Unveiling the Structural Properties of LigninâCarbohydrate

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Unveiling the structural properties of lignin-carbohydrate complexes in bamboo residues and its functionality as antioxidants and immunostimulants Caoxing Huang, Shuo Tang, Weiyu Zhang, Yuheng Tao, Chenhuan Lai, Xi Li, and Qiang Yong ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b03262 • Publication Date (Web): 04 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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ACS Sustainable Chemistry & Engineering

Unveiling the structural properties of lignin-carbohydrate complexes in bamboo residues and its functionality as antioxidants and immunostimulants Authors: Caoxing Huang †,‡, Shuo Tang †, Weiyu Zhang †, Yuheng Tao †, Chenhuan Lai †, Xi Li †, Qiang Yong † *

Affiliation: †: Co-Innovation Center for Efficient Processing and Utilization of Forest Products, College of Chemical Engineering, Nanjing Forestry University, Longpan Road 159, Nanjing 210037, China; ‡: State Key Laboratory of Pulp and Paper Engineering , South China University of Technology, Wushan Road 381, Guangzhou 510640, China Caoxing Huang, E-mail address: [email protected] Shuo Tang, E-mail address: [email protected] Weiyu Zhang, [email protected] Yuheng Tao, [email protected] Chenhuan Lai, [email protected] Xi Li, [email protected] * Corresponding author: Tel: +86 25 85427797; E-mail address: [email protected]

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Abstract Lignin-carbohydrate complexes (LCCs), a significant component of plant cell walls,

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have been found to bear biological functionality as antioxidants in food and as

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immunostimulants for living cell. In this work, a lignin-rich and a carbohydrate-rich

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LCCs preparations were isolated from bamboo residues (bamboo green and bamboo

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yellow). Each preparation was characterized by chemical composition and LCCs

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linkage types and quantities by high performance anion exchange chromatography

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(HPAEC) and NMR technologies (quantitative 13C NMR and 2D-HSQC NMR).

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Furthermore, evaluation of each LCCs preparation’s suitability as antioxidant and

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immunological substances were explored. Antioxidant assays indicated that all the

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LCCs preparations exhibited pronounced antioxidant activities for scavenging the 2,

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2-diphenyl-1-picryl-hydrazyl and hydroxyl radicals, while the lignin-rich LCCs

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outperformed the carbohydrate-rich LCCs. Immunological analysis showed that

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carbohydrate-rich LCCs could significantly inhibit the growth of breast tumor cells

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(MCF-7), while lignin-rich LCCs could stimulate the growth of macrophage cells

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(RAW 264.7). These results imply that LCCs extracted from bamboo may be used as

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novel and natural antioxidants or immunostimulants.

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Keywords: lignin-carbohydrate complexes; structure characterization; antioxidant

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activity; immunostimulant

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TOC/Abstract Graphic

Scavenging 6'

HO

1

?

2

6

2' 3'

1 OCH3

OCH3

6

2

5

Isolation

1'

O

a

3 4

5' 4'

ß

O R

5

Radicals in vitro

3

O

OCH3

4

C1-carb

O HO phenyl glyc lc) O oside (PhG Carb

OH O

C1:R=C6 in Glc,Man,Gal,C5 in Ara C2:R =C2 or C3 inMeXyl,Glc,Man,Gal,Ara O O

benzyl ? ether (BE)

6'

1'

5' ß

HO

4' O

a 1

2' 3' OC H3

6

2

5

3 4

OC H 3

O

Bamboo

21

?-eat er (Est)

lignin-carbohydrate complexes

Stimulating

Immunity

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Synopsis

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This work shows the application of lignin-carbohydrate complexes in bamboo as novel

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and natural antioxidants or immunostimulants.

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INTRODUCTION Plant cell walls are chemically described as lignocellulosic due to their biopolymer

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composition of cellulose, hemicellulose, and lignin. For lignin, it is a dimensional

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phenolic polymer which imparts rigidity and hydrophobicity to biomass.1 The chemical

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linkages naturally formed between lignin monomers include a variety of aryl ether and

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carbon-carbon linkages.2 In addition, there also exists chemical linkages (covalent

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bonds) between lignin and polysaccharides (mainly hemicellulose). These linkages are

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termed lignin-carbohydrate complexes (LCCs) linkages, including phenyl glycosides,

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benzyl ethers, and benzyl esters.3 These chemical confluences between lignin and

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polysaccharides are wholly unique in that they bridge two dissimilar biopolymers.

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To valorize these lignin and polysaccharides, it would be of great value to determine

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whether biomass derivatives containing LCCs linkages exhibit unique properties.

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LCCs are believed to negatively contribute towards chemical or biochemical

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processing of lignocellulosic materials, providing recalcitrance towards biorefinery

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operations such as pretreatment delignification and monosaccharide fermentation.4 It is

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important to isolate LCC-containing biopolymer fragments in order to learn the

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functions of these enigmatic linkages across various processes and applications. Several

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methods have been developed for LCCs isolation over the past several decades, mainly

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comprised of various configurations of solvent extractions (aqueous, organic, and mixed

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solvents).5,6 To provide some historical context, water extraction of LCCs was first

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proposed by Traynard,7 and demonstrated upon poplar. Bjorkman 8 then advanced the

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work by Traynard, introducing a LCCs isolation procedure which involved mixed

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solvent extraction (aqueous dioxane and dimethylsulfoxide) from ball-milled woodmeal.

