Some structural features of Panax ginseng C. A. Mey pectin

CarbohydrateReword2 Elscvier PublishingCompany,Amsterdam

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PtintedinBeJgium

SOME

STRUCTURAL

FEATURES

OF Panax ginseng C. A. MEY

PECTIN

T. F. SOLOV'EVA, L. V. ARSNYUK,AND Yu. S. OVODOV Institute of Biologically Actine Substances, Vladivostok 22 (U. S. S. R.)

Siberian Department.

Academy of Sciences of the U. S. S. R..

(ReceivedJune21st, 1968; in revisedform, August23rd, 1968)

ABSTRACT

The’ pectin isolated from roots of Punax gizzseng C. A. Mey, contains residues of D-galacturonic acid, D-galactose, and L-arabinose, as main components, and those of D-xylose, L-rhamnose, and an unidentified sugar, as minor components of its carbohydrate chain. On partial hydrclysis with acid, the pectin afforded a a..~galacturonan having a linear chain of (1+4)- and, possibly, (l-,5)-linked galacturonic acid residues. Pectinase digestion of the pectin furnished a neutral, pectic polysaccharide in which L-arabinofuranose residues apparently occur in the exterior chains. On partial hydrolysis with acid, the polysaccharide afforded a branched galactan and an acidic heteropolysaccharide. The galactan contains chains of (1+3)- and (146)~linked D-galactopyranose residues. The heteropolysaccharide appears to contain sequences involving D-galactose residues, and others involving residues of D-galacturonic acid, D-xylose, and L-rhamnose. INTRODUCTION

Recently, we reported1 the isolation of a polysaccharide mixture, containing a starch-like glucan and a pectin, from the roots of Panax ginseng C. A. Mey. A

preliminary investigation of the pectin showed that it contained D-galacturonic acid, D-galactose, and L-arabinose as main components, and D-xylose, L-rhamnose, and an unidentified sugar as minor components. The present paper describes the elucidation of some structural features of Parzaxginseng pectin. EXPERIMENTAL

General experimental conditions. Partition chromatography was performed on Whatman 3 MM or Leningrad factory “Goznak” paper with the following solvent systems (v/v): (A) ethyl acetate-pyridine-water-acetic acid (5:5:3:1); (B) butanone saturated with 1% ammonia; and (C) ethyl acetate-acetic acid-water (l&7:8). Thin-layer chromatography (t.1.c.) of methyiated sugar derivatives was performed on silica gel “KSK” (> 200 mesh) with chloroform-methanol (1O:l) as developer, Curbohyd.

Res., 10 (1969) 13-18

14

T. F. SOLOV’EVA,

L. V. ARSENWK,

Y. S. OVODOV

T.1.c. of sugars was performed as described elsewhere’. Detection was effected with aniline hydrogen phthalate, silver nitrate in ammonia at lOSo/ min, and cont. sulphuric acid or saturated aqueous ammonium sulphate at llS’/lO min. Gas-liquid

chromatography

(g.1.c.) was carried out as previously described3. Pretreated4 DEAE-

cellulose (phosphate form) was used for ion-exchange chromatography. Molecularsieve chromatography (m.s.c.) was carried out on Biogel P columns’. The columns were calibrated with dextrans of known molecular weights. I.r.-spectra were recorded

