl
-uecuoc Jo uotlcnpo.rd eqt JoJ ssecord 3 JoJ peau
eurll-J€,t eql tuo{ peleulStro sralro'u-oo slg
pue ugo3 {q uot1euo1lcer; eurseld Jo spoqlaqr
Ioqorle 1{qta aql Jo lueudolerrep eq1
'plro.,\A. 3r{1 u! seJlueJ
solsnJsueJl poolq lsotu uI pasn sI l" le uqo3 '{qpadola.rap leql ol J€iIttrIs ,{ra,r poqleur e '{epo1'sruauoduroc palerudes,{llelleroqdoJlcele eql
qtti\ Istlluapr fllellrurd ora'/'r qclql\ euseld eql
uroq ssollc€r; paleredos 'preare11 1€ s0lelcosse
srq ptr€ uqo3 ,(q lno pe{ro^\ ,{11uc1poq1eur ssec
-ord y 'sur1nqo13 I pue t' 'a 'utunqlu "(leuleu
'stsaroqdorlcole uo paleredes eJelA suollc?{
uralord-euseld rno; l"ql 'l€ 1o uqoS '(q pelep
-rJnla poqlaur ogl o1 JolJd pllnoJ s?^A lI 'sesod
-rnd crlnad"Joql JoJ sutelord-poolq Jo uolleJ
-edas aqt ol acrlcsrd ut elqecrldd?sl pus elllcole
-sqn oq ol PunoJ se,u. ssacord 1no-3u111es eq1
'slles leJlneu Jo sllss plc" pel€Jlueluoc Sursn
poqleu 1no-3ut11es eqt fg euseld eql tuor3 pe}€J
-edas lsrg aJOA\ sutlnqolS pue ultunqle'Xlaueu
'poolq Jo sluenlllsuoc uralord Jelqc oqtr
'poqlour
.lno'3u111es,'sotlsJluetuoc lles ut uollaIJBA
ol enp sutalord 3o ,{1r1rqn1os 3o uorleralle (11)
186I,IXX'IOA
f{
AIII SIUA^I
uorlBJnlB.l.
lurod crt]:r
3o sarlrrCc
-urrq lnoq-trald ;o -ct
-ard osle p
e'"ust1d aqt
lc .,
puB e iusE
rssq aq! lposn caq.\.'
,{ltuepuada
'osn ur q3
el{l SJA.\\Ol
,{q13ua1 at
uo uortd:JO] SSelOJ.
3I!eqt ,(q ut
f,q pesltr:q1t,n Eur4
'uruseld p
uolsnJSrr?J
IIIoq ulqr
roJ elqBll
sE ,{rolctqtYn gd a
uorl"slurltol spoJrls
JO UOISJSt
'solssqsE :
-o.rd eus.[uo{ sJeg
{I€ qEno
luaprysut
€'0g'0
z'0I'€
9'0 v'90'62 0'l
9'7, E ?
s'0 9'98'0 g',1
z'o ?'8
z'o z'tE
uaSoui:qt3urtnqol9-ruqnqolc-guIInqol9-D
urunqlv
9'I0'9z'98'iL'0
IAAI III .tIBrrrsEld uollJ€JC
'eusqd 3o er1r1/urelord;o uBur suollcEJd EItrsBId go uorllsoduroJ : t alqvJ.
l I'060'060'0
tl'0
s-9-s-t-
0v0t97,
8
8't A8'9 AI8'9 III.IIV'L I
uollBJluecuoJcruol 36'dura1 /oloqoclv 11d uotlcerg
IoqsJIV,{q uo lleuolpergEtuseld JoJ s[tolllpuoJ : € olq?I
L6
The Pharmstudent Vol. yyJ,1,
50o/o a
Add Hcl
50o,/o aa
Add a
7oo;/o a4.1 a
R"dirso;
l0o/o acr
Add acet<
chill
50o/o ace
Dilute to 2.
add acetc
Precipitatdry with
261mg;tot
98
FJ6
t)cG
o()
(A
a>' ti -:
^OiY---J-66:E.;sH .S-dB HiBE ,i'i
E -H 3€ 6-.;kir o;'5; 5{
IOd
qi
c_:-^cga
\e
t--
>llc)
,d-- .i:-lqulgh l-A
Aa
Eo, E,EA E= q.qF -gE -i \!n! ==-o 6.n G {
-6; -?: -i---: E€Rii P= ! " €s-tH fiE' ; d\iFg €!E S S
!;€ E=; nHsE de,E $=
*Ec : = S i=B'g -F s E $_E;F Ei B E ", EEtgt
=e ;' cq t ".'
