CA2002155A1 - Process for the isolation of purified gangliosides from ganglioside-containing living tissue - Google Patents

Abstract

ABSTRACT

The invention relates to a process for the isolation and purification from ganglioside-containing living tissue of gangliosides having a salic acid content of more than 30% by weight which comprises dehydrating macerated tissue, extracting the dehydrated tissue as a powder with a chloroform-methanol mixture, extracting the chloroform-methanol fraction with saline, separating from the extract thus obtained a methanol-saline fraction, loading said methanol-saline fraction on to a column filled with silica gel having non-polar functional groups, i.e. reverse-phase silica gel, packed within a solvent, washing said column with an organic solvent, eluting the de-sired gangliosides from said column employing organic solvents of different polarities and recovering the purified ganglio-sides from said organic solvents.

Description

5~

The pr~ ?nt inverltiQn relL-~tc~ tu ~-1n impr~vt~d prot ec~ f~r th~
isolatiorl and pLIri-FicL3tion fr~m :livlng tiss~le uf: g~n~ qid~?r3.

~ `c~n~lioL~ideL~ ~re ~ l~;tu Qf ~lyc~-~phins~c~lipic:l~ whit-h were first dicqcov~red by EA t~lenk in 1'~ le fc~LInd g~ngliosides L~nly in br~in tis~C.3~.e b~lt c~th~?r inv~stitaat~r~.q h~3vr? 3~qince fft~qtL~blifihf?d their e~:istenc e in th~ c e:ll memhr ane~ c~f all c~tht~r itis~L~L~q7 vi~.
the spleen, liver, nc~ rt~nal PIL~m~ membl-ane~a1 kiclneys~ ~nd ~ n~
Garl91iO5i d~ ar-e definecl by the pi-esenr;e L~l: an a~idic ~ c~e,r rall~d siali~ ac id in their t_i~rbohydrat~ t-hain~ ert~in men)br~anes r~f nE~rve cell~ are particLIl~rly rlr-h in ç~c,lnglio~idu~r;q wht E;e c:arbt~hyclrate chclin~ int l-lde mt.~re than C~rle kind t~f laialit acid. The p~tt~r n thL~lr d:~cqtr:ibL~tion v~ lL~3 1~r~om C~
~nc~ from 5pet_ie~a ttr ~peci~ a~3 ~c~mio `Y~makawa t~ Univer~ity c~
TO~YL~ gestecl in 195:2.

The mr~lecL~lar strc~ctL~re of ~anglio~ide~ hea~ been knr~wn for A
lon~ time. ~ach mole~clle ie~ shaped ~omewh~t like th~ letter L, one 1~9 c~t the L being f~rmed by ~ c~rbohydr~te chain ~r chain ~f ~imple s~lgar- re~id~le and the ~tht~r l~ by a lipid alled c eramide. The twr~ leg6 o~ the I thLl~ form tw~ di~tinct compuntant~. The car ~hy~r~te chairl i~ hydr~philic and it interacts with the w~tery mediLIm ~clrrQL~rlding the cell. The r~cond ~np4nent, i. h'~ the f~t ~ lble ceramid~ i~ hydrophobi~
~nd ~ it tends tc~ e~rlclde water molec~ e~ ne~r-byJ

Ther~ n~id~r~blt2 ~riati~rl ~ tr-LIt~lr~ betw~n the cer~mide ~nd hydr-Dcar~on chain and abo~lt 130 vari~tie~ c~f ~lyc~phing~lipid~ are now ~nown. ~uut fQrty u~ th~m c~r-e '.?

clas3i~ied into the ~o c~l:led g~1nt.~1 io ~t:r~lr ture, te~n ~r e r las~ ied intr.~ the fJlobo ~frLIctLlr-r~ hnd ~ ty ar~ c1~f~ ied into t~e 5C~ Ci~llf~CI ~r~f~lir~ ~tr~ f ~ rf~, f~f~pf~nclirlcJ r.~r~ thf* ~f~qLlf~tlt~f~ f~F tt~
s~lgarC foLInLl in the ~c~re of thf-~ r~a~bQhydrat~ chains and the n~t~lr~e r.~F th~ chf-~mi r ~1 1 .ink~ge~ br;t~er~n thf* ~ur~ars~. The remai ni ng twenty 91 ycol i pi d~ are nr~t. f30 cl ar~ i r~r~ .