on a Zeiss UR-10 spectrometer. Uranic acid contents were determined by a modified carbazole reaction6, and protein by the Lowry method’. Ash content was determinec? by heating polysaccharide samples to constant weight at 600”. The methoxyl content was determined by the Vieback and Schwappach method ‘. The samples were pretreated with water vapour to remove methanol ‘. The periodate consumption was determined spectrophotometrically lo . All solutions were concentrated in uacuo at 30-40” in a rotatory evaporator. Chromatography on DEAE-cellulose. Pectin (300 mg) ([cY]~~ +205” (in water); OMe, 5.4; uranic acid, 60.0; protein, 3.8; ash, 8.9%) in water (15 ml) was added to a column (3.6 x 40 cm) containing DEAE-celiulose in the phosphate form. The column was eluted in succession with 200 ml each of water, 0.01~, 0.025~~ 0.05~, 0.10~~ 0.25~, and 0.5~ sodium dihydrogen phosphate, and, finally, with a gradient of water-O.3hi sodium hydroxide (1 litre). The fractions (15 ml) were anaiysed by the phenol-sulphuric acid method’ ‘. After dialysis, polysaccharide material was recovered in the usual way. Three fractions were obtained: O.OlMsodium dihydrogen phosphate eluted a negligible amount of polysaccharide; 0.25~ sodium dihydrogen phosphate eluted polysaccharide (0.06 g) containing galacturonic acid, galactose, arabinose,xylose, and rhamnose residues; and sodium hydroxide eluted polysaccharide (0.10 g) having the same qualitative composition of monosaccharides. Periodate oxidation of the pectin. - The pectin (1.26 g) was added to sodium metaperiodate which was prepared from periodic acid (2.88 g) and cold acetate buffer (pH ca. 3.8). The reaction mixture was kept in the dark at 4’ with periodic shaking over 12 days. The consumption of periodate was monitored in the usual way on aliquots. When the reaction was complete, ethylene glycol was added to destroy the residual periodate. After deionization, the solution was treated with potassium borohydride (1 g) for 24 h, deionized, and evaporated. The residue (0.71 g) was hydrolysed with 2~ sulphuric acid for 10 h at 100”. The hydrolysate was examined by paper chromatography (solvent A) and t.1.c. Preparation of a galacturonan. - The pectin (8 g) in water (800 ml) was treated with 2~ sulphuric acid (80 ml) for 3 h at 100”. The hydrolysate was then poured into methanol to afford the galacturonan (2.46 g), [LX];’ +240” (water; (Found: OMe, 5.9; uranic acid, 99.0; protein, 0; ash, 1.5%). Esterzfication and reduction of the galacturonan. - The galacturonan (1.9 g) was triturated, wetted with methanol, and treated with ethereal diazomethane

overnight at 20”. The residue was filtered off and washed with ether. This product (1.88 g) was dissoIved in water (75 ml) and reduced with potassium borohydride (1 g) Curbohyd. Res.,lO(1969)13-18

