Phytochmistry, Vol. 30. No. 9, pp. 3017 u)20, 1991 Printed in Great Britam.

WATER-SOLUBLE

0031~-9422/91 s3.00+0.00 Q 1991 Pergamon Press plc

POLYSACCHARIDES JOSEF

FROM

GINKGO BZLOBA LEAVES

KRAUS

Institute of Pharmaceutical Biology, University of Regensbur& Universitltsstrasse 3 1, D-8400 Regensbur8, Germany (Receioed in reuisedjorm

1 February 1991)

Key Word Index-Ginkgo biloba;Ginkgoaceae; leaves; water-soluble polysaccharides; structural analysis. Abstract-The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GFl) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the M, distribution GFl and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GFl (M, 23 000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (M, 500000) consisted mainly of 1,2,4-branched mannose (29%), 1.4~linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of co 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,Zlinked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (M, 40000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (IS%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose. (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.

INTRODUCTION

Ginkgo biloba L. is the only surviving species of the Ginkgoaceae. Extracts from the leaves of G. biloba are known to exhibit positive effects on peripheral as well as cerebral blood circulation. These activities are mainly based on the content of flavonoids and terpenoids Cl, 21. Recently, Itokawa et al. [3] reported antitumour active long-chain phenols in G. biloba. Apart from some preliminary investigations [4] nothing is known about the water-soluble polysaccharides. With regard to the taxonomical classification, the composition and the structure of Ginkgo polysaccharides seemed to be of great interest. The present paper concerns the isolation, purification and structure elucidation of the water-soluble polysaccharides of G. biloba leaves. RESULTS

AND DlsCUSilON

Isolation, fractionation, and sugar composition A crude polysaccharide fraction from the dried leaves was obtained, after a pre-extraction with organic solvents, by extraction with water, followed by precipitation with ethanol. After dialysis the high M, fraction was freeze-dried (yield 1.1%). By anion-exchange chromatography on DEAE-Sephacel the crude polysaccharide fraction could be separated into a neutral fraction GFl (1.2%) and two acidic fractions, GF2 (16%) and GF3 (15%). respectively. The homogeneity as well as the M, dimensions were evaluated by means of gel permeation chromatography using standard dextrans. On a Sephacryl S-300 column no heterogeneity was indicated for GFl

and GF3 and the average M, was determined as 23000 and 4O@OO,respectively. GF2 could be separated on a Sephacryl S-400 column into GF2a (70%) and GF2b (30%). The average M, was determined as 500000 for GF2a and 24000 for GF2b. The sugar composition of the fractions is shown in Table 1. Besides low proportions of galactose, mannose and glucose, the neutral fraction GFI is mainly composed of arabinose indicating the presence of an arabinan. GF2a as well as GF2b are character&d by large amounts of mannose, rhamnose and glucuronic acid, and low proportions of arabinose, glucose and galactose. Due to the similarity in composition of both fractions, it was assumed that GF2a und GF2b represent the same structural type of polysaccharide mainly differing in M,. Therefore, structural investigations were only performed with GF2a. The GF3 polysaccharide was composed of a large amount of galacturonic acid and cd equal proportions of arabinose, galactose, rhamnose, and glucuronic acid. All fractions were free of nitrogen indicating the absence of protein. Structural features of GFl, GF2a, and GF3

After permethylation [S, 63 of GFl and conversion into the corresponding partially 0-methylated alditol acetates, 1,4-di-O-acetyl-2,3,5-tri-0-methylarabinitol, 1,3,4-tri-O-acetyl-2,5-di-0-methylarabinitol, 1,4,5-tri-Oacetyl-2,3-di-O-methylarabinitol, 1,3,4,5-tetra-O-acetyl2-0-methylarabinitol and 1,2,4,5-tetra-O-acetyl-3-0methylarabinitol could be identified by comparing their

3017

3018 Table

J. KRAUS 1. Sugar compusition

Arabinose Rhamnose Mannose Glucose Galactose Glucuroruc Galacturomc

of Ginkgo polysaccharide Fraction

(mol-%)*

GFI

GF2a

GF2b

GF3

91 _.

