Constitutional Studies on the Mucilage of "Yamanoimo. Dioscorea batatas Decne, forma Tsukitne. Isolation and Structure of a Mannan õ

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1 [Agr. Biol. Chem., Vol. 36, No. 5, p , 1972] Constitutional Studies on the Mucilage of "Yamanoimo Dioscorea batatas Decne, forma Tsukitne," Isolation and Structure of a Mannan õ By Akira MISAKI, Toshiko ITO* and Tokuya HARADA The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka *Kinran Women College, Suita, Osaka Received September 20, 1971 A mucilage extracted from tubers of Dioscorea batatas Decne, forma Tsukune with cold water has been found to contain a mannan associated firmly with protein. A water-insol uble mannan ([ƒ ]D-31, formic acid), isolated from the mucilage by formation of copper hydroxide-complex, is a ƒà(1 4)-linked D-mannan of _??_ 110. Methylation and periodate oxidation studies indicated that it has a small degree of branching at the C-3 positions of the mannose residues, one out of units. The oligosaccharides, separated from the enzymic digest of the mannan, were characterized by methylation analysis to be 4-O-ƒÀ-Dmannosyl-D-mannose, and O-ƒÀ-D-mannosyl-(1 4)-13-D-mannosyl-(1 4)-ƒÀ-D-mannose. The native marman ([ƒ ]D-30.6 in water), isolated from the mucilage after degrada tion of the protein moiety with u proteolytic enzyme, was homogeneous and had molecular weight 23,000 (sedimentation analysis). It is partially acetylated (O-acetyl, 14.2 ), most probably at the C-2 and or C-3 positions. On the basis of these findings, the constitution of the mucilage has been discussed. "Y amanoimo" or Chinese yam which belongs to Dioscoreasceae is native to China, and has been cultivated in the temperate zone of Japan. The tubers of Dioscorea batatas Decne. and D. batatas Decne. forma Tsukune Makino are particularly used as a mucilageous food article. These tubers contain, in addition to starch, a water-soluble mucilage that is ex tractable with cold water. In 1928, Taka hashi1,2) showed that the mucilage comprises a protein and mannan-like polysaccharide, probably as a form of glycoprotein, although the presence of a hexosamine could not be confirmeḍ Until quite recently, very little õ This study was supported by u research grant from the Ministry of Education. A part of this work was presented at the meeting of the Agricul tural Chemical Society of Japan 1969., in Tokyo, April, was known about the nature of the tuber mucilage. Sato et al.3) obtained a homogene ous fraction from the mucilage of D. batatas Decne. forma Icho by treatment with dodecyl sulfate. It contained marman 48%, protein 10% o and phosphorous 3 `4%. They sug gested that the mannan component is a linear chain consisting of ƒà-(1 4)-D-mannosidic linkages, by paper chromatographic identification of the acetolysis products." The presence of fructose and glucose was reported as the car bohydrate constituents of the mucilage from D. batatas Decne. forma tsukune.5) During investigations on the relationships between structures and physical properties of plant and microbial mucilages, our attention was drawn to the nature of mucilage of "Yamanoimo." This paper is concerned with the constitution of the water-soluble mucilage

2 762 A. MISAKI, T. ITO and T. HARADA of D. batatas Decne. forma Tsukune, particu larly the structural features of a mannan iso lated from it. Analytical EXPERIMENTAL AND RESULTS methods Paper chromatography was usually performed on Toyo-roshi No. 51 paper or Whatmann 3 MM paper by the descending method with the following solvent systems (v/v): (A) 1- butanol-ethanol-water (4:1:5), (B) 1-butanolpyridine-water (6:4:3), and (C) butanonewater azeotrope. Reducing sugars were detected by spraying the chromatograms with p-anisidine hydrochloride, and sugar alcohols were detected with alkaline silver nitrate