Specific degradation ofβ-aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes ofSphingomonas paucimobilis SYK-6 produced in recombinantEscherichia coli

Sphingomonas paucimobilis SYK-6 produces unique and specific enzymes, such as-etherases,O-demethylases, and ring fission dioxygenases, for lignin degradation. Cleavage of arylglycerol--aryl ether linkage is the most important process in the lignin metabolic pathway ofS. paucimobilis SYK-6. We reported the genes (ligD, ligE, ligF) for enzymes that cleaved-aryl ether linkage of dimeric compounds in previous studies. In this study we synthesized the fluorescent high-molecular-weight lignin (UBE-DHP) by dehydrogenative polymerization. We investigated the-aryl ether cleavage ability of these enzymes produced in recombinantEscherichia coli. When UBE-DHP was incubated with LigF, 4-methylumbeliferone was released as a result of-aryl ether cleavage of O-methylumbelliferyl--hydroxypropiovanillone (compound III) incorporated in UBE-DHP. Here, we report that-etherase ofS. paucimobilis SYK-6 can be expressed inE. coli and is able to cleave the-aryl ether linkage in synthetic high-molecular-weight lignin.     

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     

Lignans ofLinum flavum var.compactum

A new dibenzylbutyrolactone lignan 7.6-dihydroxybursehernin, together with six known lignans (pinoresinol, lariciresinol, secoisolariciresinol,-peltatin,-peltatin, 5-methoxypodophyllotoxin) were isolated from the methanol extracts ofLinum flavum var.compactum. The enantiomeric analysis of pinoresinol and lariciresinol isolated from the species, which are upstream lignans in the lignan biosynthetic pathway, indicated that they are not optically pure, which is in accordance with our recent findings on lignans occurring in other plant species.Parts of this report were presented at the 42nd Lignin Symposium, Sapporo, October 1997; and the 43rd Lignin Symposium, Fuchu, October 1998     

5α-Reductase inhibitory component from leaves ofArtocarpus altilis

The inhibitory effects of 17 samples prepared from Thailand plants on 5-reductase activity were examined. The acetone extract of leaves ofArtocarpus altilis showed potent 5-reductase inhibitory activity. Fractionation guided by 5-reductase inhibition led to the isolation of 2-geranyl-2,3,4,4-tetrahydroxydihydrochalcone from the acetone extract of leaves ofA. altilis. This compound showed more potent inhibitory effect (IC₅₀=38M) than-linolenic acid known as a naturally occurring potent inhibitor.     

Dihydroroseoside, a new cyclohexanone glucoside, from the leaves of shirakamba (Betula platyphylla Sukatchev var.japonica Hara)

A new cyclohexanone glucoside (II) and a known cyclohexenone glucoside roseoside [I, (6S, 9S)-6-hydroxy-6-(9--D-glucopyranosyloxy-trans-7-butenyl)-1,5,5-trimethyl-1-cyclohexenone] were isolated from an ethanol extract of shirakamba (Betula platyphylla Sukatchev var.japonica Hara) leaves. The structure ofII was determined to be (6S, 9R)-6-hydroxy-6-(9-O--D-glucopyranosyloxy-trans-7-butenyl)-1, 5,5-trimethyl-1-cyclohexanone by¹H-NMR and¹³C-NMR spectroscopic analyses. It was named dihydroroseoside.     

Four glucosides ofp-hydroxyphenyl derivatives from birch leaves

The extractive of shirakamba (Betula platyphylla Sukatchev var.japonica Hara) leaves was investigated. Four glucosides ofp-hydroxyphenyl derivatives were isolated, and their structures were indentified as betuloside (I), 3,4-dihydroxy-propiophenone-3--d-glucopyranoside (II), salidroside (III), and arbutin (IV). Arbutin was newly found in the leaves of shirakamba.     

