International Journal of Biological Macromolecules 122 (2019) 1163–1172

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International Journal of Biological Macromolecules

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Organic solvent fractionation of acetosolv palm oil lignin: The role of itsstructure on the antioxidant activity

Izabel de Menezes Nogueira a,b, Francisco Avelino a, Davi Rabelo de Oliveira a, Nágila Freitas Souza b,Morsyleide Freitas Rosa b, Selma Elaine Mazzetto a, Diego Lomonaco a,⁎a Department of Organic and Inorganic Chemistry, Federal University of Ceara, 60440-900 Fortaleza, CE, Brazilb Embrapa Agroindústria Tropical, Rua Dra Sara Mesquita 2270, Planalto do Pici, 60511-110 Fortaleza, CE, Brazil

Abbreviations: PPOMF, pressed palm oil mesocarp fibacetosolv lignin; MeOH-F, methanolic fraction; EtOH-F, etfraction; DPPH, 2,2‑diphenyl‑1‑picrylhydrazyl radical; LCCFTIR, Fourier Transform Infrared Spectroscopy; NMR, nucdifferential scanning calorimetry; GPC, gel permeationhydroxyanisole; BHT, butylated hydroxytoluene; PG, propyguaiacyl; S, syringyl; Cl-TMDP, chloro‑4,4,5,5‑tetramethypolydispersity index; RPM, relative proportion of mon(3,5‑di‑tert‑butyl‑4‑hydroxyhydrocinnamate; HSQC, heteroence; pCE, p‑coumarate.⁎ Corresponding author.

E-mail address: lomonaco@ufc.br (D. Lomonaco).

https://doi.org/10.1016/j.ijbiomac.2018.09.0660141-8130/© 2018 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 May 2018Received in revised form 30 July 2018Accepted 11 September 2018Available online 13 September 2018

Pressed palm oil mesocarp fibers (PPOMF) are by-products from oil palm industry and represents a potentialsource of lignocellulosic biomass. In order to add value to this agro-waste, dewaxed palm oil acetosolv lignin(DPOAL) was extracted under eco-friendly pulping method. The chemical composition and structural character-istics of DPOAL were investigated. The results showed elevated yield (48.5%) and high purity (94.3%), besides amoderate average molecular weight (1394 g mol−1) and narrow polydispersity index (1.88). Structural charac-terization via FT-IR, 1H\\13C HSQC and 31P NMR indicated that DPOAL was a typical HGS-type lignin. In addition,to increase the phenolic hydroxyl contents and improve DPOAL's antioxidant properties through a simplemethod, a fractionation process with methanol, ethanol and acetone was carried out, obtaining the methanol(MeOH-F), ethanol (EtOH-F) and acetone (ACT-F) soluble fractions. These were characterized by FT-IR, DSC,1H\\13C HSQC and 31P NMR, which showed higher values of phenolic and aliphatic hydroxyls groups comparedto DPOAL. The antioxidant activity was evaluated by the free radical scavenging activity of2,2‑diphenyl‑1‑picrylhydrazyl radicals (DPPH·) and compared with commercial antioxidants, such as BHT andIrganox 1010. Interestingly, lignin samples had significantly lower IC50 values compared to commercial antioxi-dants, what suggests a great potential as novel natural antioxidant.

© 2018 Elsevier B.V. All rights reserved.

Keywords:Biomass valorizationStructural featuresRadical scavenging

1. Introduction

In the current scenario of crescent concern about the environmentalpollution combined to the increasing prospection of alternative mate-rials, there is a growing interest in green and sustainable chemistry, spe-cifically in the lignocellulosic raw materials, as promising renewableresource for chemicals building blocks [1].

The oil palm industry plays a very important role in the world pro-duction of vegetable oils and its production reached 55.70 million tonsin 2015 [2]. This industry generates several by-products, such as pressedpalm oil mesocarp fibers (PPOMF), in which approximately 5.8 tons are

ers; DPOAL, dewaxed palm oilhanolic fraction; ACT-F, acetone, lignin-carbohydrate complex;lear resonance magnetic; DSC,chromatography; BHA, butyl

l gallate; H, p‑hydroxyphenyl; G,l‑1,3,2‑dioxaphospholane; PDI,omers; Irganox 1010, tetrakisnuclear single quantum coher-

produced from the production of 1 ton of crude oil [3]. In this context,PPOMF represent an important source of lignocellulose, which is littleexplored and whose potentialities in biorefinery processes deserve fur-ther investigations.