EUE gd; E o s s-:rj:.c iin J A t sE
- t; g -g f,i - ii ","--E -- S 3E;: -eE: 'F; ? {Fge Eri g€ B" ! ii*f,EE 3EE f,x 3' i iE
9p
Nfra-ra
-ho
rFo-c.t
.:.n to.F-iEY*FOe
.5 S=r \ .s.Y
-r - Pcr
5 b -E€!br
ps$=Fl F< ca
c)oo
oxrr)
>|
()
()
(J
A: € u s d* E-sEs *i i ;Er E;" EE€'-, 3E -A: i -3, --€E s -;,
EE --5 A:-
=:E .iEE E:__Eii-EEE_-EE*-
E"E* iEt FE Rs :EE X; s[ i* frEE ;EE
f. I'E sFa.-, . -=i *rE i6E $.,.€= E!F rS\
-;--.4n---i>(.)G)u: 9 >+G .i\t 3 FqE iF -\ HE
-.10k(.;).,
-l€>kar o-P.C)Fao';€F>O:F.sQ(6-!Eh=cdPv>{.)
kP()o
O
C)Ifr
.:tvt3FA*v
.()cE!htrUc)E3o3O!dA
6{) ._LaOAdv
BooadF Y?'EQ A.e
4 NRl-X Xgrq E€>r.=& t[iH 6H
.xig e:E x li EEav ! c6-
iL < F tr q.:-d, o ::<,:jlE-+iJi x og(Eo € E Llca r F-=rffi 9iE3
H AigE- - ts'6 ao-<'= F
'* av:..99\=6 ><
>.4- v
NOEUcqEii.Ss**9r-3 dtrd9rFU:/q)UtsELd:: S S>\{\
l-
I
t
sllun 009 isprl0s lEg'11
lueleuradns auolecv
slrungg91-sprlos i39g'7ll Hd 1'1dd euolecy
slron 969'961 'N lutol i?w 197:Jeqlo T [ouel{la qt1,tt' ,(rP
iu 1t1 d o.t 7 o po uog-a1e1tdtcar4
i
i
iI
l
II
I
tD,, -t
(r>J-\-GOl
=.SFESco N. C.f\
{1.+
3: q g:-od
- S.- --\. a
ns 5=x ' d,{ Ita
t\)
G5
--(Jl
E=oa
eEnJlrluoc
T lllqr :o/oSg otouolec€ ppe
euol33? %os qrp 1 L'z or alnucI
I
I
9 Hd :uollnloseuotecB %09
I
eEnguluec T IIIqc:9 11d:o/sOg ot euolace pPV
I e,rrlceul i8gg.glelqnlosur puolocp %0t
I
slrun 0001 'sprlos ,f973
llueleuradns auolace o,!g1I
'uorlnlos ol ouolocE %01
eolor? o/oOt yl o^lossrpallI
slrun 666'611 i3g'7y
9 gd !'rdd ouonre o/o1L
stlun 00gluralord Egy:9 gditdd auolacu o/ogg
o/oOt ot euolec€ ppvI
9Hdllueleuredns euolecu o/ogs
$run 0009 'uralord3E961 :p'1 gd
:.ldd auoterB %0SI
eAnJlJluet'sJq 9I roJ IItgJ i9 gd o1 ICH ppv
I
slueleuredns ouolece 0/o69
I
v,
o(!
rya
o
@
I
'alelydrcard lc?Jlxe-eJ ry eEn3rrlue3'srq 9I roJ plor ur puels lauolocE.I S'91 rlll&\ xlhl
'sirun 969'611 :T S'91 lV'ttld !euse14 poolg pelerlJulqdo4opuuog urues ere141 luuu8arg Jo rrolleJgrrnd
6t86t'IXX'101t Dpw,cwJD
100 The Pharmstudent Vol. XXL
8.
9.
10.
ll.12.
13.
t4.
t7.
18.
19.
Fractionation of Plasma with EtherPlasma
,tAdd Ether (llo/d at Ooc & pH 1.i._l
Supernntant I
Add Ether (l0o/o)at Ooc & pH 5.35
Precipitate(Fibrinogen)
Supernatant 2
.t
Add Ether (18.5%) at-3.50c & PH 5.5
& ionic conc. 0.035
IPrecipitate
(Prothrombin)
SuPernatant 3
Itv
Add Ether (l2o/d & Alcohol (16.50/0)
at-5oc & pH 5
.lJ
PreciPitate
JPurifiediFreeze Dried
Albumin
IPrecipitate
(Globulins).J
Add Ether (9ofi at Ooc.
pH 5 & ionic conc. 0.01II
ISupernatant
0Add Ether 1t8.sli,)at-3.50c, pH 5.75
& ionic conc. 0.025.,t
Precipitate(z-globultns).
Bibliography
l. Abraham Mazur & Benjamin Harrow (1971): Text Book of Biochemisfty l0th Edn.
Saunders.
2. Biggs, R. (Ed.) (1975) : Hulnan Blood Coagulqtiotr, Haemostasis & Thrombosrs. 2nd Edn.
Oxford : Black well.
3. British Pharmacopocia (1958, 1973) : British Pharmaceutical Press, London.
4. Blumberg, B. S., Sutnick, A. I. & London, w. T. (1969) : J. Am. Med. Ass. 207, 1995.
5. Cartland, F. G. & Nelson, J' W. (1937): J. Biol. Chem. 119,59.
6. Cboh Hao Li (1951) : ,Hormones : Anterior Pituitary', in Enc),clopedia of Chemical
Technology, Ed. Kirk, R. E. & Othmer, D. F. The Interscience Encyclopedia Inc. New
York.7. Cohn, E. J., Oncley, J. L., Strong, L. E., Hughes, W. L. & Armstrong (1944): J. Clin
Invest. 23, 417.
