Molecular design for physiologically active compounds from lignin by oxidative degradation

全文

(1)46[1] 29-32 (2021). Trans. Mat. Res. Soc. Japan. Molecular design for physiologically active compounds from lignin by oxidative degradation Keigo Mikame1*, Kurumi Watanabe1, Takashi Watanabe2 and Masamitsu Funaoka3 1 Faculty of Agriculture, Niigata University, Ikarashi 2-no-cho, Nishi-Ku, Niigata, 950-2181, Japan 2 Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan 3 LIPS (Lignophenol & Systems), Collaborative Innovation Partnership *Corresponding author: Fax: 81-25-262-6854l: mikame@agr.niigata-u.ac.jp. It is important that lignin convert to high value-added product for effective utilization. It is said that lignin has little physiologically activity, although the various polyphenols of similar structure to lignin, for example lignans, flavonoids, stilbene, have various physiologically activity. In this study the physiologically active compounds molecularly designed from lignin based on structure of high activity polyphenols. The objective structure characteristic of lignin derivatives is as follows. 1) Phenolation of lignin with 1,2-dihydroxy aromatic derivatives, 2) Conversion of native lignin to β-O-4 type lignin, 3) Control of molecular weight (Mw1000-3000), 4) Conversion to long conjugate structure. o-Dihydroxy phenolation and conversion of native lignin to β-O-4 type lignin were achieved by phase separation system with catechol. Control of molecular weight of lignin derivatives and induction of long conjugate structure were occurred by alkaline cupric (CuO) oxidative degradation. The structure characteristic of lignin derivatives through the these treatment were analyzed by PDA-GPC, LC-MS and FT-IR. The antioxidative activity of lignin derivatives were largely similar to catechin in spite of polymer, extremely useful as physiologically active compounds from lignin. Key words: lignin, oxidation, physiologically activity, antioxidant. 1. INTRODUCTION Recently increases in the carbon dioxide emissions that lead to global warming accelerate the substitution of fossil resources in energy or raw materials for chemicals. Lignocellulosic biomass, as an abundant renewable carbon source, has been recognized as a potential feedstock for energy production and the synthesis of various chemicals[1],[2]. However, the lignocellulosics must be separated into individual components before use as chemical feedstocks. Separating lignocellulosic components is extremely difficult, since the lignocellulosic have the interpenetrating polymer network structure composed of lignin, hemicellulose and cellulose[3]. Thus degradated and separated chemicals from lignocellulosics is expensive. In order to make efficient use of lignocellusic components for energy or raw materials for chemicals, it is important to produce raw materials with high added value at the same time as separation of lignocellusic components, for example, resin additives, cosmetic raw materials, food with health-promoting benefits, pharmaceutical raw material and so on. Lignin is the most abundant natural polymer next to cellulose and exists in plant cell walls as one of the major constituents. Lignin provides improved physical strength to the plant cell walls, as well as serving functions for preventing degradations by living substances and/or serving functions for controlling flowability of water by providing hydrophobicity to cell walls, and lignin acts as. protector from Ultraviolet (UV) radiation for plant materials. However, in contrast to the importance and potential of lignin in nature, lignin-based products have scarcely been in human life. This strange phenomenon is due to complicated structure and reactivity of lignin. Although lignin is phenyl propanoid type phenolic compound is the same as flavonoids and lignan, lignin have little physiological activity. However lignin holds the possibility of physiologically active compounds. Because lignin are converted into compound of similar structure to tannin, flavonoids, lignan, stilbene and so on. These polyphenols has various physiological activity. In the soil, lignin oxidative degradation by white rot fungi and demethylation of methoxyl groups by some microorganisms. Thus phenolic hydroxyl groups of lignin is increase and conjugated structure between aromatic ring and propane side chain for example α-carbonyl and α-β double bond[4],[5]. Through these structural conversion, lignin degradation compounds acquire high adsorption ability of protein and metal ion[6]-[8], that is related to bioactivity. In our previous study, it have showed that phenolated lignin “lignophenol” derived from native lignin through the phase-separation process and lignopehnols were degrades by alkaline treatment. The alkaline degradation compounds have various physiological activity, for example renal disease, diabetic disease etc [9]-[11].. 29.

