The Carbohydrates of the Jerusalem Artichoke and other Compositae

13 Pages·2005·3.2 MB·English

114 I95I
The Carbohydrates of the Jerusalem Artichoke and other Compositae
By J. S. D. BACON AND J. EDELMAN
DepartmentofBiochemistry, UniversityofSheffield
(Received 5June 1950)
The most fully investigated polysaccharide of the Estimationoftotalreducingsubstances(TRS).Themethod
Compositae is inulin (named from Inula helenium), usedwasessentiallythatofMiller& VanSlyke(1936) with
firstobtainedfromthetubersoftheJerusalemarti- slight modifications (cf. Bacon & Bell, 1948). Reducing
choke, Helianthu8 tuberou,s L. (Rose, 1804), but sugarin easilyhydrolysed combination wasdetermined by
more easily prepared from the dahlia tuber (cf. TbyRSadedsitnigma0ti1onvsolb.eoffor5e-5a%nd(awft/evr)hoyxdarloilcysaicsi,dwshoilcuthiwoanstodotnhee
McDonald, 1946). ('Artichoke' is used to indicate
sampleandheatingthemixturefor30min.inaboiling-water
Jloenrgusbaeleenmeavritdiecnhtok(ecft.hTraonurgehto,ut18t9h3ias,pab)petrh.a)tIitnuhlaisn abadtihl.uTtihoencoofnc2e0nttriamteisoonrofmocrarebwohaysdrnaeteedewdasbesfoorcehotsheenTthRaSt
may constitute only a small fraction of the total estimation;undertheseconditionsneutralizationoftheacid
carbohydrate present in the artichoke tuber, the wasunnecessary. Contrarytothepreviousexperienceofone
remainder resembling inulin in being composed ofus (J.S.D. B.; see Bacon & Bell, 1948) we found that
essentially of fructofuranose units, but having a fructose gave 1-06 times the reduction by an equivalent
greater solubility in water and in aqueous ethanol. amountofglucose.Thecorrectionrequiredwasusuallyabout
Theterm'inulide'hasbeenusedbyFrenchworkers the same as the experimental error because the ratio of
fructosetoglucoseinthewholeextractswasapproximately
(Wolff& Geslin, 1918)todescribesubstances ofthis 4: 1. TheTRSisexpressedashexose; thestandardusedis
type, which occur also in the tissues ofmanyother statedwherethediscrepancybetweenglucoseandfructoseis
Compositae (cf. Colin, 1919). Lemoigne (1941a, important.
1942) has published a comprehensive review of Estimationofglucosebyglucoseoxidase. Aspergillus niger
existing knowledge of the carbohydrates of the (NationalCollectionofTypeCultures,No.594),subcultured
artichoke. onmalt-agarslopes,wasinoculatedintoflaskscontaininga
In the course ofan investigation into the trans- liquidculture medium (0.5% NaNO3,0-1% MgSO4.7H20,
03%K2HPO4,10%glucose(wfv))andtheflasksincubated
formations offructosepolysaccharides inthetubers
wehavemadeasurveyofthecarbohydratespresent, mateas30u0r.inTghteheappeuaprtaankceeooffsmtahleleponrztyimoneswofasthefomlelodwieudmbiyn
using new methods, in particular paper partition thepresenceof0g2lucose.
chromatography. Although it was not our main The liquid was poured off, dialysed against running tap
intentiontoinvestigatethecompositionofthetubers, waterandfilteredthroughafoldedfilterpaper(J.Barcham
and some ofthe techniques employed are perhaps GreenLtd.,HayleMill,Maidstone, no.904j). The dialysed
not the most suitable for that purpose, it is clear material was neutralized with NaOH and concentrated in
from the results presented here that existing ideas vacuo in a water bath at 35 40°. The product was a clear
aboutthetubercarbohydratesmustbemodified. A yellow liquid which contained alittle invertase activity in
preliminary account of this work has been given addition to the glucose oxidase, indicated by a slow 0,
(Bacon Edelman, 1949). uptakeinthepresenceofsucrose. Glucosewasestimatedin
& 0-2M-acetate buffer(pH5) eithermanometrically (Keilin &
Hartree, 1948) or by difference in TRS before and after
METHODS incubation withtheenzyme.
disSstoalnvdeadrdin8solaUttuiroantsedofb8eungzaori8c. aGcliudcsoosleutainodn f(Froulcitno,se19w2e9r)e, theEsmteimtahtoidonooffCkoelteose(.unTphuibsliwsahsedd;oncef cBolaocroimnet&ricBeallll,yu1s94i8n)g
msuectrroys.e iNnowagltuecro,saencdoutlhdebceondceetnetcrtaetdiobnycghleucckoesedobxyidpaoslearii-n wgeisttheda tfrhuacttofsreucsttoasnedawrads. tChheroomnaltyogkreatposheicpreevsiednetncienstuhg-e
thefructosesampleused. extracts.Themethodgavetheexpectedamountoffructose
Polarimetry. The opticalrotations ofsolutions were read wthheetnuabpeprleixetdrtaoctssucbreofsoer;eiatngdavaefttehreacsiadmheydkreotloysseisv,albuuetwtihtihs
iqnuaD2rrtdyemr.sweatiungbdhettshuidsneitsnelgrimcaiesnsatotaidkoienunsm.flrTaoummbptehraesscluiwgtehrstuesrofuaccruectse..oSuatmpilnetso fcrauncntootsebceomtbaikneendaasseivniidneunlcne.that it measures accurately
withafreshweightof8-12g.weredriedfor24hr. at 1000;
Paperpartitionchromatography
afurther2thr.dryingdecreasedtheweightbyonly10-20mg.
