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香川大学農学部学術報告 第９巻第２号 正誤表
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http://www.lib.kagawa-u.ac.jp/metadb/up/AN00038339/AN00038339_9_2_e.pdf
Notice
Technical Bulletin of Faculty of Agriculture, Kagawa University
Vol.9 No.2 Errata
URL
Vbl．9，一No，．乏（1957）
SEPARATION OF AN UNKNOWN SUGAR FROM
THE HY工）ROLYZATE OF A HEM■ICELLULOSE
OF BROAD・BEAN SEEDS．
A PRELIMINARY REPORT．
TeiitiNARASAKI（Labora！oryof AgriculturalProductsTechnology）（ReceivedJuly31，1957） 105
The detection of a ribo三e−1ike sugar was reported by KAWAMURA＆NARASAKI（1）and KAWAMURA，et al．（2）inthehydr01yzateofa hellicellulo3eBlOfadehulledbroad・rbeanseedsofvariety“Naga三aya＝．．The deteニtionandtheidentificationwe：emadeonlybytheRfvaluesonpaf）erChIOmatOg工早mSbythreesets
Of different soIvent sy5temS，buヒno des〇ription was madeonthe color re：Ctionof the spot on chro−：r）aN
to即amS
If the Rf values areidenticalin atleast three different soIvents ar］din color reactions with
various spray re3gentS，gOOd evidence can be said to be pIOVided that the two sugars areiderrti〔：al However，the pre；ent Opinions are sucb thaヒpユpe：Clronatography cannot give a conclu3ive evidence
at all，and the unknown shoulユbe separ・ated and characterizeまas c‡y．Stalline derivativeぅ
Thepresent expeI‘iTnentS Were conlucted to separatetheunknown Eug3r free froTn the oth占r compo− nent sugarsforthefurtheごStudie；0≡itsidentificationSone re二u王ts contl’adictingforFlコer P工e5umption were Obtained anユthe∋e reSults are pre；enteiin the present paper preliminarily
EXPERIMEN甘A且J
l動座動感血用．／■αゐク∽まcβgJ〝Jo」Sββ1
A heTnicellulo3e Bl（2g）wasobtaineまfromlO kg of de†1ulledbro3d・be・3nごeeisofvariety‘‘Nagasaya” by a proceまure shownin Fig1
ThehemicelluユoseBIPrepafationgh6wedm′ノp〃230→▲50Oc（deこ0二nPO三ition），Wellcohcidingwith the preっeedingreちults（1’2）。Thepreparationcouldbehydrolyzedalm0Stquantitativelybyhe・atinginaselコ1ed glasstubein a boiling−Water bath with2NH乞SO墾for4hours，but a very smallamountofaninsoluble re3iduewasno七ed afterhydて一01ysiswhenalarger amountofthe pTeparation（100−500mg）wastaken for hydrolysis．Theinsolublere3idueshowelpositive Mousff re急Ction andnegative biulet reaCtion，end it seemed to be an acid−reSistan七polysaccharide
2r勅）−d言∽ク搾SgO乃αJαSCブ邦d夏作g少‘ゆ♂7Cゐ′∂椚αわg′‘ゆカッq／■∠ゐタカ）dγ∂わgβどβq／ゐタブ弗豆cggJ〝わsββ1
Pr4）aration of th3h）dYOlyzalc．The hemicellulose Bl（02g）was hydrolyzed with20mlof2NH2SO4＿ in a se31ed glass tut）e by heatingin boiling water for4hours．，The coole王filtrate was neu七ralized with BaCO8，Centrifuged，andthe neutrali2：eユsupernatant was concentIateitosyrupbe10W！OOc小Thesyrup WaSeXtraCteiwithlO mlof50％ethanol，andtheextract wasconニentIatedtolmlfoIltheappli〇ation
to paper chromatography
SoIv3ntS“ChromatogIaphy was carTied o∵lt byusing phenol‥Water（PhOH）（4：1，∇V）for the 王irst
run，and n−butanol：aCeti（ニaCid：Wa・t9r（BuOH：AcOH）（4：1‥2，Ⅴ／V）and n−butanol：pyridin〇：Water
（BuO‡i＝C3H5N）（3‥l：l5 v：Vl）foご丑esecondrunh DoubledevelopTrien七WaSmadewith BuOH：AcOH aTld BuOH：C5H5N．whi】esil明）1e developmentⅥノaS employedⅥ3th PhOH
