Chemical properties of lignin from Aralia cordata

Pergamon

Pbyrohmutry.

Vol. 37.

No

2. pp 44%

1994

Copynghht G I994Elmwin Sama Ltd Pnawdm Ora1 Briuln All n&s mewed 00119422191 s7.00+ 0.00

CHEMICAL

PROPERTIES

TAKASHIHIBINO, DAISUKESHIBATA,

OF LIGNIN FROM ARALZA CORDATA

TAKASHI

ITo,t DAISEI TsucHtYA,t TAKAYOSHI and CATERINE LAPIERRE~

Mitsui Plant Biotechnology Research Institute, TCI-AlO,

HIciucHr.*t BRIGITTE POLLET$

Sengen 2-l-6, Tsukuba, Ibaraki 305, Japan: tDcpartment

College of Agriculture and Veterinary Medicine, 3-34-l Shimouma, Setagaya-ku, Tokyo 154, Japan; :Laboratoire Biologique, INRA.

of Forestry, de Chimie

Paris Grignon, France

(Received in revised form 7 April 1994)

Key Word Index--Arolia

cordota;

Araliaceae; guaiacyl-syringyl

lignin; nitrobenzene

oxidation;

thioacidolysis.

Abstract-Lignin from Arabia cordata, was isolated and characterized spectrometrically and by chemical degradations. IR and “CNMR spectra of the dioxan lignin showed a pattern of guaiacyl (G)-syringyl (S) lignin esterified with p-hydroxybenzoate. Alkaline hydrolysis of the lignin gave p-hydroxybenzoic acid. Molar ratios of the main syringyl to guaiacyl monomers recovered from alkaline nitrobenzene oxidation and from thioacidolysis are 1.3 and 1.2. respectively, indicating that A. cordata contains a guaiacyl-syringyl lignin, although the amount of syringyl lignin is a little lower than that in normal angiosperm wood.

INTRODUCTION cordata is not a typical woody plant because its height is ca 1.5 m. Etiolated young shoots are rich in flavour and are cultivated in cellars for food in Japan. Shoots were used in previous investigations as plant material for the preparation of cinnamyl alcohol dehydrogenase (CAD) which has been used for the isolation of its cDNA [I, 23. The antisense CAD gene of A. cordara was constructed and transferred to tobacco plants; the lignin synthesized by these transgenic tobacco plants has been characterized [33. In a continuation of these previous studies, the spectrometric and chemical characterization of A. cordata lignin were conducted, as compared to poplar lignin. This paper reports on the main structural features of lignin from mature plants of A. cordara. Arab

RESULTSANDDISCUSSION After alkaline nitrobenzene oxidation of A. cordata and poplar dioxan lignins, vanillin and syringaldehyde were obtained as major degradation products. The amounts (weight percentages) of syringaldehyde and vanillin determined by GC and their molar ratio (S/V) were 34% and 1.3 in A. cordata and 42% and 1.8 in poplar lignins, respectively. These results indicate that A. cordota lignin consists of guaiacyl-syringyl lignin like that of ordinary angiosperm lignins [43. GC mass spectrometric analysis of thioacidolysis products of A. cordata and poplar dioxan lignins showed that the major lignin-derived monomers were the erythro- and threo-trithioethyl deriv-

*Author to whom correspondencc should be addressed.

atives of guaiacyl (G-CHSEt-CHSEt-CH,SEt), Gl and G2, and syringyl (S-CHSEt-CHSEt-CH,SEt), Sl and S2, series. These major isomers were derived from the thioacidolysis of the prominent arylglycerol-/?-aryl ether lignin substructures. In addition, several minor products could be identified, such as (G3, S3) and (G4, S4) identified by GC mass spectrometry (Fig. I). These mass spectral assignments (Table I) were confirmed by comparison with appropriate authentic compounds, except for G3 and S3 tentatively identified from their mass fragmentation patterns. However, the hypothetical assignments of G3 and S3 to phenylpropane compounds with rearranged side-chains were strongly supported by the fact that the occurrence of these products was particularly pronounced in the case of dioxan lignins which have been isolated in acidic medium and prone to side-chain rearrangements. On the contrary, these products represented less than 5% of the main isomers in the case of protolignins, analysed in situ. The profile of the GC of A. cordota dioxan lignin was very similar lo that of poplar lignins. The S/G molar ratios for the thioacidolysis monomers were 1.2 in A. cordato and 2.0 in poplar lignins. respectively (Table 2) [S]. Alkaline hydrolysis of dioxan lignin of A. cordara gave phydroxybenzoic acid, although the amount was smaller than that from poplar lignin. The IR spectrum of A. cordata dioxan lignin showed the characteristic pattern of angiosperm lignin [6]. Major absorptions from 1800 to 800 cm-’ and their relative intensities in A. cordata and poplar dioxan lignins were similar (Table 3). Absorptions at 1220 and 1120cm-’ related to the syringyl group were stronger than those of 1270 and 1030 cm _ ‘, respectively, but the absorption at