(2) 30. Molecular design for physiologically active compounds from lignin by oxidative degradation. In this study, we made an attempt at molecular design for physiologically active compounds from lignin with the following principle. 1) Phenolation of lignin with 1,2-dihydroxy aromatic derivatives 2) Conversion of native lignin to β-O-4 type lignin 3) Control of molecular weight (Mw1000-3000) 4) Conversion to long conjugate structure o-Dihydroxy phenolation and conversion of native lignin to β-O-4 type lignin is achievable by phase separation system with catechol. In the process, native lignin was modified by selectively grafting catechol to benzyl position, the most reactive sites, to give catechol type lignophenol (lignocatechol) that remain the original β-O-4 linkage of lignin and have high phenolic content[6],[12],[13]. o-Dihydroxy aromatic derivatives have high adsorption ability to of protein and metal ion. It is possible to control molecular weight of lignocatechol by alkaline treatment. p-Cresol type lignophenol (lignocresol) is significantly depolymerized by Neighboring Group Participation (NGP) reaction, that cleavage of β-O-4 linkage by attack phenolate ion of induced p-cresol at ortho position to β-carbon. As a results of this reaction, 30% of aryl coumaran dimers is derived from native lignin[6],[14]. But catechol is induced at ortho or para position to native lignin in lignocatechol. Although ortho type lignocatechol is depolymerized by NGP reaction, para type lignocatechol is stable through the alkaline treatment. It is expect to control molecular weight to Mw1000-3000 by this treatment. This molecular size is expect to have π-π stacking effect related to adsorption ability. Conversion of lignocatechol to long conjugate structure is due to alkaline cupric (CuO) oxidative degradation. This conjugation is result from oxidation of lignin side chain. It is expect that lignin derivatives through these conversion have high physiologically activity similar to polyphenols.. Fig.1 Depolymerization of lignocatechol by NGP reaction in alkaline solution. 2. EXPERIMENTAL 2.1 Preparation of lignocatechol through the phase-separation system For solvation of lignin with 5 mol/C9 (phenyl propane unit of lignin) of catechol dissolved in acetone was added to defatted birch (Betula platyphylla) wood meal and acetone was evaporated with stirring. Then 25ml of mix acid solution (phosphoric acid/sulfuric acid=8.5/1.5) was added to the mixture and the stirring was continued at 50℃ for 40 min. The reaction mixture was rapidly poured to excess distilled water. The insoluble fraction was collected by centrifugation, washed with distilled water until neutral and lyophilized. The dried insoluble fraction was extracted with acetone. The acetone solution was then concentrated under reduced pressure and added dropwise to an excess amount of n-hexane/benzene (1/2, v/v) with stirring. The precipitated lignocatechol was and collected by centrifugation washed with diethyl ether. Yields of lignocatechol were 88% of native lignin. 2.2 Alkaline cupric oxide oxidation of lignocatechol The dissolved 100 mg of lignocatechol in 2 ml of 1.0 N NaOH solution and 50 mg of CuO were added to stainless autoclave and heated at 180℃ for 1 hr. After cooling, CuO in reaction mixture was removed by centrifugation, and supernatant solution was acidified by the 1 N HCl. The precipitant (ACIS) was washed by de-ionized water and then dried. The molecular weight distribution and average molecular weight of derivatives from lignocatechol analyzed with gel permeation chromatography equipped with photodiode array detector (PDA-GPC). <GPC condition> Column; Shodex GPC KF-802 and 804, Eluent; THF, Flow rate; 0.6 ml/min, Temp.; 40℃ , Detect; 280 nm and 320 nm, Standard: polystyrene standard The molecular weight of derivatives from lignocatechol were determined with Shimadzu LC-MS 2010A system. <LC-MS condition> MS mode: APCI negative, Column: Imtakt Cadenza CD-C18, 3.0mm ID x 100mm, Eluent: 35% methanol, 0.25ml/min, Column temp: 40℃ FT-IR spectra of lignocatechol derivatives were determined on Shimadzu FT-IR 8400 Spectroscopy using KBr discs. The spectra were recorded from 400 to 4000cm-1. 2.3 DPPH radical scavenging activity of lignin derivatives The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability of lignin derivatives was determined according to the functionality assessment manual for food with some modifications. Catechin was used as the positive control. Radical scavenging activity was expressed as decrease rate of absorbance DPPH. DPPH radical solutions were prepared by dissolving DPPH into anhydrous ethanol with the concentration of 400 μM. The mixture solution containing 0.8 ml of 100 μM catechin, 0.5 ml of 200 mM MES (2-Morpholinoethanesulfonic acid) buffer (pH 6) and 400.