P. ginseng

PECTIN

15

in water (25 ml). The reaction mixture was kept overnight in a refrigerator, neutralized with acetic acid, dialysed, treated with Amberlite IR-120 (H+) resin, and evaporated to dryness. Methanol was twice distilled from the residue, and the esterification and reduction procedures were repeated five times to give galactan 1 (0.98 g) (Found: OMe, 17.9; uranic acid, 6.0; protein, 0; ash, 0.66%). Periodate oxidation, reduction, and hydrolysis of galactan 1.- The polysaccharide (0.3 g) was oxidised in the dark with 0.05~ sodium metaperiodate (100 ml) for 3 days at 10”. After deionization, the solution was treated with potassium borohydride (0.2 g) for 24 h, deionized again, and evaporated. The residue was hydrolysed with N sulphuric acid for 7 h to yield a mixture of threitol and glycerol which was then examined by paper chromatography in solvent CPreparation and analysis of the methylated galactan 1.- Galactan 1 (0.66 g) was completely dissolved in methyl sulphoxide by stirring overnight. The solution obtained was treater for 6 h at 20” in an atmosphere of nitrogen with a solution of the methylsulphinyl carbanion prepared from sodium hydride (0.33 g) and methyl sulphoxide (9.9 ml), as described earlier12. Methyl iodide (5 ml) was added, and the reaction mixture was stirred in a nitrogen stream overnight. Recovery of the product (0.46 g) was achieved in the usual manner13, and this was remethylated in a similar way to afford the methylated galactan l(0.2g) which had 41% of methoxyl groups. Its i.r. spectrum showed the absence of free hydroxyl groups. The methylated galactan 1 (0.2 g) was dissolved in cold 72% sulphuric acid (0.1 ml) and kept for 1 h at room temperature. The reaction mixture was diluted with water (0.8 ml) and heated in a sealed tube for 4.5 h at 100°. After neutralization with barium carbonate, the solution was concentrated to a syrup (0.175 g), an aliquot of which was diluted with chloroform and examined by t.1.c. and paper chromatography in solvent B. The residual syrup (0.17 g) was transferred to a column (2.1 x 3 I.7 cm) of silica gel and eIuted with chloroform and then by a gradient of ethanol in chtoroform to give fractions (4 ml each) which were examined by t.1.c. Three combined fractions were obtained as follows: a mixture (4 mg) of 1,2,3,5,6penta-U-methylgalactitol and 2,3,4,6-tetra-U-methyl-D-galactcse; 2,3,6-tri-O-methylD-galactose (100 mg); and a mixture (29 mg) of the previous sugar and, probably, the methyl ester of 2,3-di-U-methyl-D-galacturonic acid (trace only). Further separation and purification was achieved by preparative, paper chromatography to give 2,3,4,6-tetra-0-methyl-D-galactose (25 mg), [a]$’ + 117” (water), which was converted into the “anilide”, m-p. and mixed m.p. 188”; and 2,3,6-tri-O-methyl-Dgalactose (80 mg), [a]:’ +80” (water), which was oxidised with bromine to give 2,3,6-tri-0-methyl-D-galactono-l&lactone, m.p. and mixed m.p. 99.3”. Methanolysis of the methylated galactan 1 (0.05 g) was achieved by treatment with a mixture of 72% aqueous perchloric acid and methanol (1:lO) for 3 h at 100” in a sealed tube. After neutralization and evaporation, the residual syrup was subjected to analysis by g.1.c. Enzymic digestion of the pectin. -An aqueous extract (1 litre of 1% solution) of roots of Panax ginseng was incubated with commercial pectinase (0.3 g) for 96 h at Carbohyd. Res., 10 (1969) 13-18

16

T.

F. SOLOV’EVA,

L. V. ARSENYUK,

Y. S. OVODOV

39-40”, filtered, and subjected to amylolysis as described previously’. The solution obtained was deproteinised by the Sevag procedure14 and poured into methanol to furnish a degraded, pectic polysaccharide, [a];’ +84” (water) (Found: uranic acid, 4.95; ash, 10.35%). The polysaccharide material (50 mg) was completely hydrolysed with 2~ sulphuric acid, yielding galactose, arabinose, xylose, rhamnose, and small proportions of galacturonic acid and an unidentified sugar having the highest relative mobility on thin-layer and paper (solvent A) chromatograms. Partial hydrolysis of the degraded, pectic polysaccharide. - The material (4 g) in water (300 ml) was hydrolysed with 2N sulphuric acici (30 ml) for I h at 100”. The mixture was cooled and poured into ethanol to yield B precipitate of galactan 2 (0.6 g) and a solute of heteropolysaccharide (0.7 g) which was precipitated with acetone. The product was completely hydrolysed with 2~ sulphuric acid to afford galacturonic acid, galactose, arabinose, xylose, rhamnose, and an unidentified sugar, as shown by t.1.c. and paper chromatography (solvent A). Preparation and analysis of the methylated galactan 2. - The galactan 2 (0.6 g) was methylated twice, as described above, to yield the fully methylated product (0.114 g) which contained 33.69% of methoxyl group and contained no hydroxyl groups as indicated by the i.r.-spectrum. Hydrolysis of the material was carried out as above, and the resulting mixture of methylated sugars was examined by t.1.c. and paper chromatography (solvent B). The mixture of corresponding methyl glycosides was examined by g.1.c. and t.1.c. Partial hydrol’ysis of the heteropolysaccharide. - The polysaccharide (1 g) was hydrolysed with 2~ sulphuric acid (25 ml) for 1 h at 100”. The hydrolysate was neutralised with barium carbonate, filtered, concentrated to 6 ml, treated with Amberlite IR-120 (Hf) resin, and transferred to a column with DEAE-Sephadex (HCO,). The column was washed with water until the effluent gave a negative phenol-sulphuric acid test. This procedure yielded two neutral oligosaccharides which were separated by preparative chromatography to afford galactobiose (3 mg) and galactotriose (4 mg). Further gradient elution with aqueous formic acid furnished two acidic oligosaccharides (10.7 and 6.6 mg), each of which, on hydrolysis, yielded D-galacturonic acid and rhamnose, whilst the latter saccharide also afforded xylose. RESULTS AND DISCUSSION

