In vitro cytotoxicity studies of industrial Eucalyptus kraft lignins on mouse hepatoma, melanoma and Chinese hamster ovary cells

International Journal of Biological Macromolecules 135 (2019) 353–361

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In vitro cytotoxicity studies of industrial Eucalyptus kraft lignins on mouse hepatoma, melanoma and Chinese hamster ovary cells Oihana Gordobil a, Alona Oberemko b, Gintautas Saulis b, Vykintas Baublys b, Jalel Labidi a,⁎ a b

Chemical and Environmental Engineering Department, University of the Basque Country UPV/EHU, Plaza Europa, 1, 20018 Donostia-San Sebastián, Spain Department of Biology, Vytautas Magnus University, Vileikos 8, 44248 Kaunas, Lithuania

a r t i c l e

i n f o

Article history: Received 18 March 2019 Received in revised form 30 April 2019 Accepted 18 May 2019 Available online 21 May 2019 Keywords: Kraft lignin Chemical structure Antioxidant Cytotoxicity Cancerous cells

a b s t r a c t Kraft lignin is a polyphenolic compound generated as a by-product from the kraft pulping process in large quantities annually worldwide. In addition to its commercial availability, its structural features make it worth to be considered in the pharmaceutical area. The present study was carried out to evaluate in vitro antioxidant and cytotoxic properties of kraft lignin on mouse hepatoma MH-22A, melanoma B16 (tumor cells) and Chinese hamster ovary (CHO, non-cancerous) cells. Moreover, several analytical techniques were used in order to elucidate the chemical structure of isolated industrial lignins (FT-IR, GPC, Py-GC–MS, 2D HSQC NMR). Results revealed high phenolic content in their composition, high-condensed structure and high phenolic hydroxyls group content. DPPH and ABTS⁎+ radical scavenging assays demonstrated their strong antioxidant activity, which was higher than found for commercial antioxidant (BHT). Kraft lignins act cytotoxically inducing apoptosis- and necrosislike processes on both on tumor and normal cells. However, the results evidenced that MH-22A cells showed greater sensitive behavior than B16 and non-cancerous CHO cells, which were more tolerant of kraft lignin. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Lignin, the aromatic structural component of the cell wall of the lignocellulosic biomass, is the only-high-molecular-weight polyphenolic compound in the Earth. Regarding the chemical features of lignin, in general, it is composed of three phenolic structural units, namely guaiacyl (G), syringyl (S) and p hydoxyphenyl (H), which are linked to each other by C\\C and ether (C\\O\\C) bonds. In addition, lignin contains a great variety of functional groups such as methoxyl groups, aliphatic and phenolic hydroxyl groups and small amounts of carbonyl groups and carboxyl groups [1,2]. However, thought the composition of lignin depends mainly on the botanical source, the fractionation process of biomass has also a significant influence on the final structure of lignin [3]. Thus, there is not a single lignin molecule in nature. In spite of having been studied for many years, its chemical structure is still an unresolved puzzle. Nevertheless, current analytical techniques make easier the understanding of the molecular characteristics by the identification of its monomeric compounds, linkages between the main elemental units and functional groups, which form the lignin polymer. Although the chemical variability of this astounding aromatic compound makes challenging the development of novel and valuable applications for lignin at industrial scale, at the same time makes lignin very versatile compound. As a result, lignin has received great interest from ⁎ Corresponding author. E-mail address: [email protected] (J. Labidi).

https://doi.org/10.1016/j.ijbiomac.2019.05.111 0141-8130/© 2019 Elsevier B.V. All rights reserved.

the scientific world to exploit its advantages and today several researchers consider it as the most promising compound in the near future. Some previous studies proved the potential of lignin to be incorporated in several areas. At present, the lignin valorization is centered on three main routes; fragmentation technologies for the obtention of chemicals [4–6], development of new strategies to incorporate it in the polymeric industry, commonly by chemical modifications [7–10] and use it as an energy source [11–13]. However, lignin and its derivatives could be bear in mind for uses in the pharmaceutical area since various research works have previously confirmed the therapeutic properties of lignins such as anticancer, antimicrobial, antioxidant, antiviral or even for treatments to control diabetes and obesity [14,15]. Lu et al., 1998 examined the effect of alkalilignin on the growth and viability of HeLa cells (human cervix carcinoma) proving the ability of alkali lignin to hinder the growth and viability of cancer cells in a dose-dependent manner after 4 days of incubation [16]. Moreover, in 2015 Wang and coworkers isolated two lignin-carbohydrate macromolecules from chaga mushroom (Inonotus obliquus) by hot-water extraction and demonstrated that these lignin derivatives induced cell apoptosis and inhibited the activation of the nuclear transcription factor NF-κB in cancer cells [17]. Inonotus obliquus has been extensively used for the treatment of several diseases [18] in Russia and western Siberia and Wang el at., 2015 suggested that the lignin might be one of the active components responsible for diverse medicinal values of Inonotus obliquus. Barapatre et al., 2016 reported the