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Due to the success of Bjorkman’s procedure, it is still practiced in literature today and

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its LCCs derivatives are often referred to as Bjorkman LCCs. Some products from other

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lignin isolation protocols, such as cellulase enzymatic lignin and milled wood

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enzymatic lignin, have also been demonstrated as bearing significant LCCs character.9,10

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Unfortunately, milled wood enzymatic lignin and cellulase enzymatic lignin are not

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representative of all native LCCs linkages, as some of the original ester and glycoside

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linkages are enzymatically degraded during enzymatic hydrolysis process.4 Hence, the

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Bjorkman method is still considered the gold standard method for LCCs preparations

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from lignocellulosic biomass. In terms of structural characterization of LCCs, various spectroscopic and

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chromatographic methods are practiced for analyzing types and/or quantities. Examples

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of such methods include fourier transform infrared spectroscopy (FT-IR), nuclear

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magnetic resonance (NMR), and gel permeation chromatography (GPC).3,11 Of these

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methods, modern solution-state NMR technology is one of the most useful and

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informative technology for analyzing LCCs structures and lignin components.3,12 2D

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NMR, especially two-dimensional heteronuclear single quantum coherence (2D-HSQC),

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have been applied for observing and quantifying the amount of various LCCs linkages

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from different biomass preparations.6,13 A method using a combination of quantitative

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13

C NMR and 2D HSQC NMR was proposed and demonstrated by Zhang and

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Gellerstedt,12 which was shown to provide reliable data about the distribution and

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relative amounts of LCCs in several LCCs preparations isolated from various biomass

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by different methods.6,10

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Beyond characterization, the duality of LCCs products (hydrophobic lignin linked

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to hydrophilic polysaccharides) may impart added value in the form of antioxidant

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(lignin-derived) and biologically-compatible (polysaccharide-derived) properties.

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According to the work of Sakagami et al.,14,15 LCCs can exhibit three unique biological

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activities: 1) immunopotentiating activity, (such as induction of antitumor, antimicrobial

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and anti- parasite activities); 2) a broad antiviral spectrum; and 3) positive augmentation

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of vitamin C activity towards radical-scavenging.

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For bamboo, it can be fractionated to the fractions of bamboo node, bamboo

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branches, bamboo green, and bamboo yellow. In bamboo utilization industry in China,

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bamboo green and bamboo yellow are discarded or burned because their mechanical

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properties are weak.16,17 Hence, the efficient utilization of bamboo residues is an urgent

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issue for boosting the value of bamboo residue. It is reported that the extractives and

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compositions of bamboo showed the antioxidant and biological function.18,19,20 However,

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few work has been do to evaluate the LCCs from bamboo residues possess the unique

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ability of biological functionality as antioxidants or immunostimulants.

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In this work, carbohydrate-rich and lignin-rich LCCs were isolated from bamboo

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green and bamboo yellow according to the method of Bjorkman.8 Following isolation,

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the composition analysis was carried out by high performance anion exchange

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chromatography (HPAEC) and the structural characterizations were performed by gel

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permeation chromatography (GPC) and NMR technologies (quantitative 13C NMR and

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2D-HSQC NMR). Meanwhile, evaluation of LCCs preparations as an antioxidant and

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immunological substance was executed with hopes of demonstrating unique

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value-adding properties to LCCs from lignocellulosic biomass.

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MATERIALS AND METHODS

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Materials. Moso bamboo (Phyllostachys pubescens) residues used in this study were

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provided by a Bamboo Processing Factory in Fujian, China. Bamboo green and bamboo

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yellow in bamboo residues were ground into a particle size between 40 and 80 mesh.

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Both of the bamboo green and bamboo yellow were extracted with benzene/ethanol (2:1,

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v/v) in a Soxhlet extractor for 12 h. The main components of the extracted bamboo

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green and bamboo yellow were as follows: 42.6 % and 41.0 % of glucan, 16.2 % and

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17.3 % of xylan, and 28.20 % and 27.0 % of lignin, respectively. The extracted bamboo

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green and bamboo yellow were allowed to air dry and then stored until LCCs isolation.

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Isolation and purification of LCCs. Bjorkman LCCs were prepared from the extracted

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bamboo green and bamboo yellow according to the original method from Bjorkman.8 A

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planetary ball milling was used to mill the samples before LCCs preparation. The

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isolation and purification of LCCs were accorded to our previous work.4,11 The isolated

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LCC-AcOH from bamboo green and bamboo yellow were termed as G-LCC-AcOH and

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Y-LCC-AcOH, respectively. The isolated LCC-Bjorkman from bamboo green and

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bamboo yellow were termed as G-LCC-Bjorkman and Y-LCC-Bjorkman, respectively.

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Chemical composition analysis. Chemical compositon of each raw material and the

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LCCs samples were analyzed according to the NREL (National Renewable Energy

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Laboratory) standard analytical procedure.21 The monosaccharides in acid hydrolyzate

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were analyzed by a HPAEC system (Dionex ICS-5000, USA) equipped with a column

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of PA10 (2×250 mm) and a pulsed amperometric detector. The elution program

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consisted of an initial isocratic elution in 37 mM NaOH from 0 to 20 min, followed 200

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mM CH3COONa from 20 to 35 min, and finally equili-brated in 37 mM NaOH from 35

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to 50 min.