Absolute configuration of arylglycerol-β-aryl ethers obtained by asymmetric reduction of the correspondingα-ketonic compound with intactFusarium solani cells

When (±)--oxo-guaiacylglycerol--(vanillic acid) ether (1) is degraded byFusarium solani M-13-1, the-ketone is initially reduced to giveerythro andthreo guaiacylglycerol--(vanillic acid) ethers (2), arylglycerol--aryl ethers, both of which are enantiomerically pure. The absolute configuration in each2 was determined by Mosher's method; the products were converted to,-di-(R)--methoxy--trifluoromethylphenylacetates (MTPA esters) (3) oferythro (-)- andthreo (+)-veratrylglycerol--(methyl vanillate) ethers (3), whose¹H nuclear magnetic resonance (NMR) spectra were examined and compared with those of four di-(R)-MTPA ester (3) diastereomers from chemically synthesizederythro (±)-3 andthreo (±)-3. To assign the- and-MTPA-OCH₃ peaks, the¹H NMR scans of several compounds that have substructures of 3 and their 3,4,5-trimethoxyphenyl analogues were examined. When a racemic alcohol reacts with (R)-MTPA to give a pair of (R)-MTPA ester diastereomers, the value was defined as the absolute value of the difference in the¹H chemical shifts of the peak between the diastereomers. It was found that the values of-MTPA-OCH₃ were larger than those of-MTPA-OCH₃ owing to a shielding effect of the veratryl ring located on the-MTPA-OCH₃, and that the-MTPA-OCH₃ peaks in the 3,4,5-trimethoxyphenyl compounds shifted downfield relative to those in the veratryl compounds. On the basis of the¹h NMR data of (R)-MTPA esters, the absolute configuration of the four chemically prepared diastereomers (3) were determined. The catabolicerythro 3 [fromerythro (-)-3] andthreo 3 [fromthreo (+)-3] were identical to (R, S, R)-erythro 3 and (R, S, S)- threo 3, respectively. An hydrogen species in the fungal reduction would attack the-ketone fromre-face of both (R)-1 and (S)-1, givingerythro (S, R)-2 andthreo (S, S)-2, respectively.Part of this paper was presented at the 33rd Lignin Symposium, Tsukuba, November 1988     

Antioxidant activity of extracts from <Emphasis Type="Italic">Calocedrus formosana</Emphasis> leaf,bark, and heartwood

Calocedrus formosana Florin (Cupressaceae) is an endemic tree to Taiwan. To evaluate the antioxidant activity of the plant extracts from heartwood, bark, and leaf of C. formosana, assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion scavenging activities, as well as prevention of DNA strand cleavage were performed in this study. Similar IC₅₀ values against the DPPH radical were found for the heartwood and bark extracts at approximately 23µg/ml. Moreover, the heartwood extract exhibited the highest inhibitory activity against superoxide radicals among the test samples; a 2.3-fold lower value of IC₅₀ for superoxide radical inhibition was found in the heartwood extract relative to that of (+)-catechin. Much less effect on inhibition of DPPH and superoxide radicals was found from the leaf extract of C. formosana. More than 70% of superoxide radicals were inhibited in the presence of 10µg/ml heartwood extract, whereas only 15% inhibition was obtained from the leaf extract. The heartwood extract, at a dose of approximately 0.5mg/ml, apparently completely prevented the X174 supercoiled DNA cleavage induced by ultraviolet photolysis of H₂O₂, as judged by agarose gel electrophoresis. This report also suggests that the antioxidant activities of the plant extracts of C. formosana are in good correlation with their phenolic contents.     

Effects of stilbenes from bark of <Emphasis Type="Italic">Picea glehnii</Emphasis> (Sieb. et Zucc) and their related compounds against feeding behaviour of <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Kolbe)

The effects of stilbene glucosides and related compounds on termite feeding behavior were investigated using paper disc methods against the subterranean termite Reticulitermes speratus. The stilbene-rich fraction and isorhapontin (3-methoxy-3,4, 5-trihydroxystilbene-3--d-glucoside) from bark extracts of Picea glehnii showed avoidance by termites in choice tests. In the no-choice tests using compounds purified from the stilbene-rich fraction, the largest feeding deterrent effect was observed for piceid (3,4,5-trihydroxystilbene-3--d-glucoside), followed by isorhapontin, and astringin (3,3,4,5-tetrahydroxystilbene-3--d-glucoside), at the concentrations from 0.63 to 2.5µmol/disc. No change in activity was observed at retentions of more than 5.0µmol/disc. When the activities of isorhapontin and its aglycone derivative (isorhapontigenin: 3-methoxy-3,4,5-trihydroxystilbene) were compared with that of taxifolin (3,3,4,5,7-pentahydroxyflavanone) in the no-choice test, the stilbenes exhibited a larger antifeedant potential. Methylation of isorhapontigenin increased its termiticidal activity.Part of this study was presented at the 32nd Annual Meeting of the International Research Group on Wood Preservation, 2001     