Deproteinization. Routinely, extracts weredeproteinized TheapparatususedwassimilartothatofConsden,Gordon
byadding0.1vol.of30%(w/v)lead acetate (Pb(C2H302)2 & Martin (1944) and Partridge (1948). Strips of sponge
3H20)solution. Tothefiltratewasadded3% (w/v) sodium rubber were clipped roundthe tops ofthe drainpipes, and
oxalate solution in the proportion 2-5: 16ml. of filtrate. asheetofplateglassrestingonthisgaveasatisfactoryair-
Occasionally minimal amounts ofeachreagentwereused. tight seal. Thepipesstoodinleadoraluminiumtrays con-
VoI. 48 CARBOHYDRATES OF THE ARTICHOKE 115
tainingtheaqueousphase. Largesheetsofpaperwererunin transmitted light, often showing better contrast than the
wooden tanks lined with lead (for phenol and collidine). original chromatogram. The procedure was used routinely
Butanol-aceticacidmixturesrapidlycorrodedthelead,and because the background colour of the chromatograms in-
an aluminium tank lagged with cotton wool was used for tensifiedwithkeeping,andmadefaintspotsalmostinvisible,
thissolvent. Thetroughswereconstructedofstainless steel. particularlywiththephloroglucinolreagent.
Strips (15x60cm.) and sheets (60x60cm. or 57x46cm.) Quantitative estimation. Quantities (5pl.) ofthe solution
ofWhatman no. 1 filterpaper were used. The solvent was tobeanalysedwereputontostripsorsheetsoffilterpaper
removed from the paper by heating it in a current ofair using an 'Agla' micrometer syringe (Burroughs Weilcome
inaspeciallyconstructedoven. andCo.); themeasurement hasanaccuracyof ±1%. The
Solvent&. Inadditiontothesolventmixturesdescribedby paperwasrunforasuitabletime(usually2days)inbutanol-
Partridge (1948), i8opropanol-aqueous NH3 (9: 1, v/v), n- aceticanddriedat60 80°. Everysecondorthirdstrip,each
butanol saturated with N-aqueous NH3 and mixtures of corresponding to one application of the solution, was cut
varying proportions of n-butanol-acetic acid-water were outandsprayedwithanappropriatereagent.Thepositions
used. Ofallmixturesinvestigated,thatcomposedof40ml. ofthespotsintheunsprayedstripswerethendeducedand
n-butanol, 10ml.aceticacidand50ml. water (called here- rectanglesfromsufficientstrips(usuallytwotofour)togive
inafter'butanol-acetic') (Partridge, 1948)wasfoundtogive enough material to estimate were combined for a single
themost satisfactory results with thetuber carbohydrates, estimation. Thecarbohydratewasextractedbyheatingthe
andwas usedfor most ofthe qualitative and allthe quan- paperto75-80'for20min. inaknownvolumeofwaterin
titative chromatography. aglass-stoppered tube, andshakingthetubevigorouslyto
Reagentsfordetectionofcarbohydrates. Thedeveloped and disintegratethepaper.Theresultingsuspensionwasfiltered.
dried chromatograms were sprayed with the appropriate Theestimation was carried outon asample ofthefiltrate.
reagentsandheatedintheoven. (i)Phloroglucinol(Horrocks Thisprocedurewasfoundsatisfactoryforketoseestimation,
&Manning,1949). Spotscontainingfreeorcombinedketose asblanksonthepapergavedensityreadingsoflessthan0-01
show pink when heated at 100-110° with this reagent. innearlyeverycase; recoveriesof95-100% were obtained
Glucose is not detected. (ii) Naphthoresorcinol (Partridge, with sucrose, but lower recoveries, namely 85-95%, were
1948)andsimilarreagentsmadewithresorcinolandorcinol found with tuber extracts. Estimation ofTRS was not as
were tried. Resorcinol gave a delayed colour production accurateorconvenient.Theestimationislessaccuratethan
duringadayatroomtemperaturefollowingtheusualheating that used for fructose for amounts of material less than
intheoven; orcinolgavegreenspotsonapinkbackground. 80,ug.,somorematerialhadtobetaken. Blankswereofthe
(iii) Benzidine (Horrocks & Manning, 1949). Spots con- order of 10ptg./spot, varying with the area ofpaper, and
taining free aldose groups show a chocolate brown colour werenoteliminated bywashing thepaperwith coldwater
when heated at 100-120°, but glucose in easily hydrolysed before application of the solution to be estimated. Com-
combinationgivesadelayedcolourproductionafterseveral binedreducing sugarwas hydrolysed afterextractionfrom
days at room temperature. Fructose, either free or com- thepaperbyadding0-5ml.0-5N-HCIto40ml.ofthefiltrate
bined, isdetectedinrelatively large quantitiesonly (about contained in the tube in which the estimation was to be
100itg. or more). (iv) Benzidine-trichloroacetic acid. 05g. carriedout,andheatingitinaboiling-waterbathfor30min.
Benzidine (A.R.) in 10ml. acetic acid, 10ml. 40% (wlv) Thetubeswerecooledinwaterandtheacidneutralizedby
aqueoustrichloroaceticacid,80ml.ethanol.Thiswasfound adding0-5ml.05N-NaOH,afterwhichalkalineferricyanide
tobethemostusefulreagentforthematerialsstudied. Itis wasaddedandtheestimationcompletedbythenormalpro-
very sensitive to both free and combined glucose, giving a cedure.
moreintensebrowncolourwiththissugarthanthebenzidine RESULTS
reagent describedabove, andwilldetect less than 1,ug. A CARBOHYDRATES OF THI ARTICHOKE TUBERS
yellow colour is developed with free or combined fructose,
but the reagent is appreciably less sensitive to this sugar. SourceB ofmaterial. At first, small lots oftubers
(v)Anilineacidphthalate(Partridge, 1949). were purchased as they were required, during
Treatment with invertase on thepaper. Sugars which are November 1948, butlaterasackfulwasboughtand
attacked by invertase can be treated with this enzyme on stored in amoderately cool cellar (7-8°). Although
thepaper. Chromatogramsruninonedirectioninbutanol- boughtatdifferenttimesthesetuberswereprobably
acetic were sprayed with a4% (v/v) dilution of'invertase allfromthesame batch, varietyunknown. During
concentrate' (BritishDrug Houses Ltd.) inwaterandhung 1949 a crop was raised from some ofthese tubers,
iTnhethperoodvuecntsatof50h°ydorvoelrysaisfrweeerweasteeprarsautrefadcienftohre1u5s-u3a0lmwiany. whichwere plantedin asunnyposition on the out-
byrunningthechromatogramatrightanglestotheoriginal skirts of Sheffield. The plants grew 3-3*5 m. high,
direction. This treatment hydrolysed completely up to andalthoughflowerbudsformedtheydidnotopen.