(3) Keigo Mikame et al. Trans. Mat. Res. Soc. Japan. 110.0. Lignocresol. %T. 2839.0 90.0 2939.3. 817.8. 80.0. 1589.2 1325.0 1419.5. 3419.6. (%). Transmittance. 100.0. 1035.7. 1458.1. 70.0. 1126.4 1220.9 60.0. 1506.3. 4000.0. LC. 3000.0. 2000.0. 1500.0. 1000.0. 500.0 1/cm. 100.0 %T. Lignocatechool. 60.0. 40.0. (%). Transmittance. 80.0. 20.0. 0.0 4000.0. 3000.0 LCC(8.5:1.5). 2000.0. 1500.0. Wave length (cm - 1 ). 1000.0. 500.0 1/cm. Fig.2 FT-IR spectra of lignocresol and lignocatechol. 10.0. 27.724. UV spec. 20.0. LCC CuO ACIS. 27.674. 12.5. 17.5. 15.0. 280n m. 12.5. 7.5 10.0. 5.0 7.5. 2.5. 320n m. 5.0. 0.0. 300 300nm. 250. 15.0. 17.5. 350 350nm. nm. 20.0. 22.5. 25.0. UV spec. 100. 50. 90. 27.5. 30.0. 27.618. mV mAU 120 検出器A Ch1:280nm 27.32/ 1.00/bgnd Ch2:320nm 110 60 検出器A. 32.5. 35.0. min. LCC AL ACIS. 80 70 60. 30. 27.746. 40. 283. 3. RESULTS AND DISCUSSION 3.1 Molecular design for physiologically active compounds from lignin It is important for physiologically activity of polyphenols to have many phenolic hydroxyl group. However native lignin have only 0.1 to 0.2 mol/C9 of phenolic hydroxyl groups. Because most phenolic hydroxyl groups of lignin precursors are etherified in the steps biosynthetic process. At the first stage of activation of lignin, native lignin was phenolated with catechol through the phaseseparation system. This will increase amount of phenolic hydroxyl group. The amounts of phenolic hydroxyl group of p-cresol type lignophenol (lignocresol) be much larger than native lignin[6]. Fig. 2 is FT-IR spectra of lignocresol and lignocatechol from birch wood. The hydroxyl group absorption peak (around 3200cm -1 ) of lignocatechol is much more than lignocresol. In addition, lignocatechol is 1,2-dihydroxy structure relating physiologically activity of polyphenols. The lignocatechol has two grafting type that is ortho grafting and para grafting. The NGP reactivity of these lignocatechols is different in alkaline media (Fig. 1). Therefor cleavage of β-O-4 linkage is partiality. Although lignocresol was significantly depolymerizated from Mw=6800 to 700, lignocatechol slightly depolymerizated from Mw=5300 to 3650 in 1.0N NaOH solution at 170℃.. mV mAU 22.5 検出器A Ch1:280nm 1.00/bgnd 検出器A28.34/ Ch2:320nm. 15.0. 50. 264. μM DPPH incubated in dark at room temperature for 20 min. The absorbance of the solution was measured at 520 nm by the UV–vis spectrophotometer. The control solution contains equivalent anhydrous ethanol instead of the DPPH radical solution, and the blank solution contains equivalent anhydrous ethanol instead of catechin solution. The lignin derivatives solution were prepared the same weight as catechin.. 31. 46[1] 29-32 (2021). 280n m. 40. 20. 30. 320n m. 20. 10. 10 0. 0 15.0. 250. 300 350 300nm 350nm. 17.5. 20.0. nm. 22.5. 25.0. 27.5. 30.0. 32.5. 