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ROUTIIG AND CONTROL RECCRD

DO NOT DETACH FROM ON LOAN' DOCUMENTS 2 Hay 1955 DATE

Tranalatior of Jatsrn 24cert io3a3/ ramadotucieg of Poa sáreccivo doncent or Ergot by Artifteiel Gultveo (Japan)

Attached to. 00-A-40815

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eda Fhavi. Ine, 2427 -cho,

"The aanulacturing of nes effockive content of ergot by arti,ficial cnltnre,"

I) Tho abatract of the nabure aod objoes of cho inventton.

Tho pupposo o2 this invontion 18 prepère the Very ocloctivo nov uaterial chich pozseRscs 1€ po.orful actlon to contraet.tho uterus Pro: ar enlture erçot haeteria which pruitees rea alzalok FOC The cuneral proocduros aro a9 Collevst lirti ho bacteria, which prodnees 1 C ca the basos cuch 35 TAE juioo, 01 othe bases secord tion or the ertot alkaloid 3ae coleny cultura bace (in the lattur oaso axtraccion CaF ont c à or extraot fron the alunina which provious 1 sorbed she loid) by organie solvunts in cOlut ens cvch 20 Qther, chlorofora, benssno getranel soutono, tolnono, pyridine, carbon disalrido or carbon botraehloride, ete. are carried out.

1) The oxtraos is decelorirod by tho aetivated charcoal, ete. and thon con leaood to xive xtsais or
2) Toexe.d cach ao
chlorie acid, sulfurio hoid or bartarie auid eta. To tho acid volution, anonia, ote. (alkalino aatorial) vas alded to proczpitato Asrocl.vin fron iupurkty.

II) Dotailed cxplanation of tho

This invention is tho addendun to the original inventton in vhich tho Trod bactoria vas oultured in tho ualt juico and othor ertl riolal dulsural bases and the evlturel baso ras diveeslyextpneted with organle colvont suah as oshur, chlorolora, beasond, ete. whieh aro not niueiblo vith rator or adsorbod by adsofbont cuch as aluuina to gos aoiive ccsponeat of urjot, but alter ths fur\~ ther researoh, tho orot bacteria, waieh prodneuo hou alkaloid. Acroelavin, vao culSarud Tn cho salt juice, zuji juico or other

Japanuse devernieat

inzeatop: To:0 Saneno 11 Xasani Hikani

Tho totail of tho Patont and Invonsion ¿l70336

Patvnt ounor:

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artirtoial cultural base, Chen troi this thoro waa obtained the
ot contrcaston or utorna 3al the 93 o1 aaturial do
not eoppapond to the 10.a CPO 00 nteed 1ä23 1201。 3-20 :0 C 。うこ众 17 chlorozora) and the maxlaia abaorpbión spectes
hao tho poak a6 25a io

The poopyo 1 C20.: 738 tho colorlens clinic 1 nc 1 1 51V23 res acid or 20 2nt Ita n t ic or or C 3c and thelr soln 0:1 did C J 3 e it 1 .1 colo tion to tho sulfurà C aci i c0lu ( 721 7 5 i 2 e 1 no bansaldotyde, otc. 1-‡ i C 5 22 001. in CE t] 1 and D C ccet 0 3 C toluono 0olnt ete 973 4 4 21 1 ,000,000 dilutlon gives ed 5 ical cetion upon tho inolated ntoru3 of pabhit or nea

The oxtvaotion of Agróolavin fron tho baotorinl solory or oultural baso ahieh vas cnlturod on cultural besos oueh as aalt juico os 1 KGji Jaico op othup synthosie basos wan oarriod out ao follova:

1 nacterial colony trá culèural coluticn dy tho r 12 af: C j i 1. and oxè obur alth C cotono, 0:: 11 1
5 ote c eè C che (eco) 07 sod sho C te C C C And e solu of C nio 001 nced solat 02 shaken ého much Ha 30 àc ode ard : V vatorial een 1 C iith the OPCE 1 Vent. ; 2 052 tho
30. 1 n 560 Then 1 repoa tion solution W35 Cc 7 02 of Agroolavin soperated out.
Chon pho seid solution, whioh uau the coid oxtraot fron tho orgonio
solvent nonbalnins Agroelavin, uas ende aikalice, Agrockavin, Eaco 57
the condcnuntion of tho colution cnd poopocting to tho ayount or
the aoid used.

Tho ppt. was filtorod and vashed and dissolvod in tho orjonic solvonb, whioh wao, dvy uhen necosnary, nnd ovganie colutken vaa condansod to give tho eryutal or the pefiar or Agroelavin.

-2.

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Tho eultura-baso which yas Filtered Prou tho beotoria-coloay, vish wan entrneted vits Only or othona or vitheud condenoin! sho volwne, wator or adaorbod on tho aluáina eto. vhieh are nos aivelblo wich alkalino nadia ie was orbraotod uil tho opganic polvont and t'hen éomleased to glvo Sho cvyutal or tho poudor of Agvoelavin.