ColoYタeagfniSAAmmoniec：ilAgN〔）；；．．A mixture o亡equalvolume∋Of O1NAgNO3and5NNHlOH
Tech．Bull．Fac．Agr．KagawaUniv． 106
BrDad−bean seeds，10kg
Soakedin O2％NaOH for2days， dehulled by hand
Dehulled seeds
Soakedin water for・2days，blended by mixer， StarCh separated by filtration．．
ー【J
StaI′Cb
ExtT竺CtedwithlOlofO2％NaOHtoremove
pIotein up to biuIet reaction became negative
Soluble protein Sqgars solubilized by Takadiastase Residue Ⅵ7ashed wit壬1Water，95％C2H60H， ether，and dIied、 Crude holocellulose，100g Extractedwith5％NaOH，underN2（7・times） Extract of hemicellulose，61 Residue 「一一…▼−【
Adjusted to pH42−−4小O with CH8COOH，Centrifuged Supernatant，71 Hemicellulose A，1g Concentrated to25l， below400C．added with 5lof95％C2H50H， Centrifuged．， Soluble residue Hemicellulose B， Solution of hemicellulose B Insoluble residue Addedwithl／2volume of a mixtuIe of equal volumes of FEHLING SOlutions A and B，Cent工ifugedい Solution of hemicellulo三e Bl Washeユwith90％Ci2H50H（2times），10％HClin C2H50H （6times，tOfreefromCuii），95％C2H60H（6times）， 99％C2H60H（3times）（tofreefromC「），ether Hemicellulose Bl，2g Fig。1PrりCedureofpreparing hemicellulose Bl
107
Vol一．9，No2（1957）
Thedevelopedchromatogramswerethenwashedwith O・1N Na2S208”5H20・followed by washing
withwaterforpermanentpreservationofthedevelopedchrDmatOgramS”ThespotsonpaperchIOmatO・
gramsweresomewhatenlarged，butthewashedchromatogramswerewellpre3erVedwithoutanychange
even afterone year of storage
BAnilinehydrogen phthalate。Thereagentwasmadebyadding930mgof aniline（freshlydisti11ed）
andl6gofphthalicacidtolOO mlofwater−Sat11ratedn・・butanollThedriedpapersheet＄WereSprayed
withthisreagent andheatedfor5minutes atlO5O
ChYOmalogy郎hy．ThehydrolyzatewasappliedonT6y6No．50filterpapersheets（20×20cm），and
thepaperSheetswererunwithPhOHonetime，after developmentthepaperwaswashedwithether
toremoveresidualphenolonpaper．dried，andthenrunwithBuOH：AcOHoIBuOH‥C＄H6Nbydouble
developmentforsecond directionWashingwith ether markedlyimprovedtheumiformity of second
SOlventdevelopment
Resulis．．TypicalchromatogramsareshowninFigsl2and3l・Whitespotsinthechromatogramswere
thoseofsupplementedstandardsugars‖Color＄Ofthespotsweregivenwithanilinehydrogenphthalateasde・・
scribedinthesefiguresAllthespotsgavepositivereactionwithammoniacalAgNO8．butnocharacteristic
diffeIenCeWaS nOtedinthe colors
Unknown（dullyeuow） Ar。bin。S。
◎OR怒笥bw，
Unknown恥lIye1low） e O Arabinose 佃arkl−ed） 〉 .O （daTkred．（b還竃⑳
Xybse（dark r■ed） ． Ⅹylose他arkIed）（brown yellow） Glucose（brown yellow）
】
】 l l l 言
MH：G軋Nは皿ble devdopTh印t）臥10H：AcOH（doublcdevelopment）
Fig3．Two・・dimensionalpaper chromatogram of suga工Sinthe hydrolyzateof a hemicellu10Se Bl by PhOHBuOH：C6H6N system
Fig．2．Two−dimensionalpaper chromatogramof
sugarsinthehydrolyzateofa hemicelluloseBl
by PhOH−BuOH：AcOHsystem・・
3，5ゆαγα′豆∂循0ノ■班昭勿∽鳥肌昭朋・S聯′f〝≠カβカグd′〃Jγgαねq／αゐβ桝ダcgJJ〝hsββ1みツタ‘ゆβγCゐ′β，那f呼，1ゆカブ．
A hemicellulose Bl（1g）was hydr・olyzed as mentioned above，and the hydr’Olyzate was sub5ected
topaper−Chromatographicseparationofthelmknownsugar