reagent. Paper electrophoresis was effected on Toyo roshi No. 52 paper with 0.1 M sodium borate, at 600 volts for 3.5 hr, and electrophoreto grams were sprayed with p-anisidine trichlo roacetic acid. Gas-liquid chromatography (glc) was carried out with a Hitachi K53 Gas-Chromatograph, fitted with a flame ionization detector, using a stainless steel column (0.4cm, i.d.). Methyl sugars were converted into their corresponding methyl glycosides and separated on a column (1 m length) of 15% butanediol suc cinate polyester on Neosorb NC (Nishio Ind. Co.). Nitrogen was used as a carrier gas, at a flow rate of 60 ml/min. Retention times (Tg) are quoted relative to that of 2,3,4,6- tetra-0-methyl-ƒ -methyl-d-glucoside. Sugar alcohols were converted into their acetates by heating with pyridine and acetic anhydride and separated on a column (2 m length) of 3/o ECNSS-M on Gas Chrom Q,6) at 60 ` 190 (programed at a rate of 7 /min). Determination of carbohydrates was generally made by the phenol-sulfuric acid method." A glucose oxidase reagent (Glucostat, Wor thington Biochemical Co.) was also used for assay of glucose. Formaldehyde was determined by the method of Hanahan et al.8) Acetyl content of the native mannan was determined colorimetrically by the procedure of McComb and McCready.91 Amino acid composition of the mucilage was analyzed by a Hitachi KLA-2 amino acid analyzer, after hydrolysis in an evacuated tube with 6 N hydrochloric acid at 100 C for 16 hr. Sedimentation analysis of the native mannan was conducted in a Spinco Model E analytical ultracentrifuge. For estimation of molecular weight by the Archibald method,"' the value for specific volume of cm3/g was adopted. Infrared spectra were measured on KBr disks (polysaccharide, and the acetyl deriva tive) or in chloroform solution (methylated product), with a Hitachi Infrared Spectro photometer, Model EP 1-2. Preparation of mucilage The sequence of procedure for separation of the mucilage and the polysaccharides is shown in Scheme 1. Fresh tubers of Dioscorea SCHEME 1. Fractionation Procedure of Constituents of "Yamanoimo". batatas Decne. forma Tsukune Makino, culti vated in Kyoto prefecture, were sliced, and a portion (1 kg; 32.5 g in dry weight) was homogenized mechanically in the presence of

3 Constitutional Studies on the Alucilage of,yamanoimo," Dioscorea batatas Decne 763 water (3.5 liters) at 10 Ž for 12 hr, after which the homogenized mixture was centrifuged (10,000 rpm, 30 min). The sediment was again treated with water (2.5 liters), and to the combined clear viscous solution was added 2.5 volumes of acetone to precipitate the crude mucilage. It was dissolved in water (500 ml), centrifuged and dialyzed, and the mucilage was precipitated by the gradual addition of acetone (yield, 2.45 g). The mucilage prepara tion ([ƒ ]D,-10.8 in water) gave no color with iodine. It contained 11.32% N and 13.8% carbohydrates (as glucose). Acid hydrolysis with 2 N sulfuric acid at 100 Ž for 6hr gave mannose together with a small amount of glucose, as revealed by paper chromatography. The amino acid composition of the mucilage after hydrolysis with 6 N hydrochloric acid is shown in Table I. TABLE I. AMINO ACID COMPOSITION OF THE MUCILAGE The sample was hydrolyzed in a evacuated sealed tube with 6 N HCl, at 100"C, for 16 hr, and the hydrolyzate was analyzed with a amino acid analyzer. was thoroughly washed with water, ethanol and diethyl ether, and dried in vacuo (yield, 2.6 g). The mannan isolated through the coppercomplex was not soluble in water and in 1 N sodium hydroxide, but dissolved gradually in formic acid. It had [ƒ ]28D-(c=2, in formic acid). Anal. Found: C, 41.34; H, 5.87; N, 0.0. Calcd.: C, 41.9; H, 5.82, as (C6H10O5),,. The polysaccharide (100mg) was hydrolyzed with 90% formic acid (10ml) at 100 Ž for 3 hr, followed by treatment with 