3 26 29 5.5 1.5 29 7

9 22 30 8 2 25 4

18 15

3.5 2.5 3 acidt acidt

‘Determined tDetermined groups.

fractions

as alditol acetates by GC. as alditol acetates after reduction

16 15 30

of carboxyl

R,s and mass spectra data by GC-MS with those of authentic samples, or values in the literature [73. The molar ratio of the five methyl/acetyl arabinitols was ca 3: I :9: I :2 (Table 2) indicating a l,Slinked arabinofuranosyl backbone. On average, three in 12 arabinofuranosyl residues of the main chain are branched. two-thirds via C-2 and one-third via C-3. The observed 13C NMR chemical shift of fi 109.4 (6 108.7 [9]) for C-l of the arabinofuranosyl residues suggest an r-linkage. Although it was not possible to determine the exact nature of the side chains, the structural features shown in Fig. 1 are suggested to be present. GF2a was mainly composed of mannose (29%), rhamnose (269/o) and glucuronic acid (29%); traces of arabinose, glucose (5.5%) and galacturonic acid (6%) were also detectable. Methylation analysis was performed with both the carhoxyl-reduced and the unreduced poly-

Table 2. Methylation Partially methylated alditol acetates* 2,3,5-Me,-Arabinitol 2,3-Me,-Arabimtol 2,5-Me,-Arabinitol 2-Me-Arabinitol 3-Me-Arabinitol 2,3,4-Me,Rhamnitol 3,4-Me,Rhamnitol 3-Me-Rhamnitol 2,3,4,6-Me,-Glucitol 2,3,6_Me,-Glucitol 3,4,6-Me,-Mannitol 3,6-Me2-Mannitol 2,3,4,6-Me,-Galactol 2,3,6-Me,-Galactol 2,4,6-Me,-Galactol 2,3,4-Me,-Galactol Z&Me,-Galactol

analysis

GFI

GF2at

17.2 43.5 4.2 6.4 10.6

2.4 1.9

1.9 I.1

25.0

26.1

fractions

Fraction (mol-%) GF3li GF2a,,&

GFL:

9.5 11.4

9.1 11.8

9.2

4.x 5.2 3.5

4.3 4.1 3.0

1.3 7.3 6.3

29.3

29.5

6.4 41.1 15.2 15.9

1.3

x.2

12.3

3.2 -..

32.3

16.3

20.9

2.1 3.1 1.2

29.6 3.0 ._

15.8

15.0

5.9 42.3 2.1 12.8 I.1

traces

-

polysaccharide. partially hydrolysed

GF&§

._

-.

*Analysed by GC-MS. t Total 63.1%. $Carboxyl-reduced $Carboxyl-reduced, IiTotal 56.2%.

of Ginkgo polysaccharide

GFZa,,,:

traces traces

saccharide. The main linkage types of the neutral sugars were shown to be terminal rhamnose (25%) and 1,2,4branched mannose (29%). Apart from a minor amount of 1,4-linked glucose (3%) only traces of other linkage types could be identified. The results of the methylation analysis of the carboxyl-reduced polysaccharide demonstrated that glucuronic as well as galacturonic acid are 1,4-linked. Therefore, mannose is the only branched sugar and rhamnose represents the terminal sugar residues. Due to the absence of branched rhamnose as well as uranic acids, a rhamnoglucuronan or galacturonan structure seems to be unlikely. Partial hydrolysis with 0.5 N TFA was carried out with the intention of removing the terminal rhamnose. As a result, 70% of the rhamnose residues and low proportions of arabinose could be hydrolysed by this method. Methylation analysis of the residual polysaccharide, after reduction of the uranic acids, resulted in a decrease in 1,2,4-linked mannose and terminal rhamnose, and an increase in 1,2linked mannose and terminal glucose, which corresponds to terminal glucuronic acid. The other sugar linkages remained more or less unchanged. These results indicate that ca one-half of the terminal rhamnose is directly connected to mannose mostly via C-4. However, the rest of the terminal rhamnose seems to be bound to glucuronic acid, which is indicated by the resistance of the rhamnose residues to mild acid hydrolysis and the increase in terminal glucuronic acid residues of the remaining polysaccharide. The ‘?Z NMR chemical shifts suggest a a-linkage for C-l of mannopyranoside (696.9) [IO] and rhamnopyranoside (699.4) [j I], and a /I-configuration for C-l of glucuronopyranoside (6 103.3) [I I]. Additionally, the signals at S 17.3 and 175.4 could be assigned to C-6 of rhamnose and the C-6 of nonesterified uranic acid, respectively [ 121. Based on these results GF2a is presumed