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evaluation of the in vitro anti-proliferative/cytotoxic activities of eleven different lignin fractions isolated from the wood of Acacia nilotica by pressurized solvent extraction and successive solvent extraction methods. Only four lignin fractions depicted higher cytotoxic potential towards breast cancer cell line but were ineffective against normal primary human hepatic stellate cells evidencing their specificity (cytotoxic activity) towards cancer cells [19]. Hence, the biological activity of lignins cannot be ignored and research on this topic is necessary in order to open new perspectives for the application of lignin in the pharmaceutical industry. However, though being nowadays kraft lignin the most available commercial lignin worldwide, there are not studies about the cytotoxic effect of kraft lignin on cancerous cells. Kraft lignin is generated as a by-product during the delignification of the wood chips for the pulp manufacture [20]. The kraft process produces a residual stream, called black liquor where small water/alkali-soluble fragments of lignin are solubilized [21]. At present, the vast majority of black liquor is concentrated by evaporation and combusted for the production of energy while only a small fraction is exploited for low-value commercial applications such as additives for concrete admixtures, dust control, feed and food additives, dispersants or binders. Industrial kraft lignin usually contains sulfur because of the pulping process that consists of the treatment of wood chips with a mixture of sodium hydroxide (NaOH) and sodium sulfide (Na2S) at elevated temperature (150–170 °C). Some of this sulfur is organically bound as aliphatic thiol groups (R-SH) and some as elemental sulfur, constituting, at times, important impurities that can have consequences for further valorization [22]. In addition, kraft process usually generates lignin with high-condensed structures owing to the kraft delignification leads to the destruction of ether bonds in lignin and the formation of carbon carbon bonds during the last stage of the process [9]. Therefore, this type of lignin results in having high phenolic hydroxyl group moieties which affect in a positive way to the antioxidant properties [23,24]. Therefore, the specific characteristics of kraft lignin, its high industrial availability together with previous reported biological activities of the lignin molecule were the main reasons that motivated this research work. Thus, the objective of the present study was the assessment of in vitro cytotoxic activity of industrial kraft lignins precipitated at two different pH on mouse hepatoma MH-22A, melanoma B16 and Chinese hamster ovary (CHO, non-cancerous) cells. 2. Materials and methods 2.1. Kraft lignin isolation from industrial black liquor The lignin samples used in this study were isolated by precipitation with sulfuric acid at two different pH (2 and 6) from the kraft liquor produced as residue in a local company Papelera Guipuzcoana de Zikuñaga (Hernani. Spain). This company uses Eucalyptus chips for manufacturing of cellulose pulp. The black liquor had the following characteristics: ρ = 1.03 ± 0.03, pH = 12.7 ± 0.01, total solids = 11.8 ± 0.18, inorganic material = 7.9 ± 0.6, organic material = 3.9 ± 0.6 and lignin content = 31.5 g/L.

equipped with a refractive index detector (RI-2031Plus) and a photodiode array detector (MD-2018Plus). The column operated at 50 °C and the mobile phase (0.005 M H2SO4 prepared with 100% deionized and degassed water) pumped at a rate of 0.6 mL/min, the injection volume was 20 μL. The ash content was calculated according to procedure TAPPI T211 om-02, Ash in wood pulp paper and paperboard: combustion at 525 °C [25]. Finally, Euro EA Elemental analyzer (EuroVector) determined the sulfur content. 2.3. Molecular properties of kraft lignins The weight-average (Mw), the number-average (Mn) and polydispersity (Mw/Mn) of kraft lignins were determined by GPC (Jasco LCNet II/ADC), equipped with a RI-2031 Plus Intelligent refractive index detector, PolarGel-M column (300 mm 7.5 mm) and PolarGel-M guard (50 mm 7.5 mm). 0.25 mg of lignin sample was dissolved in 5 mL of N,N dimethylformamide (DMF) with 0.1% of lithium bromide and 20 μL of solution was injected. The column operated at 40 °C and eluted with N,N dimethylformamide (DMF) with 0.1% of lithium bromide at flow of 0.7 mL/min. Polystyrene was used as a standard for the molecular weight of lignin. FT-IR analysis of kraft lignins was carried out on a PerkinElmer Spectrum Two FT-IR spectrometer. A total of 64 scans were accumulated in a transmission mode with a resolution of 8 cm−1. The spectrum was recorded from a range of 4000–600 cm−1. Lignin samples were characterized by Py-GC–MS using a 5150 Pyroprobe pyrolyzer (CDS Analytical Inc., Oxford, PA). The identification of the pyrolysis products was accomplished using a GC–MS instrument (Agilent Techs. Inc. 6890 GC/5973 MSD). The Py-GC–MS was carried out following the method described by Herrera et al., 2014 [26]. A quantity between 400 and 800 mg was pyrolyzed in a quartz boat at 600 °C for 15 s with a heating rate of 20 °C/ms (ramp-off) with the interface kept at 260 °C. The pyrolyzates were purged from the pyrolysis interface into the GC injector under inert conditions using helium gas. The fused-silica capillary column used was an Equity-1701(30 m × 0.20 mm × 0.25 μm). The GC oven program started at 50 °C and was held for 2 min. Then it was raised to 120 °C at 10 °C/min and was held for 5 min after that raised to 280 °C at 10 °C/min. was held for 8 min and finally raised to 300 °C at 10C/min and was held for 10 min. The compounds were identified by comparing their mass spectra with the National Institute of Standards Library (NIST) and with compounds reported in the literature [27–32]. For the 2D HSQC NMR, around 50 mg of lignin was dissolved in 0.5 mL of DMSO d6. 2D HSQC NMR spectra were recorded at 25 °C in a Bruker AVANCE 500 MHz equipped with a z-gradient double resonance probe. The spectral widths were 5000 and 12,300 Hz for the 1H and 13C dimensions, respectively. The number of collected complex points was 1024 for the 1H dimension with a recycle delay of 1.5 s. The number of transients was 64, and 256 time increments were always recorded in the 13C dimension. The 1JCH used was 145 Hz. Prior to Fourier transformation, the data matrices were zero filled to 2048 points in the 13C dimension. Data processing was performed using MestReNova software. The central solvent (DMSO) peak was used as an internal chemical shift reference point (δC/δH 39.5/2.49).