1 N sulfuric acid for 0.5 hr to hydrolyze the formyl ester. After neutralization with barium carbonate, the hydrolysate was evaporated to a syrup, from which D-mannose was crystallized, [a]' (c=2.5 in water, after 24hr), mp and mixed mp 130 `132 Ž. D-Mannose was converted into its phenylhydrazone by the usual manner, mp and mixed rap 199 ` 200 Ž. Since paper chromatography (solvent B) showed that the mother liquor of the crys talline mannose contains small quantities of glucose and galactose, the acid hydrolyzate of mannan (50mg) was subjected to paper electrophoresis, which separated mannose (MG 0.7) and a mixture of galactose and glucose (MG ). The latter components were extracted from the paper with water, and after removal of borate as methyl borate by evaporation with methanol, the mixture was fractionated by paper chromatography. The colorimetric determination of glucose and galactose after separation on paper chromato a) Expressed as percent to total amino acids. Isolation of mannan The above mucilage (20 g) dissolved in water (1 liter) was treated with Fehling solution (1.5 liters), and the precipitated mannancopper hydroxide complex was washed with water, after which the complex was decomposed by addition of 1 N hydrochloric acid until the blue color disappeared. The white precipitate grams indicated that the contents of glucose and galactose are 1.8 o and 0.9%, respectively. The mannan preparation showed similar absorption peaks in the infrared spectrum similar to those for a known plant mannan,11) including those at 810 (type 3), 867 (type c), and 890 cm-1 (type 2b). Methylation analysis of mannan The mannan was methylated by the Hako mori method.12) The polysaccharide (1g) was

4 764 A. MISAKI, T. ITO and T. HARADA stirred in methylsulfoxide (30ml) at 60 C for 1 hr. Since it was not solubilized completely, stirring was continued for further 2 hr at 80 C under a nitrogen stream. After cooling, methylsulfinyl carbanion (10ml), prepared by the reaction of sodium hydride and methyl sulfoxide according to the procedure of Sand ford and Conrad13) was added under a nitro gen stream, and the resulting alkoxide was treated with methyl iodide at 20 C. The re action mixture was dialyzed, and the methy lated mannan in the nondialysable solution was extracted with chloroform in the usual manner. To ensure complete methylation, the methylated product was further methy lated twice with methyl iodide (25ml) and silver oxide (2g) by refluxing for 18 hr. After evaporation of methyl iodide, the methylated mannan was dissolved in chloroform and precipitated by addition of peteroleum ether (yield, 600 mg). The resulting powder showed no absorption band at 3400 `3600 cm-1 of the infrared spectrum. The methylated mannan (300 mg) was meth anolyzed by refluxing with 4.2%, methanolic hydrogen chloride (6 ml) for 24 hr. After neutralization with silver carbonate, the meth anolysate was evaporated to a small volume. As shown in Fig. 1, gas-liquid chromatography revealed the presence of the methyl glycosides of 2,3,4,6-tetra-O-methyl-D-mannose (Tg 1.00), 2,3,6-tri-0-methyl-D-mannose (Tg 3.32) and a di-0-methyl-d-mannose (Tg 7.20), together with a very small peak (Tg 2.38) corresponding to methyl 2,3,6-tri-0-methyl-ƒÀ-D-glucoside. The mixture of glycosides (260mg) was hydrolyzed with 1 N sulfuric acid at 100 C for 24 hr. After neutralization (BaCO3), the hydrolysate was evaporated to a syrup (234 mg). For identification of the methyl sugars, the hydrolysate (125mg) was fractionated by thick paper chromatography (Whatman 3MM paper, solvent C). Each component was eluted with water from the paper, and con centrated to a syrup. The methyl sugars were identified as follows: (a) 2,3,4,6-Tetra-O-methyl-D-mannose (9.7mg). Rf 0.78 (solvent C); [ƒ ]25D+14.4 (c, 1.4 in water). The aniline derivative had mp and