Thf* pre~3ence of g~.lngliof;irle3 in ~b~lndilnce hil~ been e~3tablir3hecl in the rJr-ey ~nd ~-Jhi te matter o-F bovinf~ brclin~ sp:lr~en and li~f~r, human brain~ ne-lronal plas~ma membran~ nd the like~
S~ch ganglio~3ifies po~e3~ ~ nL~mber r~f applic2tion~ in the ~if-~ldf3 oF medical ancl biochemi~el sciences an~ in the~ c~sm~tic~
indLIstr-y~ For this rea23c)n~ it ha long b~en e~n DbJectivr~ of researcher~ to de~velop new and mor~ efficir*nt' m~th~d~ r th~
preparati on o~ br~th s~lbst~nti ~11 y pL~re ganc~l i r~!3i dr~ a~ wel 1 a~
tho~e possesC~ai ng a hi~h s;iali ~cicl content~

~ nLImber r~f meth~ds for the i~ol~tion ~nd purific~tior) of ganglio~ides fro~ livin~ tir~s~le have b~erl de~cr-ibed in the ~rt.
Some of the~e meth~ds ~re refer-red to hereaft~r by way of reference.

One method fDr the e~:tr~ction ~nd p~lriflratiDn of gangliosiderj from bovine hrain~ wa5 developed by F~l~h-Pi et ~1 (J. E1iol. Cllen~ ?~ 4977 1957~ and wa~ perferted by S~lz~lki, K
~J~ ~e~lrochem. 7 127 ~7 1~5~ 7his method involved the e~traction wf minced bo~ine br-~in with chlDrofDr-m-lrl~th~nol~
~hloro~orm-m~th~nol e~:tr~ct wa~ then 5~lb jected to Folch partition with saline~ The rneth~l)DI Ralille fr~ction w~ls separ~ted, . .

r ~nCentr~tE^d al1~ di~1Y~CI aCJC-tinSt di~illed w~ter at 4~C, ~nci th~
aialy~ed s;c~mple wa~ lyophili~d to c~tain cr~e c3anc31ic)~i Cl~5 i n powd~r form~ The c~ntent of ~ lic acid in th~ crud~ g~nglio3ide pr~parstion wa~ foL~nd to be 7% t~ . by w~i~ht and th~ cun~nt ~f neutral lipids, proteirl~ anli 1QW mQleC~ r weiyht imp~ritie~

w~ m-~ch higher-.

~ nDther prior art m~tho~ i-3 th~ ~ne cl~scrib~d by L~ 5venr~erholm ~cta ~hrmic~ ~c~and~, 17, ~9~ . Thi~ m~thu~
~ims ~t th~ furth~r- p~u-ificatlon Df the crude pr~paratic~n of g~ng}io~id~ obt~ined by thca SL~2~Ci m~thod ~e~rib~d abov~. The Svennerholm method involved the c~ mn chrum~togr~phy Df the ~rud~ q~n~lic~ici~ prup~r-b~tion un ~ ic ~cid ~lpp~rt. ~r-~
g~nglio~ide~ were di~solved in a chlc~r-oform-methanol mi~tL~re and appli~d on to a silicic acid ~ul~mn pack~d in ~hloro~orm-methanol ~5:5~. ElLltiion oF ~n~lic~ides wa~ achi~ved hy lncre~cï~ing t~
c~no~r.tration cF l~e~hanol in th~ hlor~fortn-m~thanol mi~tLIre.
The ~ontent of Qi~iC ~cid in the purified ganglio~ide~
preparation wa~ found to ~e 15% to ~O~f. by weight and the con~ent Df DthF~r impLIrities wa~ lower than that obtained by Su~u~ci's method.

Later Dn~ ~ever~al Dther chrotllatographic ~upport~, vi~.
ana~il, flori~il, al-~mina~ iatro~eads ~n~ uni~il were prQpo~ed f~r the purific~tion ~cJ ~Jan~llo~id~. Eurth~rmnr~ kin~3 ~dvantage o~ th~ pre~ence ~ ~ninnic ~ruup~ in g~nglio~ide~
~v~r~l anion e~changer~, such a5 DE~E~-~eph~d~, DE~E-cellulose~
TE~E-~llul~ E~E-eilic~ E~E-CP~ and ~o onl were ~ o L~d f~r th~ purificati~n of g~n91i~ide~ How~ver~ the low 2~2~ 55 ~apacity an~ difficLIltles in re~er)~-ra-tiul-, of i~n-~chHn~t~ col~lmns re3tritet~ their u~e in pr~od~lcin~ pur-ifie~ g~n~1ir~itd~