polysaccharide.

Polysaccharides from Ginkgo biloba

3019

a-Ara

I i

a-Ara

+5)

-Q-Ara- (1

5) -a-Ara-

(1

) -a-Ax-i-

(1

5) -a-Ara-

(1

i 1 a-Ara

-2)

-a-Man-

repeating unit of GFl polysaccharide.

(142)

-a-Man-

$ Rl

4 a-Rha

(142)

-a-Man-

(l-

i R2

a-Rha-(l+)-,9-GlcA-(l+)-/I-GlcA-(l+)-fl-GlcA-(la-Rha-(1-_)4)-~-GlcA-(1--+4)-/3-GalA-(1~4)-~-G~cA-(1a-Rha-(1-4)~fi-G~CA-(ld)-fl-GalA-(la-Rba-(lFig. 2. Proposed

structural features of GF2a polysaccharide.

have a 1,2,4-branched mannose backbone with ca one-half of the side chains present as single rhamnose residues. The 1,4-linked glucuronic and galacturonic acid residues represent the residual side chains, which carry rhamnose as terminal sugar. The structural features shown in Fig. 2 are thus suggested to be present. The most acidic fraction GF3 is composed of galacturonic as well as glucuronic acid, rhamnose, galactose and arabinose and seemed to homogeneous according to M, distribution. By means of methylation analysis of both the original as well as the carboxyl-reduced polysaccharide, the following linkage types could be identified: terminal and 1,Slinked arabinose, terminal, 1,2-linked and 1,2,4-branched rhamnose, 1,3-linked and 1,3,6-branched galactose as well as 1,4-linked galacturonic and glucuronic acid. The molar ratio of the identified partially methylated alditol acetates could be determined as 2: 2: 1: 1: 1: 1: 3 : 6: 3. These results indicate that the main chain of the polysaccharide is probably formed by 1,4linked galacturonic (and/or glucuronic) acid and 1,2linked rhamnose residues. About half of the rhamnose residues are branched via C-4 with side chains consisting of branched arabinogalactans. In order to confirm the proposed arabinogalactan structure of the side chains, mild acid hydrolysis using 0.05 N TFA was performed. This led to an almost selective and complete cleavage of both the terminal and the 1,5-linked arabinose residues. After methylation, the remaining polysaccharide showed an increase in terminal as well as 1.6~linked galactose, whereas the 1,3,6-branched galactose disappeared. All other sugar linkages remained unchanged. These results clearly demonstrate that the terminal as well as the 1,5linked arabinose residues are connected to 1,6-linked galactan units mainly via C-3. The fact that the amount of 1,2,4-branched rhamnose and the one of terminal galactose is ca equal, suggests the presence of arabinogalactan side chains. Additionally it could be demto