200jug. ofsucrose and didnot interfere with RF values or A fewtubers were taken out ofthe ground in mid-
colourdevelopment. September,whentheywerestillverysmall, butthe
Photographyofchromatograms. Aftercolourdevelopment majority were left there until April 1950 when the
the chromatograms were photographed: a sheet ofKodak bulk were harvested and some small tubers re-
ReflexContactDocumentpaperwasplacedfacedownwards planted. In the autumn of 1949 another sackful of
inclosecontactwiththechromatogram,thetwobeingheld tubers, indistinguishable from the previous year's
ebleetcwtreiecnlpaimepces35ofcmp.lataeboglvaess,thaendchirlolmuamtionagtreadm.byWiat4h0eWx.- batch, waspurchased; thesewerenamed 'Bedford-
posuresrangingfrom20to40sec.satisfactorynegativeswere shire Pinks' and had probably been grown in the
obtained from which positives (cf. Fig. 2) were made by Midlands.
8-2
116 J. S. D. BACON AND J. EDELMAN I95I
Extraction oftubers. The tubers were thoroughly seemedinourexperiencetobeadisadvantage,since
scrubbed inrunning coldwater, cutintopieces and itencouraged the extraction ofgelatinous material
disintegrated in a Waring blender with about half insolublein 50% (v/v) aqueousethanol.
their weight ofwater. The proportion ofwater re- Deproteinization. When these experiments were
quired depended to some extent on the losses of begun, the use oflead acetate and sodium oxalate
waterwhichthetubershadsufferedinstorage.When wasthoughtsufficientbothtoremoveproteinandto
about 10% water had been lost the tubers became destroyanyenzymnicactivity,butitwasdiscovered
rubberyinconsistencyanddisproportionatelylarge thattheopticalrotationoffreshextractsclearedin
amounts of extra water were needed to get satis- this way fell slowly when they were kept at room
factory blending. The dry weight of tubers was temperature;simultaneouslythefreeTRSincreased.
approximately20%ofthefreshweight(seeTable1). Ifthe extracts were boiled before deproteinization
During the blending, and afterwards, the mash theyshowednosuchchangeinrotation.Thechange
darkened, presumablythroughtheactionofphenol couldnotbeascribedtoareactionbetweencyanide
oxidases, but this couldbelargelyprevented either andfreereducingsugarintheextracts; a2% (w/v)
byaddingpotassiumcyanidetoafinalconcentration solution of fructose in 0-005M-potassium cyanide
ofabout 0-005M, orbyremovingtheouterlayersof showedno change inrotationwhenkeptfor 2 days
thetubers. A combination ofthese twoprocedures atroomtemperature. Itseemscertainthatahydro-
gave almost colourless preparations. The mash lytic enzyme system was responsible, because later
obtained was squeezed through gauze or mada- experiments (Edelman & Bacon, 1950) demon-
pollam, yielding an opalescent liquid. A few ex- stratedthatthehydrolyticsysteminthetuberswas
tracts, which are mentioned below, were made in relativelystableinthepresenceofleadacetate. All
other ways. extracts to be used for analyses were accordingly
heated in a boiling-water bath for 10 min. as soon
Examination ofextracts
aspossible after their preparation, usually reach-
pH. The pH ofall extracts was about 6-5, irre- ing 80° within 10-15min. of the beginning of
spective ofthematurityandageofthetubers. The blending.
addition ofcalcium carbonate to the tubers during Optical rotation. The rotations of deproteinized
theextractionprocesstoprotect thecarbohydrates extractsvaried considerably; Table 1shows typical
from hydrolysis was therefore not necessary. Its results, with details ofthematurity and age ofthe
presence when the mash was boiled with water tubers.
Table 1. Compositionandopticalrotationoftuber extracts
(Allextractions were carried outasdescribed underMethods, withtheexception ofthat dated 8 Feb. 1949whichwill
be described later (Edelman &8 Bacon, 1950). Where dry weight determinations hadnot been made, the calculations of
g. TRS aftermildhydrolysis/100 g. freshtuberweremadeassuming80%water.)
Total reducing
substances Optical
Dry (TRS) after Ketoseas rotation [adD
matter mildacid percentage corrected calculated
(g./100g. hydrolysis ofTRS toundiluted onTRS
Date Date Date fresh (g./100g. after juice after
bought dugup extracted tuber) freshtuber) hydrolysis (2dm.tube) hydrolysis
8Nov. 1948 17Nov. 1948 15-5 -4.030* -10-40*
22Nov. 1948 23Nov. 1948 0.620
1 Dec. 1948 Dec. 1948 15*7t
1
18Dec. 1948 18Dec. 1948 14-8 78 -0*780 - 2.520
3Feb. 1949 3Feb. 1949 +1.640*
8Feb. 1949 8Feb. 1949 12*3 79 +1.590 + 5.150
8Feb. 1949 19Mar. 1949 +3.280
8Feb. 1949 4Apr. 1949 20*6 14*4 78 +0-13° + 0.380
8Feb. 1949 9May 1949 20-3 14-2 78 +1.020 + 2.850
28Sept. 1949 6Oct. 1949 18-4 14-4 91 -7.780 -22.10
28 Sept. 1949 7Nov. 1949 21*1 16-0 86 -4-150 -10.30
28Sept. 1949 24Nov. 1949 25-0 19-4 85 -3.540 - 6.840
24Nov. 1949 24Nov. 1949 16-6 14-4 73 +1-02° + 2.940
24Nov. 1949 24Nov. 1949 22-0 18*9 83 -3.870 - 8-030
5 Apr. 1950 1 May 1950 15-3 10-6 85 -0*360 - 1.430
5Apr. 1950 1 May 1950$ 14*9 10-9 83 +0.050 + 0.190
* Theseextractswerenotboiled beforedeproteinization, buttherotationswerereadimmediatelyafterwards.
tt TThhies[eOxC]tDraocftthweassumgaadrelibbyerattherdowbiyngmidlidceadcitdubheyrdsroilnytsoisbowialisng-w6a4t.e8r0.andblendingthewholewhen cold.
