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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.

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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,

;

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90

equent extractions may be required. Tbe extract

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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

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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

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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

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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

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100 The Pharmstudent Vol. XXL

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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

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at-5oc & pH 5

.lJ

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JPurifiediFreeze Dried

Albumin

IPrecipitate

(Globulins).J

Add Ether (9ofi at Ooc.

pH 5 & ionic conc. 0.01II

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& 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.

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21.

25.

26.

27.

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29.

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