mixed mp, C. (b) 2,3,6-Tri-O-methyl-D-mannose (80.5 mg), Rf 0.56; [ƒ ]25D-8.8 (c, 2.7). The aniline deriva tive had mp and mixed mp, 122 `123 Ž. This component was shown by glc to contain a trace of 2,3,6-tri-O-methylglucose. (c) 2,6-Di-O-methyl-D-mannose (8.3 mg). The dimethylmannose fraction having [a]u+19.5 (c, 2) gave a single spot on paper chromatography and electrophoresis (Rf 0.27; MG 0.08, and a red color with p-anisidine spray reagent), identical to that of a synthetic 2,6-di-O-methyl- D-mannose kindly gifted by Dr. Perry.14) On glc the methyl glycoside (Tg 7.20) of this sugar was indistinguishable from the methyl FIG. I. Gas-liquid Chromatogram of the Metha nolysate of Methylated "Yamanoimo" Mannan. Conditions: 15% butandiol succinate polyester on Neosorb N.C., 175 (I) Methyl 2,3,4,6-tetra- 0-methyl-D-mannoside, (II) possibly, methyl 2,3,6- tri-o-methyl-ƒà-d-glucoside, (III) methyl, 2,3,6-tri- O-methyl-D-mannoside, and (IV) methyl 2,6-di-Omethyl-D-mannoside. glycoside of 2,6-di-O-methyl-mannose. The molar ratio of tetra-, tri- and dimethyl mannose was 1: 8.9:0.9 on a weight basis, and the ratio of tetra- and trimethylmannose estimated by glc was 1 : 9.2. Periodate oxidation The mannan (40 mg) was suspended in water

5 Constitutional Studies on the Mucilage of "Yamanoimo," Dioscorea batatas Decne 765 (60ml) and 0.1 N sodium periodate (40ml) was added, the oxidation being conducted with mechanical stirring at 5 Ž in the dark. At suitable intervals, the consumption of perio date and the production of formic acid were determined by arsenite method and by titra tion with 0.01 N sodium hydroxide, respective ly. Extrapolation of the data on a graph shown in Fig. 2 indicates that the polysac charide consumed 0.95 mole of periodate per sugar residue with production of 0.10 mole of formic acid. was distilled off as methyl borate. Examin ation by paper chromatography (solvents A and B) revealed the presence of erythritol (major), glycerol and mannose. The colori metric determination of these compounds after separation on a paper chromatogram showed that the molar ratio of glycerol, ery thritol and mannose was 1.0: 9.6: 1.1. For analysis by glc, a portion of the hydrolysate (15mg) was reduced with sodium borohydride (5mg) to convert the mannose into mannitol. After decomposition of the excess borohydride by the addition of Amberlite IR-120 (H+form) followed by removal of boric acid as methyl borate, the reduced product was acety lated by heating with pyridine (0.2 ml) and acetic anhydride (0.2 ml) at 100 C for 1 hr."' The reaction mixture was concentrated to a syrup, which was dissolved in a small volume of chloroform, and then injected into a gas chroinatograph, equipped with a column of 3% ECNSS-M on Gas Chrom Q. The separa tion of the compounds are shown in Fig. 3. The molar ratio of glycerol, erythritol and mannitol was found to be approximately 1.0: 9.2: 1, in good agreement with the result by paper chromatographic determination. FIG. 2. Rates of Periodate Consumption and Formic Acid Production during Oxidation of Mannan, with 0.04 M Sodium Periodate at 5"C. Smith degradation15) The mannan (200 mg) was oxidized with 0.05 M periodic acid (200 ml) for 10 days in the manner described above, and the oxidized mannan was reduced with borohydride at room temperature. The resulting mannan polyalcohol was hydrolyzed with 1 N sulfuric acid at 100 C for 6 hr. The hydrolysate was neutralized (BaCO3), filtered, and the filtrate was passed through a column of Amberlite IR-120 (H+-form). The acidic solution was evaporated and the boric acid in the residue FIG. 3. Gas-liquid Chromatogram of Acetyl Deriva tives of the Reduced Products after Smith De gradation of Mannan. Condition: 2% ECNNS-M on Gas Chrom Q, 60 `19 (programed at a rate of 7.5 /min). (I) Glycerol, (II) erythritol, and (II) mannitol.