Gangli~ e~ puri~fied by the priorA art methods ref~rred t~
ab~ve ~wffer from the drawback in that they pos~e~ lic acirJ
content in the r~n~e Df no more th~n lS% to r~O% by weight.
Moreover, all the reported prior art meth~ds require3dic~lysi~ c~
the gangliD~ide fra~tion~ ~bt~ined ~fter Folch partitiot7 of the ~hloroform-methanol e~ttr~t ~f living ti~u~ wed by concentration, ~lrther dialy~is and ~t le~st twrJ ch~m~tQrdr~phic step~ in ~rd~r ~ ~bt~in ganglio3ide~ h~ving a slalic ~cid contQnt nf 1uSt abQut i5~: to 2C!~. by weight~ A~ a rr~L~lt of the~e ~ditional, ne~t~s~ary C~tep~, the prior ~rt m~thod~ b~com~
e~tremr~ly cumberssom0 ~nd tim~ con~L~min~.

It is th~refore ~ primary object of thr~ ir-vention tc providræ
an improved proce~a~ for the manuf~tur~? ~rD~n ve~rinu~i living ti~ Uy containing ~anglic~ ec~ Qf p~ri~i~d ~an~lio~id~ corlt~ining n~t 1 ess than ;;C~% by ~E~i ght of ~i al i c aci d ~nd i n whi ch the content of i mpur i t i ~ 91~ e~t 1 y reduc ed ~

~ rnore ~pecific ob~ert of the lnvention rE~ le~ in the proYi ~ on of ~uch an i mproved proce~ f or- the manuf act~r~ of puri f i ed galngl i r~&~i des con t~i ni ng not 1 es~ than 30% by wei ght uf ~ialic acid in which khe kedic~ t ~ ma~ cc~r~ ming ~tep2~ uf clialy~is and concentr~tion are not invulved.

In tr~ditic~n~l ~d~ rption chromatography perfl~r me?d on a pol6~r ~,tationary ph~e ~3ilica ge~ norl--pol~r mol~ pha~e i~

2~12~55 ,ed. In ad~urpitiQn chrnm~togr~phy, polar ~olLct~ L~re tightly adsor-bed to the polar C~t~tionary pha~e and car~ ther~ef~r~ eluted 1 ater than non-pol ar 9;01 Llte~ ' compoL~nd~ c:~n b~ t~ f r~n~
th~ ~tæt~on~ry phas~e by increa~ing the p~larity of the mobil~
phase. In rever L~e pha~e ~hrc~mato~raphy, ~ implied, the prtc:e~
ia~ rever~ed. Th~ s~tationary ph~ non-pc~l~r ~nd the mobil~
pha~e i :~ pol ar . Now i t i '~ the nrln-pol ar or I i pc)phi 1 i ;:
interaction~ which determine the migratior- vigl~l:ity alc~n~ the ~tati~n~ry ph~se, ~nd a~ one ~;peCtB, pc~lar L~lutes ~re nuw eluted earlier frQm the! culclmr- in prE?f~r~nc~ tc~ nc)n~p~lhr 5!~1LIteE~ E1LltiOn o-f ~ partir ~llar compDund in thi~ e isa rlOW
effected by reducing the pul~rity of the m~bi1e pha~ u~ually by the additi Dn of or yanic solver~t~. .

Th~? applicanl:s hav~ c3b~Yrved th~t th~ ~ilanol functi~ncalitiej pr ent in ailica gel hre~ r-e~ponYiibls fur the~
polar natur~ of the ~ilica 5el matri~ th~e pc~lar~
functionalitie~ are ~ul~stituted with non-pc31~r grr~p~ thE?n ~he silic~ gel matri~: will bf~com~ n~n-pol~. Th~ applicarlt~ h~v~
f ound that the use ~ B non-pol ar si l i c~ 9~l ~r-E?vers30d phaoed silica g~ll column with a meth~nol-s~line fr~ctiQn ubt~ine~d ~ftei!r Fs:lch p~rtition o-f ~ chlr~rr~fDrm-meth~l701 el tr~ct of ~c~ton~?
powster o~ living ti~sueç3 can aYoid the mo~t time coneiumin~ ~nc:l 1 abouri ous; step~ of di al y~i ~ and cDncentrati r~n i n ysn~71 i o~i d 3?
pUri~iCAtiOn. tvloreover, a ~ingle r~ver~ed phase chrDm~togr~phic st:E~p rr~sul~ in hirdh pur~lty ~an~liL7~idQ~ ar3~ very much rcduce~
th~ cor3tent of impw- itl~.