2.2. Chemical composition of kraft lignins 2.4. Total phenolic content (TPC) and antioxidant activity The purity of kraft lignins was assessed taking into account the carbohydrate content, Klason lignin (AIL), as well as the ash and sulfur content. For the carbohydrate quantification, lignin samples were subjected to acid hydrolysis with 72% w/w H2SO4 for 1 h at 30 °C followed by second acid hydrolysis carried out by diluting the samples to 12% (w/w) H2SO4 and autoclaving them for 1 h at 121 °C. After hydrolysis, the samples were filtered and the remaining solid phase was considered as Klason lignin. An aliquot of the hydrolysate was analyzed using High Performance Liquid Chromatography (HPLC) in a Jasco LC Net II/ADC with an Aminex® HPX-87H column (BIO-RAD) (300 × 7.8 mm)

The total phenolic content (TPC) of kraft lignins was determined by the Folin–Ciocalteau spectrophotometric method using gallic acid as reference compound and dimethyl sulfoxide as solvent, in a Jasco V630 spectrophotometer equipment. The total phenolic content of kraft lignin samples was expressed as μg gallic acid (GAE) per mg of dry lignin [33]. For the antioxidant activity evaluation, two methods were carried out: DPPH and ABTS⁎+. Trolox and BHT were taken as positive controls. The inhibition percentage of the DPPH and ABTS radicals was calculated following the equation reported in a previous work [33].

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2.5. Cytotoxic assays 2.5.1. Preparation of solutions To obtain lignin solutions of different concentrations 0.1 g of sterile lignin was dissolved in 1 mL of dimethyl sulfoxide (DMSO), diluted with 9 mL of complete culture medium and was used as a stock solution. The stock solution was then diluted with the medium to a range of concentrations between 0.0001 and 0.01 mg/mL. The medium for growing of mouse hepatoma MH-22A, melanoma B16 and Chinese hamster ovary (CHO) cells consisted of Dulbecco's modified Eagle's medium (DMEM) (cat. no. D5546, Sigma-Aldrich Chemie, Steinheim, Germany) supplemented with 9% fetal bovine serum (cat. no. F7524, SigmaAldrich Chemie), 1% L glutamine (cat. no. G7513, Sigma-Aldrich Chemie), 100 U/mL penicillin, and 100 μg/mLstreptomycin (cat. no. P0781, Sigma-Aldrich Chemie). 2.5.2. Propagation of cells Three types of cell lines were used: tumor cells (mouse hepatoma MH-22A and melanoma B16) and non-cancerous (CHO) cells. Cells were grown in monolayer cultures in 25-cm2 (60–70-mL) flasks (Greiner Bio-One, Frickenhausen, Germany) at 37 °C in a humidified 5% CO2–95% air atmosphere in water-jacketed incubator IR AutoFlow NU-2500E (NuAire, Plymouth, MN, USA). All manipulations that required sterile conditions were done in vertical laminar flow cabinet Aura Vertical SD4 (BIOAIR Instruments, Siziano, Italy). Cells were resuspended in a DMEM Eagle at a concentration of about 1 × 106 cells/mL as described previously [34]. Cell lines maintained at 2–8 × 105 cells/mL under sterile conditions at 37 °C and 5% CO2, using DMEM general culture medium (Sigma-Aldrich, USA), supplemented with 2 mM L glutamine, 100 μg/mL streptomycin, 100 IU/mL penicillin and 10% FCS.