6 766 A. MISAKI, T. ITO and T. HARADA Enzymic degradation of mannan and characteriza tion of the products Since a preliminary experiment showed that the mannan was solubilized by a crude cellulase preparation from Aspergillus niger (Miles Chemical Co.), probably owing to the presence of a ƒà-d-mannanase, the mannan (3g) suspended in water (75ml) was incubated with the cellulase (100mg) in 0.1 M acetate buffer (ph 5.4, 75 ml) at 37 Ž for 40 hr. The enzymic digest was centrifuged, and the supernatant fluid was deionized by passage through columns of Amberlite IR-120 (H+form) and Dowex I (OH--form) resins, the neutral solution being concentrated to a small volume. Paper chromatography (solvent B, two developments) and paper electrophoresis (5ml) was added, and the methylated product was extracted with chloroform (10mlx5), and the extracts were washed with water, dried over with MgSO4, and concentrated to a syrup. After methanolysis by heating with 2.5% methanolic hydrogen chloride, the mixture of methyl mannosides was examined by glc using a butandiol succinate polyester column at 175 C, which showed an equimolar pro portion of methyl 2,3,4,6-tetra-O-methyl, and methyl 2, 3, 6-tri-O-methyl-D-mannoside. A portion of the mannobiose (25mg) was con verted into the crystalline phenylosazone, which showed mp 187 `189 Ž (lit. 203 ` 206 Ž)."' The above results indicate that the mannobiose is 4-0-ƒÀ-D-mannopyranosyl-Dmannose. revealed the presence of mannose (R -N, 1.0; predominant), mannobiose (RM 0.64), manno triose (RM 0.34) and probably mannotetraose (R,1 0.12), together with a trace amount of glucose. For fractionation of these oligosac charides, the digestion products were dissolved in water (10 ml) and subjected to chromato graphy on a charcoal-celite column (3 ~25cm). After elution of the bulk of monosaccharide (1.86g) with water (750ml), oligosaccharide components were eluted successively with 5 to 15% aqueous ethanol, yielding the following fractions: fraction I (260mg; eluted with 5% ethanol, 500ml) which contained manno biose (major) and mannose, fraction II (110mg; 0-ƒÀ-D-Mannopyranosyl-(1 4)-O-(3-D-mannopyrano syl-(1 4)-D-mannose This fraction had [ƒ ]D-20.6 (c, 1.7, water), RN, 0.30 and Me, Methylation followed by methanolysis gave methyl 2,3,4,6-tetra-, and 2,3,6-tri-O-methyl-D-mannoside in the approximate ratio of 1:2, as revealed by gic. This indicates that the trisaccharide is ƒà-(l 4)-linked D-mannotriose. In addition to the mannotriose, a small amount of an oligosaccharide, having [ƒ ]D (c, 1.5, water), R, and MG 0.57, was isolated. This component was presumed to be a ƒà-(1-4)-linked D-mannotetraose. eluted with 10% ethanol, 500ml) which con tained mannotriose and -tetraose; no sugar was obtained with 15% ethanol. Fraction I and II were further fractionated on thick papers using solvent B, and each oligosac charide was characterized as below. Degree of polymerization of the mannan The mannan (76.7mg) was suspended in water (3ml) and reduced with sodium boro hydride (30 mg in l ml water) at room tem perature. The excess of sodium borohydride was decomposed by the addition of acetic 4-O-ƒÀ-D-Mannopyranosyl-D-mannose This fraction had [ƒ ]D-8 (c, 1.3, water), Rat 0.64 (solvent B) and Me A portion (17mg) was dissolved in methylsulfoxide (1ml), and methylated with methylsulfinyl carbanion (0.5ml) and methyl iodide (0.4ml), as de scribed previously. After methylation, water acid (ph 5.5), and the reduced mannan was oxidized with 0.05 NI sodium periodate (20ml) at 5 C for 48 hr. To a portion of the reaction mixture (5ml) was added 10,0 sodium bisulfite (1ml) and then ethanol (5ml) to precipitate the oxidized polysaccharide."' The mixture was centrifuged, and aliquots (1 ml) of the