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Molecular weights analysis. The molecular weights (Mw and Mn) of the LCCs

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samples were determined with GPC equipped with a PL-gel 10 mm mixed-B 7.5 mm i.d.

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column and an ultraviolet detector. 3~5 mg of LCCs was dissolved in 2 mL of

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tetrahydrofuran, and then 20 µL of the lignin solution was injected into the column. The

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column was operated at ambient temperature and eluted with THF at a flow rate of 1.0

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mL/min. Monodisperse polystyrene was used as the standard.

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Characterization of LCCs preparations by NMR. The quantitative 13C NMR and

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2D-HSQC NMR of LCCs preparations were analyzed by Bruker AVANCE 600 MHz

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spectrometer. For the quantitative 13C NMR experiments, 100 mg of purified LCCs was

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dissolved in microtube containing 0.5 mL DMSO-d6 and 40 µL of Chromium (III)

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acetylacetonate (0.01 M). For the 2D-HSQC NMR experiments, 40 mg of LCCs was

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dissolved in 0.5 mL DMSO-d6. The amount of various lignin substructures and LCCs

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linkages in LCCs preparations were calculated from the NMR spectra according to the

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work of Wen et al.22 and Yuan et al.23

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Antioxidant activities of LCCs in vitro. The antioxidant property of LCCs

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preparations were analyzed by scavenging the 2, 2-diphenyl-1-picryl-hydrazyl (DPPH)

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radical and hydroxyl radical. DPPH radical and hydroxyl radical scavenging assays of

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LCCs preparations were performed according to the reported method in the work of Niu

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et al.24

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Biological function as immunostimulants. To gauge LCCs biological function as

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immunostimulants, the LCCs preparations was used to culture the breast tumor cells

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(MCF-7) and macrophage cells (RAW 264.7). RAW 264.7 cells and MCF-7 cells were

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cultured in RPMI 1640 medium supplemented with penicillin (100 units/mL),

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streptomycin (100 units/mL) and 10% (v/v) fetal bovine serum at 37 °C in a humidified

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atmosphere with 5% CO2. The cells in the logarithmic phase were cultured in a 96-well

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flat-bottom plate (2×104 cells/well) for 24h. After the dnoted culture time, various

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LCCs concentrations (25~800 µg/mL) and 100 µL culture medium (control) were added

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to the well to culture the MCF-7 cells (72 h) and the RAW 264.7 cells (24 h). Then, 20

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µL Tetrazolium salt solution was mixed with cultured cells at room temperature, and

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finally kept in darkness place for 4 h. After 4 h, the supernatant of the cell was removed

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and added 150 µL dimethyl sulfoxide solution to the well. The absorbance of mixture

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(dimethyl sulfoxide and cells) was measured at 595 nm using a microplate reader. The

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increasing rate of LCCs for macrophage cell growth and the inhibition rate of LCCs on

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breast tumor cell growth were calculated as the following formula.

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151 152

ODcontrol - ODsample ×100 % ODcontrol ODsample - ODcontrol Increasing rate (%) = ×100% ODcontrol Inhibition rate (%) =

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(1) (2)

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Where ODcontrol is the value for cell culturing with no LCCs and ODsample is the value for

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cell culturing with various LCCs concentrations. All the results of ODcontrol and ODsample

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are obtained from the average number of four replicate experiments.

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RESULTS AND DISCUSSION

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Composition analysis of LCCs preparations. As discussed in the introduction,

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Bjorkman LCCs preparations are considered to be the leading LCCs preparation

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protocol. This is due to the abundance of native covalent bonds present in the

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preparation which originate from the wood cell wall. In the protocol of Bjorkman

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procedure, LCC-AcOH preparation can also be obtained for analyzing the valuable

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information of LCCs linkages.10 Therefore, in this work, both Bjorkman-LCC and

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LCC-AcOH were obtained from bamboo green and bamboo yellow, and subjected to

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comprehensive analysis. Preparations are termed as G-Bjorkman-LCC and

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Y-Bjorkman-LCC, and G-LCC-AcOH and Y-LCC-AcOH. The carbohydrate

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constituency and isolation yields of each LCCs fractions were shown in the Table 1.

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From Table 1, it can be seen that the isolation yields (based on the lignin content in

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extracted bamboo) of LCC-AcOH from bamboo green and bamboo yellow were 1.9%

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and 2.1%, respectively. For the LCC-Bjorkman from bamboo green and bamboo yellow,

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the yields were 6.4% and 5.9%, respectively. Table 1 showed that G-LCC-AcOH and

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Y-LCC-AcOH contain greater amounts of lignin (69.1% and 67.3%, respectively)

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compared to the Bjorkman LCCs. G-Bjorkman-LCC and Y-Bjorkman-LCC were indeed

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found to be carbohydrate-rich, comprised of 55.8% and 53.4% total carbohydrate,

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respectively. As shown in Table 1, both araban, galactan, mannan, and xylan were

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linked to lignin, specially enriched in Bjorkman-LCC preparation. Xylan can be

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observed to be the predominant sugar constituent in the all LCCs preparations, with

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amounts ranging from 69.4%-81.8%. For example, the xylan proportion in the

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carbohydrate in carbohydrate-rich LCCs was ~81%, while the xylan proportion in the

