Structure of small lignin fragments retained in water-soluble polysaccharides extracted from birch MWL isolation residue

To analyze the structural features of lignin in the vicinity of lignin–carbohydrate linkages, water-soluble lignin–carbohydrate complex (LCC) with low lignin content was prepared from residual birch wood meal after the extraction of milled wood lignin (MWL). The molecular weight distribution of lignin in this LCC appeared together with carbohydrate in the relatively high molecular weight region of the gel permeation chromatogram. This result was consistent with our previous results obtained for the same fraction of Japanese cedar (sugi); however, after treatment with polysaccharide-degrading enzyme, the molecular weight distribution of carbohydrate and that of lignin shifted significantly to the lower region. These results demonstrated that molecular size of this LCC is determined by carbohydrates while lignin is present as a minor fragment in this fraction. The syringyl/guaiacyl (S/V) ratio of this LCC was higher than other lignin fractions. Ozonation analysis implied that this LCC has a relatively high number of β-1 structures. It is likely that lignin that exists near lignin–carbohydrate linkages has more endwise-type features than other lignin fractions.This paper was presented in part at the 48th Lignin Symposium, Fukui, Japan, October 2003 and at the 12th International Symposium on Wood and Pulping Chemistry, Madison, USA, June 2003     

Molecular properties of lignin-carbohydrate complexes from beech (Fagus crenata) and pine (Pinus densiflora) woods

Summary Lignin-carbohydrate complexes (LCC) were isolated from pine (Pinus densiflora) and beech (Fagus crenata) milled-wood lignins. The LCCs were treated with enzyme to obtain precipitates (A-P, B-P) and water soluble fractions. The water soluble fraction from beech LCC was subjected to gel filtration to give LCC fragments (B-E-I). In order to protect the phenolic hydroxyl group, B-E-I and B-P were methylated with diazomethane, resulting in nonphenolic LCC fractions B-E-Ip and B-Pp, respectively. On treatment of B-E-I and B-P with sodium hydroxide, a remarkable amount of xylose and a trace of arabinose were detected as monomeric sugars. However, the same alkaline treatment of B-E-Ip and B-Pp gave only a faint trace of xylose. With DDQ treatment of acetylated B-E-I and B-P, monomeric sugars were released in the same amount as those obtained on alkaline treatment. Methylation of the monomeric sugars gave monomethylated xylose. From these results, it was concluded that xylose residues participate in lignin-carbohydrate linkages, and that lignin is linked to xylose at the 0–2 or 0–3 positions through an alkali-labile benzyl ether bond. The molecular-weight distributions of the lignin moieties, measured by HPLC, indicate that the lignin moieties of beech LCC are 100 times larger but less frequent than those of pine LCC. This nonuniformity of distribution of lignin and carbohydrate moieties in hardwood LCC molecules would result in less hydrophobic interaction in aqueous solutions.     

Lignin characterization of triploid clones of Populus tomentosa Carr.

In order to understand the structural characteristics of lignin in triploid clones of Populus tomentosa and its changes in the processes of pulping and bleaching, milled wood lignin (MWL), lignin carbohydrate complex (LCC) and the residual lignin from kraft pulp (KP) and sulfite pulp (SP) were isolated and analyzed by Fourier transform infrared (FTIR) spectrum and ¹³C nuclear magnetic resonance (NMR). The most diagnostic peaks were assigned and the differences were discussed. The spectral patterns reveal that triploid P. tomentosa shows the specific features of hardwood from temperate areas, but in the spectrum of FTIR, the strength ratio of A 1270 cm⁻¹ to A1226 cm⁻¹ is 0.88, higher than the average of hardwood from temperate areas, which will make the lignin delignification more difficult during pulping and bleaching. The LCC from triploid P. tomentosa is mainly composed of xyloglucan and glucuronic acid, and other glucides have much lower ratio. In LCC FTIR, there are three peaks at 1 427, 1 329 and 1 046 cm⁻¹, indicating that both semi-cellulose and cellulose could exist in LCC, and that there might be relationships between cellulose and lignin. Compared with the residual lignin from KP and SP, the condensed structure in KP is more than that in SP.     