7 Constitutional Studies on the Mucilage of "Yamanoimo,' Dioscorea baratas Decne 767 supernatant fluid was used for colorimetric determination of formaldehyde. The average of triplicate measurements showed that the DP of mannan is 110, assuming that the re ducing end unit is linked by a (1 A) bond. Isolation of a partially acetylated mannan from Pronase-digested mucilage The mucilage, extracted from the tuber with water, contains both protein and man nan, and was soluble in water, but when the mannan was separated from the mucilage as its copper complex it became insoluble in water. Therefore, an attempt was made to isolate the mannan by a milder procedure. The mucilage (ca. 1g) was dissolved in water (100 ml), to which was added Pronase-P (4 45,000 P.U.K./g, 100 mg) dissolved in 0.2M citrate buffer (ph 7, 100 ml). The mixture was incubated at 40 Ž. The viscosity of the solution decreased gradually and became con stant after 90 hr (relative viscosity, , measured at 27 C). The digest was heated at 80 C for 10 min to inactivate the enzyme, and was treated again with Pronase (50 mg) for 48 hr. The digest was centrifuged to re move a small amount of insoluble material, and the supernatant fluid was dialyzed. The non-dialysable solution was concentrated to a small volume, and then subjected to gel fil tration using a Sephadex G-50 column (2:40 cm). The carbohydrate component which was eluted with water showed a symmetrical the spectrum was very similar to that of the chemically acetylated mannan by the reaction with trifluroacetic acid,19) except OH-band at 3400 `3600 cm-1, as shown in Fig. 4. Colori metric determination of O-acetyl groups in dicated that the water soluble (native) man nan contained acetyl groups. This partially acetylated mannan gave a FIG. 4. Infrared Spectra of (A) Mannan Isolated via Copper Complex, (B) Chemically Acetylated Mannan, and (C) Native Mannan. peak. It was precipitated by the addition of 80% ethanol (yield 173 mg). The polysaccharide, so obtained, was readil) soluble in water, and showed intrinsic viscosity, [ƒå] 1.28 at 25, and [ƒ ]25D-30.6 (c=1.7). Ele mental analysis showed it to contain neither nitrogen nor phosphorous, but it yielded on acid hydrolysis traces of ninhydrin positive compounds, in addition to the major com ponent, D-mannose. In infrared spectrum, the polysaccharide showed absorption bands at , and 1240 cm-1, characteristic to acyl groups, and FIG. 5. Sedimentation Diagrams of Native Mannan. Measurements were made at a concentration of 5 mg/ml of polysaccharide in 0.1M NaCl. The photographs were taken at (A) 90 min, and (B) 150 min after attaining top speed (59800 rpm) at 20 C, with a Spinco Model E Ultracentrifuge (sedimented from right to left).

8 768 A. MISAKI, T. ITO and T. HARADA single symmetrical peak (S20,w=1.4S) in ultra centrifugation, and its average molecular weight was calculated to 23,000. The structural investigations of the Descorea batatas mannan were performed on the man nan isolated via its copper complex. The mannan, obtained in a 13% yield from the DISCUSSION The mucilage obtained from the tubers of D. batatas Decne, forma Tsukune in yield (dry weight basis) was free of starch. This preparation ([a]1,-10.8 ) contained approxi mately 71.5% protein (N, 11.3%) and carbohydrate (estimated as glucose); it is readily soluble in water to give a viscous so lution. Acid hydrolysis yielded mannose and small amounts of other sugars such as glucose, and various amino acids. The major amino acids are aspartic acid ( ), glutamic acid ( ) and leucine (7.23 0) as seen in Table I. The amino acid composition in the present preparation is somewhat different from the results obtained by previous studies.2,3,5) In addition, analysis of the amino acid frac tion on the autoanalyzer revealed the presence of a trace amount of a glucosamine-like com ponent; however, it is not yet clear whether the compound is the true amino sugar or an artifact. It is interesting that there has been some dispute as to the presence of amino sugars in the mucilage, since the first sugges tion by Oshima and Tadokoro in 1912,201 but direct chemical evidence is still lacking. The residue of the water-extract of the tubers was treated with 1 N sodium hydroxide in the cold, yielding a water-soluble starchlike polysaccharide. Its chemical constitution will be reported elsewhere. The mannan was isolated from the watersoluble mucilage by two different methods. The first procedure involved the formation of insoluble mannan-copper hydroxide com plex, which gave a water-insoluble mannan. The second procedure which involved removal of the protein moiety by treatment of the mucilage with Pronase-P gave a water-soluble, partially acetylated mannan, designated as native mannan. mucilage, is insoluble in water and in dilute alkali, but like other plant mannans is soluble in formic acid. It had [ƒ ]D-31 (formic acid), and gave on acid hydrolysis D-mannose, iden tified as its crystalline solid and phenylhydro