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carbohydrate in G-LCC-AcOH and Y-LCC-AcOH was ~69%. The xylan was the main

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carbohydrate in LCCs preparations could be explained by the fact that xylan is the

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predominant constituency in hemicellulose in bamboo cell walls.25 Furthermore, most

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hemicellulose is located in the secondary wall, which has been found to contain the

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greatest amount of LCCs.25,26 In this work, the results revealed that the LCCs

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preparations from moso bamboo are comprised similar types of carbohydrates, which

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was in accordance to the work of You et al.13

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Molecular weight distributions. It necessary to estimate molecular weights of the

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LCCs preparations for inter-sample comparison, as that the molecular weight of any

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LCCs preparation heavily depends upon its isolation protocol.27 In this work, the

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average molecular weights (Mw and Mn) and polydispersity index (PDI) of

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G-LCC-Bjorkman, Y-LCC -Bjorkman, G-LCC-AcOH and Y-LCC-AcOH analyzed by

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GPC are presented in the Table 2. It shows that the molecular weight of four LCCs

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preparations ranged from 5970 g/mol to 14920 g/mol. Lignin-rich LCCs were of a

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lower molecular weight than that of carbohydrate-rich LCCs, indicating more

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carbohydrate linked to lignin in LCCs possessing high molecular weight. Moreover, all

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four LCCs preparations possessed relatively narrow molecular weight distribution(PDI).

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Specifically, PDI values across all preparations ranged from 1.2 to 1.7. This property is

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likely to serve as beneficial towards NMR analysis due to improved solubility

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parameters in the NMR solvent (DMSO-d6), where we expect clearer signals from

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NMR spectra due to the narrow distribution of molecules in terms of molecular mass.13

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13

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characterize structures of LCCs preparations procured from bamboo green and bamboo

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yellow. The spectra of these LCCs preparations are presented in Figure 1, and the

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signals in spectra are assigned according to the published work.11,22

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C NMR analysis. Quantitative 13C NMR was employed as an analytical technology to

In Figure 1, it can be seen that the four LCCs preparations showed relatively

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similar peaks in the spectra, with strong signals for syringyl (S) units, guaiacyl (G) units,

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and p-hydroxyphenyl (H) units, carbohydrate structures as well as inter-lignin unit

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linkages in the LCCs preparations. Specifically, from the region between 103-153 ppm,

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the signals for syringyl units (152.3 ppm (C-3/C-5), 138.3 ppm (C-4), 134.5 ppm (C-1),

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and 104.4 ppm (C-2/C-6)) , guaiacyl units (149.3 ppm (C-3), 147.2 ppm (C-4), 134.5

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ppm (C-1), 119.5 ppm (C-6), 115.3 ppm (C-5), and 111.2 ppm (C-2)), and

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p-hydroxyphenyl units (128.2 ppm (C-2/C-6)) can be detected. The observable

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similarities in this region indicated that Bjorkman-LCC and AcOH-LCC from both

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bamboo contained lignin featuring H, G, and S monomeric constituency. The strong

12


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resonance between 90-102 ppm and 50-86 ppm region are attributable to both

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carbohydrate structures as well as inter-lignin linkages in LCCs. Due to overlapping

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signals presented in the 13C spectra, identifying the signals of lignin-carbohydrate

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linkages in LCCs is very difficult. To overcome this problem, multi-dimensional NMR

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spectroscopic techniques, such as 2D-HSQC and HMBC, are proposed to directly

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acquire the various lignin-carbohydrate linkages in LCCs preparations.

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2D-HSQC NMR analysis. 2D-HSQC NMR technique were used to quantify and

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describe the distribution of chemical linkages present in LCCs preparation. The

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2D-HSQC spectra of the four LCCs preparations are presented in Figure 2 with the

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main substructures depicted in Figure 3, and the main lignin cross-signals in HSQC

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spectra are listed in Table S1. The signals of LCCs preparations in the 2D-HSQC

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spectra are assigned according to the published work.6,23,28

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As shown in Figure 2, all the side-chain regions (δC/δH 50-90/2.5-6.0) of the

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2D-HSQC spectra showed strong signals of methoxy groups (δC/δH 55.7/3.72) and

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lignin substructures. Among these different substructures, β-O-4 linkages (A, δC/δH

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71.8/4.88, δC/δH 83.6/4.32, and δC/δH 85.8/4.12) are the prominent lignin linkages in the

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entire LCCs preparations. In addition, the signal located at δC/δH 86.0/4.11 and

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83.9/4.30 were attributed to Cβ-Hβ signal of β-O-4 structure that linked to syringyl units

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(S) and guaiacyl units (G)/p-hydroxyphenyl units (H), respectively. This suggested that

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both S-type lignin and G/H-type lignin are associated with carbohydrates in the

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carbohydrate-rich LCCs and lignin-rich LCCs. Moreover, resinols (β-β) and

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Phenylcoumarans (β-5) substructures were observed in the all LCCs preparations.

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Specifically, signals at δC/δH 84.8/4.69, 53.6/3.05, and 71.3/4.18,3.82 were attributed to

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the Ca-Ha, Cβ-Hβ, and the double Cγ-Hγ correlation signals in β-β substructures (B) and

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δC/δH 86.8/5.49 and 53.1/3.49 were attributed to the Ca-Ha and Cβ-Hβ correlation signals

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in β-5 substructures (C).