Erythro/threo ratio ofβ-O-4 structures as an important structural characteristic of lignin. I: Improvement of ozonation method for the quantitative analysis of lignin side-chain structure

Ozonation as a quantitative tool to analyze the stereo structures of arylglycerol--aryl ether linkages was examined using wood meal, milled wood lignin, and a lignin model compound, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (veratrylglycerol--guaiacyl ether, VG). The procedure was improved. When mild postreduction was conducted for ozonation products, the total yield of erythronic and threonic acids from this model compound was 74%, which is 15% higher than the yield without postreduction. A decrease in the recovery of these two acids under prolonged ozonation treatment was successfully suppressed by postreduction. Theerythro/threo ratio of VG determined by the ozonation method with postreduction is in good agreement with the ratio determined by¹H-nuclear magnetic resonance. Excellent reproducibility of the yield was obtained by adopting a procedure that included trimethylsilylation of ammonium salts of ozonation products using a dimethylsulfoxide-hexamethyldisilazane-trimethylchlorosilane mixture and subjecting it to gas chromatography analysis. It was concluded that arylglycerol--aryl ether structures comprise at least 35% of the C₃-C₆ structure in birch wood meal, with anerythro/threo ratio of 2.8.This paper was presented at the 43rd Lignin Symposium, Fuchu, Japan, October 1998; and at the 10th ISWPC, Yokohama, Japan, June 1999     

Formation and chemical structures of acid-soluble lignin II: reaction of aromatic nuclei model compounds with xylan in the presence of a counterpart for condensation,and behavior of lignin model compounds with guaiacyl and syringyl nuclei in 72% sulfuric acid

To elucidate the formation and chemical structures of water-soluble material in acid-soluble lignin (ASL), lignin aromatic nuclei model compounds of creosol (I) and 5-methoxycreosol (II) were reacted with xylose or xylan in the presence of apocynol as a counterpart for condensation in 72% sulfuric acid (SA). The reaction of I gave mainly condensation product. However, the condensation reaction of II with apocynol was suppressed because of steric hindrance from the methoxyl group, and II yielded a C-xyloside after refluxing in 3% SA together with condensation products. To obtain information on CHCl₃-soluble material in ASL, model compounds of arylglycerol--aryl ethers with guaiacyl (VIII) and syringyl (X) nuclei were treated by the Klason procedure. VIII gave only insoluble polymerized product, while X gave insoluble polymerized product and CHCl₃-soluble low molecular weight products, which were dissolved in 3% SA. These results prove earlier views that water-soluble material in ASL consists of condensation products formed from syringyl lignin and monosaccharide units in hemicellulose. In addition, the CHCl₃-soluble material in ASL appears to be composed of low molecular weight degradation products from SA treatment of Klason lignin with the syringyl nucleus.Part of this report was presented at the 51st Annual Meeting of the Japan Wood Research Society, Tokyo, April 2001 and at the 47th Lignin Symposium, Fukuoka, October 2002, and was reviewed in Mokuzai Gakkaishi (2002) 48:55–62     

Structure of small lignin fragment retained in water-soluble polysaccharide extracted from sugi MWL isolation residue

In order to analyze the structural features of small lignin fragments that are closely associated to polysaccharides, lignin carbohydrate complex (LCC) with low lignin content was extracted with water from the residual wood meal (sugi, Cryptomeria japonica) of milled wood lignin (MWL) isolation. This LCC exhibited almost the same neutral sugar composition as those extracted by other LCC solvents (dimethylformamide, dimethylsulfoxide, and others) but the lignin content was only 5.3%, which was much lower than others. Gel filtration chromatography demonstrated that lignin in this LCC was found together with carbohydrates at the higher molecular weight region, but after the treatment with carbohydrate-degrading enzymes the apparent molecular weight of both lignin and carbo-hydrates decreased significantly. Using a mild alkaline treatment, the apparent molecular weight of lignin also decreased while that of polysaccharide was not affected. These data indicated that lignin in this LCC is present as small fragments attached to high molecular weight polysaccharide at least partly by alkali-unstable linkages. Structural analysis by ozonation method revealed that the lignin in this LCC was lower in erythro/threo ratio of -O-4 structure and relatively richer in the threo type structure carrying C-aryl linkages at -position (-5 and/or -1) than other lignin fractions present in MWL, LCCs extracted by other solvents, and their extraction residues. It was suggested that the chemical structure of lignin closely associated to carbohydrates was different from that of the main part of lignin.This paper was presented in part at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, Japan, April 2002, at the 47th Lignin Symposium, Gifu, Japan, October 2002, and at the 12th International Symposium on Wood and Pulping Chemistry, Madison, USA, June 2003     