Thehydroly2＝ateWaSintroducedalongahori2＝Ontal1ine5cmfromthebotomof T6y6Nol・50filter
papersheet40×40cm‖ThesheetwasirrigatedwithBuOH：AcOHsoIventbythetriple＞deve10pment
technique…Threestripswerecutfr・omthedriedsheet as a guide strip，and developedwithaniline hydrogenphthalateTheareacontainingtheunkn0wnSugar・WaSCutby referringtothethreedeveloped
stripsandextracteabyrefluxingwith50mlofwater
Theext工aCt OfunknownsugarshowednegativeBIAL，phloroglucinolandorcinolreactions，andonlyone spothavingRfvaluesOfO55withBuOH：AcOH，0156with BuOH：C5H6N，and O59with PhOH was
detectedby paper chromatography
4．Pr密aYalionqfosazone．Theunknownsugar solution（20mi）。WaS COnCentratedtoabout2mlin vacuobelow400c，．The concentrated sugarsolutionwasaddedwith．50mgphenylhydrazinehydrochloride
108 Tec‡1＝Bull…Fac‥Agr．．Kagawa Univい
and75mgsodium acetate，and themixturewasheated on a boiling−Water bath for aboutlhourT壬1e ・reSultingye1low crystalline productwas filtered，WaShed、with water，dissolvedinto O．5mlofhotethanN
Oh and recry＄tallized by adding about2 mlof coid water．Yellow needles obtained showed m．p 15ト90（uncorrected）‖D・・Ribosephenylosazonewas prepared from standard D−ril⊃OSe by the similar
procedure，Whichshowed mp160・N2O（uncorrected）”Mixedmelting pointwas1550（uncorrected）of thetwoosazones′Showinga cleardepressionofthemelting point
5q′Sleine−SuFfuYicacidreaciionqfthe s＠aYatedunknou）nS〟gay．DISCHEfound（8）thathexoses，PentOSeS， methylpentoses，2・deoxypentose，and heptoses produce substances with very characteristic absorption SpeCtrainhis cysteine・・Sulfuric acid工eaCtion（4）…Thesesugars seem to be differentiatedfrom eachother
duetothefactthatthedevelopmentofthe colors，aSWellas their・ensuing destTuCtion，depends upon
thetypesofsugarsIThus the cysteine−SUlfuricacid reaction byDISCHEWaSapPlied to the separated
unlてnOWn SugaI
Theunknownsugar solution（Ohlml）was diluted toユml，addedwith4mlof conc．H2SO4＝ While COOlingintapwaterlThemiⅩtureWaSkeptatroom temperatureforlhour・with fIequent Shaking
Then Ohlmlof 3％cysteine hydrochloride was added and themixture wasshaken Colorintensitiesi
Were readat5minutes−intervalin the firs；t30minutesafter the addition of cysteine solution at room
temperature，andthenfromtimetotimeupiOthe24thhourby a photoelectric colorimeter at430mJ＾ The resultsa‡eShowninTableI，Whichshowedtha七thegreatestcolorintensity wasdevelopedafter
about6hoursaftertheadditionof cysteinesolutionatroomtemperature
A ratioof anabsorption at430mFL tO thatat530mJIWaS about5Oatthebegining，anditdecreased
to aslow asl．1in6hou工S
TableIChangeof colorintensity of七he unknown sugar by
the cysteine・・Sulfuric acid reaction with tiTrle
Time（hT）
0 05 10 20 4“0 80 18 24
0pticaldensity O．26046060 0“フ5 086 0．88 078 0．78 at430m〃
DISCUSSION
Theunknown sugar，Which was reported as rit：0二eiコth．e preceeding papers（1，2），WEiS Separatedir）．tO apureeolution・Thepurityofthesoユution
SpOtOf Rf values coinciding with thoseof unknown sugユrin the prec3eding reports by t・he three sets