zone. In addition, the mannan contained small proportions of D-glucose (1.8 0) and D- galactose (1 0), nevertheless, it should be re garded as a true mannan according to the definition of Aspinall.21) Since complete methylation of the mannan could not be achieved by the Haworth method it was methylated by the Hakomori and Purdie methods."' Gas-liquid chromatography of the methanolysis products from fully methylated mannan revealed the presence of 2,3,4,6-tetra-O-methyl-,2,3,6-tri-O-methyl- and a di-o-methyl-d-mannose; the molar ratio of tetramethyl- and trimethyl-mannose was 1: 9.2. The di-o-methyl sugar was characterized as 2,6-di-O-methyl-D-mannose by paper elec trophoresis and from its retention time on gas-liquid chromatography. The above results indicate that the mannan has a backbone of ƒà-(1 4)-linked D-mannopyranose residues, sim ilar to other plant mannans, such as Ivory nut mannan,17,23) coffee bean mannan24) and the mannan of a green seaweed."' However, the isolation of 2,6-di-O-methyl-D-mannose indicates that the present mannan possesses a low degree of branching with an average re peating unit of 10 `11 sugar residues, branching occuring at the C-3 positions of (1 4)- linked mannose residues. The degree of polymerization, 110, estimated by chemical method indicates that the mannan has a longer chain length than other plant mannans. For instance, Ivory nut mannans, A and B, both of which are linear molecules, have degrees of polymerization and 38-40, respectively.23) In addition to the above methyl mannose fragments, a very small

9 Constitutional Studies on the Mucilage of "Yamanoimo," Dioscorea batatas Decne 769 amount of 2,3,6-tri-O-methylglucose was de tected by gas-liquid chromatography, suggesting the possible presence of (1 4)-linked glu cose residues in the mannan molecule. The branched structure of "Yamanoimo" mannan was supported by periodate oxidation and the Smith degradation. On periodate oxidation the (1 4)-linked mannan produced formic acid corresponding to one non-reducing end for every ten mannose units. The Smith degradation, which involves periodate oxida tion, borohydride reduction and hydrolysis with acid, gave glycerol, erythritol and mannose. Since the glycerol and erythritol must fied further on paper to give di-, tri- and tetrasaccharides. The disaccharide and trisac charide were characterized as 4-0-ƒÀ-D-man nosyl-d-mannose [I,n=0], and 0-ƒÀ-D-mannosyl- (1 4)-0-ƒÀ-D-mannosyl-(1 4)-D-mannose [I, n= 1], respectively. The tetrasaccharide was not characterized completely, but the paper chromatographic and electrophoretic examin ation indicated it to be ƒà-(1 4)-linked D- manno-tetraose [I, n=2]. Although a branched oligosaccharide was riot obtainable, the above results confirm that the mannan chain comprises mainly 13-(l 4)-linked D-mannose residues, as suggested by methylation study. have arisen from the non-reducing terminals and the (1-4)-linked D-mannose residues, re spectively, and the mannose from the branch points substituted at C-1, C-4 and C-3 posi tions, the ratio of these compounds, 1.0: 9.2: 1.1, is in good agreement with that of tetra-, tri- and di-0-methyl sugars obtained from the methylated mannan. It is not possible to say whether the side chains are built up from single sugar residues or from several sugar residues, but the fact that the mannan preparation is insoluble in water and in dilute alkali suggests that the side chains must be very short, probably with single sugar unit. Consequently, the linear portions of the molecules will be able to as sociate with each other through hydrogen bond, as the case of unbranched ƒà-(1 4)- linked D-mannans. Additional information on the structural units of the mannan was provided by isolation and characterization of the oligosaccharides from the enzymic digest. When the mannan was incubated with a crude preparation of cellulase from Aspergillus niger, the polysaccharide was gradually solubitized with release of monosaecharides (mannose and a trace of glucose), and a series of D-manno-oligosac charides, indicating that the enzyme prepara tion contains ƒà-d-mannan hydrolase. The digestion products were fractionated by char coal-celite column chromatography and puri The fact that the tuber mucilage containing approximately 10% of D-mannan is solu ble in water, whereas the mannan isolated via its copper-complex