Note: Descriptions are shown in the official language in which they were submitted.
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
'.?
S5
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
S5
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 :
1 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~ .