Of soIventsystems…The color reactionof the spot was duliyellow with aniline hydrogぅn phthalate The result suggests that the unkr−OWn Sugar CarInOtbe ribo3e，for ribose shows daTk red colorifit
appearsonpap？rChromatogramsatalll・Thecolorof七hespotwas very similar to that of methyl− pentosesL士hespot，Whichwasreportedas rit）OSebyHoNMA＆Al（EIAGAWA（5）detectedfromferment−
edsoybean paste，Showed also dullyellow color’．Various color reactions for pentoses were allnegative
With the separatedunknown sugar・SOlution．There土ore，it seems to be veryimprobable that the
ur）known sugar can be ribose
Measurementof melting pointof the phenylo∋aZOne Ofunkr）OWnSugar COuldrI．Otgive any conclusive evid3nC∋forth3id3ntificationofthe sugar，but the results alsose〇mtO unfavor the former assumption
thatthe unknown sugaris ribose
The reaction productof pento；eS by the cysteine−Sulfuric acid reactionshows the greatest absorption after15一20minutes after the addition of cysteine solution at room temperature at390m／］，ar）d then
itbegins to decrease slowly HeKOSeS develop maximum colorintensities more rapidly at412p4miL，
and the coloris stable at room teTnperature．Therefore，the present results coincide with those of neither pentoses nor・hexoses‥ While，heptoses produce an o工aI唱e COlorwith an absorption maximum
Vol．．9，No2（1957） 109
ar・O11nd430m］｝，and the compour）dis unstable and transforhedslowlyintoa pur・Ple compoundwith an absorption maximum at510mlL；the greatest colorintensityis reached after severalhoursl・The colorreactioご10ftheuI血OWnSugarSeemS tO Shov a verysimilarbehaviorwithheptosesdescribed above”Methylpentoseち give themaximum colorintensity only after 24 hours，and 2−deoxypentose3 glVethe finalreaction product only after24hourslTherefore，it seemsle言SprObablethat theunknown Sugarisone of these2kindsoE sugarsby仕e resultsof cysteine−Sufuric acid TeaCtion
SIJI沌〕M A R Y
（1）A hemicelluloseBIWaS prepareユfrom de！っu111ed broad−bean seeds of vaIiety‘●NEgaSaya”，and
hydrolyzed with sulfuric acidto study theincludedunlくnOWn Sugar
但）Theunlmownsugarwasseparate且fromthe hydroly2；ateOfahemicelluloseBlbypaper chroma’
togr・aphywithBuOH：AcOHsolventonalarge paper sheet（40×40cm），and extractedwith water to obtain an unlrnown sugar solution
（3）Theseparatedsugar showeヱnegativeB‡AL，Phloroglucinol，andorcinolreactionsThephenylosaz oneoftheunknow11Sugar meltedat15「l−9Oc．（decomposition）
俸）Cysteine−Sulfuric acidre二Ctionsuggestelthatthe unknownsugaTismost prot）able tot：e One Of
heptoses．Some possibilities seemedto remainthat thesugarcanl〕emetI〕ylpentose or2−deoxypentose，
but thesugar seemedtobeneither pentose nor hexose
ACⅨNOⅥrLEDG朋：ENT
Theautho工wishestothank Prof．S，KAWAMURA，Laboratoryof BiologicalChemistry，inthisUni・−
VerSity，for his kind guidance
REFEREⅣCES
（1）KAWAMURA，S”，NARASAKI，T，：Bull．Agr （3）DISCHE，Z：TBiol．．Chem，204，983−997 Cゐク沼．．50C．J（ゆα粥，20，289−290（1956） （1953）”
（2）MP，一−，Kusu‡‡A王iA，R，SAIO，T：7セCh 佐）鵬：J。Biol。Chem”，181，379−392（1949） Bm．Fbc“Agy．Kbgawa Univ，8，20l・−211 （5）HoNMA，N，AKEてAGAWA，T”：，／… Fbmenia・
（1957） ≠査∂〝7セCゐ乃OJ。，33，490−493（1955）．