.

O

2~

E~ssE~d on thi'~ discc~veryl th~ c-~pplicantc3 have~ dev~l~p~d procr ~s for the prQvl~ n of high p~lr i ty 9angliQ~iide~ high in c~ialic ~cid cont~nt and low in n~n~lipid ~nd non~ lic ~cid lipid content from 1ivincJ ti~L~L~eLa ~r~h as bDvin~? br ~in, human brain and the like employing e~ilica c~el h~vir-~3 non~polar f L~ncti c~nal groups~ ag ~ s~lppor t i n the cDl ~mn ~Dr the s puri ~i c6ltl onof pho~pholipi d~, cerebro23id~, nucleoside der ivative~-.3 arld th~
li~te. M~reove?r, 3Llch prur ess permitct the prep.arEItion o~ r-ge t~atche~ o~ ganglios;ide~ by employinci batch c3p-3ratic~n u~in~3 the 5ai d si 1 i ca ~el ~

Pccordingly, the prE~e?nt invention providE~-a a prr~ce-as for the isolation and p~rification frQm yarlglio-~;id~-c:~ntæ,inin~i livlns3 t~ss~;e c~ 3angl jDsiidE~L~ hæving a ~alic acid content o~ m~re than 3~. by w~ ht whi~:h compr-ic~e~ de~hydr-atin~ m2~c~rat~ecl ti~
eY~tr~lctinci thE~ ciehydrcatel ti 55LI~ as a powd~!r with a chl~roform-m~thanol mi~:t~r~, extracting the ~hlorof~rm-methanQI fr~ction with saline, ~par hting frum the ~tr~r~t ~hu~ o~tain~d msthanol-3aline fr~ction, lo~din~ ~aid meth~nol-~aline fraction on to ~ cDl~lmn filled with silica gel h~ving non-pol~r functi~n~l group~ reverse?-pha~ ~3ilica gel, pac~tls?d within a sc~lv~nt, w~shing ~aid r olumr) with an org~nir.: solvent, ~lLIting th~ rJeired gdngl i osi de~ from ~al d ~ol umn employing ~rg~ni~ ~olvent~ of di f~erent polarities c-lnd recovering the p~ri fied gangliosid~s 4rom ~aid or~nic solvents~ .

The living ti~.sue employed in the procr~ f the invention ~n b~ c~el~ct~d from ~Dvine br~in, human br~in, human 2~ 5 ~r- ~thr-c~ tl~13, I)C ~ p~ n~t~r ~ ?~ J t~r~ Jr dehyclr-atinrJ the mlrer~t~d tis~LIe~ the prr ferrecl clehyrJI atil-lcJ ~Igent i ~ acetone~

Prefer2.bly, the tiei~L~e elllpluye~d i~ firr~t of L~ll wa~her;l Wi th culd watr~r ~o r emove e :traneo~ erlti t:ies~ ci~ch a3 bl~r~rJ hefc~re~
b ei n c~ mac er ~ t ~d .

~ hr- r~~tic~ of Lhl t~r of orm to ll7eth~lnrl1 in thr chlor-c)form-methanol mi~: ture r ~n YaY-y fl"OII) ~ tU 1 : 2.

Thr non-pc7lar- f~ll7~tional gro~p~ in th~ ~ilic~ C7--1 e mploy~d in the ~ol~.lmn chr oinatog.-2phy cit~p ~ b~ ~r~lect~d from c~l kyl grr~Llpei crjnt~ininrJ 1 to 3CI r ar-brln atum-i, tr-iall.ylr~ilyl in whir:h t~;e ~ yl gr~L;p~ cQnt~tin 1 tc7 l~ al-bc~n atc)ms~ ar)ri the 1 i)ie. l~he contell-t r7~ Gar-~c7n in the non~pol~r silic-~ gel c~n vary from l~ t 3i~-~. t~y wei ght ~

mplçs r~f the al~y~l f~nc:tiDnal grc~upai r~f the ~ a ir:cl~lde methyl ~ ethyl, L7LItyl ~ h~>~yl, o~tyl ~ cle?c.yi ~ dod~cyl end the l i ke.