2.5.3. Determination of surviving fraction Ten microliters of fresh stock solution were added into the tubes with 90 μl of warm, fresh serum-free media with the cells to obtain final lignin concentrations in the range 0.01–1 mg/mL such that there were sixfold tubes per dose including the untreated control. The cells were mixed every 10 min during 20 min and 1 h and then plated in 34-mm internal diameter (9.2 cm2 growth surface) Petri dishes (93,040, TPP Techno Plastic Products AG, Trasadingen, Switzerland) containing 2 mL of the culture medium at low densities (200–400 cells per dish) and allowed to grow at 37 °C and 5% CO2 for 5–6 (CHO and B16 cells) or 9 days (MH-22A cells). To reveal the rupture of cell membrane (morphologic expression of necrosis) cells were stained with Trypan Blue solution (Sigma-Aldrich, USA) after 120 min of lignin treatment. To see the colonies clearly, they were fixed with 96% ethanol (Stumbras, Kaunas, Lithuania), stained with a gram's crystal violet solution (cat. no. 94448, Fluka Chemie, Merck, Germany), and counted under a binocular light microscope (MBS-9, LOMO, St. Petersburg, Russia). Colonies that were too tiny (b50 cells) were not counted. Cell morphology was investigated with the help of Luminera USB Scientific Camera (Canada).

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Student's t-statistic calculation was used to compare the surviving fractions of cell lines. 3. Results and discussion 3.1. Purity of kraft lignins Lignin purity and chemistry are extremely important for their use at an industrial scale. The purity of precipitated kraft lignins expressed as Klason lignin content was 75.4 ± 0.3% for KL pH 2 and 83.7 ± 0.2% for KL pH 6. Moreover, it was found low polysaccharides contamination with values around 1.5% for both kraft lignins. Regarding the ash content, KL pH 2 presented 1.2 ± 0.2%, while KL pH 6 had 2.2 ± 0.3%. Because of lignins are obtained from the industrial kraft process. The sulfur content was determined by elemental analysis and it was found 3.7% for kraft lignin isolated at pH 2 and 3.9 for KL pH 6. 3.2. Molecular properties of kraft lignins The weight-average (Mw), number-average (Mn) molecular weights as well as polydispersity degree (Mw/Mn) related to polystyrene standards were determined by GPC and the results are collected in Table 1. KL pH 2 had lower molecular weight and polydispersity indicating more uniform lignin fractions than KL pH 6. As can be observed, KL pH 2 contained around 60% of molecules with around 3548 g/mol and 40% of molecules with molecular weight lower than 450 g/mol. In the case of KL pH 6, three different fractions were clearly noticed in the chromatogram. The higher one, which corresponds to 50% of the sample, presented values of 6756 g/mol. In addition, it is remarkable the high amount of low molecular weight fractions in both lignins, ~40% for KL pH 2 and 27% for KL pH 6. These molecular weight properties are widely related to their origin since the black liquor comes from an industrial aggressive process where the lignin of the wood chips is solubilized in water-alkali media as a result of the degradation of βaryl ether linkages of the lignin structure [35]. Moreover, several analytical techniques such as FT-IR, Py-GC–MS and 2D HSQC NMR were used in order to obtain information about the chemical structure of kraft lignins. Both kraft lignins revealed similar spectroscopic patterns of hardwood lignins, as can be appreciated in Fig. 1. A wide absorption band centered at 3400 cm−1 is originated from the O\\H stretching vibrations in aromatic and aliphatic hydroxyls groups [35,36], whereas bands at 2930 and 2840 cm−1 arose from the C\\H stretching and asymmetric vibrations of methyl (CH3) and methylene (-CH2-) groups, respectively [37]. The structure of lignin was evidenced by the presence of aromatic groups given by the typical aromatic skeletal vibrations (peaks observed at 1600, 1510 and 1425 cm−1) [38]. The most significant difference between both spectra is that KL pH 2 presented a pronounced band at 1710 cm−1, associated to nonconjugated carbonyl stretching vibration [39], which showed that KL pH 2 contained a significant amount of carbonyl groups from ketone and aldehyde groups, compared to lignin sample precipitated at pH 6. In addition, it is remarkable the clear presence of the bands related to C\\O of syringyl units (S) [39], which can be observed at 1330, 1120

2.6. Statistics The experiments were repeated at least three times. All data received were processed with the help of the MedStat software (version 3.0, № MS000027) through analysis of variance. Numerical data were represented as arithmetic average and standard deviation (x±σ). The differences were considered statistically meaningful at P b 0.05. The calculation of the required number of samples was performed by comparing two frequencies or two arithmetic averages. The data obtained from the experiments were checked for the normality. Further analysis of the collected numerical information was carried out depending to the accordance to the normal law of Gauss distribution. The method of comparing the arithmetic averages of two independent samplings with

Table 1 Molecular weight distributions of precipitated kraft lignins.