Attempts to fractionate the pectin by using ethanol, Cetavlon, precipitation with calcium chloride, and formation of cupric complexes were unsuccessful. However, by chromatography on DEAE-cellulose, the pectin was separated into two fractions that were acidic polysaccharides having the same quaIitative monosaccharide composition. Chromatography on DEAE-cellulose could not be used for separation of the pectin on a large scale; considerable loss of product occurred during the process, and the alkali used for elution degraded the pectin lg. On the above basis, the nonfractionated pectin was seIected for structural studies. The pectin consumed 0.76 mol CarbohydRex, 10(1969)

13-18

P.ginseng

17

PECTIN

of periodate per sugar moiety, and when the product was reduced with potassium borohydride and then hydrolysed, galactose and small proportions of arabinose and galacturonic acid were produced, suggesting the presence of (l-3)-linked sugar residues and branch points. It is known that the methylation of polysaccharides containing a high proportion of uranic acid residues is often difficult to achieve and may be accompanied by some degradation ’ ‘. Therefore, the pectin molecule was subjected to partial hydrolysis. A galacturonan was the first fragment obtained on hydrolysis of the pectin with 0.9% sulphuric acid for 3 h. The polysaccharide was converted, by esterification with subsequent reduction’ 6, into a corresponding galactan in order to facilitate methylation studies. The galactan 1 thus obtained was permethylated according to the method of Hakomori13 to give a product having a methoxyl content of 41%. Acid hydrolysis gave 2,3,6-tri-(mainly), 2,3,4,6- and 2,3,5,6-(trace)-tetra-O-methyl-D-galactose, 1,2,3,5,6-penta-O-methylgalactitol, and an unidentified sugar, possibly the methyl ester of 2,3-di-O-methyl-D-galacturonic acid. D-Galactose derivatives having a lower extent of methylation -werenot detected, indicating the absence of branch points in the polygalacturonan chain. It should be noted that 2,3,6-tri-o-methyl-D-galactosides were present in the methanolysate as pyranose and furanose forms in the molar ratio ca. 3:2 (as shown by g.1.c.). The above data demonstrated that galactan 1 contained a linear chain having (1+4)- and, possibly, (1+5)-linked D-galactose residues. Therefore, the galacturonan had a similar structure_ However, there are indications that a furanose form of D-galactose appeared on esterification and subsequent reduction of a polygalacturonan “, or it might be expected to arise under the conditions used in methanolysis of the methylated galactan. This phenomenon requires more-detailed investigation. The above structural features of galactan 1 were confirmed by periodate oxidation followed by reduction with borohydride and hydrolysis, yielding threitol and glycerol. The high, positive, specific rotation of the galacturonan demonstrated the predominant CL-Dconfiguration of the glycosidic bonds. A degraded, pectic polysaccharide was obtained by pectinase digestion of the pectin. The material contained D-galactose, D-galacturonic acid (small proportion only), L-arabinose, D-xylose, L-rhamnose, and an unidentified sugar (traces) having the highest mobility on chromatograms. Molecular-sieve chromatography of the degraded, pectic polysaceharide showed a wide distribution with regard to molecular size. Trace amounts of the degraded, pectic polysaccharide were eluted from DEAEcellulose with sodium phosphate. The main polysaccharide material appeared in effluents having higher pH-values, when elution with alkali was effected. These data demonstrated the acidic character of the polysaccharide4 and suggest a possible occurrence of bonds between acidic and neutral fragments of the pectin. Mild hydrolysis of the above polysaccharide with 0.9% sulphuric acid for 45 min resulted in the complete liberation of arabinose, suggesting that residues of this sugar were probably present in the furanose form in the exterior chains. The degraded pectic polysaccharide was subjected to partial hydrolysis with acid to afford Curfmhyd.Res., 10 (1969)

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18

1.