Role of caffeic glucoside esters in defense-repair processing of trees II: Synthesis of 2-(3,4-dihydroxyphenyl)-ethyl 3-α-L-rhamnopyranosylβ-D-glucopyranoside

The reference compound (9), with a partial structure of acteoside, was synthesized to elucidate the relation between structural features and the precipitation or solubility of the oxidation products of acteoside: 2-(3,4-dihydroxyphenyl)-ethyl 3-O-(-L-rhamnopyranosyl)--D-glucopyranoside (9). The glycosyl acceptor 2-O-benzoyl-3-O-(2,3,4-tri-O-acetyl--L-rhamnopyranosyl)-4,6-O-benzylidene--D-glucopyranosyl trichloroacetimidate (7) was prepared from allyl 2-O-benzoyl-3-O-(2,3,4-tri-O-acetyl--L-rhamnopyranosyl)-4,6-O-benzylidene--D-glucopyranoside (4) via isomerization of the allyl group with an iridium complex to the 1-propenyl group and its hydrolysis with HgCl₂lHgO, followed by treatment with CCl₃CN and DBU in a 65.5% overall yield. The glycosyl acceptor 3,4-diacetoxyphenethyl alcohol (16) was prepared from homoveratric acid via demethylation with 57% HI and its acetylation with Ac₂O and 85% H₃PO₄, followed by selective reduction of the carboxyl group to the alcohol with a borane-tetrahydrofurane complex in a 61% overall yield. The glycosylation of7 with16 in dichloromethane promoted by BF₃-Et₂O gave a 74.3% yield of8. Hydrolysis of8 with 90% CF₃COOH gave the debenzylidenated product, which was treated with NaOMe to afford a 32% overall yield of the desired compound9. This compound9 was identical with the natural specimen.This study was presented in part at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 3–5, 1996     

The effect of strain, growth stage, and cultivating condition of Ganoderma lucidum on 5α-reductase inhibition

The inhibitory effects of 102 methanol extracts of 40 mycelia, 9 culture fluids, and 53 fruiting bodies of 40 strains of Ganoderma lucidum on 5-reductase were investigated. The methanol extract of the fruiting body of each strain was found to show the strongest 5-reductase inhibitory activity among the extracts tested.     

Soaking faba bean seed in some bio-agents as prophylactic treatment for controlling<Emphasis Type="Italic"> Meloidogyne incognita</Emphasis> root-knot nematode infection

Soaking faba beans or treating their soil with the bio-agents Abamectin (a fermentation product from Streptomyces avermitilis), a strain of Bacillus thuringiensis, Nemaless (containing strains of Serratia marcescens), and Sincocin-AG were compared to the nematicide Oxamyl and significantly (P 0.01 and/or P 0.05) reduced the population density of M. incognita with increasing the measured plant growth . On this basis, seed soaking was recommended as an economical, easy and pollution free method for managing M. incognita.     