Usually, the PPOMF are used only as solid fuel for boilers, which rep-resents an underutilization considering its biorefinery potential. Severalstudies have been developed in order to add value to this material, in-cluding the production of ethanol, butanol, hydrogen, xylose and or-ganic acids [4–7]. However, the use of lignin, which represents up to30 wt% of the fibers, is little explored [8]. The low value and abundanceof the lignin makes it an attractive feedstock for many researchers toevaluate its use in different fields [9].

Lignin is the second most abundant natural polymer after cellu-lose [10]. Chemically, it is a complex amorphous polyphenolic mol-ecule, composed mainly of three different monolignol monomers,incorporated into lignin aromatic cores in the form ofphenylpropanoids, namely p‑hydroxyphenyl (H), guaiacyl(G) and syringyl (S) units. [11].

Among the several methods for lignin extraction, the organosolvprocess represents an excellent alternative to obtain high quality lignin.This method has as main advantage the use of green solvents, such asacetic acid, generating high purity and low molecular weight lignins

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[12], which are desirable and attractive features for the development ofnew materials.

Lignin, as a polymeric polyphenol, is considered a promising naturalantioxidant agent and several studies have been developed in the senseto explore this property [13–18], since antioxidant compounds arewidely applied to food, medicine and chemical industry [19]. Due totheir purity, non-toxicity, high security and strong antioxidant capacity,natural antioxidants are more favored in the present life [20], whencompared with the most used synthetic antioxidants, such as butylhydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gal-late (PG).

Lignin has a complex structure and limited solubility in organic sol-vents, then some approaches must to be used in order to decrease itsmolecular weight and increase its phenolic hydroxyls and methoxylcontent. The result is the generation of high reactivity fragments withhigh capability to neutralize free radicals. Therefore, it is noticeablethat the chemical structure of lignin plays a key role in the antioxidantactivity. Then, a good structural elucidation of its structure is crucial,as well as the choice of a method that provides these aforementionedstructural features.

Several studies have been developed in the sense of improving theantioxidant properties of lignin. Chemical modifications in its structure,such as depolymerization [21], insertion of new reaction sites [22] andmodifications in hydroxyl groups [23] have been shown to be efficientmethods in improving its capacity to eliminate free radicals. However,most of these methods require harsh conditions, such as the use ofhigh pressures and temperatures or toxic and hazardous reagents.

An alternative, simpler, cost-effective and eco-friendlymethod is theorganic solvent fractionation of lignin, which has also been shown to beefficient for improving its antioxidant properties. This simple method isbased on the selection of specificmolecularweight fragments by the useof solvents with different polarities, yielding fractions with differentamounts of phenolic hydroxyls, which have shown improvements intheir antioxidant activity [16,24].

Generally, the literature reports the use of green solvents such asacetone, ethanol, ethyl acetate and organic aqueous solutions. Thechoice of the solvent will directly influence on the structural featuresof the lignin fractions obtained, such as methoxyl, phenolic hydroxyls,double bonds and lignin-carbohydrate complex content and molecularweight distribution.

Therefore, the main objective of this work was evaluate the influ-ence of the structural features of DPOAL and its soluble fractions ontheir antioxidant properties, comparing their performance with that oftwo commercial antioxidants, such as BHT and Irganox 1010, as aform to add value to a large produced agro-waste that is PPOMF.

2. Materials and methods

2.1. Materials

The following chemicals were usedwithout any further purification:glacial acetic acid (Synth), hydrochloric acid (37%, Synth), acetone(Synth), ethanol (Synth), methanol (Synth), dimethyl sulfoxide‑d6(99.96%, Sigma-Aldrich), HPLC-grade tetrahydrofuran (Chromasolv,Sigma-Aldrich), 1,4‑dioxane (99%, Sigma-Aldrich), chloroform‑d(99.96%, Sigma-Aldrich), pyridine (99%, Sigma-Aldrich), chromium(III) acetylacetonate (99.99%, Sigma-Aldrich), cyclohexanol (99%,Sigma-Aldrich), 2‑chloro‑4,4,5,5‑tetramethyl‑1,3,2‑dioxaphospholane(95%, Sigma-Aldrich), 2,2‑diphenyl‑1‑picrylhydrazyl (Aldrich),2,6‑di‑tert‑butyl‑4‑methylphenol (≥99%, Aldrich) and pentaerythritoltetrakis(3,5‑di‑tert‑butyl‑4‑hydroxyhydrocinnamate) (Irganox 1010,98%, Aldrich).

