April 5, 1959
CONSTITUTION OF GLUCOMANNAN OF KONJAK FLOUR
tachment of an ether linkage to a double bond is known t o cause a high frequency shift toward 1250 ern.-' of one of the stretching vibrations associated with this linkage.g The band contour of the aromatic C-3 conjugated glucosiduronate G is clearly distinguished by a relative increase in intensity and a comparative lack of resolution between 1100 and 1065 ern.-'. The superposition of this band contour with that of the C-17 conjugated Compound H is evident
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from a better resolutioii near 1090 crn.-l. The strikingly similar band contour of the enolglucosiduronates I, J and K shows also a relative intensity increase near 1075 cm.-’. The band complex is distinguished by the resolution of an absorption peak a t 1070 an.-‘ and by narrowing of the major absorption band a t 1038 cni.-l both of which appear to be due to a loss of band components near 1025and 1050 cm.-l. BOSTON18, MASS.
[CONTRII3UTION FROM TFIt{ DEPARTMENT Olr I\(;RICIJI.TURAL BIOCl[LMISrRY, [JNIVERSITY O F hlINNESOTA]
Constitutional Studies on the Glucomannan of Konjak Flour’ 1 3 F. ~ SMITH AND H . C. SRIVASTAVA RECEIVEDOCTOBER 4, 1958 Methylation of the glucoinannan isolated from the bulbs of Antorphophallus konjac yields the corresponding methylated derivative which gives upon hydrolysis a mixture of the 2.3,4,6-tetramcthyl ethers of D-glucose and D-mannose ( 1 mole), the 2,3,6-trimethyl ethers of D-glucose and D-inannose (11 moles) and the 2,6-dimethyl ethers of D-glucose and D-mannose ( 1 mole). These data are in accord with the results of periodate oxidation of the polysaccharide. The structural significance of the results is discussed.
Amorphophallus konjnc C. Koch ( S y n . Conophnl- and 2,3,6-tri-0-methyl-~-mannoseand based on lus konjak Schott), a member of the family Araceae these results a formula for the polysaccharide was is the source of the so-called “konjak flour” a advanced. Since the proof of the structure of the popular article of food in Japan. The bulbs of components of the hydrolyzate of the methylated three year old plants are cut into thin slices which glucomannan did not appear to be entirely satisare then dried and powdered to give konjak flour. factory, i t seemed desirable to re-examine the conPreliminary studies2-B on the konjak flour showed stitution of konjak glucomannan using the more that the polysaccharide was composed of glucose modern techniques that were not available to the and mannose residues but results differed with earlier workers. This paper is concerned with the regard to the relative proportions of the two coni- results of methylation and periodate oxidation ponent sugars. studies on a glucomannan isolated from a comDegradation of the konjak polysaccharide by a mercial sample of konjak flour. sporulating bacterium isolated from konjak flour The glucornannan was isolated from konjak was reported3 to produce a trisaccharide, “laevi- flour by precipitation from aqueous solution as dulin” ([CYID -11.5’), and a similar trisaccharide, the copper complex following the procedure adopted “laevidulinose” ([cY]D - 15O), composed of D- for the Iles glucomannan.* The polysaccharide mannose (2 parts) and D-glucose (1 part), was ob- was regenerated from the copper complex by adding tainede by the action of Takadiastase on konjak dilute hydrochloric acid, the last traces of copper flour. However, the structure of these oligosac- being removed by means of Versene (ethylenecharides was not established. Acetolysis7 of the diaminetetraacetic acid). Kon jak glucomannan konjak glucoinannan followed by saponification was obtained as a white powder which showed afforded a trisaccharide ([a]=-16’ in water) [(Y]D -38’ in water and which upon hydrolysis which was shown to be composed of mannose (2 gave rise to a mixture of D-glucose and D-mannose moles) and glucose (1 mole). The structure of this in a molar ratio of 2 :3. oligosaccharide, which appeared to be identical Methylation of the glucomannan first with with laevidulin or laevidulinose, was not estab- methyl sulfate and alkali, and then with silver oxide lished. Hydrolysis of methylated konjak mannan and methyl iodide yielded the fully methylated was also reported’ to give a mixture of 2,3,4-tri- polysaccharide which showed [ a ] ~19’ in chloro0-methyl-D-glucose, 2,3,4,-tri-@-methyl-~-mannoseform. Fractional precipitation of the methylated polymer from a solution in acetone with ether and ( I ) Paper No. 3921, Scientific Journal Scries. Minnesota Agricultural Experiment Station. The matrrial of this paper forms part of a thesis petroleum ether indicated that the substance was submitted b y H. C. Srivastavn to the Graduate School of the Uniessentially homogeneous. Upon methanolysis folversity of Minnesota in partial fulfillmrnt of the requirements for the lowed by hydrolysis, the methylated glucomannan degree of Ph.D., 1956. gave a mixture of methylated sugars which were (2) C. Tsuji. Bull. Coll. nor. I m p . Uniu., I’okyo. 1, 115 (189B). (3) M. Mayeda, J. Biochcm. Tokyo,1, 131 (1922). shown by coluniri chromatographic analysis9 t o (4) K. Goto, ibid., 1, 201 (1922). 2,3,4,6consist of 2,3,4,6-tetra-O-methyl-~-glucose, ( 5 ) S. Miyake, J . Coll. Agri. I m p . Uniu., Hokkoido, 11, lG4 (1927). (6) T. Ohtsuki, Acta P h y b c h i m . ( J a D o n ) , 4, No. 1, 1 (1928).