(l-

i a-Ara Fig. 1. Proposed

= Rl R2 = = =

-a-Ara-

onstrated that no arabinose is directly attached to rhamnose. The 13C NMR chemical shifts in the area of anomeric carbon atoms suggest an a-linkage for arabinofuranoside (6 108.2) [9], galacturonopyranoside (697.8) and rhamnopyranoside (699.4) [13], and a /?-linkage for galactopyranoside (6103.3) [93. The shifts at 6170.2 and 174.1 could be assigned to the carboxyl group of esterified and non-esterified uranic acid. Because of the complexity of the ’ %ZNMR spectra, the remaining more or less broad peaks can hardly be assigned to the various sugar residues. Based on these analytical data the structural features shown in Fig. 3 are thought to be present. Polysaccharides like GFl as well as GF3 represent common structures for water-soluble polysaccharides of higher plants [19-211. However, the polysaccharide GF2a with a 1,2,4-branched mannose backbone and the rhamnose and glucuronic acid side chains seems to be unique for G. biloba and may become an important criterion for the chemotaxonomical classification of the Ginkgoaceae. EXPERIMENTAL General. All evapns were performed under red. pres. at a temp. of 40” or below. TLC was performed on silica gel 60 F2s4 with MeJCOCN-Hz0 (17: 3) and detection with diphenyaminophosphoric acid reagent [14]. Dialysis was done against Hz0 with continuous stirring at 4”. N content was determined by elementary analysis. All chemicals were of analytical grade. Isolation of polysoccharides. Dried plant material (Fa. Schwabe, Karlsruhe, F.R.G.) was ground (0.02 mm) and successively extracted with CH2CII, MeCOEt and MeOH under reflux in a Soxhlet apparatus until each extract was colourless. The air-dried plant residue was then extracted with Hz0 for 24 hr at 20” (cont. 5%) and polysaccharides pptd with EtOH (final cont. 75%) and kept overnight at 4”. The ppt. was dissolved in HZO,

3020

J. KRAUS

44)

-a-GalA-

(l-2)

-a-Rha-( 4

1

) -a-Ga

lA-

) -a-GlcA-

(1

(1 3

I B-Gil6

(3-l)

-a-Ara-

(5&-l)

-a-Ara

(3+1)

-a-Ara-

(5&l)

-a-Ara

(34--l)

-a-Rha

I 8-Gil7 fl-G:l-

Fig. 3. Proposed

repeating

dialysed (MWCO 3500 D) and finally freeze-dried yielding ca I, 1% referred to as starting material. Fractionation ofpolysaccharides. The crude polysaccharide fr. was dissolved in Hz0 and applied IO DEAE-Sephacel (3.5 x 2Ocm) which had been equilibrated with K-Pi buffer (pH 6.0) and washed with H20. Elution was started with Hz0 (GFI), followed by 0.1 M (GF2) and 0.5 M (GF3) K-Pi buffer (pH 6.01, successively. Fractionation was monitored by using the anthrone reagent [IS]. Homogeneiry and M,. Homogeneity and M, were investigated by means of GPC. GFI as well as GF3 were applied to Sephacryl S-300, GF2 was applied to Sephacryl S400 (1.6x 87 cm). All samples were prepd as 20 mg ml- ’ solns and elutton was performed wtth 0.1 M NaCI. k‘rs of 2 ml were collected at a flow rate of 20 ml hr- i. Each fr. was tested usmg the anthrone method [ 151. For M, estimation, calibration was performed using dextran standards of known M,. Sugur analysis. Complete acid hydrolysis of polysaccharides was achieved by treatment with 2 N TFA for I hr at 120- and I atm. TFA was removed by repating evapn (x 5) 10dryness. Neutral sugars were converted to the corresponding alditol acetates according to the method of ref. [I63 and analysed by GC. GC was performed with FID detection and a fused silica column (DB-225, 0.25 mm;30 m) at 0.8 N, ml min- ’ and 220 [9]. Total uranic acid content was determined according to the method of ref. [ 173 with o-hydroxydiphenyl reagent. After reduction of uranic acids to the corresponding neutral sugars a differenttal estimation could be achieved by GC as described before. Merhplation analysis. Polysaccharide (1-5 mg) dissolved in fr. dist. and dried DMSO (200 ~1) was methylated with methylsulphinyl carbanion (2oOgl) and Mel (150~1) according to the method of ref. [S], modified as in ref. [6]. After hydrolysis methylated sugars were converted to the corresponding partially methylated alditol acetates. GC-MS was performed on a DB-1701-30W (0.25 mm,‘30 m) fused silica column at l70-210(linear I - min _ ‘) coupled to a selective mass detector. Reduction oJ carboxyl groups. Reduction was performed according IO the method of ref. [IS]. Polysaccharide (30 mg) was dissolved in 10 ml H20, I-cyclohexyl-3-(2-morpholino-ethyl)carbodiimde-metho-ptoluenesulphonate (CMC, 432 mg) was added and the pH maintained at 4.75 by addition of 0.01 M HCI for 2 hr. 2 M NaBD, soln (20 ml) was added dropwise while the pH was maintained at 7 by addition of 4 M HCI under continuous stirring. After 2 hr the pH was adjusted IO 6.5 with HOAc, the mixt. ultraliltered (Amicon, YM 2) and the non-dialysable fr. lyophthsed. Sugar analysis and methylation was performed as described above. Partial hydrolysis. Polysaccharide (50 mg) was treated with 10 ml 0.05 N (GF3) or 0.5 N TFA (GF2a) at 100’ for 1 hr. After