F. SOLOV’EVA,

L. V. ARSJZNYUK,

Y. S. OVODOV

galactan 2 (15.5%) and an acidic heteropolysaccharide (17.5%). The latter consisted of residues of D-galacturonic acid, D-galactose, D-xylose, L-rhamnose, and the unidentified sugar- mentioned above. The galactan 2 was permethylated and hydrolysed to yield 2,3,4,6_tetra, and 2,3,4-, 2,3,6-(traces only) and 2,4,6-t&, and 2,3-di-Omethyl-D-galactose, as shown by paper, thin-layer, and gas-liquid chromatography. These data indicate a branched structure for galactan 2 containing (l-+6)- and (1 +3)-linked D-galactose residues. On partial hydrolysis, the heteropolysaccharide yielded a mixture of mono- and oligo-saccharides, which were fractionated on DEAESephadex into acid and neutral fractions. Further separation was carried out by chromatography on filter sheets and gave a galactobiose and galactotriose, as well as two acidic oligosaccharides, respectively containing galacturonic acid and rhamnose, and gaIacturonic acid, rhamnose, and xylose. A more-detailed study of these oligosaccharides was not carried out because of the small amounts available. ACKNOWLEDGMENTS

We thank L. I. Glebko and J. I. Ulkina for microanalysis, and A. F. Pavlenko for g.1.c. analysis_ REFERENCES I Yu. S. OVODOV AND T. F. SOLOV’EVA, Khim. Prirodn. Soedinen., (1966) 299. 2 Yu.S. OVODOV, E-V. EW-USHENKO,V. E.VASKOVSKY,R. G. OVODOVA,ANDT. F.SOLOV'E~A, J. Chromatog., 26 (1967) 11 I. 3 Yu. S. OVODOV AND A. F. PAVLENKO, 3. Chromatog., 36 (1968) 531. 4 H. NEUKOM, H. DEUEL, W. J. HERI, AND W. K~NDIG, Helu. Chim. ACM, 43 (1960) 64. 5 D. M. W. ANDERSON AND J. F. STODDART, Curbohyd. Res., 2 (1966) 104. 6 T. BIITER AND H. M. MUIR, Anal- Bfochem., 4 (1962) 330. 7 0. H. LOURY, N. J. ROSEBROUGH, A. L. FASR, AND R. I. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 F. VIEBBCK AND A. SCHWAPPACH, Ber., 63 (1930) 2818. 9 Z. I. KERTE~Z, The Pecfic Substances, Interscience Pubs., New York-London, 1951, p. 238. 10 G. 0. A~PINALL AND R. J. FERRIER,Ckem. Znd. (London), (1957) 1216. 11 M. DUBOIS, K. A. Grrres, J. K. HAMILTON, P. A. REBERS, AND F. S&IITH, Anal- Chem., 28 (1936) 350. 12 E. J. COREY AND M. CHAYKOVSKY, J. Amer. Chem. Sot., 84 (1962) 866. 13 S. HAKOMORI, J. Biochem. (Tokyo), 55 (1964) 205. 14 M. S. SEVAG, Biochem. Z., 273 (1934) 419. 15 K. HUNT AND J. K. N. JONES, Can. J. Chem., 40 (1962) 1266. 16 G. 0. ASPMALL AND A. CARAS-RODRIGUEZ, J. Chem. Sot., (1958) 4020. 17 G. N. BEAVEN AND J. K. N. JONES, J. Sot. Chem. Znd., 58 (1939) 363. 18 H. NEIJKOM AND H. DEIJEL, Chem. Znd. (London), (1958) 683.

Curbohyd. Res., 10 (1969) 13-18