Example!s cf the tr-i ~lkyl~ilyls for-ming the fLInctional grDLIp of the ~ilica ~?1 inc:l~lde triethylc~ilyl, tr~imethyl ~3ilyl ,and l:.he Prefera~ly~ the lQaded silica gel colLImn i~ washecl with one bed vul~lme ~f th~? wasshin~ sol ~er)t~

f~cc~rding to a f:.lrther fE.!?atLIrE' Clf thE in~elltiQn, the ~rganic ~cllvent E~mpl~yed for washin~ th~? 1Qaded siilice~ g~3l CO1LImn can hE

r th~ me ~ t~ c3r ~3,~1~ i c s~l v~ . t~n~pl ~ d -f (3r p.~ i n~ 3i i t~ t h ~a c c~

The c~ulverlt emplL3yecl lor- pLl('kil-c"~ t:he si 1ica gr-~1 wikhil-l tJ~1e col~lmn ancl whict- c~7n al~jr~ be empluyecl aci the wa~hint"~ ~L~lver7t may he sjeler tr!?cl fr r,~m ~hll~rlr3Fi3r-m, mc~th.lnr31 7 d~ hlOr-C)me'tth-:.)rlL~, dichloroethane, r~th.~oc31, chlL7rc3fQrm--me.hanr~l 7 Ciir~ Or'C3el:hL7ne methanol, dichlorr7methc~nE~ methanol, me$hanol-w~7tE?r7 ethanul-w;~tc.~r, l ~tr~h~drof~lran, c~l: etuni tr~i l ~ c-~toni tr i l e-water i sopropanol and the 1 i keA

The pr-ef_~r-recl solvent For- el~tillc,l thr~ 9anrJlio&iid~ i6 chlorQform-methanol in which the ratio uf chlurt\fc~r m to mc th~r7ctl can var-y frLtn~ ~: i tr,3 ;~

Conveniel7tly~ the prDportiol- of the ~3ilicc~ 9E?l in the col~lmn tct the living tisC~lt! empl~ye?d in the prue;C~3 of th~ inv~ant~on is~
apprD~ nately 10 9 of 3ilica ~el fQr e?vri~ry l4~) 4 rtf tissLIe~

The invention wil 1 nctw t~ described io gre~ter d_?tai 1 in the folIowing ~a::an~ples which shoLIl'd not tte c,ctn~tr ~ d ~ itative of th~? 3c~tpe c~ f i nvE?n 1: i L1r) .

~ r~l ,E, 1, Rovine br~i~t W&1.'3 ~IrC~lrld i~ltd ~(tra(::t~!lJ Wi th ~ce~4n~. rhe c~cetr~nE~ powder was lsolat~ad by ~iltr~tiLtn l~ir~ chn~r fLInn~
T~e ac~torte powcier thu~3 obt~ine.~d w2~ e,:~r~c:t~d wi th chlc~roform~
methanol ~ ): 1 and 1: r;~ and the solirJ me.terial w~ tliscarcJetl.
Th~ r~s~llting ._hlor~t~4rm n~e?thartctl r~itr~r:t wa~ ~b je~cted to Frolch par ti ti on wi th ~al i ne and the ~nethanol -~sal i ne phRF~e? w~lS

,:,,.

5S : .

.
ieparated. The sep~r a-t:e l m~thal~Dl ~ 7e fr actiorl w.;~ clire~tly applied on 1:~ a b~ltyl-silic:a gel c:olL~mn p.~cke~l withirl a J.~ter--rllethanul sr~ tiurl. The cc~ mt7 wa~ w.~,hr3li wi th ~rlri bE~rl vol~me c~f a ~jimil.-tr 1 ~ I water-methanol ~ol~ltion therel~y elutir79 fr-Dm the bLItyl--~jil ic~ yel r~ mn the rhlDrDfQrm-meth~ l fr~ctior containing 4anglic~side~s. Thereaftel-, tn~ r~r7~ id~ w~r~;~
i sol ated f rDm the chl L~r-~f Dr m- mE~th.~l lol -racti Dn by known me~hr~d~i.