unit of GF3 polysaccharide.

cvapn of acid the reaction mixt. was dissolved In HsO, applied to a Biogel P-2 column (1.6 x 90 cm) and eluted with HsO. Elution was monitored by testing frs (2 ml each) with anthrone reagent [ 151. The monosaccharide fr. was analysed by TLC and GC; the residual polysaccharide was investigated as described in the text. were dissolved in D20 13C NMR. Polysaccharides (20 mg ml _ ‘) and spectra recorded at 62.89 MHz at 306 K with TMS as ext. standard. Chemical shifts are given in 6 values. AcknowledgementsThe author thanks the “Fonds der chemischen Industrie” for financial support and Mrs H. Schmid for Technical assistance.

REFERENCES I. Schtlcher. H. (1988) Z. Phytorher. 9, I 19. 2. Schennen, A. (1988) Thesis, Universitiit Marburg. 3. Itokawa. H., Totsuka. N., Nakahara. K., Maezura, M., Takeya, K.. Kondo, M., Inamatsu. M. and Morita, H. (1989) Chem. Pharm.

4. Hollriegl,

Bull. 31, 1619.

H., Koehler,

H. and Franz

G. (1986) Sci. Pharm.

54, 321.

S. (1964) J. Biochem. 55. 205. 5. Hakamori. 6. Harris, P. J., Henry, R. J., Blakeney, A. B. and Stone, B. A. (1984) Carbohydr. Res. 127. 59. 7. Jansson, P.-E., Kenne, L., Liedgren. H., Lindberg, B. and Lonngren, J. (1976) Chem. Commun. Stockholm Linio. 8, 1. 8. Bjdrndal, H., Hellerqvtst. C. G.. Lindberg, B. and Svensson, S. (1970) Anger. Chem. 82, 643. 9. Cartier, N.. Chambat, G. and Joseleau, J.-P. (1987) Carbohydr. Res. 168, 275.

10. Gorin. P. A. J. (1975) Carbohydr. Res. 39, 3. 1I. Defaye, J. and Wong, E. (1986) Carbohydr. Res. lM, 221. 12. Miiller, B. M., RoDkopf, F.. Paper, D. H., Kraus, J. and Franz, G. (1990) Pharmazie (in press). Regensburg. 13. Miiller, B. M. (1989) Thesis, Universitat 14. Bailey, R. W. and Bourne. E. J. (1960) J. Chromatogr. 4,206. 15. Morris, D. L. (1948) Science 107, 254. 16. Blakency, A. B.. Harris. P. J., Henry, R. J. and Stone, B. A. (1983) Carbohydr. Res. 113, 291. 17. Blumenkrantz. N. and Asboe-Hansen, G. (1973) Anal. Biothem. 54, 484.

18. Taylor. R. T. and Conrad, H. E. (1972) Biochem. 11, 1383. 19. Miiller, B. M., Kraus, J. and Franz. G. (1989) PIanta Med. 55, 536.

20. Kraus,

J. and Franz,

G. (1987) Dtsch. Apothoker

Ztg.

127,

665.

21. Stephen, (Aspinall,

A. M. (1983) in The G. 0.. ed.). p. 122.

Polysaccharldes

Vol. 2.