KL pH 2

KL pH 6

Mn

Mw

Mw/Mn

683 1942 397 172 991 4422 1140 390

2285 3548 448 174 3773 6756 1224 447

3.3 1.8 1.1 1.0 3.8 1.5 1.1 1.1

% 60.7 34.3 5.0 49.9 23.1 27.0

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Fig. 1. (a) FT-IR spectra of kraft lignins, (b) Magnified region of FT-IR spectra.

and 830 cm−1. However, it was not appreciable the characteristic band of the guaiacyl units (G) which is usually discernible at 1260 cm−1 [40,41]. This result indicated the predominance of S units over G units in the kraft lignins. In addition, analytical pyrolysis (Py-GC–MS) at 600 °C on kraft lignins was performed in order to study their phenolic compounds and determine the syringyl/guaiacyl ratio (S/G). Pyrolytic product distributions from lignin pyrolysis usually involve phenols, acids, esters, and aldehydes [28]. Previous work demonstrated that at 600 °C achieves the maximum yield of phenolic compounds [28]. In this work, the identification of 33 phenolic compounds, which represents 91–94% of the samples, released during the pyrolysis is listed in Table 2. The identified pyrolysis products were classified into four categories according to their aromatic structure: phenol-type compounds (H), guaiacyl-type compounds (G), syringol-type compounds (S), and catechol-type compounds (Ca). The results confirmed slight difference between both lignin samples but in both cases the majority of compounds formed during the pyrolysis derived from syringyl units, as expected for coming from hardwood. Both precipitated lignins contained traces (b2%) of phenol derivatives. Moreover, it was found between 8 and 10% of catechol derivatives. Other works proved that catechol derivatives come from the degradation of S-type compounds when pyrolysis was carried out at elevated temperatures [28,42,43]. Therefore, the syringyl/ guaiacyl ratio (S/G) was calculated by dividing the sum of peak areas from syringyl units (including catechol derivatives) by the sum from the peak areas of guaiacyl derivatives presented in Table 2. The S/G ratio was 3.8 and 4 for KL pH 2 and KL pH 6, respectively. The isolated kraft lignins were subjected to 2D-HSQC in order to provide detailed information about lignin structure and linkages between elemental units. Fig. 2 shows 2D-HSQC spectra of kraft lignins, which were divided into side-chain region (δC/δH 50–90/2.6–6) and aromatic region (δC/δH 100–150/5.5–8). The signals were assigned according to previously reported data [44–47] and the results are summarized in Table 3. As can be observed, few cross-signals were detected for kraft lignins, especially in the case of KL pH 6. The most prominent signal in both spectra was associated with methoxyl groups (δC /δH 56.4/3.74). In addition, the results of the side-chain demonstrated the presence of β-aryl-ether linkage (β-O-4′), β-5′ bond in phenylcoumaran substructure and β-β′ in resinol substructure. KL pH 2 showed the presence of β-O-4′ alkyl-aryl ether linkages and γ-acetylated β-O-4′ alkyl-aryl ether linkage, while in KL pH 6 structure only γ-acetylated β-O-4′ linkage was found. The relative abundance of the main inter-unit linkages was calculated according to Wen et al., 2013 [48] following the proposed semi-quantitative

method which uses integral values of the side chain region. The results are represented in Table 4. As expected, both kraft lignins presented low amount of β-aryl ether linkages in their structure, with values around 30% and 12% for KL pH 2 and KL pH 6, respectively.

Table 2 Identification of the pyrolysis products from kraft lignins by their mass fragments. Compound

Origina

Area percentage (%) pH 2

Phenol o-cresol m-Cresol p cresol 2,6 Xylenol 2,3 Xylenol 2 Ethylphenol m xylenol Total Catechol 3 Methoxycatechol 4 Methylcathechol Total Guaiacol 3 Methylguaiacol 4 Methylguaiacol 5 Methylguaiacol 4 Vinylguaiacol 4 Ethylguaiacol Isoeugenol Vanillin Acetoguaiacone Guaiacyl acetone Total Syringol 3,4 dimethoxyphenol 4 Methylsyringol Acetosyringone 4 Vynilsyringol 4 Ethylsyringol 4 Propylsyringol 4 Propenylsyringol Syringaldehyde 4 Allylsyringol Total Sulfur derivatives S/G

H H H H H H H H Ca Ca Ca G G G G G G G G G G S S S S S S S S S S

0.29 0.43 – 0.41 0.18 0.34 0.19 0.18 2.0 0.30 7.70 0.46 8.5 3.25 0.90 5.21 – 5.58 2.51 0.44 0.22 0.99 – 19.1 16.43 3.72 19.98 0.65 5.58 10.51 1.18 2.59 0.37 2.45 64.1 0.33 3.8

Mass fragments

pH 6 0.38 – 0.32 0.43 – – – – 0.8 0.84 8.43 – 9.3 9.34 0.90 1.76 0.14 4.62 – 0.48 – 0.39 0.35 18.0 38.15 0.78 6.86 0.29 8.60 4.19 – 1.95 0.56 1.72 63.4 0.25 4.0

94/66/55 108/107/79/77/90 108/107/77/79/90 107/108/77/79/51 122/107/77 107/122/77 107/122/77/138/121 122/107/121 110/64 140/125/97 124/123/78 109/124/81/53 123/138/77/95/67 138/123/95 123/138/95 150/135/107/77 137/152/122 164/77/149 151/152/81 151/166/123 137/180/122 154/139/111/96 154/139/111 168/153/125 181/196/153 180/165/137 167/182/168/77 167/196/168/123 194/91/151 182/181/111/167 194/91/179/119

a H: Phenol; G: 2-methoxyphenol (guaiacyl); S: 2.6-dimethoxyphenol (syringyl); Ca: 1.2-benzediol (catechol).