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In the aromatic region of LCCs 2D-HSQC spectra, S, G and H units were clearly

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observable. The signal at δC/δH 104.1/6.74 was attributed to the C2,6-H2,6 correlations of

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S units in lignin. G units showed the signals at δC/δH 111.0/7.01, 114.4/6.73, and

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119.0/6.82, which were attributed to C2-H2, C5-H5, and C6-H6 correlations. H units in

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lignin showed the signal for the C2,6-H2,6 correlations at δC/δH 127.8/7.22. It should be

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point out that the C-H correlation signals at δC/δH 104.9/7.05, 99.0/6.21, 94.4/6.57, and

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104.3/7.31 were also observed in the aromatic region. According to the work of Lan et

247

al,29 these signals were corresponded to the C3-H3, C6-H6, C8-H8, and C2',6'-H2',6' in tricin.

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For tricin, it has been identified as an authentic lignin monomer that participating in the

249

lignification reactions in the cell wall of gramineae biomass.29,30 Hence, it can be

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speculated that the tricin maybe also participate in lignification reactions involving

251

genesis of LCCs due to its presence in our LCCs preparations.

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For LCCs preparation, it is important to identify and quantify the LCCs linkages,

253

which are knows as phenyl glycoside (PhGlc), benzyl ether (BE), and γ-ester (Est). It is

254

reported that the C1 signals of PhGlc can be observed in δC/δH 102.6-101.4/5.17-4.94.10

255

In addition, if there are different kinds of carbohydrate associating with lignin to form

14


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LCCs, three signals of C1 in PhGlc will be appeared in δC/δH 100.2-98.4/5.03-4.9

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(PhGlc1), δC/δH 100.6-100.3/4.85-4.65 (PhGlc2), and δC/δH 101.9-101.5/4.86-4.79

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(PhGlc3).23,31 In this work, the correlation signals of PhGlc1, PhGlc2, and PhGlc3 in

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G-Bjorkman-LCC, Y-Bjorkman-LCC, G-LCC-AcOH and Y-LCC-AcOH can be

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identified at δC/δH 100.1/5.09, 100.9/4.63, and 101.9/4.92, respectively. This indicated

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that the phenyl glycoside linkage are both in the lignin-rich LCCs and carbohydrate-rich

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LCCs. For benzyl ether linkage, its existence in the LCCs structure can be divided into

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two parts: (1) C1-linkages between the a-position of lignin and primary OH groups of

264

carbohydrates, which shows the signals at δC/δH 80-81/4.5-4.7 (BE1); (b) C2-linkages

265

between the a-position of lignin and secondary OH groups of carbohydrates, which

266

shows the signals at δC/δH 80-81/4.9-5.1 (BE2). In this work, both BE1 and BE2 showed

267

the Cα-Hα signals in the lignin-rich LCCs and carbohydrate-rich LCCs spectrum, which

268

were at δC/δH 81.4/4.64 and 81.2/5.06, respectively. For the ester bonds in LCCs, the

269

Cα-Hα correlations and Cγ-Hγ signals in the 2D-HSQC spectrum were showed at δC/δH

270

77-75/6.2-6.0 and δC/δH 65-62/4.5-4.0, respectively. In this work, both lignin-rich LCCs

271

and carbohydrate-rich LCCs did not show signals at δC/δH 77-75/6.2-6.0. While, their

272

Cγ-Hγ signals were identified at δC/δH 65-62/4.5-4.0. However, the structure of

273

γ-acylated β-O-4 aryl ether shows the similar signals for γ-esters (Est), which can not be

274

distinguished by the present spectrometer (600 MHz).3 Hence, the signals of γ-esters

275

(Est) are always reported with the overlapping signals from γ-acylated β-O-4 aryl

276

ether.10 This lack of distinguishing can be overcome through use of high-resolution

15



277 278

NMR spectroscopy with CryoProbeTM technology. In addition, the signals of associated carbohydrates in LCCs preparations, such as

279

β-D-xylopyranoside units (X) and arabinofuranoside units (Ara), could be clearly found

280

in the 2D-HSQC spectra.13,23 For example, the C2-H2, C3-H3, C4-H4, and C5-H5

281

correlations from X units were observed at δC/δH 72.5/3.02, 73.7/3.22, 75.4/3.60 and

282

62.6/3.40, respectively. The cross-peaks at δC/δH 81.6/3.89, 77.1/3.72, and 61.9/3.52

283

were attributed to the C2-H2, C3-H3, and C5-H5 correlations from Ara units,

284

respectively.The signals from 4-O-methyl-a-D-glucuronic acid units (U) units were

285

observed at δC/δH 97.2/5.18 (U1) and 81.1/3.11 (U4). All of these results were in

286

accordance with the carbohydrate analysis results in the Table 1.

287

Quantification of substructures and linkages in LCCs. A quantification method that

288

coupling quantitative 13C NMR and 2D HSQC NMR was applied according to the

289

method proposed by Zhang and Gellerstedt.12 As shown in the Figure 1, the aromatic

290

carbons region (163-103.6 ppm) was assigned as 600, which was selected as the internal

291

standard reference for other structural moieties calculation. The integral values obtained

292

from 103-96, 90-78, and 64.5-58.5 ppm were used to quantify the lignin structures and

293

LCCs linkages in quantitative 13C spectra.23 The quantification of lignin substructures

294

and LCCs linkages in four LCCs preparations are shown in Table 3, expressed as per

295

100 monomeric lignin units (100Ar).