V01. 48 CARBOHYDRATES OF THE ARTICHOKE 117
It isprobable that some enzymic hydrolysis had Itwouldseemfromtheseandsimilarexperiments
takenplaceduringextraction,causingasmallchange that 'inulin'wasnotpresentinsignificantamounts
ofrotation in the negative direction. Experiments inextractsofthetubersobtainedinNovember 1948.
inwhichthetimeofextractionintheWaringblender It is possible that the solubility of inulin in cold
wasvariedindicatedthatdifferencesoftheorderof water (initselfnotaverywelldefinedproperty, cf.
-0.50 (calc. for undiluted juice) might be ascribed Lemoigne, 1941b) is increased by the presence of
tothiseffect,whichisthussmallcomparedwiththe inulides, but in a qualitative experiment it was
seasonalvariations. Alternativemethodsofextrac- foundthatacommercialsampleofinulindissolvedin
tion,e.g. byfirstthrowingdiced tubersinto boiling tuberextract,would stillprecipitate whenthe solu-
water,andlaterblendingthem,gavesolutionswith tionwasfrozenandkeptcool.
morepositiverotationsbutotherwiseverysimilarin Extracts made from the batch oftubers dug up
composition (cf.thetwoextractsmade 1May1950). inAugust 1949showedadepositionofinulinalmost
Itisbynomeanscertainthatthewholeoftheoptical atonce; solutions preparedforpolarimetryandleft
activity ofextracts is due to carbohydrate. overnight had to be heated to dissolve the hazy
On hydrolysis the rotation always became more precipitate which had formed even at room tem-
negative, andthe 'differential' [M]Dcalculatedfrom perature. Fresh extracts showed a precipitation
the change in rotation and the change in TRS for whenfrozen,butafterhavingbeenheatedtoboiling
complete hydrolysis with oxalic acid at 600 was pointtodestroythehydrolyticenzymesystemthey
-63.5°; that calculated for inulin (Eoc]D -40.0°) is showedamoredelayeddepositionofpolysaccharide.
-53.40,andforsucrose -83.00. Itseemspossiblethatthe 'inulo-coagulase'effectof
The[X]Dofthemixtureofsugarsproducedbymild Wolff(1916)maybeduetotheadditionofnucleiof
acid hydrolysis of one extract was -64.8°, which aggregatedpolysaccharidetoboiledextracts.
correspondsto amixture offructose andglucose in Extractsmadefromtubersgrownunderthesame
theratio 4-3: 1. Thiscalculation doesnottakeinto conditions, but dug up in a more mature condition
accounttheprobable formationofdifructoseanhy- several months later, showed a slight precipitation
dridesduringhydrolysis (Jackson& Goergen, 1929). of inulin, but extracts made from the tubers
Spontaneous precipittae.. Although 'inulin' is harvestedinApril 1950resembledthoseusedinthe
almost insoluble in cold water it can be extracted winterof1948-9ingivingnoprecipitate.
fromplanttissueswithouttheuseofheat,andunder In the absence of any method of determining
these conditions it later appears as a dense white 'inulin'theseobservationsmaybetakentoconfirm
precipitate on the walls of the vessel holding the earlierobservations (Thaysen,Bakes&Green, 1929)
extract. No such precipitation was noticed with that the amount of inulin in the tubers decreases
extracts made by the procedure described above during the autumn andwinter, bothrelatively and
during the period from November 1948 to the absolutely.
summerof1949. Anextractmadebythemethodof Precipitaion by ethanol. Although many experi-
Green (1888),i.e.byboilingdicedtuberswithwater ments were carried out with a view to preparing
and concentrating the extract in an evaporating polysaccharide byethanolprecipitation, to serve as
basin,depositedasmallratherflocculentprecipitate a substrate for metabolic studies, it was clear that
inthecourseof2months.Theprecipitatewascentri- fractionation by this means was at best very un-
fugedoff,washedwithwater,anddissolvedinwater satisfactory. Additionofethanoltoaconcentration
bywarmingtoabout 800. Onfreezing,thematerial greater than 50% (v/v) leads to the appearance of
reprecipitated; it could be presumed to be inulin a heavy flocculent precipitate, but after this has
(ketose was present), but insufficient was available been removed by filtration or centrifugation, pre-
to characterizeitfurther. cipitation continues for somemonths at roomtem-
Material resembling inulin was obtained from perature. Forexample, of23-4g. oftotalketose in
extractstowhichethanolhadbeenadded: anequal an extract only 6-6 g. was precipitated by the
volume ofethanol was added to fresh extract and addition of an equal volume of ethanol; but later
the precipitate allowed to settle overnight before the filtrate deposited a precipitate from which a
being filtered off. The clear filtrate, left for several substance resembling inulin was prepared (see
months at room temperature, slowly deposited a above). This slow 'after precipitation' appears to
translucentlayerofcarbohydrateonthewallsofthe be characteristic ofthe carbohydrates ofthe tuber
flask. When an attempt was made to dissolve this (cf. discussion of the depolymerization of inulin,
material in a little cold water it did not dissolve Lemoigne, 1941c), and would seem to make it im-
completely,andtheinsolublefractionprovedtohave possible to define any fractions as being soluble or
the properties ofinulin, i.e. it was awhite, ketose- insoluble in a given concentration of ethanol (but
containing, substance, soluble in water at 800, but see, nevertheless, Tanret (1893a, b), who also notes
slowlyseparatingasacompactprecipitate afterthe the considerable effects of temperature on the
solutionhadbeenfrozenandleftintherefrigerator. solubility offractions).
118 J. S. D. BACON AND J. EDELMAN I95I
Precipitation byacetone. Theadditionof7-20vol. Paperpartition chromatography
acetone to extracts produced an immediate floccu-
Qualitative. Theextractsshowedaseriesofspots,
lentprecipitate, andanopalescencewhichpersisted
alldetectedwitheitherphloroglucinolorbenzidine-
for several days. Chromatographic analysis (see
trichloroaceticsprays(seeFig.1).Freehexoses,when
below) of the precipitates and supernatant fluids
indicatedthatmanysubstancesare commonto the
two fractions.