PPOMF (Elaeis guineensis) was produced in the town of Tailândia(Pará, Brazil) and kindly supplied by Embrapa Amazônia Oriental.They were milled in a knife mill (Fritsch pulverisette 19) using sieveswith pores diameter of 0.5 mm and characterized based on TAPPI

T203 cm-99, T204 cm-97, T211 om-02, T421 om-01 and T222 om-2for determination of alpha-cellulose, extractives, ash, moisture and lig-nin, respectively. The composition of PPOMF was cellulose (22.6 ±1.5%), hemicellulose (20.0 ± 1.5%), lignin (35.0 ± 0.7%), extractives(9.3 ± 0.1%), and moisture (8.3 ± 0.2%).

2.2. Lignin extraction and purification from palm oil by acetosolv method

The extraction was performed using a 93% (w/w) aqueous aceticacid solution with 0.3% (w/w) HCl as catalyst, which was added to10 g of milled palm oil fibers in a ratio of fiber to solvent of 1:10 (w/v). The systemwas kept in reflux in a flat-bottomed flask at 115 °C dur-ing 3 h, under atmospheric pressure and magnetic stirring.

At the end of the extraction, the sample was allowed to cool downfor 10 min at room temperature. Then, the black liquor was isolatedfrom the cellulosic pulp residue by filtration using filter paper (averagepore diameter of 28 μm), which was washed with acetic acid (60 °C).The resultant black liquor was concentrated under reduced pressureand precipitated in deionized water (pH ≈ 5.0) at 80 °C, using a blackliquor to water ratio of 1:10 (v/v) and allowing the mixture (pH≈ 3.0) to rest at room temperature for 24 h. The precipitated ligninwas then vacuum filtered on Buchner funnel (filter average pore diam-eter 8 μm) and washed with distilled water until the pH of the filtratewas the same of that of the distilled water. The lignin obtained wasdried at 105 °C for 24 h affording 1.7 g of acetosolv lignin (POAL)(48.5%).

In order to remove the fatty acids present in lignin, 15 g of POALwaswashed with hexane (1:10 g/mL ratio of POAL to hexane) for 3 h underreflux and vigorous magnetic stirring. After this process, the materialobtained was filtered and dried at 70 °C for 24 h, yielding 14.2 g ofdewaxed palm oil acetosolv lignin (DPOAL) (98.6%).

2.3. Solvent fractionation of dewaxed palm oil acetosolv lignin (DPOAL)

DPOAL was fractionated by extractions with acetone, ethanol andmethanol. The fractions obtained were abbreviated as ACT-F, EtOH-Fand MeOH-F, respectively.

DPOAL (1.0 g)was added to 10mLof the respective solvent and con-tinuously stirred at room temperature for 30 min. After this time, theundissolved material was removed by filtration and soluble fractionwas concentrated under reduced pressure. The yields of the fractionsare expressed in percentage and were calculated based on the initialweight of DPOAL.

2.4. Characterization of DPOAL and its soluble fractions

2.4.1. Fourier Transform Infrared Spectroscopy (FTIR)FTIR analyses were performed using a Perkin Elmer spectrometer

model FT-IR/NIR FRONTIER usingKBr pellets, inwhich the same amountof lignin was used for all pellets. The spectra were recorded between4000 and 400 cm−1 with resolution of 4 cm−1 using the arithmetic av-erage of four scans.

2.4.2. Nuclear resonance magnetic (NMR) spectroscopy1H\\13C HSQC spectra were recorded at 25 °C on a Bruker Avance

DPX 300 spectrometer (operating at 300MHz for 1H nucleus), equippedwith a 5-mm One Probe with z-gradient coils. Lignin samples (30 mg)were solubilized in 500 μL of DMSO‑d6. The central solvent peak wasused as internal reference (DMSO δH/δC 2.50/39.5). A semi-quantitative analysis of the volume integrals of the HSQC cross-peakswas performed using Top Spin 3.5 pl 7 NMR software.

2.4.3. Gel permeation chromatography (GPC)Gel permeation chromatography (GPC) analyses were performed in

a Shimadzu LC-20AD (Kyoto, Japan) at 40 °C using a setup comprisingtwo identical analytical GPC columns in series (Phenogel 5 Linear/

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Mixed columns, 7.38 mm× 300 mm, Phenomenex, Torrance, CA, USA),a flow rate of 1.0 mL min−1, HPLC-grade THF as eluent, and monitoredby a UV–Vis detector (Shimadzu SPD-M20A) at 280 nm. DPOAL andits soluble fractions (2 mg) were dissolved in 2 mL HPLC-grade THFand then were filtered using a 0.22 μm PTFE filter. Thus, 20 μL of the fil-tered solutionwas injected into the GPC system at 40 °C at a flow rate of1.0 mL min−1. Standard calibration was performed with polystyrenestandards (PSS standards, PSKYTH, Allcrom, Mw range 362–2.52× 106 g mol−1).

