Hideo Ohashi

NMR analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins

Peroxidase/H2O2-mediated radical coupling of 4-hydroxycinnamaldehydes produces 8-O-4-, 8-5-, and 8-8-coupled dehydrodimers as has been documented earlier, as well as the 5-5-coupled dehydrodimer. The 8-5-dehydrodimer is however produced kinetically in its cyclic phenylcoumaran form at neutral pH. Synthetic polymers produced from mixtures of hydroxycinnamaldehydes and normal monolignols provide the next level of complexity. Spectral data from dimers, oligomers, and synthetic polymers have allowed a more substantive assignment of aldehyde components in lignins isolated from a CAD-deficient pine mutant and an antisense-CAD-downregulated transgenic tobacco. CAD-deficient pine lignin shows enhanced levels of the typical benzaldehyde and cinnamaldehyde end-groups, along with evidence for two types of 8-O-4-coupled coniferaldehyde units. The CAD-downregulated tobacco also has higher levels of hydroxycinnamaldehyde and hydroxybenzaldehyde (mainly syringaldehyde) incorporation, but the analogous two types of 8-O-4-coupled products are the dominant features. 8-8-Coupled units are also clearly evident. There is clear evidence for coupling of hydroxycinnamaldehydes to each other and then incorporation into the lignin, as well as for the incorporation of hydroxycinnamaldehyde monomers into the growing lignin polymer. Coniferaldehyde and sinapaldehyde (as well as vanillin and syringaldehyde) co-polymerize with the traditional monolignols into lignins and do so at enhanced levels when CAD-deficiency has an impact on the normal monolignol production. The implication is that, particularly in angiosperms, the aldehydes behave like the traditional monolignols and should probably be regarded as authentic lignin monomers in normal and CAD-deficient plants.

Degradation mechanisms of a nonphenolic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole

Abstract Past studies have shown that Trametes versicolor laccase catalyzed Cα-Cβ cleavage, Cα-oxidation, β-ether cleavage and aromatic ring cleavage of a nonphenolic β- O -4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I), in the presence of 1-hydroxybenzotriazole. In this study, we investigated the detailed degradation mechanism for these pathways of substrate I by laccase/1-hydroxybenzotriazole couple, based on incorporation experiments from H 2 18 O and 18 O 2 . Oxidation of substrate I in H 2 18 O resulted in the incorporation of 18 O into three aromatic ring cleavage products and a β-ether cleavage product. These results were identical with those for lignin peroxidase. We inferred that these cleavages of substrate I proceed via the β-aryl cation radical intermediate of I. However, the Cα-Cβ cleavage degradation product of I by the laccase/1-hydroxybenzotriazole couple was 4-ethoxy-3-methoxybenzoic acid, whereas it was 4-ethoxy-3-methoxybenzaldehyde for lignin peroxidase. It is clear that one atom of 18 O from 18 O 2 was incorporated into 4-ethoxy-3-methoxybenzoic acid during the oxidation of substrate I. To confirm the reaction intermediate, we carried out the chemical reaction of 2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)- 3-hydroxypropanone with alkaline hydrogen peroxide, since the expected hydroperoxide intermediate is quite similar to that formed from the reaction between the benzylic radical of I and O 2 . The result showed that the main degradation product was 4-ethoxy-3-methoxybenzoic acid. Thus, we propose a novel Cα-Cβ cleavage via a kind of Baeyer-Villiger reaction for nonphenolic β- O -4 lignin model dimer by laccase/1-hydroxybenzotriazole couple.