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Fig. 2. Side-chain and aromatic regions of HSQC NMR spectra of isolated kraft lignins.

Consequently, the main lignin substructures found for isolated lignins were β-β′ resinol type and β-5′ phenylcoumaran type. It is common since kraft lignins come from a severe chemical process where the pulping conditions causes the cleavage of ether linkages in order to remove lignin from the wood chips [21]. Moreover, during the last stage of the pulping several C\\C bonds are generated providing to kraft lignin high condensed structure [9]. Regarding the

main cross-signals in the aromatic region, the presence of S-unit was observed at δC/δH 104.1/6.64 which was related to C2,6-H2,6 correlations. However, signals of the oxidized syringyl unit (Cα = O) (δ C/δH 106.7/7.25) and C5 -H5 in guaiacyl units (δ C /δ H 115.6/6.77) were only appreciable in KL pH 2. Finally, signal of C β–Hβ of cynnamyl acetate end-groups was only found also in the case of lignin precipitated at pH 2.

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Table 3 Assignments of 13C\ \1H cross signals in the HSQC spectra and their presence isolated kraft ligninsa. Label

δC/δH (ppm)

Assignments

KL pH 2

KL pH 6

Fγ

129.9/7.04

✓

✗

G5 S′2,6

115.6/6.77 106.7/7.25

✓ ✓

✗ ✗

S2,6 Bβ Bγ

104.1/6.64 85.7/4.64 71.6/3.81 and 4.18 63.7/3.18

Cβ–Hβ of cynnamyl acetate end-groups (F´) C5-H5 in guaiacyl units (G) C2,6-H2,6 in oxidized (Cα = O) syringyl unit (S′) C2,6-H2,6 in syringyl unit (S′) Cα–Hα in β-β′ (resinol) substructures Cγ–Hγ in β-β′ (resinol) substructures

✓ ✓ ✓

✓ ✓ ✗

✓

✓

✓ ✓ ✓

✓ ✗ ✓

A′γ

63.7/3.92 Cγ Aγ 59.6/3.41 -OCH3 56.4/3.74

Cγ–Hγ in β-5 (phenylcoumaran) substructures Cγ–Hγ in γ-acetylated β-O-4 Cγ–Hγ in β-O-4 C-H in methoxyl groups

a

Presence and absence of the corresponding assignment were denoted as ✓and ✗, respectively.

3.3. TPC and antioxidant activity of kraft lignins The results of Folin-Ciocalteau assay indicated high content of phenolic hydroxyl groups compared to lignins in other works [19]. Besides, kraft lignin precipitated at pH 2 contained a higher amount of phenolics than KL pH 6 with values of 488.8 ± 2.7 μg GAE/mg dry lignin and 439.1 ± 0.1 μg GAE/mg dry lignin, respectively. This result was also reflected in the FT-IR spectra where the O\\H band is significantly higher in the case of KL pH 2. Moreover, the content of phenolics is also widely related to the molecular weight of lignin, since TPC increases significantly with the reduction of the molecular weight [49]. Furthermore, the low molecular weight and high phenolic content of this type of industrial lignin are due to the cleavage of β-aryl and α-aryl ether linkages in its chemical structure during the last stage of the kraft pulping process [21]. The phenolic hydroxyl groups have a high influence on the antioxidant activity of lignins [49,50]. However, the radical power of phenolics not only depends on the formation of the phenoxyl radical but also on its stability. Therefore, other functional groups like methoxyl groups and conjugated double bonds can stabilize phenoxyl radicals by resonance providing a positive effect on the antioxidant behavior of lignins [51,52]. Moreover, CH2 in the α-position of the side chain can increase the antioxidant activity [23]. Nevertheless, the content of conjugated carbonyl groups can be a negative factor [19,53]. Hence, in order to assess the inhibitory effect of isolated lignins from the industrial black liquor two spectrophotometric tests were performed and the results were compared to two commercial antioxidants, Trolox and BHT, which were taken as positive controls. Fig. 3 shows the correlation between lignin concentration and inhibitory effect of kraft lignins against ABTS⁎+ and DPPH radicals together with the values of Efficient concentration (IC 50 ) in each case. As known the lower IC 50 value the higher is the antioxidant capacity. The results demonstrated a potent free radical scavenging activity (IC50) at very low concentrations, 3–5 μg/mL for ABTS ⁎+ and 9–11 μg/mL for DPPH. No significant difference was found between both kraft lignins. The

Table 4 Relative abundance of the main inter-unit linkages from integration of 13C\ \1H correlation signals in the HSQC spectra. Sample

β-O-4 (%)

β-β´ (%)

β-5(%)