becomes insoluble in water, prompted further investigation of the nature of mannan. In order to remove the protein moiety the mucilage was subjected to digestion with Pronase, after which the digest was dialyzed, and the non-dialyzable fraction was purified by gel-filtration on Sephadex G-50, which gave only a single peak of carbohydrate. The native mannan precipitated with ethanol is soluble in water and gives viscous solution. It gave a single symmetrical peak on ultracentrifugation (Fig. 5), and had a molecular weight of 23,000, calculated by the Archibald method. The infrared spectrum of the native man nan shows absorption peaks at , and 1240 cm-1, due to ester groups, and is similar to that of the acetylated mannan, except OHband (3400 `3600 cm-1). The chemical analy sis indicated that it contains acetyl groups,

10 770 A. MISAKI, T. ITO and T. HARADA FIG. 6. A Possible Structure of "Yamanoimo" Mannan. corresponding to approximately one acetyl group per two sugar residues. An attempt to show the exact location of the O-acetyl groups by use of protective reagent such as methyl-vinyl ether26) was unsuccessful because of the insolubility of the partially acetylated mannan in methyl sulfoxide. Analysis of the products by the Smith degradation was em ployed to provide further information. The native mannan was subjected to prolonged periodate oxidation (0.1 N periodic acid at 5 C for 21 days), followed by borohydride reduc tion and the resulting polysaccharide polyalcohol was completely hydrolyzed with 1 N sulfuric acid. The resulting polyhydric alco hols were acetylated and subjected to gas liquid chromatography, giving the molar proportions of glycerol, erythritol and mannose (mannitol): 13%, 29%, and 58%; under the same condition, the deacetylated mannan gave these compounds in the proportions, 8.9%, 82%, and 9.1%. From these results it can be deduced that the O-acetyl groups are attached in such a manner that they block periodate cleavage of the (3-(1 4)-linked D- mannose residues, most probably at the C-3 or C-2, or at both positions. In this connec tion, it may be pointed out that the acetyl groups in naturally occuring acetylated xylans, which is water-soluble, are mostly linked at C-3, and the remainder at C-2 positions of Ĉ-(1 4)-linked D-xylose units.27) The optical rotation of the native mannan (-30.6, water) is consistent with that of the insoluble mannan (-31, formic acid)., Moreover, the molecular weight, 23,000, measured by sedimentation analysis, is very close to 20,900, the calculated value, by assuming that the mannan of DP 110 contains acetyl groups. In Fig. 6, a possible structure of "Yamanoimo" mannan is illustrated. The solubility properties of the native man nan may be attributed to the presence of a small degree of substitution which confers ir regularity to essentially linear chains, this resulting in water solubility of the mannan. In the process of separation of the mannan from the water-soluble mucilage via the copper complex, the O-acetyl groups most probably are hydrolyzed under the alkaline condition, and the deacetylated mannan would no longer be soluble because of molecular association of its linear chains through hydro gen bonds. Satoh et al." reported the presence of acetyl groups in the mucilage of D. batatas, forma Echo, and they suggested that the acetyl groups are linked to the mannan moiety. From the present results it is clear that the acetylated mannan is chiefly responsible for the viscosity characteristics of the mucilage. However, it is not possible at this time to say whether the mannan is linked covalently with the protein moiety, or present in the form of the polysaccharide-protein complex by certain type of molecular association, although the findings that the native mannan isolated by use of Pronase digestion was free from nitrogen and also free from phosphate seem to be in favour to the later system. Further investigations on the nature of the mucilage are being pursued. Acknowledgements. The authors wish to thank Dr. M. B. Perry, National Research Council, Ottawa, Canada, for LL gift of 2,6-di-O-methyl-D-mannose. Thanks are also due to Dr. A. Kakinuma, Takeda Chemical Industry, Ltd., for sedimentation analysis of the mannan. REFERENCES 1) T. Takahashi, Nippon Nogeikagaku Kaishi, 4, 191 (1928).

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