Aromatic ring cleavage of a non-phenolic β-O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole

The novel cleavage products, 2,3‐dihydroxy‐1‐(4‐ethoxy‐3‐methoxyphenyl)‐1‐formyloxypropane (II) and 1‐(4‐ethoxy‐3‐methoxyphenyl)‐1,2,3‐trihydroxypropane‐2,3‐cyclic carbonate (III) were identified as products of a non‐phenolic β‐O‐4 lignin model dimer, 1,3‐dihydroxy‐2‐(2,6‐dimethoxylphenoxy)‐1‐(4‐ethoxy‐3‐methoxyphenyl)propane (I), by a Trametes versicolor laccase in the presence of 1‐hydroxybenzotriazole (1‐HBT). An isotopic experiment with a 13C‐labeled lignin model dimer, 1,3‐dihydroxy‐2‐(2,6‐[U‐ring‐13C]dimethoxyphenoxy)‐1‐(4‐ethoxy‐3‐methoxyphenyl)propane (I‐13C) indicated that the formyl and carbonate carbons of products II and III were derived from the β‐phenoxy group of β‐O‐4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase‐1‐HBT couple could catalyze the aromatic ring cleavage of non‐phenolic β‐O‐4 lignin model dimer in addition to the β‐ether cleavage, Cα‐Cβ cleavage, and Cα‐oxidation.

Screening antiacne potency of Indonesian medicinal plants: antibacterial, lipase inhibition, and antioxidant activities

Indonesian medicinal plants were screened as potential sources of antiacne agents. The screening methods were performed using antibacterial assay against Propionibacterium acnes, lipase inhibitor assay, and antioxidant assay. The results showed that from 40 plant materials extracted with methanol and 50% ethanol in water, Caesalpinia sappan was the best extract based on the combined activities: antibacterial (minimum inhibitory concentration 0.13 mg/ml; minimum bactericidal concentration 0.25 mg/ml), lipase inhibitory [50% inhibitory concentration (IC50) 120.0 μg/ml], and antioxidative (IC50 6.47 μg/ml). Another prospective extract is Intsia palembanica based on its lipase inhibitory activity (IC50 4.1 μg/ml) and antioxidant activity (IC50 3.87 μg/ml).

Lignans of Chamaecyparis obtusa

Heartwood ofChamaecyparis obtusa contains significant amounts of a dibenzylbutyrolactone lignan, hinokinin (8). This investigation demonstrated that the contents of 8 and a norlignan, hinokiresinol (12), were higher in the heartwood region than in the sapwood, indicating their nature of being heartwood extractives. Eleven lignans — xanthoxylol (1), 7-oxohinokinin (2), savinin (3), dihydrosesamin (4), isoactifolin (5), sesamin (6), piperitol (7), hinokinin (8), pluviatolide (9), haplomyrfolin (10), and rnatairesinol (11) — were isolated from young shoots ofChamaecyparis obtusa cv. Breviramea. Eight lignans (1, 2, 4, 5, 7, 9,10, and11) were isolated from this plant for the first time. Chiral high-performance liquid Chromatographie analysis showed that8, 9, 10, and11, were found to be levorotatory and optically pure (>99% e.e.). Based on the chemical structures of the isolated lignans, possible biosynthetic pathways of8 are discussed.

Brazilin from Caesalpinia sappan wood as an antiacne agent

In screening experiments for antiacne activity, methanolic and 50% ethanolic extracts of Caesalpinia sappan wood showed the most potent activity out of 28 species of plants extracts. These extracts showed inhibition of Propionibacterium acnes growth, lipase inhibitory activity, and antioxidant activity. In order to isolate the active compound from C. sappan, separation of the extract components was performed by column chromatography and preparative high-performance liquid chromatography (HPLC). Brazilin, protosappanin A, and sappanone B were isolated from methanolic extracts. Brazilin showed better antibacterial activity [minimum inhibitory concentration (MIC) = minimum bactericidal concentration (MBC) = 0.50 mg/ml] than protosappanin A (MIC = MBC = 1.00 mg/ml) and sappanone B (MIC = MBC > 2.00 mg/ml). The 50% inhibitory concentration (IC50) for lipase inhibition was lowest for brazilin (6 μM), which showed strong inhibition compared with protosappanin A (100 μM) and chloramphenicol (677 μM, positive control). The antioxidant activity of brazilin (IC50 8.8 μM) was not significantly different from protosappanin A (9.1 μM) and (+)-catechin (10.2 μM). The antioxidant activity of brazilin and protosappanin A were higher than sappanone B (IC50 14.5 μM). Brazilin is considered to have suffi ciently potent activity for use as an antiacne agent.