Precipitationbyaqueousbaryta(cf.Tanret, 1893a). Spotn
Aqueousbarytaaddedinexcessprecipitated 15and
2%,respectively,ofthetotalketoseoffreshextracts,
andofthematerialsolublein 50% (v/v)ethanol.
FreeTRS. ThefreeTRSofdeproteinizedextracts
amountsusuallytoonly2-3%ofthetotalTRSafter
mild acid hydrolysis. Since reducing sugar is
liberated from the carbohydrates by a hydrolytic
enzyme system in the extracts (Edelman & Bacon, Spot 5
1949) the exact value ofthe free TRS is probably
not ofmuch significance. Values have been found
Spot4
ranging from 0-25 to 0-75 g./100 g. fresh weight of
tubers. Insome extractsthere has been chromato-
graphicevidenceforthepresenceofmonosaccharides,
butinothersitseemsthatthebulkofthefreeTRS
must either be non-carbohydrate in character, or Spot 3
alternatively due to reducing end groups on some
ofthe polysaccharide present.
Combined TRS. The greater part, possibly all, of
thecombinedreducingsugarisinaformliberatedby
mild acid hydrolysis. An experiment was carried Spot2
outtodiscoverwhethermoredrasticacidhydrolysis
would liberate further glucose in the extracts (see
Table 2). From these results it seems that only an
insignificantamountoftheglucosecanbepresentin
combinationwithpyranosidiclinkages,orwithnon-
carbohydrate residues.
After mild acid hydrolysis the ketose content of
the extracts is unchanged, and corresponds to
80-85%oftheTRS,theexactpercentagedepending
onthematurityandageofthetubers (seeTable 1). SpotI
The remaining TRS liberated was accounted for as
glucose by the use ofglucose oxidase (see Tables 2
and 3).
It is evident from these data that the carbo-
Fig.1. Typicalchromatogramofextractofartichoketuber.
hydrates consist essentially of non-reducing sac- Runinbutanol-aceticfor3days;sprayedwithbenzidine-
charides,inwhichthemajorityoflinkagesareofthe trichloroacetic acid. The position of spot 1 corresponds
furanosidic type. withthat ofsucrose(seeFig. 3). (Second positiveprint.)
Table 2. Comparison ofeffecto ofweak and more drasticacidhydroly8i8 onliberation ofglucose
fromartichoke-tuber extract
(2.0ml.extractheatedwith2-0ml.oflON-H2SO4inboiling-waterbathfor2hr. Cooled,andneutralizedwithlON-NaOH.
Further 2-0ml. heated with 04ml. 5.5% (w/v) oxalic acidin boiling-water bath for 30min. Both hydrolysates diluted
to25ml. withwater. 2-0ml. ofeachsolution incubated at400in Warburg cupswith 1-2ml. 0-6M-acetate buffer (pH5),
0-3ml. glucose oxidase and0-1 ml. catalasesolutions until 2uptake ceased. Values calculatedperml. originalextract.)
Glucose Glucose (by
02atN.T.P. calculated TRS Ketose difference)
(MA.) (mg.) (mg.) (mg.) (mg.)
Weakly hydrolysedextract 1510 23-4* 97-5 74-5 22-6
Moredrasticallyhydrolysedextract 1520 23.6*
* 1mg. glucoseundertheconditionsoftheexperiment gave 02uptakeof64-5pl.
VoI. 48 CARBOHYDRATES OF THE ARTICHOKE 119
Table 3. Estimation ofglucose inhydrolysedartichoke-tuber extractbyglucoseoxidase
(1-0ml. hydrolysed extract (diluted x20) incubated at 400 in Warburg cup with 1-0ml. 0-2m-acetate buffer (pH5),
0-2ml. glucose oxidaseand 1 drop catalase (horse-kidney extract), untilO uptakeceased. Estimations doneon contents
ofcupdilutedto50ml. Valuescalculatedperml. oforiginalextract.)
Glucose Glucose
calculated Non-ketose calculatedas
02uptake from O; (by disappearance
(pS. at uptake Ketose TRS difference) ofTRS
N.T.P.) (mg.) (mg.) (mg.) (mg.) (mg.)
Untreated 79.5 101 21-5
Treatedwithglucose oxidase 1510 22.5* 80-5 78-2 22-8
* 1mg. glucose underthe conditions oftheexperimentgave 02uptakeof67-5pl.
Table 4. Distributionofketo8eamongthe componentsofthetubercarbohydrate
(Wherethedates correspond these analyses refertothe extracts describedin Table 1. The figures forindividual com-
ponentsareinsomecasestheresultsofasingleketoseestimation, butinothersrepresentthemeanofseveral,e.g. spots 1,
2 and3for8Feb. 1949areeachthemeanoftenestimations.)
TRS Ketose Ketoseas %totalketoserecovered
after asper- from chromatogram
hydrolysis centage r-
(g./lO1 g. ofTRS Fruc-
Date Date Date fresh after tose Spot Spot Spot Spot Spot Spot
bought dugup extracted tuber) hydrolysis spot 1 2 3 4 5 6
8Feb. 1949 8Feb. 1949 12-3 79 10-8 10-5 11-7 10-9 10-8
16Sept. 1949 16Sept. 1949 2-2 2-5 2-5
28Sept. 1949 6 Oct. 1949 14-4 91 1-5 3-7 3-0 2-8 2-8 3-0
28Sept. 1949 17 Oct. 1949 13-3 86 4-2 5-4 3.3 4-4 4-1 4-6
28Sept. 1949 24 Oct. 1949 14-2 90 2-2 7-2 5-3 6-6 3-8 4.3 4-9
28Sept. 1949 31 Oct. 1949 15-3 82 2-6 6-3 3-9 5-0 5-3 5-8
28Sept. 1949 7Nov. 1949 16-0 86 1-3 6-6 4-2 6-3 5.5 5.9
28Sept. 1949 24Nov. 1949 19-4 85 7.7 5-0 6-5 6-6 6-7
27 Oct. 1949 23Nov. 1949 7.3 7-3 8-4 8-0
24Nov. 1949 24Nov. 1949 14-4 73 9-4 8-7 10-7 9-9
24Nov. 1949 24Nov. 1949 18-9 83 4.9 4-7 6-6 6-3 6-2
24Nov. 1949 4Jan. 1950* 7-9 7-1 9-1
24Nov. 1949 14Feb. 1950* 82 1-3 8-8 9.3 10-5
24Nov. 1949 3Mar. 1950* 1-4 9-2 7.9 9-2
24Nov. 1949 15Mar. 1950* 2-1 9.9 8-8 8-8
5Apr. 1950 1 May 1950 10-6 85 - 11-5 9-9 10-4
5Apr. 1950 1 May 1950 10-9 83 11-5 10-1 10-6
* Tuberspeeledbeforeextraction.