The ~;,i,.llic ac~d rc~ntwrlt in the i~ tt~4d ~lnyllcs~ldt~ l^J4 fotlnd to be mDr-e than 3C~% by weight. The protein cr~ntr}~r7t ther ~of wa~3 foLlrid to t~e O.C15 g pr~r g ~iali~ ar id of the g.-tnglio~ide~
The cDntent of L~ther- imp~lr ltie~ wa~; very mLIch re:iured ir) ~c)mparison with gar r31iosicl~ pr e!par~d by known m~th~d~. Thi~
established that the g~nglir~ ide~, reF,ultinrl fr ~m the~ pr~ r~f the invention ~rr~ of va~r y h~h p~lrit~.

~;,X~.M~.. E'i!~ E^.. ~ :E................... .

The procedLIrrQ c~f E?;ampl~? I w~3 folluwed ~Ip to th~ ~paration r:~f the methanDl-~alinE~ fr actiDn. Ther~af ter, the s,epar ated meth~n-ol~ line frartiorl wa~ dir ~ctly appli~d c:~n t~ n ethyl-siii.ca gel cr~ n)n pacl;Rd withih d' 2: 1 water-methandl sDlution.
The - column wa~ w~ Qd with on~ be~ vc~lum~ of a ~aiml~a~. ~ . J
w~ter-meth~nol s~ol~ltion thræreby el~ing fram thæ ~?thyl-~ilica oel .
cQI l.lmn . a ~; : . 1 chl r,~rof orm-m~ ol ~r~Qti on . ec~n~i ni r~
g~ng.llasi~es. Thereaft~r~ the:gan~ sid~ were rerov6?recl ~rQm .the: ~r;Qan~c ph~ y ~nown ~n~thQtl~. . . -.. . . . .. . . .. . . . . . . . .

; - Th~? s~ c ~cid crnt~nt clf ehe ~i~ol~t~d gan~lio~lde~ w~

! , ' ' . ' ` . ' ' ` , ,.
,, , . . ,, , ' ,' . : ', , ' ',. . .
., , , .. ' , ` .. . . ,' . ' f~ a 55i fD~-nd to be grr ate~ than ;~ . by weight. Th~ contr~r~t ~F protL~in~
~ncl other impuritie~ Wclra ~ol.lnd l:r~ b~ much r~e.~d~c~d in cc1mp~ri~or with ganra1iwsidr;~i pt~eparerd t~y e~ .til~r,~ met:hDrl~. OncE~ ~g~in7 thi~; e~tablished the hic3h pur ity c:~f thr-~ prepared ~3~nr,,~1ios3irJe1~

Th,r-~ procr~dure of E,:ample X wa followed L~p ta the ~;eiparation . .
of the me?~h~nDl--5al inE? ~ ctiL~n. ` Ther e~t~r~ th~ sep~r~te?d methanr~l -sal i nr* fracti on wa~ &~ppl i ed on trJ ~ tr-i is?thyl s3i I yl ated-~
a. gel rolumn p~cl.erJ within'a 1 ~ 1 w~ter--m~th~nol ~iolL~tion.
The c:ol umn was~ then wa~hr-~d wi I:h r~ne bed vr~l umr~ of 21 ~i mi 1 ar 1 : 1 w2~ter-methanGl ~301utir~n thereby eluting frQm the r Qlumn a ~ : 1 chloroform-Methanol fr-ac tion conta.ininrl cJanc,71ioside~. Thereafter, the ganc11i ClEiC~ rr~ c)le.t~d frc~m th~ chlc1rs:)~Qrm--methanL~l fraction by ~cn0wn m~thocl~. -The ~ialic acid content of the i~lated yarl~liosides was rnL~nd to ~e gr-eat~r than 3C)% by weic,Jht. Th~ cunterlt uf pr-otein d ~ther imp~ritie~ wa~ ~L~L~nd to be m~ch redL~cer~ in c~mparison ~Jith ~anyl i o~i de~ prep6~l~-ed l~y e~: i sti n~} methr~. Once a~in7 thi~ e~ta~ hEd the hi~h pLlri ty oF the pr-ep.lr~d ~an~liosidea.