296 297

As can be seen from Table 3, G-LCC-AcOH and Y-LCC-AcOH exhibited similar structural features. For example, the abundance (100Ar) of main lignin substructures in

16


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298

G-LCC-AcOH and Y-LCC-AcOH were 25.1 and 27.8 (β-O-4), 4.2 and 4.5 (β-β), and

299

3.1 and 2.8 (β-5), respectively. For G-Bjorkman-LCC and Y-Bjorkman-LCC, the

300

abundances of main lignin substructures were also similar. Moving to LCCs

301

quantification, the abundances of benzyl ether, phenyl glycoside, and ester LCCs

302

linkages in G-LCC-AcOH, Y-LCC-AcOH was 3.1/100Ar and 3.7/100Ar, 1.9/100Ar and

303

2.8/100Ar, 6.1/100Ar and 7.2/100Ar, respectively. For G-Bjorkman-LCC and

304

Y-Bjorkman-LCC, the amount of benzyl ether, phenyl glycoside, and ester LCCs was

305

1.1/100Ar and 0.9/100Ar, 4.2/100Ar and 4.5/100Ar, 4.1/100Ar and 3.8/100Ar,

306

respectively. These results demonstrate that the quantity of phenyl glycoside in

307

Bjorkman-LCC is higher than in LCC-AcOH, while the amount of benzyl ether were

308

lower in Bjorkman-LCC than in the LCC-AcOH preparations. These difference might

309

explainable by the observation that Bjorkman-LCC were carbohydrate-rich LCCs, in

310

which carbohydrate may be preferred to link with lignin by glycoside. For

311

G-LCC-AcOH, Y-LCC-AcOH, both of them were lignin-rich LCCs, in which

312

carbohydrate prefer to link with lignin by ether. You et al.13 also found the LCC-AcOH

313

from Arundo donax Linn contained more benzyl ether LCCs structures than the

314

Bjorkman-LCC (same LCCs preparation protocol as employed in this work).

315

Antioxidant activities in vitro of LCCs preparations. Free radicals and reactive

316

oxygen species are commonly believed to be root causes of many life-threatening

317

diseases. For this reason, free radical and reactive oxygen species sequestration in vivo

318

has become an important research topic in relevant medical sciences.32 To evaluate

17



319

whether LCCs can function as antioxidants, DPPH radical and hydroxyl radical

320

scavenging assays were performed using the LCCs preparations. Results from both

321

assays (displayed as curves) are shown in Figure 4A and Figure 4B.

322

From Figure 4A and Figure 4B, it can be seen that both LCCs preparations were

323

effective scavenging ability for DPPH radical and hydroxyl radical, and their

324

scavenging ability improved with higher dosages of LCCs. Niu et al. 24 also reported

325

that LCCs isolated from mushroom exhibited pronounced reductive power and strong

326

scavenging activities on DPPH and hydroxyl radicals in vitro. Focusing upon

327

LCC-AcOH, it was found that both preparations showed similar ability to scavenge

328

radicals. For example, the IC50 (the concentration required for 50% scavenging the free

329

radical) for DPPH radical scavenging of G-LCC-AcOH and Y-LCC-AcOH was 0.18

330

mg/mL and 0.20 mg/mL, respectively. Comparatively, the radical scavenging ability of

331

LCC-AcOH was much higher than that of LCC-Bjorkman. For example, the IC50 for

332

DPPH radical and hydroxyl radical scavenging of G-LCC-AcOH were 0.18 mg/mL and

333

0.71 mg/mL, whereas the IC50 for these radicals scavenging of G-LCC-Bjorkman were

334

0.29 mg/mL and 0.98 mg/mL, respectively. Hence, it can be speculated that the LCCs

335

preparation containing higher carbohydrate character contains lower antioxidant activity.

336

The reasons for this observation may be attributed to several causes. Firstly, the

337

effectiveness of lignin as an antioxidant can be lower when that lignin is in the presence

338

of polar molecules (i.e. polysaccharides), whose presence also decreases the

339

concentration of antioxidant-reactive phenolic functional groups.33 A second

18


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340

explanation may be that phenol-containing biomolecules (i.e. lignins) of lower

341

molecular mass are significantly more effective as antioxidants.34 This can be attributed

342

to molecular mobility, where higher molecular weight lignins tend to be less mobile in

343

solution due to their branched/network chemical structures. This belief is in line with

344

what was observed in our work, where the LCC-Bjorkman had higher polydispersity

345

indices (1.6-1.7) than LCC-AcOH (1.2-1.3). Generally, the relatively higher antioxidant

346

activity of the G-LCC-AcOH and Y-LCC-AcOH preparations suggests that these LCCs

347

would be more suitable for consumption as in vivo antioxidants.

348

Immunological activities of LCCs preparations. It has been reported that LCCs have

349

diverse pharmacological activities (beyond antioxidants), such as antitumor,

350

anti-microbial, and anti-viral properties.14,15 We tried to study the potentially

351

immunological activities of LCCs isolated from bamboo green and bamboo yellow. The

352

effects of each LCCs preparation on the activation of macrophages cells and the

353

inhibition of breast tumor cells are shown in Figure 5A and Figure 5B, respectively.