present,occupiedpositionsbelowsucrose, cf. Fig.2. series. With butanol-acetic andphenol no evidence
It is convenient to refer to the spot in the sucrose wasfoundforheterogeneity;thisisconfirmedbythe
positionas'spot1',totheoneaboveitas'spot2'and behaviour of fractions of higher RF (mentioned
soon,thespotwiththelowestRFbeingreferredtoas below) originally separated by the use of phenol,
'spotn'.Therewasverylittlemonosaccharide,none which showed the same number of components
ofit pentose. Spot 1 had an R, indistinguishable whether run in that solvent or in butanol-acetic.
fromthatofsucroseinallsolventmixturesused. It These observations do not exclude the possibility
gavethebrowncolourcharacteristic offree orcom- that the spots represent mixtures of similar sub-
binedglucosewithbenzidine-trichloroacetic. Using stances,particularlythoseoflowerR,, whichmust
thisreagentthecolourofthehigherspotswasmore be assumed to consist of higher oligosaccharides.
and more yellow as the series was ascended to The spots occupy the same relative positions in
RF=0,suggestingahigherfructosetoglucoseratio. phenolasinbutanol-acetic.
Totestthehomogeneityofthespots,two-dimen- Quantitative. The ketose content ofthe spots was
sional chromatograms were run. Using butanol- estimatedinanumberofextracts (seeTable 4).
aceticinbothdirectionsthe spotswere foundto lie The results demonstrate the even distribution of
in a straight line, and there was no resolution of ketose among the components of higher R,, in-
any spot into two or more components. This ex- dependentlyoftheircontributiontothetotalketose.
cludes the possibility that the R,'s ofthe spots are The most noticeable deviation in this respect is
influenced bythe presence ofothermembers ofthe spot 3, whichseems always to have ahigher ketose
120 J. S. D. BACON AND J. EDELMAN I95'
contentthanspots2or4. Estimationsofketoseand action ofpurified preparations ofinvertase cannot
ofcombinedTRSinspots1-3weremadeasdescribed be taken as evidence for the presence ofsucrose in
under'Methods'(seeTable5forsometypicalresults theextracts (cf. Colin, 1919).
with one extract). It willbeseen thattheratios of Partialseparationofthecarbohydrates. Astreakof
fructosetoglucoseinspots2and3,calculatedonthe 1-00ml. ofboiledextract, 45cm. long, was applied
assumption that the substances are non-reducing, 9-5cm. from one edge of a sheet of filter paper
vary according to the basis of calculation. Small (57x47 cm.). This was done by hand in four suc-
errors in the calculation of combined TRS have cessive applications from a graduated pipette with
relativelylargeeffectsontheratio,butanalternative a finely drawn tip, the applications being of0-225,
explanation of the discrepancies may be found in 0-33, 0-375and 0-07ml. Guidespotswereplacedat
slight hydrolysis duringthe dryingprocess. Onthe eachend ofthestreak. Thesheetwasrunfor 92hr.
other hand, the assumption that these spots are with phenol saturated with water, and the guide
non-reducingmaybeincorrect; theymayrepresent stripscutout andsprayedwithorcinol. Because of
mixtures ofreducingandnon-reducingsugars. thelargequantityofmaterialputon(101 mg.TRS)
Table 5. TRSandketosedeterminations onindividualcomponents ofthetubercarbohydrate
(Anextract made on 14Feb. 1950from tubers bought24Nov. 1949, deproteinizedwithPb, wasused. 5 or7 pl. spots
were placed on chromatograms, andthese were run togetherin groups A, Band C, all in butanol-acetic for 3 days. The
extract wasanalysedusingordinarypipettes, andalso usingthe 'Agla' syringe: 81-8 (ordinary), 81-1 (Agla) mg. TRS/ml.
after hydrolysis; 67-2 (ordinary), 65-7 (Agla) mg. ketose/ml. All results are expressed in ,g./50ju. original extract; TRS
iscalculatedas ,ug. hexoseon basisofafructosestandard.) ,Q r
1'.KO OI
blanks
from
same
TRS TRS sheet Ratio Ratio
before after Glucoseas corrected Glucoseas fructose/ fructose/
Chroma- hydrolysis hydrolysis Ketose 1-06(q-p-r) forarea 1-06(q-r-t) glucose glucose
Spot tograms (P) (q) (r) (8) (t) (u) from (8) from (u)
1 A 602 270 76 271 1-0
B 147 658 316 199 1-6
C 178 668 260 245 166 258 1-1 1-0
2 A 494 282 67 156 1-8
B 125 521 328 68 4-8
C 158 542 284 106 124 141 2-7 2-0
3 A 489 322 56 118 2-7
B 118 533 356 60 5.9
C 113 532 315 111 104 121 2-8 2-6
Treatment with invertase. When two-dimensional only the spot in the sucrose position hadseparated
chromatograms oftuberextracts were treatedwith cleanly and this occupied the region between 220
invertasebetweenthefirstandseconddeveloprm,ents, and 290mm. from the point of application. The
spots in the glucose and fructose positions were remaining streak was arbitrarily divided into four
obtainedfromeverycomponentexceptthematerial zones by cuts 9, 45, 155 and 220mm. from the
with R. approximately zero. In this case fructose startingline. The corresponding areas fromthe un-
appeared, but glucose could not be detected with sprayed sheet were then cut out. The strips were
certainty. This was not unexpected owing to the extractedwithwaterinvacuoat40-45O inaSoxhlet
small proportion of glucose in this material (see extraction apparatus, each being extracted six to
Table 6). Faint spots alsoappeared withRFvalues ninetimes. Thevolumeofwaterwassuchthatonly
higherthanthatoftheoriginalspotfromwhichthey about 1 ml.wasleftintheflaskatthelastevapora-
derived, but similar to original components with tion. The final liquid remaining in the extraction
higher BR. Thus, for example, spot 2 gave rise to a chamber was tested for fructose by the Seliwanoff
very faint spot inposition 1, andspot 5to spots in methodwithnegative oronlyveryslightlypositive
positions 4and 3. Manysuch 'daughterspots'were results,indicatingalossoflessthan 1%ofthetotal
slightly retarded in position when compared to fructose at this stage ofthe recovery. The concen-
original componentswithsimilarR,, and, although trated extracts, which were yellow in colour, were
it appears that invertase produces shorter-chain eachmadeupto5ml. Chromatogramsshowedthat
oligosaccharides from each component, further fractionA (R, correspondingtothatofsucrose)was
evidence is necessary to prove them identical with pure,thatBcontainedtwospots,andthatC,Dand
those occurring in the tuber extract. It is obvious E wereprobably more heterogeneous. An analysis
that the appearance ofreducing sugar through the ofthesefractionsisgiveninTable 6.