Inhibition ofTrichoderma cellulase activity by a stilbene glucoside fromPicea glehnii bark

The stilbene glucoside isorhapontin (5,4-dihydroxy-3-methoxystilbene-3--D-glucoside) is the major constituent of the ethyl acetate extracts fromPicea glehnii bark. Isorhapontin inhibited the hydrolytic activity ofTrichoderma cellobiohydrolase I (CBH I) for both bacterial microcrystalline cellulose and the soluble cellooligosaccharide celloheptaitol. The inhibitory effect for celloheptaitol, however, was more drastic than that for bacterial microcrystalline cellulose. The hydrolytic activity of the CBH I core domain for celloheptaitol was also inhibited by isorhapontin to a similar extent, suggesting that the interaction between isorhapontin and the core domain of CBH I is the reason for this phenomenon. The inhibition of CBH I activity by isorhapontin showed mixed noncompetitive and uncompetitive types in a concentration of the inhibitor of less than 125M. TheK _i andK _i values were estimated to be 57.2 and 33.3M, respectively. Whereas isorhapontin strongly inhibited CBH I activity, its aglycone isorhapontigenin (3-methoxy-3,5,4-trihydroxystilbene) showed almost no inhibition. Consequently, both the stilbenic and the-glucosidic structures in isorhapontin are essential for the inhibitory effect on CBH I activity. Isorhapontin also inhibited the activity ofTrichoderma endoglucanase I for celloheptaitol, whereas almost no effect was observed for the activities of both endoglucanases II and III.An outline of this work was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Antioxidant activity of liquors from aqueous treatments of Pinus radiata wood

Non-isothermal, aqueous treatments of Pinus radiata wood were carried out to reach maximal temperatures in the range 210–240°C, and the liquors from the treatments were analysed for phenolic content and antioxidant activity using several tests (,-diphenyl--picrylhydrazyl (DPPH) radical scavenging, hydroxyl radical scavenging, Trolox equivalent antioxidant capacity, -carotene bleaching and reducing power). Increased severity resulted in increased yields in phenolics (up to 1.5 g phenolic compounds/100 g oven-dry wood) and improved antioxidant activity. The DPPH radical scavenging capacity of compounds present in autohydrolysis liquors and the -carotene bleaching ability of the compounds extracted with ethyl acetate from liquors were higher than those of synthetic antioxidants. The fractions solubilised in autohydrolysis treatments were also effective as hydroxyl and ABTS radical scavengers. The autohydrolysis liquors and their ethyl acetate soluble fraction showed reducing power in the range reported for extracts from other natural sources.     

Simple method for synthesizing phenolicβ-O-4 dilignols

A modified synthetic method for phenolic-O-4 lignin substructure model dimers was developed involving protection of the phenolic hydroxyl group of acetophenons with benzoyl chloride, bromination with 4-dimethylaminopyridiniumbromide perbromide, condensation with phenols in the presence of 18-crown-6-ether, condensation with paraformaldehyde, reduction with NaBH₄ and debenzoylation. This method results in shorter reaction times and increasing yields without the application of strict anhydrous and drastic conditions or chloric solvents. This alternative route could be applied to the-O-4 dilignol syntheses of four combinations of guaiacyl and syringyl derivatives.     

Mechanical relaxation processes due to sugars in cane (Arundo donax L.)

The storage modulus and the mechanical loss tangent of untreated, extracted, and sugar- impregnated canes (Arundo donax L.) were measured over a temperature range of –150° to 0°C at low frequencies. Two relaxation processes, labeled and , were detected in the ranges –60° to 0°C and –120° to –100°C, respectively. The and processes shifted to lower temperatures with increasing moisture content. The process was detected only in the canes containing sugar. The magnitude of its loss peak increased with an increase in sugar content. It was speculated that the process was due to some interactive molecular motions of the adsorbed water and sugar. The process, detected in all of the canes, was attributed to the motion of the adsorbed water in the amorphous cell wall substances.     

Synthesis of dihydroxyphenacyl glycosides for biological and medicinal study: β-oxoacteoside from Paulownia tomentosa