35.0. min. Retention time (min). Fig.3 PDA-GPC analysis of LCC CuO ACIS and LC CuO ACIS As a results of LC-MS analysis, although many cresol type arylcoumarans (guaiacyl: m/z=285, syringyl: m/z=315) were formed from lignocresol[14], small amount of catechol type arylcoumarans (guaiacyl: m/z=287, syringyl: m/z=317) from lignocatechol were formed. These results show that para type grafting to benzyl position of lignocatechol is takes priority than ortho type (Fig.1). As a purpose of conversion of lignocatechol to long conjugate structure, lignocatechol was oxidatively degraded with CuO in 1.0 N NaOH solution at 180℃. The yield of acid insoluble fraction (ACIS) containing oligomer and middle range molecular weight lignin from CuO oxidative degraded lignocatechol was 77.5%, on the other hand, the yield of ACIS from lignocresol is 51.6% due to the difference of NGP effect. In PDA-GPC analysis, molecular weight of ACIS fraction alkaline CuO oxidative degraded lignocatechol (LCC CuO ACIS) and alkaline treated lignocatechol without CuO (LCC AL ACIS) are Mw=2900 and 3650 respectively, because of oxidation catalysis. In addition, ultraviolet absorptivity of LCC CuO ACIS in long-wavelength region was higher than LCC AL ACIS (Fig. 3). Therefore it was showed that LCC CuO ACIS is contain many long conjugate structure relating physiologically activity of polyphenols than lignocresol (LC) CuO ACIS. To confirm attribution of long conjugate structure, lignocatechol, LCC AL ACIS, LCC CuO ACIS and LC CuO ACIS were analyzed by FT-IR (Fig.1 and Fig. 4). The absorption of carbonyl (approximately 1700cm -1 ) of lignocatechol and LCC AL ACIS were little in the spectra. However the carbonyl absorption peak was slightly appeared in birch original LC CuO ACIS, and the peak was strong in LCC CuO ACIS. It was thought that carbonyl groups were formed at side chain of lignin and catechol ring of lignocatechol was convert to quinoid structure by CuO oxidation, and these.

(4) 32. Molecular design for physiologically active compounds from lignin by oxidative degradation. 100.0. LCC AL ACIS. %T 90.0. LCC AL ACIS. Transmittance (%). 2742.6. LC CuO ACIS. 80.0 2839.0. 70.0. 414.7 480.2. 2939.3. 1028.0 1423.4 1604.7. 60.0 3427.3. 1514.0. 4000.0 3500.0 3000.0 2500.0 2000.0 LCC(8.5,1.5)AL ACIS. 1750.0 1500.0. 1250.0 1000.0. 750.0. 500.0 1/cm. LC CuO ACIS. Transmittance (%). 2841.0 2937.4. 2341.4 2360.7. 812.0. 3417.6. 1035.7. 75.0. 1423.4 1610.5. 70.0. 1114.8 1461.9 1490.9 1515.9 1213.1. 65.0 60.0. 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 LC CuO ACIS. 750.0. 500.0 1/cm. 100.0 %T 90.0. LCC CuO ACIS. Transmittance (%). 80.0 416.6 70.0. 2841.0 1031.8. 2939.3. 60.0 50.0. 1423.4. 3408.0. 1461.9 1604.7 1514.0. 40.0 4000.0. 3000.0 LCC(8.5,1.5) CuO ACIS. 2000.0. 1500.0. 1217.0 1114.8 1000.0. wave length(cm-1). 20. 40. 60. 80. 100. Decrease rate of absorbance(%). 95.0. 80.0. Catechin. 0. 1218.9. 105.0 %T 100.0. 85.0. LC AL ACIS. Controll 1120.6. 1458.1. 50.0. 