T. HIBINO er ol.

446

G3

Gl*G2 cH2sl3

I

CH SEa

OTMS

0 TMS

OTMS

OTMS

Fig. 1. Compound

Table

structures

of thioacidolysis

I. Structure and mass spectral characteristics of thioacidolysis products of A. cordara dioxan lignin

OTMS

products

of lignin identified

by CC-MS.

and 105.0 ppm with those of guaiacyl ones at 150.7. 148.7, 120.5. 116.1, 112.0, I I I.5 ppm, and phydroxybenzoate carbons, at I2 I .O and I 15.4, respectively 173. The spectra

product (TMSi derivative) Cl G2 G3

[M] and prominent fragments, m/z (rel. inc.) l

G-CHSEt-CHSEt-CH,SEt

t EtSH,C-

H-CH(SEt), 1,

G4

G-CH,-CH(SEt),

Sl .S-CHSEt-CHSEt-CH,SEt s2 1 S3

ETSH,C-

H-CH(SEt), F

S4

S-CH,-CH(SEt),

418 (I), 403 (2). 295 (3). 269(100), 235(9), 75(19). 73 (23) 418 (4). 403 (6). 283 (74). 222 (621. 192 (20). 135 (100). 75 (58). 73 (52) 344 (l6), 329 (5). 283 (4), 224(3). 209(32). 192(13), 179 (9). 135 (1001. 73 (37) 448 (3). 325 (4). 299 (100). 265 (8). 204 (4). I61 (2). 73(18) 448 (9). 388 (2). 313 (100). 252 (53). 222 (21). I79 (6). I35 (74), 75 (55), 73 (47) 374 (37). 313 (5), 252 (1 I), 239 (62). 222 (12). 209 (IO), 179 (6). 135 (ICO), 73 (38)

also showed peaks corresponding of guaiacyl

and syringyl

These results indicated guaiacyl-syringyl lignin.

lignin as found in ordinary

but contains

hydroxybenzoate

showed

that

A.

cordata

lignin

consists

syringyl Iignin typical of angiosperms The “C NMR

of guaiacyl-

[6].

spectrum showed peaks typical of aro-

matic syringyl carbons at 153.5, 148.3, 138.6, 135.6, 107.0,

carbons (Table

4).

that A. cordata lignin consists of

a smaller

amount

angiosperm

of esterified

p-

[7]. EXPERIMENTAL

Plant murerial. Lignified grown in the Kyoto Takabe.

Department

Kyoto University,

of Wood

air-dried sieve.

20 m, used as control was kindly

by Dr S. Chiba,

Improvement,

Science and Technology,

Kyoto, Japan. Poplar (Populus maxim-

owiczii) wood, diameter provided

plants of A. cordata

mature

area were kindly provided by Dr K.

Kuriyama,

Oji Institute Hokkaido.

for Forest Tree

The plant was cut.

and pulverized in a Wiley mill lo pass a a-mesh

The

pulverized

EtOH-benzene

material

was

extracted

(2: 1) in a Soxhlet extractor

with

for 24 hr.

Determination of lignin and holocellulose. Contents of lignin (A. cordata. 22.l%,

1460 was almost equal lo that at 1510 cm - I. The pattern

lo side-chain

lignin substructures

poplar,

21.3%)

lose (A. cordata, 74.5%. poplar, 76.0%) by the Klason respectively

method

and sodium

and holocellu-

were determined chlorite

methods,

[S].

Prepuration ofdioxan lignin. Dioxan (150 ml) containing 0.2 N HCI was added to IO g of extractive-free

plant

Lignin from Arolia cordafa

441

Table 2. Yield of thioacidolysis monomers(G1-4) and (S14k from A. cordata and poplar dioxane lignins (Pmol g- ’ lignin) Gl+G2

G3

G4

Sl+S2

s3

S4

G total

S total

S/G

398 422

69 55

31 38

501 523

84 63

35 41

498 515

620 627

1.24 I .22

41 40

752 760

92 103

41 46

446 455

885 909

1.98 2.00

A. cordata

Run I Run 2

Populus maximowiczii

Run I Run 2

361 360

44 55

Total amounts (S +G) and S/G ratio in thioacidolysis products of dioxan lignin of A. cordata and P. maximowiczii were ll30+ 17, 1.23+0.01, 1348+23 and 1.99+0.01 pmol, respectively.