Characterization of lignin-derived products from Japanese beech wood as treated by two-step semi-flow hot-compressed water

Japanese beech (Fagus crenata) wood was treated by two-step semi-flow hot-compressed water (the first stage: 230 °C/10 MPa/15 min, the second stage: 270 °C/10 MPa/15 min), and produced lignin-derived products in the hot-compressed water-soluble portions at the first and second stages, and the final residue of the second stage was characterized with alkaline nitrobenzene oxidation method and gel permeation chromatographic analysis. As a result, the lignin-derived products at the first stage, where hemicellulose was also decomposed, consisted of lignin-based monomers and dimers and oligomers/polymers in the water-soluble portion. A large part of the oligomers/polymers was, however, recovered as the precipitate during 12 h setting after hot-compressed water treatment. By the analysis of nitrobenzene oxidation products, there were relatively higher contents of ether-type lignin in the precipitate at the first stage than in original beech wood. Since the ether linkages of lignin are more preferentially cleaved by this hot-compressed water, lignin-based polymeric fractions were flowed out from the porous cell walls from which hemicellulose was removed. On the other hand, at the second stage condensed-type lignin remained in the precipitate and residue. Based on these results, decomposition behavior of lignin in Japanese beech wood as treated by the two-step semi-flow hot-compressed water was discussed regarding the topochemistry of lignin structure.     

Biochanin A triglycoside from Andira inermis

A new isoflavonol triglycoside, biochanin A 7-O-β- -apiofuranosyl-(1→5)-β- -apiofuranosyl-(1→6)-β- -glucopyranoside (1), was isolated from Andira inermis roots in addition to the known compounds genistein 7-O-β- -apiofuranosyl-(1→6)-β- -glucopyranoside and lanceolarin.     

Lignin characteristics of bast fiber and core in kenaf, bark and wood of paper mulberry and mulberry

The structural features of bast fiber and core lignins in kenaf (Hibiscus cannabinus), bark and wood lignin of paper mulberry (Broussonetia papyrifera (L.) Vent × Broussonetia kazinoki Sieb.) and mulberry (Morus bombycis) were characterized by alkaline nitrobenzene oxidation, ozonation and methoxyl group determination. Bj?rkman lignins were isolated from bast fiber and core, and bark and wood fractions of the plant samples, and structural characteristics were investigated by ¹H NMR and ¹³C NMR spectroscopies. Kenaf bast fiber gave very high molar ratio of syringaldehyde to vanillin (S/V) of alkaline nitrobenzene oxidation products, while methoxyl content was about the same as that of the core fraction. Results of ¹H NMR and ¹³C NMR of Bj?rkman lignin suggested the presence of aliphatic fragments in lignins isolated from paper mulberry and mulberry bark, but not in kenaf bast fiber. The lower yield of alkaline nitrobenzene oxidation products from bast fiber and bark might be due to the higher content of condensed structure of lignin compared to core fraction. Total yield of erythronic (E) and threonic (T) acids of ozonation products and the molar ratio of erythronic acid to threonic acid (E/T) of the bast fibers and bark were lower than the corresponding core and wood fractions, suggesting that the contents of arylglycerol-β-aryl ether intermonomer linkages in the bast fiber and bark lignin were lower than those of the core and wood fractions. Methoxyl content of bark lignin was lower than the corresponding wood lignin. The methoxyl content of the extract-free kenaf bast fiber was similar to that of the core fraction, while the values of paper mulberry and mulberry bark were about one-half of the corresponding wood fractions, respectively. In bark lignins, the methoxyl contents of Klason lignin and Bj?rkman lignin from bark were lower than those of the extract-free barks. This result suggests that the purity of Klason lignin and Bj?rkman lignins of bark may be rather low.     

A new flavonoid from the aerial part of Thalictrum atriplex

A new flavone glycoside, identified as kaempferol 3-O-[3′″-acetyl-α- -arabinopyranosyl-(1′″–6″)]-β- -glucopyranoside (1), has been isolated from the aerial part of Thalictrum atriplex.     