The protected structure of -oxoacteoside (tomentoside A), 2-oxo-2-(3,4-dihydroxyphenyl)ethyl 3-O-(2,3,4-tri-O-acetyl--l-rhamnopyranosyl)-4-O-caffeoyl--d-glucopyranoside 14 was synthesized in 14% overall yield in 11 steps, starting from d-glucose for biological and medicinal studies of phenylpropanoid glycosides. The first step was the preparation of a 3-O-rhamnopyranosyl disaccharide sugar core 2 from a suitably protected rhamnosyl trichloroacetimidate 10 and glucose derivative (diacetone-d-glucose 1) in 71% yield. To the glucose moiety of this sugar core, several protection/deprotection procedures were performed sequentially to obtain a fully acetylated sugar core 7 with a 4-OH group on the glucose moiety, in 57% yield in five steps. Thereafter, to the 4-OH group of the glucose moiety, selective 4-O-caffeoylation was achieved by proton-transfer esterification with 3,4-di-O-allylcaffeic acid 16 to give the caffeoyl disaccharide 11 in 97% yield. Then, it was converted to trichloroacetimidate 13 for a glycosylation donor in 90% in two steps. Finally, anomeric glycosylation was conducted with 2-oxo-2-(3,4-di-allyloxyphenyl)ethyl alcohol 19 with catalytic amounts of BF₃·Et₂O to give 2-oxo-2-(3,4-di-allyloxyphenyl)ethyl 2,6-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl--l-rhamnopyranosyl)-4-O-(3,4-di-allyloxycaffeoyl)--d-glucopyranoside 14 in 60% yield. Deprotected intermediates of compounds 2, 11, 14, and 19 which were obtained in high yield would be useful for biological and medicinal studies of phenylpropanoid glycosides.Part of this study was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April, 2002     

Compounds inhibitory to rat liver 5α-reductase from tropical commercial wood species: resveratrol trimers from melapi (Shorea sp.) heartwood

5-Reductase inhibitory activity of methanol extracts of the heartwood of 13 tropical wood species were examined. Strong 5-reductase inhibitory activity was observed withShorea species. From melapi (Shorea sp.), two known resveratrol trimers, vaticanol A and ampelopsin C, and two novel trimers were isolated as active compounds. The structures of the two novel resveratrol trimers were elucidated by one- and two-dimensional nuclear magnetic resonance spectroscopic analyses, including¹H-¹HCOSY, HMQC, and HMBC. The compounds were named melapinol A and melapinol B. There were no significant differences among the 5-reductase inhibitory activities of the four resveratrol trimers, which were significantly stronger than those of-linolenic acid and epigallocatechin gallate, known 5-reductase inhibitors.     

Length of cuttings in juvenile development of a hybrid poplar clone

The influence of length of cuttings on establishment of a short rotation plantation was investigated over a period of two years using direct field planting of unrooted Populus Rasumowskiana cuttings. The longer the cuttings, the higher were the survival and dry mass production. Survival at the end of the second growing season varied from 75% to 97%. In two years, the two longest cutting lengths (40 cm and 50 cm) produced about 100 g dry mass per living cutting; but owing to the higher survival rate of the longest cuttings, there was a difference per area unit: 360 g/m² for 40 cm and 400 g/m² for 50 cm-long cuttings. The shortest (10 cm) cuttings produced only 115 g/m².     

Provenance variation in growth characters of four subtropical pine species planted in Mexico

In 1995 a species/provenance test was established in the state of Puebla, Mexico to assess and compare the growth of Pinus greggii var. australis, P. maximinoi, P. patula and P. tecunumanii, including 10, 5, 11 and 4 provenances of each species, respectively. Each provenance test lot consisted of seed from 7 to 10 individual trees collected in natural stands. Range-wide samples were included in the test for P. greggii var. australis and P. patula. Material of P. maximinoi and P. tecunumanii from Guatemala, Honduras and Nicaragua, as well as Mexico was established in the test. The test site is in a mountainous area at 1440m elevation, near 20°N latitude. At 5 years of age significant differences were seen at both the species and provenance levels in survival, total height, diameter and volume. The overall test survival was 93%. Variance among species was three time greater than among provenances, comprising 25–33% of the total variation. P. maximinoi had the greatest height, diameter and volume (62dm³) followed by P. tecunumanii (53dm³). P. greggii var. australis had an average volume of 43dm³ and P. patula produced 30dm³. Although P. maximinoi had the lowest survival rate, the San Jeronimo provenance was the most productive of all 30 provenances tested; 98% survival and 73dm³ volume. This was equivalent to 2.8cm annual diameter increment and 2m in height growth per year. Significant growth differences among provenances within species indicate that potential gain exists using provenance selection.