354

Macrophage cells, like RAW 264.7, have been frequently used in studies

355

investigating macrophage activation by polysaccharides and polysaccharides

356

derivative.35 Figure 5A shows both G-LCC-AcOH and Y-LCC-AcOH presented

357

activation on RAW 264.7 macrophage cell viability at the concentration from 25 µg/mL

358

to 800 µg/mL. In comparison, G-LCC-Bjorkman and Y-LCC-Bjorkman just showed

359

activation on the cell viability at the 25 µg/mL to 200 µg/mL. Similar results were

360

shown by Niu et al.,24 who demonstrated that LCCs isolated from mushroom had

19



361

significant effects on activating macrophage cells. In addition, it was found that the

362

activation ratio of lignin-rich LCCs was higher than carbohydate-rich LCCs. For

363

G-LCC-AcOH and Y-LCC-AcOH, the maximum increasing rate on activation of RAW

364

264.7 macrophages was 50.7% and 33.7% at 200 µg/mL, respectively. Meanwhile,

365

G-LCC-Bjorkman and Y-LCC-Bjorkman showed maximum increasing rate with 17.4%

366

and 27.8% at 200 µg/mL, respectively. This indicates that the lignin-rich LCCs and

367

carbohydrate-rich LCCs have different extents of influence on macrophage activation,

368

while still both serving as macrophage activators. Martinez et al.36 reported the

369

immuno-stimulating properties of LCCs is closely related to the linkage types between

370

lignin and carbohydrate. As shown in Table 3, the total LCCs linkages of G-LCC-AcOH

371

and Y-LCC-AcOH were 11.1/100Ar and 13.7/100Ar, respectively. While, the total

372

LCCs linkages of G-LCC-Bjorkman and Y-LCC-Bjorkman were 9.4/100Ar and

373

9.2/100Ar, respectively. The higher LCCs linkages in G-LCC-AcOH and Y-LCC-AcOH

374

may be attributed to their stronger performance as macrophage activators.

375

The antitumor effects of each LCCs preparation was investigated using MCF-7

376

cells (breast tumor cells). Results are shown in the Figure 5B. It can be seen that each

377

LCCs preparation demonstrated antitumor activities at concentrations between 25-800

378

µg/mL, with inhibition rates from 3.1%-65.2%. Similar results using LCCs have also

379

been reported, such as inhibition of oral cavity cancer cells and gastroenterological

380

tumors.14,15 It is found that the carbohydrate-rich LCCs showed strong anti-proliferation

381

effects on MCF-7 cells compared to lignin-rich LCCs. Specifically, the maximum

20


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382

MCF-7 inhibition rate by G-LCC-Bjorkman and Y-LCC-Bjorkman was 65.2% and

383

51.9% at 400 µg/mL, which was significantly higher than G-LCC-AcOH and

384

Y-LCC-AcOH (11.0% and 12.1% at 400 µg/mL). The significantly poorer activity of

385

G-LCC-AcOH and Y-LCC-AcOH towards inhibition of MCF-7 cellular growth may be

386

due to their lower carbohydrate contents. As Sakagami et al.14 pointed out, when the

387

carbohydrate abundance in LCCs is low, the LCC’s antitumor activity for hepatoma is

388

significantly reduced. As shown in Table 1, the total contents of carbohydrate in

389

G-LCC-Bjorkman and Y-LCC-Bjorkman were 55.79% and 53.41%, respectively. The

390

values of carbohydrate contents are nearly double in G-LCC-AcOH and Y-LCC-AcOH

391

(25.99% and 29.39%, respectively). Hence, the higher carbohydrate in

392

G-LCC-Bjorkman and Y-LCC-Bjorkman may be attributed to their higher performance

393

for antitumor activity.

394

In this work, lignin-rich LCCs contained more benzyl ether linkages while the

395

carbohydrate-rich LCCs contained more phenyl glycoside linkages. Both the LCCs

396

preparation showed antioxidant activities, with one being superior over the other

397

(attributed to differences in lignin content). Meanwhile, the influence of different LCCs

398

preparations on macrophage and tumor cell behaviors was demonstrated, revealing

399

immunological activities of all LCCs preparations. The results suggested that the LCCs

400

from bamboo may reduce the incidence of breast tumor and increase the macrophage

401

activity, demonstrating strong value-added use as immunostimulants.

402

Acknowledgements

21



403

This work was supported by the Natural Science Foundation of Jiangsu Province

404

(BK20180477), National Natural Science Foundation of China (31570561), State Key

405

Laboratory of Pulp and Paper Engineering (201812), Priority Academic Program

406

Development of Jiangsu Higher Education Institution (PAPD) and Student Innovation

407

Training Program of Nanjing Forestry University (2018 for Weiyu Zhang).

408

Notes

409

The authors declare no competing financial interest.