KL pH 2 KL pH 6

29.7 11.4

49.4 10.7

20.9 77.9

high antioxidant properties of kraft lignins can be explained by its chemical structure, besides having a very high content of phenolic hydroxyl groups, both are composed mainly by syringyl units (~64%), as has been reflected by the Py-GC–MS analysis, and therefore contains a high amount of methoxyl groups in their structure. Py-GC–MS technique is an acceptable tool to relate the antioxidant activity of lignins to their chemical structure since the presence of some functional groups can be easy detected [23]. In this study, several phenolic compounds were detected which can have an important influence on the antioxidant behavior of isolated kraft lignins. The existence of CH2 in the α-position of the side chain can be associated with 4 methylcathechol, 4 methylguaiacol, 4 ethylguaiacol, 4 methylsyringol, 4 ethylsyringol and 4 propylsyringol compounds. Moreover, carbonyl groups in the α-position of the side chain can be found in vanillin, acetoguaiacone, acetosyringone, and syringaldehyde. 3.4. In vitro cytotoxicity assay in murine hepatoma, melanoma and control Chinese hamster ovary cells The cytotoxic activity of two kraft lignin samples towards mouse hepatoma MH-22A, melanoma B16 and Chinese hamster ovary (CHO, non-cancerous) cells at different lignin concentrations in DMSO (0.01–1 mg/mL) were evaluated and the results are presented in the Fig. 4. The use of DMSO as solvent did not affect the number of cell colonies, so the reported data describes correctly the lignin influence on cell cultures. Results revealed that the cytotoxic action of kraft lignin solutions is concentration-dependent. Low concentrations of kraft lignin (0.01 mg/mL) did not significantly influence on cell proliferation, where surviving fraction was N95% after treatment of lignin solutions at 20 and 60 min. At higher concentrations (N0.1 mg/mL) and 20 min of incubation, higher cell proliferation inhibition was observed, especially for cancerous cells. KL pH 2 showed the highest cytotoxic potential presenting IC50 values of 200 μg/mL for MH-22A, 400 μg/mL for B16 and 1000 μg/mL for CHO non-cancerous cells, at 20 min of incubation. Studied cells required higher KL pH 6 dosage to present the same cytotoxic effect than KL pH 2 (IC50 = 400 μg/mL for MH-22A, 450 μg/mL for B16 and 1100 μg/mL for CHO). Therefore, the surviving fraction was lower in the case of KL pH 2 at the same concentrations. It can be related to its physicochemical properties [19]. KL pH 2 had lower molecular weight and higher content of phenolic compounds, which were detected by pyrolysis, than KL pH 6. Moreover, KL pH 2 presented the less condensed structure and higher content of phenolic hydroxyls groups. Moreover, the results evidenced a lower number of cell colonies for MM-22A after 60 min of incubation with kraft lignin solutions, than found for B16 and CHO. The IC50 values for KL pH 2 at 60 min of incubation were 150 μg/mL for MH-22A, 320 μg/mL for B16 and 350 μg/mL for CHO while in the case of KL pH 6 were 200 μg/mL for MH-22A, 350 μg/mL for B16 and 350 μg/mL for CHO. These values indicated that B16 and CHO cells presented similar behavior against kraft lignin samples at high incubation times. However, the highest kraft lignin solution concentration (1 mg/mL) at 60 min of incubation could have induced apoptosis- and necrosis-like processes for the majority of both tumor and normal cells. Morphological properties of cells obtained after cultivation of lignin-treated cells are shown in Fig. 5. These data evidenced little differences between the proliferation of cancerous and noncancerous cells after kraft lignin treatment. Mouse hepatoma cells were the most sensitive to kraft lignins treatment and non-cancerous cells CHO were the most tolerant to lignin samples. During cultivation after kraft lignin treatment (1 mg/mL), cells had characteristics of apoptosis, which consist in cell shrinkage, changed cell morphology, pyknosis, vacuolization and the separation of cell fragments into apoptotic bodies during a process called “budding”. In addition, cells had morphological characteristics of necrosis that is cell swelling and rupture of the cell membrane. Cell viability values, regarding the rupture of the cell membrane, after 120 min of incubation were, 40 ± 4% for

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Fig. 3. Antioxidant activity of lignins against (a) ABTS⁎+ and (b) DPPH and their comparison with commercial antioxidants (Trolox and BHT).

cancerous MH-22A cells, 62 ± 4% for cancerous B16 cells, 85 ± 5% for normal CHO cells and 94 ± 5% for cells without of kraft lignin. Wang et al., 2015 also showed that the isolated two lignin–carbohydrate complexes induced cell death in a concentration-dependent manner [17]. Owing to there is not a unique lignin molecule, is not possible to compare the results of the present work with those were published by

other authors. However, previous research studies in which lignin was studied in this field, positive results were achieved. Nagasawa et al., 1992 conducted an in vivo test where mice bearing spontaneous mammary tumors were treated by injecting intravenously lignin-related cone extract of pine. The treatment was performed in three cycles each with consecutive 3 days of treatment and 4 days of interruption

Fig. 4. Surviving fractions of cancerous hepatoma (MH-22A), melanoma (B16) and non-cancerous CHO cells: A, B – for KL pH 2 after 20 min and 60 min of treatment with lignin solutions, respectively; C,D – for KL pH 6 after 20 min and 60 min of treatment, respectively (x ± σ, n = 6).