V01. 48 CARBOHYDRATES OF THE ARTICHOKE 121
Table 6. Analysis offractionsseparatedchromatographicallyfromtuberextract
(Fordetailsofexperimentsseetext, p. 120. Values calculated/ml. originalextract.)
TRS* after Glucose (by Glucoseas % total
hydrolysis Ketose difference x1-06) monosaccharide
Fraction (mg.) (mg.) (mg.) afterhydrolysis
A 15-8 7-7 8-6 52-5
B 20-75 13-0 8-2 38-7
C 28-15 22-35 6-15 21-6
D 23-25 21-0 2-4 10-3
b 10-9 10-8 0-1 (1)
Totalrecovered 98-85 74-85 25i-45
Totalin original extract 101 80-0 22'0O
Recovery 98% 94% -
* Calculated ashexose onbasisoffructosestandard.
Leaves. Theamountsoffructoseintheupperparts
CARBOHYDRATESINOTHERPARTSOFTHE of the stem, and in the petioles and leaves, were
ARTICHOKE PLANT lower than those in the other parts ofthe plant, so
Source ofmaterial. Plantsraisedfromthe tubers that concentration oftheextractswasnecessary as
describedabovewereused. Leavesweretakentothe apreliminaryto chromatographing them. The con-
laboratoryasquicklyaspossibleina cooledvacuum centratedextractsfailedtogivesatisfactorychroma-
flask; the stems, which were long and heavy, were tograms, the whole ofthe carbohydrate remaining
cutintoshortlengths. asabloborstreak,withfewsignsofdifferentiation.
Extraction and deproteinization. The stems and This effect (cf. Consden et al. 1944) could be repro-
roots were extracted and the extract deproteinized duced artificially to some extent: 7,uI. spots of M-
byessentiallythe sameprocedures aswereusedfor potassium dihydrogen phosphate solution were
tubers. Leaves were extracted and deproteinized placed on the starting line ofa strip ofpaper, and
simultaneously in the Waring blender. A typical aftertheyhaddried, 5pl.spotsofacontrolmixture
extraction was as follows: 50g. of lamina were ofsugarsandofatuberextractknowntogivesatis-
blendedwith 100ml. of35% (v/v) aqueousethanol factory chromatograms were applied at the same
(containingpotassiumcyanide)and35ml.ofchloro- point. When run in butanol-acetic the chromato-
form, N-hydrochloric acid being added cautiously gramsshowedretardationanddistortionofthespots,
drop by drop to make the mash react green with thoughnotaspronouncedasthatseenwiththeleaf
bromocresolgreen.Thewholemashwascentrifuged extracts. Application of 7 ,l. spots of 30% (w/v)
and the upper layer (yellow with a slight tinge of lead acetate (Pb(C2H302)2.3H20) and 40% (w/v)
green) wasfilteredwiththeadditionofalittlefilter barium acetate (Ba(C2H302)2.H20), which it was
aid (Hyflo-Supercel, Johns-Manville Co. Ltd., thought might precipitate interfering substances,
London). Such extracts gave no precipitate with hadno effect on the R,'s ofthe control mixture of
trichloroacetic acid. They were adjusted to pH 7 sugars,norofthetuberextract. Itwouldthusseem
before concentration in vacuo with a bath tem- that it is not salt concentration alone that is
perature of500 orless. responsible for the failure to achieve satisfactory
Roots. These showed the same series of com- separation (cf. Partridge, 1948).
ponents asthetuberswhenchromatographed. Attempts to remove salts from the leafextracts
Stems. The stem, particularlyinthe lowerparts, bytheuse ofion-exchangeresins (inwhichwe were
containedasubstantialpith,whichcouldbecutout greatly helped by Dr R. E. Kressman and the
without much difficulty (Thaysen et al. 1929). Permutit Company) hadonlylimited successinthe
Chromatographic examination showed a series of summer of 1949. The cations were removed readily
components similar to those of the tuber, both in byZeo-Karb217,buttheresultingacidsolutionwas
extracts of the pith, and of the upper stem (see broughttowardsneutralityonlyslowly,evenbythe
Fig. 2). As judged qualitatively there was more of more basic resins of the 'Deacidite' series (up to
thecomponentsoflowR., andinulinseparatedfrom 'F'). The treated solutions, which had been cycled
extracts keptintherefrigerator. repeatedly through columns of the two types of
Inner and outer regions of immature tubers. The resin gave improved, but not entirely satisfactory,
youngtubersbrokenaturallyintoanouterlayerand chromatograms, in which 'spot 1,, glucose and
an inner core. There was no qualitative difference fructose were always seen. In one or two extracts
betweentheminrespectofcarbohydrate (Fig. 2). indicationwasgivenofmaterialwithlowerRFthan
122 J. S. D. BACON AND J. EDELMAN I95I
sucrose,butinnonewasthereevidenceofaseriesof family (cf. Willis, 1931) were represented (see
componentsresemblingthetuberextract. Starchis Table 7).