Decomposition behavior of woody biomass in water-added supercritical methanol

The chemical conversion of Japanese beech (Fagus crenata Blume) in water-added supercritical methanol was studied for a wide range of water content using a batch-type reaction vessel to obtain chemicals from lignocellulosics. It was consequently found that addition of water enhanced the decomposition of wood cell wall components; cellulose, hemicelluloses, and lignin. In cases of high water content, however, it resulted in low solubility of lignin-derived products causing an increase in the mass of the residue. The water content was thus optimized to be around 10% (v/v) for the decomposition of wood. Concomitantly, the yields and selectivity of the chemicals from wood could be regulated by the addition of water, especially for the lignin-derived products. As a result, the monomeric compounds of lignin, coniferyl alcohol and sinapyl alcohol, were recovered as their γ-methyl ethers in the presence of water in higher yields than those obtained without addition of water.     

Recent progress in the chemistry and topochemistry of compression wood

Summary A review of the chemistry and topochemistry of compression wood with 200 references. Compression wood contains on the average 30% cellulose, 35–40% lignin, 10% galactan, 9% galactoglucomannan, 8% xylan, and 2% of a 1,3-glucan (laricinan). The cellulose is less crystalline, and the xylan has fewer arabinose side chains than in normal wood. The lignin is composed of guaiacylpropane and p-hydroxyphenylpropane units. It is more condensed, has a higher proportion of carbon-carbon bonds, and contains fewer arylglycerol--aryl ether structures than a normal conifer lignin. The ray cells and the primary wall of the tracheids have the same chemical composition in normal and compression woods. The galactan is largely located in the outer region of the secondary wall. Only 5–10% of the lignin in compression wood tracheids is extracellular. The middle lamella is less lignified than in normal wood, while the S₁ and inner S₂ layers have a lignin concentration of 30–40% which is twice as high as in normal wood. The lignin content of the S₂ (L) layer is equal to or higher than that of the intercellular region along the wall. The review is concluded with a brief reference to areas where present information is incomplete or lacking.A portion of an Academy Lecture of the International Academy of Wood Science, presented at the International Symposium on Wood and Pulping Chemistry (Ekmandagarna 1981), held in Stockholm, Sweden, June 9–12, 1981. Reprints of the unabridged review, published under the title Recent Progress in the Chemistry, Ultrastructure, and Formation of Compression Wood in the preprints of the symposium (SPCI Report 38, Vol. 1, p. 99–147) are available from the author. I wish to express my gratitude to my colleague Professor Robert A. Zabel for generous travel assistance     

Effect of the presence of lignin on the structure and reactivity to hydrolysis of lignincarbohydrate complexes of poplar wood obtained by sulfate pulping

Summary Lignincarbohydrate complexes (LCC) of poplar wood obtained by sulfate pulping and containing lignin in amounts of 0.9–8.3% have been studied by X-ray diffractometry. Heterogeneous hydrolysis methods have been used to determine their reactivity. The content of the crystalline fraction in LCC depends on the lignin removal and increases with the decrease of the lignin content. The degree of crystallinity of cellulose in sawdust and in LCC is fairly high and changes slightly with the lignin removal. The presence of lignin plays a blocking role in the heterogeneous hydrolysis and decreases the rate constant. It is suggested that lignin is bonded to cellulose on the outer surfaces of the crystalline part of the cellulose fibril. Some possible variants of these bonds are discussed.     

A new chlorosesquiterpene lactone from Ambrosia maritima

A new chloro-pseudoguaianolide-type sesquiterpene lactone, 11β-hydroxy-13-chloro-11,13-dihydrohymenin (1), was isolated from the Egyptian medicinal plant Ambrosia maritima. The structure was determined by spectroscopic methods, particularly high-resolution ¹H, ¹³C-NMR and 2D ¹H–¹H and ¹H–¹³C COSY NMR analysis.     

A new cytotoxic casbane diterpene from Euphorbia pekinensis

A new cytotoxic casbane diterpene, named pekinenal, was isolated from the roots of Euphorbia pekinensis. Its structure was elucidated as 5α-hydroxy-1βH,2αH-casba-3Z,7E,11E-triene-18-al by a combination of 1D- and 2D-NMR techniques and confirmed by X-ray crystallography. Pekinenal showed cytotoxic activity against all four human cancer cell lines tested.     