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Fig. 5. Morphological properties after cultivation of lignin-treated cells (1 mg/mL, 1 h): A – cancerous mouse hepatoma MH-22A cells, B – cancerous melanoma B16 cells, C – non-cancerous CHO cells; and untreated control (0 mg/mL, 1 h): D – cancerous mouse hepatoma MH-22A cells, E – cancerous melanoma B16 cells, F – non-cancerous CHO cells. Crystal violet staining.

and they observed that DNA synthesizing enzymes, were decreased in mammary tumors of treated mice compared to those of the control mice bearing tumors without treatment [54]. Another research study carried out in 1990 disclosed the capability of lignin derivatives to activate macrophages which affected to shape of human urinary bladder carcinoma cells (HUB-15), suggesting that activated macrophages directly attack tumor cells [55]. Having done various investigations about cytotoxic effects of lignin towards both cancerous and normal cells, the exact mechanism of the biological behavior of lignin, is still uncertain. Just a few works have been able to relate the cytotoxic behavior of lignin to some biological processes. Wang et al., 2015 evidenced that lignin derivatives inhibited the activation of the nuclear transcription factor NF- κB in cancer cells [17]. This protein complex is in an inactive form in almost all animal cell types, however, in nearly all cancer cells is activated, keeping the cell proliferation and preventing cell apoptosis [56]. Evidently, the molecular characteristics together with its composition, rich in phenolic compounds, are the main causes of the biological activity of this natural molecule. Nevertheless, the lack of accurate information regarding the exact biological processes involved lignin makes still challenging the use of lignin in pharmaceutical applications. 4. Conclusions This work reports the study of in vitro antioxidant and cytotoxic activity of two isolated industrial kraft lignins as well as the analysis of their composition and main structural characteristics. Aside from presenting low molecular weights, Eucalyptus kraft lignins showed highcondensed structure with a high content of phenolic hydroxyl groups. These structural features together with their chemical composition, rich in phenolic compounds, contribute to their potent antioxidant activity. Furthermore, the cytotoxic test revealed that kraft lignins induced apoptosis- and necrosis-like processes acting cytotoxically on both tumor and normal cells. However, it was observed that mouse hepatoma cells (MH-22A) presented the highest sensitivity to kraft lignin samples, while melanoma B16 and non-cancerous CHO cells had similar behavior showing more tolerant to kraft lignins treatment even at high

concentrations. In spite of having demonstrated the biological activity of industrial kraft lignins, the lack of exact biological mechanism on studied cells, still makes the use of kraft lignins for pharmaceutical applications continue being a challenge and, therefore, more investigations are required to be carried out in the near future.

Acknowledgements The authors are grateful for the financial support received from the University of the Basque Country (post-doctoral grant of Ms. Gordobil DOCREC18/29). In addition, the authors would like to acknowledge the General Research Services (SGIker) of the University of the Basque Country for carrying out the analysis of the sulfur content of kraft lignins. References [1] W.O.S. Doherty, P. Mousavioun, C.M. Fellows, Value-adding to cellulosic ethanol: lignin polymers, Ind. Crop. Prod. 33 (2011) 259–276, https://doi.org/10.1016/j.indcrop. 2010.10.022. [2] A. Berlin, M. Balakshin, Industrial Lignins: Analysis, Properties, and Applications, Elsevier, 2014https://doi.org/10.1016/B978-0-444-59561-4.00018-8. [3] S. Laurichesse, L. Avérous, Chemical modification of lignins: towards biobased polymers, Prog. Polym. Sci. 39 (2014) 1266–1290, https://doi.org/10.1016/j. progpolymsci.2013.11.004. [4] M.P. Pandey, C.S. Kim, Lignin depolymerization and conversion: a review of thermochemical methods, Chem. Eng. Technol. 34 (2011) 29–41, https://doi.org/10.1002/ ceat.201000270. [5] L. Hodásová, M. Jablonsky, A. Skulcova, A. Haz, Lignin, potential products and their market value, Wood Res. 60 (2015) 973–986. [6] A.L. Macfarlane, M. Mai, J.F. Kadla, Bio-based chemicals from biorefining: Lignin conversion and utilisation, Adv. Biorefineries, Elsevier 2014, pp. 659–692, https://doi. org/10.1533/9780857097385.2.659. [7] C. Wang, S.S. Kelley, R.A. Venditti, Lignin-based thermoplastic materials, ChemSusChem 9 (2016) 770–783, https://doi.org/10.1002/cssc.201501531. [8] D. Kun, B. Pukánszky, Polymer/lignin blends: interactions, properties, applications, Eur. Polym. J. 93 (2017) 618–641, https://doi.org/10.1016/j.eurpolymj. 2017.04.035. [9] A. Duval, M. Lawoko, A review on lignin-based polymeric, micro- and nanostructured materials, React. Funct. Polym. 85 (2014) 78–96, https://doi.org/10. 1016/j.reactfunctpolym.2014.09.017.

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