(7) K . Nishida and H. Hashima, J. Dcpl. 0, 277 (1930).
Agr.
Kyushu I m p . U n i v . .
(8) P . A. Rcbers and F. Smith, THISJOURNAL, 76, 6097 (1954). (9) J . D Gwrdes. Bertha A. Lewis, R Montgomery and F. Smith, Annl Chcin
, 9 6 , 2(,4 ( I q X ) .
F. SMITH AND H. C. SRIVASTAVA
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Vol. 81
tetra-0-methyl-D-inannose, 2,3,(j-tri-o-inethyI-D- that certain of the D-glucose and D-mannose resi2,G-di-O- dues are immune to periodate oxidation.lb glucose, 2,3,6-tri-~-tnethyl-~-mannose, methyl-D-glucose and (?) 2,G-di-O-methyl-~-man- The molar ratios of the tetra-, tri- and di-0nose. The niolar ratio of the tetra-, tri-, and di- methyl sugars indicate that the repeating unit 0-methyl sugars was 1:I 1:1 indicating an average contains on the average about 13 hexose residues repeating unit of about 13. The major proportion whereas the periodate oxidation data indicate a of the hydrolyzate consisted of a mixture of 2,3,6- value of approximately 11 residues. tri-0-methyl-D-glucose and 2,3,G-tri-O-methyl-~Further support for the type of structure promannose in which the molar ratio of the D-glucose posed above for the konjak glucomannan was afto the D-mannose derivative was 2 :3, a finding in forded by the observation that the polyaldehyde good agreement with the ratio of the two sugars resulting from the periodate oxidation of the glupresent in the original glucomannan. comannan yielded a polyalcohol, upon reduction The tetra-0-methyl derivatives of D-glucose and with sodium borohydridel6.16, which when hyD-mannose could not be separated, but their drolyzed with acid was found to give rise to glycpresence was established by demethylation with critol, erythritol, D-glucose and D-mannose; the hydrobromic acidlo to the parent sugars, D-glucose molar ratio of glyceritol to erythritol was found to and D-mannose, which were readily separated by be 1:15. This value, which also corresponds to paper chromatography and by paper electrophore- the average repeating unit16 of the polysaccharide, sis.ll is in fair agreement with the results of methylation The 2,3,G-tri-O-riiethyl-~-mannose was obtained and periodate oxirlation. as a liquid, showing [ff]D -2' in water, and charSince the konjak glucomaiinan and its methylated acterized as its 1,4-bis-p-nitroben~oate.~ The 2,3,- derivative display a relatively low specific optical G-tri-0-methyl-D-glucose readily crystallized, m.p. rotation, -38 and - 19' respectively, i t is believed 118-120°, [ff]D 70°(equilibrium value in water), that the majority of the glycosidic linkages are of the and was further identified as the Il4-bis-p-nitro- @-D-type. benzoate.8 Experimental The di-0-methyl sugars were separated by paper chromatography and the 2,G-di-O-methyl-~-glu- The following solvents were used for the partition chrocose was characterized as its lI3,4-tris-p-phenyl- matography of sugars and their derivatives: ( A ) pyridineacetatewater (1:2.5:3.5, v./v., upper layer)"; azobenzoate.12 The identity of the second di-0- ethyl (B) butanone-water azeotrope18 and (C) benzene-ethanolmethyl sugar was not established unequivocally, water-ammonia (200: 47 :14:1, upper layer) ,I* Unless stated but since i t proved by paper chromatography to be otherwise all evaporations were carried out in Vacuo at 30quite different from all the isomeric di-0-methyl- 40'. The konjak mannan flour was obtained from a local as a pale yellow, granular powder. D-glucoses and identical with the product formed merchant Isolation of the G1ucomannan.