,E ~MP,LE__IV, The proced~tr-e of E~ctmple I ~ wed L~p to th~ ~p~tratlom of ,the methanol-~aline Frr~tction. Thereafter7 the ~p~r-ated methanDl-saline -~r-aLtion wt~ dire~tly applied on to .t de~yl-~3ilica gel c~ mrl ~a!~ d wi~ ;in a ~ ater---m~than~l a~l~ltiun.
The ~l Llmn Wc~ w~t~he~ ~Ith Dne hed vol ~me o~ i mi 1 ~r 1 :

s~

atllr -mettlL~r)ol sol~lticn tht-~r eby ~_!ILltinCJ t rQln ttle C~lLlmrl o~ 3 ù
chlor~Qfor-m~-mi!thdno1 fra,c~ticn c (~ t,lirlit~g gLurlcJlic~sid~
Ther~ec~fter~~ Ll)e c3angliociid~s ~Yere isol.~tf~tJ fronl th~ rh1c~roform-meth~no1 fr-ac:tion by knclwn methodc~.

Tt1L~ 3 i ~31~ , Ir~ 0l ~ t El l t~ c~ Ç t: t) e? i !~r~ l C.1 t c.~rJ rJ ,.ilrl cJ l :i c~.3 i (:1 Lb5 Ir~f,~L,b fo(.lrld to t.7e gre,-lte?l I:harl ;,to~: t~y weic,3ht.. The tont~nt r~f prc~tL,~ins~
al~d o-ther i Inp~lr-i ti e~; ~Yari f o~lnd to be ml.lch recJuc ed i n c 0l1lpar-i ~ orl wi~h gangliusiicies prepar~e~d by e.~ ,tin(3 methr7~3. 'r'hi s establicihed the hirJh p~.lri ty of the prepat-ed c,~anc,31io!~id~c.a~

~ X ,Ç~ ,~l,F,,~=E, ,,,~
The pr-or ecl~lr e of Exampl~ I was fc~ werJ ~lp ko the ~epar~cion of the m~thar701-~a1ine fl~tlc:tion with on~ xCeptit7r~ in that in~teali Qf ho~,ine br-ain~ the ~;~tarting material L~mployed w~i hLlmL~n l~r`ythroc`ft(~C3. 'T'h~rl~lf tel~ lf'~ I~r'oc~f`~ lrl~ '~c~l lQ~ci w.a~ id~ icà~1 to that o~ Example II e~bovfe~

.
The si al i c aci i n t~r7 t ~7f th~ i ~ol b~to~ :I g~,~r~l i r~l elr~ w~
found to be greater thar~ ~!;C~% by ~eight~ Thf~ conter~t of prc~teir-~Llnd othr~r impuri t.ie.~ wa5 ~uLIncl tr~ be m~lch r edLIred in compO~r-isul7 with ganglic~iides prepar~r~d by e?.~iYtin(.7 m~tho~ic~. Thic~
e~tab1ishecJ the hicJh pur-ity r~f the pr-epar-ed r~ngl.i~c~id~.

~X.~ LE ,~I
.
The procedLlre of E,:~rnple I wc-~i fDl lc~werl up to thE~ ~;epar atir~n of th~ m~thallQl-~aline fr a~tiQrl with (~r~e ~ pti~r~ in t.h~t:
instead ~f bc~vine br~c-~in~ th~l C~tar-t.in~ mater~ial emplr~yed ~a~ humOIr~
br-e~in~ Th~r-eafter, the pr-Qcedur-e folluwed wa~ iclentic~l tQ thal:

.~ .

2C~ 5S

c3f E~:c.lnlp1r . III .:~b~ave.