Analysis of the structure of lignin-carbohydrate complexes by the specific13C tracer method

In the present study the specifically¹³C-enriched lignin precursors of biosynthesis (i.e., coniferin-[side chain–¹³C], coniferin-[side chain-–¹³C] and coniferin-[side chain-–¹³C]) were synthesized and administered exogeneously to ginkgo shoots (Ginkgo biloba L.) to obtain¹³C-enriched lignin-carbohydrate complexes (LCCs). The specifically¹³C-enriched LCCs were isolated from the newly formed xylem of ginkgo shoots administered with the¹³C-enriched precursors and degraded by enzymes. Lignin-rich fractions, so called enzyme-degraded LCCs (EDLCCs), were obtained. By determining their¹³C-NMR spectra, information related to the chemical structure of lignin building units and linkages between phenylpropane units of lignin and carbohydrates were obtained. It was found that these precursors were incorporated in natural lignin successfully. Three lignin-carbohydrates linkages (i.e., ether type, ester type, ketal type) were found at the C -position of the side chain of phenylpropane units in ginkgo LCC. No lignin-carbohydrate bond at the C- or C-position of the lignin side chain was observed in the¹³C-NMR spectra of the¹³C-enriched LCCs. This fact indicates that a specific¹³C tracer technique can be useful in NMR study of the chemical structure of LCCs.Part of this paper was presented at the 5th Pacific Polymer Conference, Kyongju, Korea, October 1997     

Cyclisation of farnesyl pyrophosphate into sesquiterpenoids in ginger rhizomes (Zingiber officinale)

The atomic ratios (¹⁴C/³H) obtained in ar-curcumene, α-zingiberene, β-bisabolene obtained from the essential oil of rhizomes of Zingiber officinale which were fed with [2-¹⁴C, 2-³H₂], [2-¹⁴C, 4R-³H₁] and [2-¹⁴C, 5-³H₂]mevalonic acid and [1-³H₂]farnesylpyrophosphate (FPP) revealed that (a) (2E,6E)-isomer of FPP is isomerised to (2Z,6E)-isomer without loss of epimeric hydrogen that means without a redox process; (b) (2Z,6E)-FPP is cyclised to bisabolyl cation which is the penultimate precursor of α-zingiberene, ar-curcumene and β-bisabolene; and (c) two 1,2-hydrogen shifts take place during the formation of α-zingiberene whereas one 1,2 shift has been observed during the formation of ar-curcumene.     

Cycloartane saponins from Astragalus peregrinus as modulators of lymphocyte proliferation

From Astragalus peregrinus, four cycloartane-type saponins have been isolated and their structures elucidated by spectral means as 20(R),24(S)-epoxy-9β,19-cyclolanostane-3β,6α,16β,25-tetrol 3-O-β- -glucopyranoside (1), 20(R),24(S)-epoxy-9β,19-cyclolanostane-3β,6α,16β,25-tetrol 3-O-α- -rhamnopyranosyl-(1→4)-β- -glucopyranoside (2), 20(R),24(S)-epoxy-9β,19-cyclolanostane-3β,6α,16β,25-tetrol 3-O-α- -rhamnopyranosyl-(1→2)-β- -glucopyranoside (3) and 20(R),25-epoxy-9β,19-cyclolanostane-3β,6α,16β,24(S)-tetrol (24-O-acetyl)- 3-O-α- -rhamnopyranosyl-(1→2)-(6′-O-acetyl)-β- -glucopyranoside (4). Compounds 2 and 3 showed to stimulate the proliferation of mouse splenocytes and were not significantly cytotoxic.     

A new flavone glycoside from the seeds of Shorea robusta

A new flavonoid, 3,7-dihydroxy-8-methoxyflavone 7-O-α- -rhamnopyranosyl-(1→4)-α- -rhamnopyranosyl-(1→6)-β- -glucopyranoside (1), was isolated from the seeds of Shorea robusta.     

Cytotoxicity of withanolides isolated from Acnistus arborescens

A new withanolide identified by spectroscopic analysis as 12β-acetoxy-4-deoxy-5,6-deoxy-Δ⁵–withanolide D and Withanolide D, were isolated from the leaves of Acnistus arborescens. Cytotoxic activity of these two compounds against human tumor cell lines HT-29, MCF-7, MKN-45, HEp-2, HeLa, U-937 and two human normal fibroblast cultures, Fib04 and Fib05, were assessed. Withanolide D presented in vitro cytotoxic activity against tumor cell lines at the low micromolar range (LC₅₀:1.0 to 1.69 µM) and showed a slightly lower activity against Fib04, suggesting moderated selectivity among tumoral and normal cells. No cytotoxic effect was observed for 12β-acetoxy-4-deoxy-5,6-deoxy-Δ⁵–withanolide D.