Simple method for synthesizing phenolic β-O -4 dilignols

A modified synthetic method for phenolicβ-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 theβ-O-4 dilignol syntheses of four combinations of guaiacyl and syringyl derivatives.

Photodiscoloration of western hemlock (Tsuga heterophylla) sapwood III Early stage of photodiscoloration reaction with lignans

The reaction during the early stage of photodiscoloration of constituents in western hemlock [Tsuga heterophylla (Raf. Sarg., Pinaceae] sapwood was investigated with chemical methods. The main photodiscoloring constituents, hydroxymatairesinol, allohydroxymatairesinol, α-conidendrin, and oxomatairesinol, were used as substrates for light-irradiation experiments in vitro. The structures of photodiscoloration reaction products were elucidated by isolation and instrumental analyses and/or co-high-performance liquid chromatography analyses with authentic specimens. The experiment was undertaken to distinguish each series of liquid phases using chloroform, water (both including a trace of methanol), and methanol, and the solid phase. The reaction products allohydroxymatairesi (2), oxomatairesinol (3), α-conidendrin (4), allo-7′-methoxymatairesinol (5), 7′-methoxymatairesinol (6), and vanillin (7) were isolated or detected in the reaction mixture of a hydroxymatairesinol system. The reaction products hydroxymatairesinol (1), 3, 4, 5, 6, and 7 were confirmed in the reaction system of allohydroxymatairesinol, which was an epimer of hydroxymatairesinol. Product 3 was confirmed from the α-conidendrin system, and reaction product 7 was confirmed from oxomatairesinol. The photodiscoloration reaction of western hemlock sapwood could be initiated by the formation of phenoxy radicals from the respective constituents. The reaction was then presumed to progress via formation of a quinonemethide intermediate in many of them. It was suggested that the reactive species, such as phenoxy radical or quinonemethide intermediate, formed by lightirradiation might be converted to quinone derivatives and colored oligomers. Products 1, 2, 3, 4, and 7, formed from substrates such as hydroxymatairesinol, allohydroxymatairesinol, α-conidendrin, and oxomatairesinol, were the same as the original metabolic constituents of western hemlock. Therefore it was concluded that the photodiscoloration of western hemlock depends not on the quantitative level of a few respective metabolites but, rather, on the coexistence of many metabolites.

Characterization of physiological functions of sapwood fluctuation of extractives in the withering process of Japanese cedar sapwood

In order to characterize the physiological functions of sapwood of needle-leaved tree, ten definite sugar and phenolic constituent contents were periodically determined among outer, middle and inner sapwoods of three Japanese cedars (Sugi, Cryptomeria japonica D. Don, Taxodiaceae) for 150 days after cutting. The fluctuation of these constituents in the withering process was characteristic in the portions of sapwood

Identification of Thuja occidentalis lignans and its biosynthetic relationship

Abstract Five lignans, (8R,8′R)-(−)-matairesinol, (8R,8′R)-(−)-thujaplicatin methyl ether, (8S,8′S)-(−)-wikstromol, 8-hydroxy-thujaplicatin methyl ether and epi-pinoresinol were isolated from Thuja occidentalis branch xylem, besides the previously identified (8R,8′R)-(−)-4-O-demethylyatein. Chiral HPLC analyses of matairesinol, thujaplicatin methyl ether, wikstromol and 4-O-demethylyatein indicated that these compounds were optically pure. A neolignan, dihydrodehydrodiconiferyl alcohol, was isolated from the acid-hydrolyzed extracts of T. occidentalis leaves. The lignans pinoresinol and secoisolariciresinol were also identified by GC–MS analysis of the extracts of xylem and leaves, respectively. Feeding experiments of [9- 2 H 2, OC2H3]coniferyl alcohol into T. occidentalis young shoots showed that two molecules of coniferyl alcohol were incorporated into pinoresinol and lariciresinol.