presentintheleaves (Brown &Morris, 1893). Undergroundorgans (100g.) (afterremovalofthe
X 0 0
E 0 L- 4 a +j 40Jt
0 rO
.00 a) O 4 L+
:3 13I--
Spot 3
Spot2
SpotI
Glucose
Fructose
Fig. 2. Chromatograms of extracts of various parts of artichoke tubers and stems. Run in butanol-acetic for 40hr.;
sprayedwith benzidine-trichloroacetic acid. Extracts madefrom plant 16Sept. 1949 unlessotherwise indicated (see
Fig. 3). (Firstpositiveprint.)
fibrous roots) were disintegrated in the Waring
TESCOMPOITHER MEMERSblender with an equal weight of 0O01M-potassium
cyanide for about 5 min. In most cases some diffi-
The storage organs of other arbitrarily selected cultywasexperiencedintheblendingbecauseofthe
speciesoftheCompositaewereinvestigatedchroma- fibrous or woody nature ofthe material. The most
tographically. Members of both subgroups of the notable exception (except for the dahlia tubers
VoI. 48 CARBOHYDRATES OF THE ARTICHOKE 123
Table 7. DetaiB ofplant8examined
(AllfromtheSheffieldarea.)
Dateof
Partofplant collection
Subfamily Systematic name Commonname examined (1949)
Tubuliflorae Dahliasp. Gardendahlia 'BabyRose' Tuber 1 Sept.
Tussilagofarfara L. Coltsfoot Rootstock 15June
Helianthustuberosus L. Jerusalem artichoke All vegetative organs
Liguliflorae Lactuca8ativaL. Lettuce, 'TomThumb' Taproot 16August
Leontodonautumnali8 L. Autumnalhawkbit Rootstock 20July
26August
Sonchusoleraceus L. Commonsowthistle Rootstock 20July
Taraxacum officinaleWeber Dandelion Rootstock 27 April
Tragopogonpratenss8 L. Goatsbeard Taproot 4July
which resembled artichoke tubers in their ease of Tanret (1893b) thathis 'helianth6nine' and 'synan-
handling) was Leontodon autumnalwi which had thrine' were chemical entities, since in his analysis
crisp,parenchymatousrootstockswithlittlefibrous thesetwofractionsandsucroseoccurredinaratioof
material. Theextracts obtained aftersqueezingthe approximately 1: 8: 2, the three constituting two-
material through gauze were light-brown or yellow thirdsofthetotalcarbohydrateoftubersstoreduntil
in colour, of pH about 6-0-6-5 and more or less June.
opaque. They were heated in a boiling-water bath It is evident that considerable possibilities exist
for 15 min. as soon as possible after preparation. for variation in over-all composition through
Theseboiledextractswere concentratedinvacuoto variations in the relative proportions ofthe many
athicksyrupandtakenupinwatertogivesufficient components. Inpractice this wouldseemto be the
concentrationofcarbohydratetoallowofchromato- explanation for the variations in fructose, as per-
graphic investigation, namely 5-10% (w/v) total centage oftotal carbohydrate, and it seems likely
fructose. When extracts so concentrated were kept thattheopticalrotationofthemixturewilldepend
intherefrigeratortherewasprecipitation (toalesser on similar variations, the components with higher
or greater degree) of material, presumably inulin, R.probablyhavingmorepositivespecificrotations.
soluble inhot butnotincoldwater. Theamountof It is not clear, however, whether any considerable
this precipitate was small, except in one case. The change could occur within a given mixture of
wholeconcentratedextractofLeontodonautumnalis, inulin, inulides and sucrose without an accom-
however, setsolid, evenatroomtemperature, anda panying change either in the ratio of fructose to
relatively large quantity of polysaccharide was glucose,orintheproportionoffreehexosepresent.
isolated from it, the yield from 70ml. of original Itis difficulttoavoidtheassumptionthatallthe
unconcentratedextractbeing0-7g. Inspiteofcon- carbohydrates present in the tuber bear a relation
siderable salt effects (especially in the case of toinulin;thattheyrepresentaseriesterminatedby
Sonchu8 oleraceu8), it was obvious that a series of inulin at one end, and bounded by sucrose at the
carbohydratescomparabletothatinartichoketuber other, all members having the typical 1:2-linked
extractswaspresentineverycase (seeFig. 3). fructofuranoside grouping and bearing a gluco-
pyranoside as anon-reducing endgroup (I). Hirst,
DISCUSSION McGilvray & Percival (1950) have reported the
isolation of 2-2 of tetramethyl glucopyranoside
Thecarbohydrate8 oftheartichoketuber frommethylated%dahliainulin,andhavepostulated
These results demonstrate the heterogeneity ofthe a sucrose-like terminal group for the molecule. In
tuber carbohydrates; they alsoprovide a basis for addition they found circumstantial evidence for
theseparation and estimationofthecomponents of 2-6% of 2:4:6-trimethylglucose, which would in-
higher R.. It is to be noted that the latter con- dicate the presence ofglucose within the chain (cf.
stitute acQnsiderable proportionofthetotalcarbo- Irvine & Montgomery, 1933). The total ofmethy-
hydrate: thefivespotswithR.'s rangingfromthat lated glucose agrees well with the percentage of
ofsucrosetoabout0-025(inbutanol-acetic) contain glucose estimated by their chromatographic pro-
morethanhalfthefructose ofsomeextracts, andif cedure,i.e.5-7%,butisconsiderablyhigherthanthe
their higher relative content ofglucose is assumed figuresgiven by others (cf. McDonald, 1946; Bell &
fromthequalitativeandquantitativedataavailable Palmer, 1949) whichlie between 1-5and 2.5%. We
they would comprise an even higher proportion of have not been able to trace the source ofthe attri-
the total sugar. The even distribution of fructose butionbyLemoigne (1941a)toKilianiofaformula
among these components in all the extracts ex- for inulin, (C6H1005)4C12H22011, in which the group
amined would seem to contradict the claims of C12H22011isintendedtorepresentsucrose.