Lignin-carbohydrate complexes (LCCs) were extracted from botanical waste, Quercus acutissima, rice bran, peanut husks, tofu refuse, and used green tea leaves. The contents of the lignin and phenolic hydroxyl groups in the LCCs taken from these samples were positively correlated. The capacity of scavenging reactive oxygen species was evaluated by employing an electron spin resonance method for its superoxide dismutase (SOD)-like activity. The lignin content and the SOD-like activity of LCC from Quercus acutissima were much greater than those of other LCCs. These findings suggest that the SOD-like activity of LCCs depends on the lignin content

Noda, Hiroyuki

Toh, Kazuko

Yokoyama, Hidekatsu

English

Wood and Fiber Science (E-Journal)

Wood and Fiber Science, 37(2), 2005, pp. 270–274© 2005 by the Society of Wood Science and TechnologyEVALUATION OF THE ABILITY OF AQUEOUS LIGNIN-CARBOHYDRATECOMPLEX EXTRACTED FROM BOTANICAL WASTE TO SCAVENGEREACTIVE OXYGEN SPECIES BY USING AN ELECTRON SPINRESONANCE METHODKazuko TohResearcherHidekatsu YokoyamaSenior ResearcherandHiroyuki NodaSenior ResearcherInstitute for Life Support Technology2–2-1, MatsueiYamagata 990–2473, Japan(Received January 2004)abstractLignin-carbohydrate complexes (LCCs) were extracted from botanical waste, Quercus acutissima, ricebran, peanut husks, tofu refuse, and used green tea leaves. The contents of the lignin and phenolic hydroxylgroups in the LCCs taken from these samples were positively correlated. The capacity of scavenging reac-tive oxygen species was evaluated by employing an electron spin resonance method for its superoxide dis-mutase (SOD)-like activity. The lignin content and the SOD-like activity of LCC from Quercus acutissimawere much greater than those of other LCCs. These findings suggest that the SOD-like activity of LCCsdepends on the lignin content.Keywords: Lignin-carbohydrate complexes, SOD-like activity, botanical waste, ESR.introductionLignin, a structural component of the cellwalls of vascular plants, is a natural polymerabundantly found in the biosphere. It is taken upas a dietary fiber when vegetables and fruits areconsumed. Although most of the lignin is ex-creted without being digested, it is thought thatpart of it has some effect on the human body. Infact, the antitumor and antimicrobial activities ofsome types of lignin have been reported (Sak-agami et al. 1991, 1998).In this research, the ability of lignin obtainedfrom botanical waste to scavenge reactive oxy-gen species (ROS) was evaluated. ROS is a gen-eral term for highly reactive molecules derivedfrom oxygen, such as superoxide radicals, hy-drogen peroxide, and hydroxyl radicals. It isthought that the overproduction of ROS mayplay a role in various diseases, including inflam-mation, cancer, and epilepsy (Oyanagui et al.1988; Kensler et al. 1983; Yokoyama et al.1992). Therefore it is important to investigatethe ability of lignin to scavenge ROS.A superoxide radical is a typical ROS. Super-oxide dismutase (SOD) is a specific enzyme thatdismutates this superoxide radical. The dismuta-tion rate by SOD occurs more rapidly than in thespontaneous process of becoming disproportion-ate (McCord and Fridovich 1969). Based on thesuperoxide radical concentration in a mixture of asample and a solution that includes a superoxideToh et al.—SOD-LIKE ACTIVITY OF LCC FROM BOTANICAL WASTE 271generating system, the superoxide scavenging ac-tivity of the sample can be estimated for a “SOD-like activity” (Kaneyuki et al. 1999). Theelectron spin resonance (ESR) method is aunique technique for the specific detection ofelectron spins of radicals. The hypoxanthine(HPX)/xanthine oxidase (XOD) system is com-monly used for generating a superoxide radical.Because the HPX/XOD system is used in a watersystem, target samples should be water-soluble;therefore the SOD-like activity of a water-solublecomponent of lignin, the lignin-carbohydratecomplex (LCC) from botanical waste, was evalu-ated by using an ESR spectrometer.experimentsPreparation of lignin and lignin-carbohydratecomplexLCCs were