410

Supporting Information

411 412

Assignment signals of substructure and LCCs linkages in 2D-HSQC spectra of the LCCs preparations

22


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Flammulina velutipes. Carbohyd. Polym. 2018, 183, 207-218, DOI:

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Table 1 The composition of LCCs preparations from bamboo green and bamboo yellow (%) Isolation yield a

Lignin content b

G-LCC-AcOH

1.9

69.1

G-LCC-Bjorkman Y-LCC-AcOH

6.4 2.1

48.6 67.3

LCCs preparations

The percentage in total carbohydrate c

Total carbohydrate b 26.0

Glucan 19.7

49.9

8.0

81.4

6.0

19.6 7.0

69.4 81.8

7.6 6.5

29.4 53.4 Y-LCC-Bjorkman 5.9 46.2 a: Based on the lignin content in extracted bamboo b: the amount in LCCs sample c: Glucan+Xylan+Arabinan+Galactan=100%

29


Xylan Arabinan 69.6 7.6

Galactan 3.1 4.6 3.4 4.7


Page 30 of 38

Table 2 Molecular weight (g/mol) and polydispersity of LCCs preparations a

G-LCC-AcOH G-LCC-Bjorkman Y-LCC-AcOH Y-LCC-Bjorkman

Mw a 7510

Mn a 5670

Polydispersity b 1.32

14920

8580

1.74

5970 10700

4830 6860

1.24 1.56

a

Mw: weight-average of molecular weight; Mn: number-average of molecular weight

b

Polydispersity: Mw/Mn

30


Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3 The amount of lignin substructure and LCCs linkages in LCCs preparations (100 Ar) characteristics G-LCC-AcOH lignin interunit linkages β-O-4 aryl ethers (A) 25.1 resinols, β-β (B) 4.2 phenylcoumarans, 3.1 β-5 (C) LCCs linkages benzyl ether (BE) 3.1 phenyl glycoside 1.9 (PhGlc) Est a 6.1 b Total 11.1

Y-LCC-AcOH

G-Bjorkman-LCC

Y-Bjorkman-LCC

27.8 4.5

16.9 0.7

19.4 0.9

2.8

1.2

0.7

3.7

1.1

0.9

2.8

4.2

4.5

7.2 13.7

4.1 9.4

3.8 9.2

a

Sum of LCCs γ-esters (Est) and γ-acylated β-O-4 aryl ether substructures

b

Sum of the amount of BE, PhGlc, and Est

31



Figure captions Figure 1. The 13C NMR spectra of LCCs preparations Figure 2. 2D-HSQC NMR spectra of LCCs preparations Figure 3. Main structures in the LCCs preparation Figure 4. The DPPH radical scavenging ability (A) and hydroxyl radical scavenging ability (B) of LCCs preparations isolated from bamboo green and bamboo yellow Figure 5. The abilities of activation of macrophages cells (A) and the inhibition of breast tumor cell (B) of LCCs preparations isolated from bamboo green and bamboo yellow

32


Page 32 of 38

Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

33



34


Page 34 of 38

Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

35



R

Page 36 of 38

O OMe

HO

6'

MeO

O

γ HO

α

β

5'

1'

4'

2'

O

HO

1'

4'

2' 3'

O

5'

1' 6' 2' 5'

(OMe)

6'

γ

HO

β α

6

(MeO)

OMe

O

5 4

O

6

1

4

3

5

(MeO)

1 2

O

OMe

4' α

γ'

3'

β

β'

(OMe)

4

4'

1' α'

γ

3

(MeO)

O

2' O

O

2

5 OMe

4

β

6

3

5

3'

5'

1

(OMe) 2

(MeO)

α

3'

1 6

6'

MeO γ

O

2

3 O

OMe

OMe

A

A'

C

B O

HO

O

HO

3'

9

7

γ OMe

5' 2

6'

OH

5

10

1

6

2

6

5

3

5

2 3 OMe

4

4

OH

OH

PCA

FA

3

6

β

α

1 OH

β

4'

1'

O

α

O

2' 8

HO

β

α

OMe

O

γ

γ

4 O

T OH

α OH

α 2 3

6 5

α

1

1

2 3

6 5

MeO

O

O

1

4

1 2 3

6 5

2 3 MeO

OMe

OMe

4 O

O

G

H

α

6 5 OMe

4

4

O

OH

S'

S

HO

6'

HO

γ

1 6

2

5

3

R

OH O

5'

1'

4'

2'

Me

β

O

O Carb

O

α

O

6'

O

γ

3' OCH3

β

HO

4

OCH3

O C1 -carb

1'

5'

1 6

2

5

3

O

α 1 OCH3

4

4'

OCH3

2

6

O

3

5

phenyl glycoside (PhGlc)

4 O

C1:R=C6 in Glc,Man,Gal,C5 in Ara C2:R=C2 or C3 in Xyl,Glc,Man,Gal,Ara benzyl ether (BE)

γ-eater (Est)

Figure 3

36


2' 3'

OCH3

Page 37 of 38

90

90

(A)

80

(B)

80

70

70 Scavenging ability (%)

Scavenging ability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 IC50

50 40 30

G-LCC-AcOH Y-LCC-AcOH G-LCC-Bjorkman Y-LCC-Bjorkman

20 10

60 IC50

50 40 30


20 10

0

0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

Concentration (mg/mL)

0.4

0.6

Concentration (mg/mL)

Figure 4

37


0.8

1.0


80

80

(A)

(B) 70

60

60

40

Inhibition rate (%)

Increasing rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 38

20 0 -20 -40

50


40 30 20 10

G-LCC-AcOH

Y-LCC-AcOH

G-LCC-Bjorkman

Y-LCC-Bjorkman

-60

0

25

50

100

200

400

800

25

50

Concentration (µg/mL)

100

200

Concentration (µg/mL)

Figure 5

38


400

800

Unveiling the Structural Properties of LigninâCarbohydrate

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