Th C-J si .~1 i L ~ i L 1.-:011 C. el I t U f tht:: :i !.D I ~1 t.~?d Ll .ll`)91 i Cl~ii d ~ 3 w,~s -fo~nd to be r.lrL~at:er thar~ l3y ~r;~iyht~ The cc3nte~nt c~f pr o1:(-in c~nrJ other impLIri ties was fc:~L\ncJ tu b~ ml.lLh r c~duc.r~c~ in cc~n)p~ri~r)rl ~ith gang1ioE~ide~-~ prc.~pareti t3y e. isciny m~?thode~. Thi C5 ~stc~blish~c~ the hiCJh p~lr i ty uf the prr~ ar ~Jd rJanLJliu~;irJeb~
, Th2 ma jor ldv~r~tage u-f the prOL'C-.~ i ol the pres~nt invt ntian r~~E~.idec; in the ~c~ct th~t s3~lch proc ess yi~:~lcJs3 a hi~h c-ontent of extremr~ly pLlr-e t~anglioc~ les wil:houl: neLe~ cinr~ the employlnent nf the tedioLI~; ~nd time-cc.)nwl.lmirlg ~t~?p~ uf di~lyE~is ~rld concentration with r-e~;pLc t tu ttie meth2,n~ 1inr~7 fr-c~ction c~t3tainr d af ter Fc lctl par-titiDn~ ~)part Fr r~m this, thr~ yleld of ganglic3~ide-s frc~m the r~?vt~r~e pha~3e ~ilica gL~l cc31~lmn i~ over '?~
wi ttl thE~ 9~1l9l i osi LiE~~ pc3~ es~lsi ng ~ si al i c 2,ci ci c-unt~nt i 1~ C~!35 o~ % by w~;?ight FLIrthel-mc3rel the pr otein content th~ non--1 ipid cont~nt anLi the nt3n-s;ialic 2c id I ipid c3nt~nt oF the pLlri f i ~d yan-~l i o~i de~ pr~p~red by -the i nventi ve pror~3 are consi clerabl y 1 e95 than i n garlgl i c3si dE~s prep~red by hi thE~r to knDwn me~thod~ .

Claims (16)

1. A process for the isolation and purification from ganglioside-containing living tissue of gangliosides having a salic acid content of more than 30% by weight which comprises dehydrating macerated tissue, extracting the dehydrated tissue as a powder with a chloroform-methanol mixture, extracting the chloroform-methanol fraction with saline, separating from the extract thus obtained a methanol-saline fraction, loading said methanol-saline fraction on to a column filled with silica gel having non-polar functional groups, i.e. reverse-phase silica gel, packed within a solvent, washing said column with an organic solvent, eluting the desired gangliosides from said column employing organic solvents of different polarities and recovering the purified gangliosides from said organic solvents.

2. A process as claimed in claim 1 wherein the living tissue is selected from bovine brain, human brain, human erythrocytes, neuronal plasma membranes and the like.

3. A process as claimed in claim 1 or 2 wherein the macerated tissue is dehydrated with acetone.

4. A process as claimed in any one of claims 1 to 3 wherein prior to being macerated, said tissue is washed with cold water to remove extraneous entities such as blood.

5. A process as claimed in any one of claims 1 to 4 wherein the ratio of chloroform to methanol in the chloroform-methanol mixture for extraction of the dehydrated tissue varies from 2 : 1 to 1 : 2.

6. A process as claimed in any one of claims 1 to 5 wherein the non-polar functional groups in the silica gel employed in the column chromatography step are selected from alkyl groups containing 1 to 30 carbon atoms, trialkylsilyls in which the alkyl groups contain 1 to 10 carbon atoms, and the like.

7. A process as claimed in claim 6 wherein said alkyl groups include methyl, ethyl, butyl, hexyl, octyl, decyl, dodecyl and the like.

8. A process as claimed in claim 6 or 7 wherein said trialkylsilyls include triethylsilyl, trimethylsilyl and the like.

9. A process as claimed in any one of claims 1 to 8 wherein the carbon content in the non-polar silica gel varies from 1 % to 30%
by weight.

10. A process as claimed in any one of claims 1 to 9 wherein said loaded silica gel column is washed with one bed volume of the washing solvent.

11. A process as claimed in claim 10 wherein the organic solvent employed for washing the loaded silica gel column is the same as the organic solvent employed for packing the silica gel within the column.

12. A process as claimed in claim 11 wherein the solvent employed for packing the silica gel within the column and the solvent employed for washing the loaded silica gel column is selected from chloroform, methanol, dichloromethane, dichloroethane, ethanol, chloroform-methanol, dichloroethane-methanol, dichloromethane-methanol, methanol-water, ethanol-water, tetrahydrofuran, acetonitrile, acetonitrile-water, isopropanol and the like.

13. A process as claimed in any one of claims 1 to 12 wherein the solvent for eluting the gangliosides is chloroform-methanol, with the ratio of chloroform to methanol varying from 2 : 1 to 3 : 1.

14. A process as claimed in any one of claims 1 to 13 wherein the proportion of silica gel in the column to the living tissue employed in the process of the invention is approximately 10 g of silica gel for every 1400 g of tissue.

15. A process for the isolation and purification from ganglioside-containing living tissue of gangliosides having a salic acid content of more than 30% by weight substantially as herein described in any of the foregoing Examples.

16