extracted from botanical waste,the woody part of Quercus acutissima, rice bran,peanut husks, tofu refuse, and used green tealeaves with the stalk.The samples were extracted in a powder formby using Soxhlet’s extractor with 100% acetone(Wako Pure Chemicals Co. Japan) and with a90% (volume/volume, v/v) aqueous acetone so-lution. Each extraction was continued for 48 h.The residue was dried and extracted also byusing the Soxhlet’s extractor with an 80% (v/v)aqueous dioxane (Wako Pure Chemicals Co.Japan) solution for 72 h. The residue resultingfrom lignin extraction by these procedures wasagain extracted with hot water (60°C) for 1 h.The extract was precipitated from five volumesof ethanol to yield the LCC.The LCCs of Quercus acutissima, rice bran,peanut husks, tofu refuse, and green tea are re-ferred to here as QLCC, RLCC, PLCC, TLCC,and GLCC, respectively.Estimation of contents of lignin and phenolichydroxyl groupThe contents of lignin were determined by anacetyl bromide method (Johnson et al. 1961).The contents of the phenolic hydroxyl groupwere estimated by ionization difference spec-trum (Goldschmid 1954). The percentage ofphenolic hydroxyl groups in LCC was obtainedfrom the formula: (17∆αmax)/4100 • 100, where∆αmax is the differential absorbance for each g/l • cm at 280 nm. For these measurements, aspectrophotometer (U-2000, Hitachi, Japan) wasused. The error level of each determination wasless than 10–2.Estimation of SOD-like activityAs a superoxide generation system, solutionof 2 mM of HPX (Sigma Chemical Co. USA)and 0.4 unit/ml of XOD (milk origin, Roche,Germany) was prepared in a phosphate buffersolution (PBS, 0.1 M, pH 7.4). The XOD solu-tion was kept on crushed ice. SOD (human ery-throcytes origin CuZn-SOD, 3900 units/mg,Sigma Chemical Co. USA) was dissolved inultra-pure water in concentrations of 1.50, 2.25,3.00, 3.75, and 4.50 units/ml. The samples werealso dissolved in ultra-pure water. The con-centrations of TLCC were 6, 10, 15, 20, and 30 mg/ml; for PLCC, 0.2, 1.0, 2.0, 5.0, and 10.0 mg/ml; for RLCC, 1.0, 2.0, 3.3, 5.0, and 10.0 mg/ml; for GLCC, 0.10, 0.25, 0.50,0.75, and 1 mg/ml; and for QLCC, 0.01, 0.02, 0.03, 0.05, and 0.10 mg/ml. As a spin-trapping reagent, 5-dimethyl-1-pyrroline-1-oxide (DMPO, 9.2 M, Labotec. Co. Japan) wasused.An XOD (50 l) solution was added to amixed solution of DMPO (15 l), HPX (50 l),SOD or sample (35 l), and PBS (50 l). Imme-diately after the mixture was stirred, it wasplaced in a capillary tube at the center of the cav-ity resonator of an ESR spectrometer (JES-TE200, JOEL, Japan). ESR measurements werestarted 60 s after XOD was added. The condi-tions for measurement were: microwave power,8 mW; center magnetic field, 336.8 mT; mag-netic field sweep width, 10 mT; magnetic fieldsweep time and time constant, 120 s and 0.1 s;and magnetic field modulation width, 0.1 mT at100 kHz.The ESR spectrum thus obtained was identi-fied with a DMPO-superoxide adduct (DMPO-OOH) by measuring the hyperfine coupling272 WOOD AND FIBER SCIENCE, APRIL 2005, V. 37(2)constant, which was aN  1.42 mT, aH  1.14mT, and aH  0.13 mT (Fig.1). The ESR signalintensity was derived from the ratio of the peakheight of the low magnetic field component ofan ESR spectrum (Is in Fig. 1) to that of an exter-nal standard marker (IM in Fig. 1). The ESR mea-surements were also made without the SOD orthe sample solution. “I” and “I0” were defined asESR signal intensities with and without the SODor sample solution, respectively. The relation-ship between “I0/I-1” and the SOD or the sampleconcentration was plotted. SOD-like activitywas obtained on the basis of the rate of concen-tration of SOD to that of the sample, which gaveI0/I-11 in the plotted curve.results and discussionAs shown in Fig. 2, the contents of lignin(weight/weight, w/w) for TLCC, PLCC, RLCC,GLCC, and QLCC were 9.8%, 4.4%, 9.4%,10.7%, and 30.1%, respectively. As shown inFig. 