Or it paa nhaken utth tho ccid and the neid solution was nado alkalinc vith aonia or other alkaline naterial so ppt. Acroclavin Evee Traa she diparisy. Tie ppb. vas Sllserod and tachod and chon discolved in she orzanle soivunt sueh ae other, bensone, acotono or nethanol cnd tho solation vas dried when necescary and condonzed to sivo tho orystal or tho pordor of Agroclavin.

The ppt., erystallino poudor or tho erystal of Asroclavin obtainod fron tho bactaris-oolony or cultuvo-baso as aontioncd becoro vao discolvod in tho wator-oiseibls opganic colvent suah ao noshanol or seotone, ote. for she pooryobailisntlon and tho solution wan 1 riltoved and condansed il recossary.

Tha crystal ras obtainad ahen water vas addod to its solution

Experiaental proceduro vas as Polloya:

prolucos Acroclnvin, vas placed in tho at 2 7 and aa paivoriedd cnd C C to sho con t 2 n :1 in cad troated 7ish chc2\~ c0 thorlal volntion vas placed in Cha desaicae conl. 2:0 condcauod echor al colution. tox to soparato the groelavin troa. the

Tho colleetod eryetallina pouder van diosolved again in ethor and Che colutiea wao dohydrated and dosolorined and then condonsed to givo tho eryutalline Asvoolavin (Osh g. of eryotal pop every l00 5 ε. of dofassed poudor). 1

2. 5.0g of cuerose, O.lg or potnsoiunhydrogon phosphate, O.033 nagnesiun sulfate, O.og of annonina ruceinato in vater ves modo pl 5.2 dy eddation of ouccinio acid.

2) " ą calturoanot 11

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2 - artrtotn) multuro-hasa aan placod tho aanplo eraet bae 八in tovta, which produesa Agroelavin, d a aa aae 1 for h) dara. Thon Sho colony vas Tilcertd Crot tha bare and prosted cnd dyled ad lov teaporasuve to cbtain the poadav, dhich shan dozastod aad vet with stionta watur and then extracted irlth boneene. Tho cordenned bease..s solution vas extvseted vith Snall aueunt or 1 A tartuvie acid solation repeatedly. Tho er:- bined acid selation vaa made slk-Waa by dropaiso eldition of annonicmater to pps. Agroelsvin Iveo fron tho Anpurity. The filtorod ppt. was veshed with dil. cesenie celution and then dlosolvod in uther. Tho dehydrabod oshorial solution vas con denrod to givo eryotallino Acroelavin (0.5 8. Eron uvery l09 g. ot becteriacolony). Tho nother liquop, afder ppt.atind tho alkaloid, was exbraoted with othor and the ethorial solution vas dehydrated and doeolorised and condoused to give cvystalliuo pou dor of Agroelavin.

pot 3. The 5 ofun aquoous 0olution o ) ウ consists-o2 ato 0.03: : of 53 of 2 tol, and 0.1% 1.0 of of and JES 6.14 cod 0 :1 on To this 1 au i 30 1735 24 D 3 cit ¿ch prodLens 11 and at 25° C 2or 17 dayo and thon at 2h0 0 for 12 ye itionally

On tho 33rd day tho cultured-beco way filtored fron tho daateria
colory and she 00 20 tronbed wi th L C 0 1 tho 2 0 doted 92 e theT 210 6:1:1 2 n cordeu 2. 7 02 eractod vith 1 C 17 it To 3202 il Cn :: 2 14 4 000 zed in 20c t th one
13 eondonsed and i th Co V ion o chen 12 C 13V 1. to eopara to tho volution
vas plccod in the oo zoen to 2 in the C ine Acreelavin
(0.2 g. of ovyotal por J CC e2 eulturo-haso

Tho Relations to this Inventien

This iuvontion is tho addondun to tho pat. l62,939 aud h tho originat invoneion tho ergot beotorin vas enlsured in ralt juieo, KUji juico or other artiricinl eulsura-basoa aad tho baso vas oxbracted divootly wish tho orssnie colveud oueh as other, Cidz or bonzone, otc. or it was troosed vith cluina to adsorb the

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… Iavontlon vas the ioprovuient ths origanal 1.1 ttt Ai.4 し,し
ant alie1e d Aroo n 2 vas- eultured in tio DT ∴0 sho
PRPSH C !.
sol7ont, che hl ino 3: C CC。 Tho solntion cccdeno, ui. J 153 v0 chareoal, ote. and iò in tho varioua ackda substaneo to Croo to obtaán tho orystal
liuo not pd. igvoolavin has very povorful aotion and con
trnet utarun.

Tha doroign covered dy dhla pabent

7 1 cü3. ön 3 the cui P ic 17en 35 added 20:19123 ion 0 s-' ne and 22 ATOO ir91

Tho patont covens shu abovo-auntloued othod to chtatn che noy autivo eo iponent, Azroolavin, of srgot.

5.