90.0. LCC CuO ACIS. 500.0 1/cm. Fig.4 FT-IR spectra of LCC AL ACIS, LC CuO ACIS and LCC CuO ACIS. structure contributed to long conjugate structure of LCC CuO ACIS. Therefore LCC CuO ACIS have a target feature described above, 1 ,2-dihydroxy aromatic, β-O-4 type and long conjugate structure and middle range molecular weight, relating to various physiologically activities. 3.2 Antioxidative activity of lignin derivatives As one of the physiologically activity evaluation, DPPH radical scavenging assay of lignophenol derivatives were carried out. The decrease rate of absorbance of DPPH treated with LC AL ACIS, LC CuO ACIS, LCC AL ACIS and LCC CuO ACIS were compare to catechin having high antioxidative activity and similar to lignocatechol degraded compounds. As shown in Fig.5, lignocatechol degraded compounds and CuO oxidated compounds were showed high antioxidative activity. Especially the decrease rate of absorbance of DPPH with LCC CuO ACIS was largely similar to catechin in spite of polymer. Thus LCC CuO ACIS is extremely useful as physiologically active compounds from lignin.. Fig.5 DPPH radical scavenging activities of lignin derivatives 4. CONCLUSION The physiologically active compounds molecularly designed from lignin for high-value-added product derived lignin. o-Dihydroxy phenolation and conversion of native lignin to β-O-4 type lignin were achieved by phase separation system with catechol. Control of molecular weight of lignin derivatives and induction of long conjugate structure were occurred by alkaline cupric (CuO) oxidative degradation. The lignin derivatives were largely similar to catechin in spite of polymer, extremely useful as physiologically active compounds from lignin. 5 References [1] I. S. Goldstein, Forest Products J., 31, 63-68 (1981) [2] F. Parisi, Adv. Biochem. Engin./ Biotechnol. 38, 53-87 (1989). [3] G.J. Clydesdale, G.W. Dandie, H.K. Muller, Immunol. Cell. Biol., 79, 547–568 (2001) [4] B. VENKATESAGOWDA, Fungal Biology Reviews, 33, 190-224 (2019) [5] K. K. PandeyA. J. Pitman, International Biodeterioration & Biodegradation 52, 151–160 (2003) [6] M. Funaoka, Polymer International, 47, 277-290 (1998). [7] D. Parajuli, K. Inoue, K. Ohto, T. Oshima, Reactive & Functional Polymers 62, 129–139 (2005) [8] D. Parajuli a, K. Inoue, H. Kawakita, K. Ohto H. Harada, M. Funaoka, Minerals Engineering 21, 61–64 (2008) [9] S. Sato, Y. Mukai, J. Yamate, T. Norikura, Y. Morinaga, K. Mikame, M. Funaoka, S. Fujita, Free Radical Research, 43, 1205-1213 (2009) [10] S. Sato, Y. Mukai, Y. Tokuoka, K. Mikame, M. Funaoka, S. Fujita, , Environmental Toxicology and Pharmacology, 34, 228-234 (2012) [11] S. Sato, T. Norikura, Y. Mukai, S. Yamaoka, K. Mikame, Chemico-Biological Interactions, 318, 108977 (2020) [12] K. Mikame, M. Funaoka, Polymer Journal, 38, 585-591 (2006) [13] K. Mikame, M. Funaoka, Polymer structure of, Polymer Journal, 38, 592-596 (2006) [14] K. Mikame, M. Funaoka, Trans. the Materials Research Society of Japan, 33, 1149-1152 (2008) (Received November 30, 2020; Accepted January 14, 2021; Published Online March 1, 2021).

(5)