3, the contents of the phenolic hydroxylgroups for TLCC, RLCC, GLCC, and QLCCwere 0.011%, 0.016%, 0.016%, and 0.077%, re-spectively. Nothing was detected for PLCC. Thecontents of phenolic hydroxyl groups and ligninwere positively correlated (correlation coeffi-cient  0.999).As shown in Fig. 4, the SOD-like activities ofTLCC, PLCC, RLCC, GLCC, and QLCC were0.200.01, 0.730.03, 4.040.06, 6.040.50,and 209.324.88 units/mg, respectively (valuesare mean  standard error from 5 independentdeterminations). The value for QLCC is muchgreater than those of the other LCCs. Catechin,tannin, and flavonoid groups are well-knowncomponents with SOD-like activity in plants(Kaneyuki et al. 1999; Kim et al. 1995; Fukudaet al. 2003). Although LCC obtained by the pres-ent method includes monosaccharides (such asarabinose, xylose, and mannose) as impurities(Björkman 1956; Watanabe et al. 1987), theFig. 1. An example of an ESR spectrum of DMPO-OOH. The ESR signal intensity was derived from the ratioof the peak heights of the low magnetic field component ofan ESR spectrum (IS) to an external standard marker (IM).Fig. 2. Lignin contents of LCCs.Fig. 3. Contents of phenolic hydroxyl group in LCCs.Toh et al.—SOD-LIKE ACTIVITY OF LCC FROM BOTANICAL WASTE 273components with the SOD-like activity de-scribed above were excluded during the LCCpreparation because they had been dissolvedwell in ethanol. We think that the SOD-like ac-tivity shown in this study is due to the structureof LCC.In the SOD-like activity of polyphenol, suchas flavonoid, the activity increases with the num-ber of hydroxyl groups in a benzene ring (Husainet al. 1987; Puppo 1992; Yoshiki et al. 1995). Ifit is assumed that the number of phenolic hy-droxyl groups in a benzene of lignin in LCC af-fects the SOD-like activity, one may expect theSOD-like activity of the lignin to be low becausethe number is one. In fact, the SOD-like activityof LCC was very low (except for QLCC, Fig.4).These results show that the SOD-like activityand the content of lignin for QLCC were muchgreater than for the other LCCs (Figs. 2 and 4).As noted above, the contents of the phenol hy-droxyl group for LCC increases as the lignincontent increases. These findings suggest thatthe SOD-like activity of LCC was influenced bythe amount of lignin (i.e., quantity of the phenolhydroxyl groups). The highly concentrated phe-nol hydroxyl group in LCC may produce a simi-lar effect on multiple phenol hydroxyl groups ina benzene ring because of the complex three-dimensional structure of lignin in LCC.The SOD-like activity of extracts from a man-grove plant (Ceriops decandra (Griff.) DingHou), maple (Acer nikoense Maxim), and thornapple (Crataegus Cuneata Sieb et. Zucc.) has al-ready been reported (Sakagami et al. 1998;Satoh et al. 1998a; 1998b). In those reports, theSOD-like activities of ethanol, hot water, and al-kaline extracts in the HPX/XOD system were in-vestigated by using the ESR spectrometer. It isbelieved that, based on the extraction methodsdescribed in those reports, their extracts were amixture of catechin, lignin, tannin, and LCC.Furthermore, it was not clear exactly how muchlignin was in the extracts, and it was not certainwhich components in those extracts contributedto the SOD-like activity. In this study, the rela-tionship between the lignin and the SOD-like ac-tivity could be clarified by excluding catechinand tannin from the samples and investigatingthe concentration of lignin in LCC.referencesBjörkman, A. 1956 Studies on finely divided wood. I. Ex-traction of lignin with natural solvents. Sven. Pappersidn.59:477–485.Fukuda, T., H. Ito, and T. Yoshida. 2003. Antioxidativepolyphenols from walnuts (Juglans regia L.). Phyto-chemistry 6 :795–801.Goldschmid, O. 1954. 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Evaluation of the Ability of Aqueous Lignin-Carbohydrate Complex Extracted from Botanical Waste to Scavenge Reactive Oxygen Species by Using an Electron Spin Resonance Method

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Evaluation of the Ability of Aqueous Lignin-Carbohydrate Complex Extracted from Botanical Waste to Scavenge Reactive Oxygen Species by Using an Electron Spin Resonance Method

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