Table 3. Relative intensities of absorption bands (cn~ ‘) in IR spectra of A. cordara and poplar dioxan lignins A. cordata 1720, shoulder l5lO= 14&-l 1325. strong 1270< 1220 II202 1030 1090. weak 915, weak

Table 4. “C Chemical shifts and signal assignments for A. cordata dioxan lignin. Solvent, acetone-d,-D,O (9: I)

P. maximowiczii 1710, strong lSlO< 1460 1325. strong 1270< 1220 1120> 1030 1090, weak 915, weak

material in a 500 ml flask. The reaction mixt. was rcfluxed for 1 hr under N,. The reaction mixt. was then filtered. and the filtrate adjusted to pH 6 and coned in uacuo. The coned dioxan soln was added dropwise with stirring into 50 times its vol. of distilled H,O. The lignin was collected by centrifugation and purified according to the purification procedure for milled wood lignin [lo]. Alkaline nirrobenzene oxidation. Extractive-free plant material (100 mg) or dioxan lignin (20 mg) was subjected to alkaline nitrobenzene oxidation at 165” for 2 hr. Acetovanillone (5 mg) was added as int. standard to the reaction mixt. which was extracted with EtOAc to remove excess nitrobenzene and its reduced compounds. The reaction mixt. was then adjusted to pH 3 with dilute HCI. Aromatic aldehydes were extracted with EtOAc and acetylated with Ac,O-pyridine and analysed by GC [4]. A Shimadzu Hicapcolumn (30 m, 0.25 mm i.d.) was used. Carrier gas, N,; column temp. 200”. Thioacidolysis. Extractive-free plant material (IS mg) or dioxan lignin (5 mg) was subjected to thioacidolysis in a dioxan soln of ethanethiol-0.2 N BF, etherate using a glass tube fitted with a Teflon-lined screwcap at loo” for 4 hr with occasional shaking [5]. The reaction mixt. (IO ml) and 5 ml of H,O to rinse the reaction tube was added to 50 ml of CH &I,. Tetracosane (I mg) was added as int. standard for GC. The pH of the aq. layer of the reaction mixt. was adjusted to 4 with aq. NaHCO, and the mixt. extracted x 3 with 50 ml CH,CI,. The combined extracts were dried (Na,SO,) and the solvent

Signal no.

ppm from TMS

Assignments

I 2 3 4 5 6 7 8 9 IO II I2 I3 14 I5 16 I7 I8 I9 20 21 22-25 26 27 28 29 30 31 32

195.2 153.5 150.7 148.7 148.3 138.6 135.6 121.0 120.5 116.1 115.4 112.0 111.5 107.0 105.0 104.0 88.4 86.8 86.4 85.9 85.4 73.3-73.0 72.5 64.3 61.3 60.8 56.6 56.3 55.0

CHO in cinnamaldehyde C3/C5 in S p-O-4 e C3 in G e unit C4 in G e unit C3/C5 in S p-0-4 ne Cl in S b-O-4 e and ne C4 in S j-O-4 e and ne Cl in H benzoate unit C6 in G units e and ne C5 in G units e and ne C3/CS in H benzoate units C2 in G unit C2 in G-G stilbene units C2/C6 in S with aC =0 or aC =C C2/C6 in S in general C2/C6 in /3-b Cfi in S b-O-4 threo Ca in j-5 C/3 in S ,!I-O-4erythro Ca in /I-/I C/I in G b-O-4 threo Ca in G. S b-O-4 erythro, three Cy in /Y-/3 Cy in B-5 and b-O-4 with C =0 Cy in G, S b-O-4 erythro, three Cy in G, S /l-O-4 erythro. tbreo Aromatic OMe in G and S Aromatic OMe in G and S Cb in B-8

G: guaiacyl, H: phydroxyphenyl,

S: sytingyl, e: etheritied,

ne: non-etherifial.

evapd in uucuo. The products were dissolved in CH,Cl, and silylated with N.O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine for GC-MS analysis. A polydimethylsiloxane fused-silica capillary column (30 m, 0.25 mm i.d.) was used. Carrier gas, He; column temp. 160-260” at +2”min-‘.

448

T. HIBINOet al.

Alkaline hydrolysis. Dioxan lignin (100 mg) was dissolved in 10 ml of I N NaOH and the soln kept under N, at room temp. for 24 hr. The soln was acidified to pH 4 with dilute HCl and extracted x 3 with EtOAc. The combined extracts were dried (Na,SO,) and evapd in rucuo. The extract was dissolved in a small amount of MeOH and analysed by TLC (Kieselgel 60 F254. CH,CI,-MeOH, 5: 1). p-Hydroxybenzoicacid wasdetected from both lignins as a dark spot under UV light and identified by comparison of R, values with that of the authentic compound. IR analysis. Dioxan lignin (I mg) was well mixed with 200 mg of dried KBr powder in an agate mortar and a disc was prepared from the mixt. for IR analysis. “C NMR unalysis. Dioxan lignin was dissolved in acetone-&-D,0 and the spectrum was recorded with a Joel GX-400 NMR spectrometer using TMS as int. standard. Acknowledgements-We gratefully acknowledge Dr Atsushi Kato, Forestry Research Institute, Tsukuba, Japan for t ‘C NMR analysis of dioxan lignin. This study was supported by a Grant-in-Aid for Scientific Research (No. 04454090) from the Ministry of Education of Japan.

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4. 5. 6. 7. a.

9. 10.

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