IPrecipitate(p-globulins)
15.
16.
20.
:1.
::.
;
I
Ii
11.
21.
25.
26.
27.
28.
29.
.l
I
P
i
5
l1
{
\
'roErqad T ee1'(3o1o1owaop p)!u!D : Qge)'14 'eqorqlutl6' oraodocowtoqtr salus Pallu n
.Qtei s1uog poolg lo uollolcossy Ltautawv aqt lo satnpnord T spoqQW lDrluqoal
'sJeqsrlqnd >ltel{' up3 qts I' s a cu a Iz S' lo il ! na zo ut t o q 4 s,uo t S ulw ay
ul 's.EnJq cr3o1o1rrua11 p se1,{1o.r1co1g 'splnlC (Poolg, : (Sfe t) 'W'C 'loue8rng
'I 'ro^ sulato.td uDLunH lo t.ololg lolnralow: (ggot)'g'1 'suuureri#tl$;?;;ff;'tIg '09 'rlsel[ 'rrs 'pmv 'u"u 'cord : (gsOt) 'ni 'V 'ecurl4
'llrH ^^€rgcl I
"l€I 'upg qryl'tttsluraqS pa1Eo1o1s,tt14 s,>1uo11 : (SqOt) '1 'g 'resg
'WS 'lL'cos 'tuoqJ 'ruv '{
: GVA) ';A1 '4 'ssorg T 'Ayf 'uoJow€J "V 'O 'lraqcry ''W 'uIcIAI "1 '1 'felcug
'ELZ'Z 'r€alrsgd 'urrsqd 'f : (0S6I) 'tr I'oou?N
'ltg'Z 'cBIuJ"qd 'urr"qd 'f : (0961) 'W 'D 'ppol4' 'U 'D'oullry
'[[ear{cqg
! progx6 'upg tttg 'aulclpary p?lullJ u1 uopntsuory poolq : (7,16) "I 'd 'uoslllol I
'LZ'01'llng 'pen 'lpg : (p9611 '1 '6 'uostJ1o1A1
'uopuoT 'llepull oJotII€g '('pg) 'V 'E 'sut1,t'rug 'upg ql8 's4|nacow'rot14 fo tloog
7xa1 s,tapua€r uI '.selnlpsqns €tnseld T slcnpoJd poolg' :Qt6t) 'V,p'I'4coc'{e141
'{JoA ^leN
'cul
apadolc,{cug truelcsrelul aql 'J 'O 'ranqlg ?'g 'U '{rty 'pg'(Eoloutlral ID4waqJ
lo olpailolc|ntg ar. '.xasT leoluoc-ouerpv : sauou:o11, :(1961) 'A'H'fure1pq4 ceyq
'uopuo'I '982'oN sauas
Ttoday plcadg llzunoJ q?nasaa Pqpan: (lsot) 'g ';4 'delcehl ilv 'u '{cl^d{ex
'{ro; ,^aeN 'cu1 erpedopfcug ecuensrelul
oq1, 'g'g 'rauqlo T 'A'U '{rl) 'pg 'r6o1out7tal P)nuaq) lo olpadolctcug tr
.snoeuelllJsrl4l .pro:fqlere4 'f:eltnlr4 JolJolsod ',{azrrn5 : sauoluJoH, : (tEOt) '6 'urwe;1
'Ll;'01'npg'l3S'ssv'tsr€Ild'reIrIV'f : (lS6I)'C'J uosutlof T'H'f 'IulH'uolllpg u€Isv uaznrel l 'spa ql9I
't.t7s1mat131oc13o1o1s{t14 lo uatday : (UAi 'y'6'sa,{ep1T 'N'A'llo'lrpo1 "V 'H'redru11 '('l
'6W'Zz 'l?ua€H 'f 'l1rfl : (ZtA1|r'g '3 'ezztg T 'g 'ila,ttplg "U '1ty9 'e{lq 'lI'uo!llpll leuoIlBIr
-:a1u1 ,{1e1r1tr 'up3 pr€ tulsyuatlcorg lo sa4flno : (Ztel)'Y '4 tdunls ?8 'iI 'A 'uqoC
'gzl'E etluetradxg : (ne}'[ 'A 'uqoC
'6gV 'gg'cos-rueg3 'urv 'f : @Vei ''I 'H 'rolf€I 4'
hl ,ulleni ,'N 'f 'ql:oarqsv "I'g'p.lo31nry ''T'ry1'seq8n11 ''g '1 'Euorls ''f 'A'uqo3
t86t'IXX 'lo/
'67,
AL
,LZ
'9(,
u[3 'f : (tt6
&roN 'JuI Blpl
ptpuat13 lo
's6il 'l0z 's
'upg puz'.ils(
'upg q41 L
(surlnqolE-$
alotldlcat4I
I0'0'co0
tuapwsutD
'se
'ie'€z
i
I
I
+
'Ll
'91
'91
L'
'tc'02
'61
xt
'vl'€l
'0I,6
'8
I0l