-To a solution of konjak by epimerizationla of 2,6-di-O-methyl-~-glucose, flour (20 g.) in 10% sodium hydroxide (2 liters), Fehling i t is believed to be 2,6-di-O-methyl-~-mannose. solution B (450 ml.) and Fehling solution A (450 ml.) were The above results reveal the general structural added with stirring, in this order. The copper complex of the polysaccharide, which precipitated, was separated by features of the konjak glucomannan. Thus, the centrifugation and washed with Fehling solution. The isolation of the 2,3,4,6-tetramethyl ethers of D- complex was decomposed by suspending it in cold ( s f ) glucose and of D-mannose shows that the glucoman- water and adding dropwise cold (5') 2 N hydrochloric acld nan possesses terminal non-reducing residues of until the solution was acid. The regenerated polysaechawas recovered by pouring the acidic solution into 95% D-glucopyranose and D-mannopyranose. I n this ride ethanol. The copper ions were partially removed from the respect i t bears a resemblance to the glucomannan polysaccharide by trituration with a mixture of acetic acid isolated from the urediospores of wheat stem rust and ethanol. The residual green color was removed in the manner. To a suspension of the polysaccharide (Puccinia graminis tritici).'d Contrary to a pre- following in water, a solution of the disodium salt of ethylenediaminevious suggestion' it is now apparent that the major- tetraacetic acid (Versene) was added and after stirring, fhe ity of the non-terminal residues of the glucomannan polysaccharide was centrifuged off. The polysaccharide of konjak flour, like those in Iles glucomannan,* was then triturated with methanol-concd. hydrochloric acid (1OO:3 V./V.), centrifuged and washed successively with are composed of 1+4-linked D-glucose and D- absolute ethanol, ether and petroleum ether. The glucomannose residues. This follows from the isolation mannan was a white granular powder, yield 17.2 g., [ a ] ' * D and characterization of 2,3,6-tri-0-methy~-~-glucose -38" in 20% sodium hydroxide (c 0.7). Hydrolysis of the G1ucomannan.-The glucomannan (50 and 2,3,G-tri-O-methyl-~-mannose. mg.) was dlssolved in N sulfuric acid (2 ml.) and the solution The identification of 2,G-di-O-met!iyl-~-glucose heated (sealed tube) for 24 hours in a boiling water-bath. and the tentative characterization of 2,6-di-0- The solution was neutralized (BaCO,), filtered, and evapomethyl-D-mannose prove that the glucomannan rated to a sirup. Paper chromatographic analysis of this sirup using solvent A and silver nitrate spray reagent% repossesses a branched chain structure and that the two spots having R fvalues corresponding to those of D-glUCOSe and D-mannose residues involved in the vealed and omannose. The ratio of D-glucose t o Dbranching are joined through CI, C3 and Cq. o-glucose mannose was determined by separating the mixture on *a This deduction is supported by the observation paper chromatogram and, after elution from the paper m
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(IO) I-. IIoiigh, J , R. N . Jones and W. 11. Wadman, J . Chcm. Soc., 1702 (1950). (11) D. R . Brigge, 13. F. Carncr, R. Montgomery and F. Smith, Anat. Chcm., 28, 1333 (19W). (12) K . Freudenberg and G . Hull, Rer., 74, 237 (1941). (la) N. Prentice, L. S. Cuendet and F. Smith, THIS J O U R N A L , 78, 4439 (1956). (14) N.Prentice. L. S. Cuendct, W.17. Geddrs anti F. Smith, i b i d . 81,884 (1959).
(15) M. Abdel-Akher, J. K. Hamilton, R. Montgomery nnd 1. Smith, ibid., 74, 4970 (1952). (16) J. K. Hamilton and P. Smith, ibrd., 78, 5907 (1958). (17) E . P. McParren, Kathleen Brand and H . R. Rutkowski, A n d . Chcm.. 23, 11415 (1951). (18) L. Boggs. L. S. Cuendet, I. Ehrenthal. R . Koch and P. Smith, Nature, 166, 520 (1950). (19) C,. A . Adams, Con.J . C h r m . , 3 3 , 5 0 (1!>35). ( 2 0 ) S.h l . 1';rrtrirlge aud I