2.4.4. 31P NMR spectroscopyIn order to determine the amount of aliphatic, phenolic and carbox-

ylic acids hydroxyl groups present in DPOAL and its soluble fractions,31P NMR experiments were performed based on the methodology de-scribed by Granata and Argyropoulos with slightmodifications [25]. Be-fore sample preparation, all lignins were dried at 105 °C during 5 h.Then, samples were weighted (30 mg) and dissolved in 500 μL of sol-vent mixture (C5H5N:CDCl3, 1.6/1 v/v ratio). To the resultant solutionwas added 100 μL of chromium (III) acetylacetonate solution(5.0 mg mL−1) and 100 μL of cyclohexanol solution (10.85 mg mL−1).Finally, 100 μL of 2‑chloro‑4,4,5,5‑tetramethyl‑1,3,2‑dioxaphospholane(Cl-TMDP) was added to the mixture, followed to the addition of 250μL of solvent mixture in order to reach the mark of 1 mL of solution.The flask was tightly closed and shaken to ensure the complete dissolu-tion of all components. The spectra were recorded on a Bruker AvanceDPX500 spectrometer (operating at 202.4 MHz for 31P nucleus). Allchemical shifts reported are related to the hydrolysis reaction of Cl-TMDP, which produces a sharp signal in C5H5N:CDCl3 at 132.2 ppm.Quantitative analysis using cyclohexanol as internal standard was per-formed based onprevious literature reports [26]. Asamanner toestablisha pattern of integration, the signalswere integrated according to the follow-ing ranges of chemical shifts: internal standard (145.39–144.97 ppm),aliphatic‑OH (150–145.50 ppm), C5‑substituted‑OH (144.5–141.2 ppm),guaiacyl‑OH (141–138.50 ppm), p‑hydroxyphenyl‑OH(138.4–137.20 ppm) and COOH‑OH (136.5–133.34 ppm). The amount ofhydroxyl groups (U – OH) was calculated based on the Eq. (1):

CU−OH ¼ nU−OH

WS¼ CIS � VIS � AU−OH

MWIS � AISð1Þ

where CU–OH is the amount of hydroxyl present in each lignin unit inmmol g−1; nU-OH is the number of moles of the hydroxyl per lignin unit inmmol;Ws is the sampleweight inmg;CIS is the concentrationof the internalstandard inmgmL−1; VIS is the volume of the internal standard in μL; AU-OH

is the peak area related to the lignin unit hydroxyl; MWIS is the molecularweight of the internal standard in g mol−1 and AIS is the peak area relatedto the internal standard hydroxyl.

2.4.5. Differential scanning calorimetry (DSC)DSC analyses were carried out using a Mettler-Toledo DSC 823e, in

which samples (10 mg) were placed in closed aluminum pans with apinhole lid. The experiments were performed under nitrogen flow(50 mL min−1), where the samples were heated to 90 °C and annealedfor 10 min, then cooled to 0 °C (3 min) and heated to 200 °C at 10°C min−1 (second cycle). The glass transition temperature (Tg) wasassigned as the inflection point of heat flow change present in the sec-ond cycle.

2.4.6. Antioxidant activityThe DPPH free radical scavenging assay of DPOAL and its soluble

fractions were determined using a Cary 60 spectrophotometer (AgilentTechnologies), operated via UV Probe, version 2.42, based on the meth-odology described by Lange and co-workers with some modifications[24]. Initially, a 1 mg mL−1 solution of DPOAL and its soluble fractionswere prepared in 90% (v/v) dioxane-water. A stock solution of DPPHwas prepared at a concentration of 10 mgmL−1 that was subsequently

diluted to a working concentration of DPPH in 90% (v/v) dioxane-waterof 0.04mgmL−1. Each different aliquot from the lignins' solution (6, 15,30, 75, 150, 300 μL)was added to 1350 μL ofworking solution ofDPPH in90% (v/v) dioxane-water before the sample was brought to a final vol-ume of 3000 μL with 90% (v/v) dioxane-water. After each addition, thesample was incubated in a dark place at room temperature for 30 min.In order to compare the antioxidant capacity of DPOAL and its solublefractions there were performed assays with two commercial antioxi-dants, namely BHT and Irganox 1010. The same aforementioned proce-dure was used for these experiments, except that the aliquots from theBHT and Irganox 1010 solution were different than those of lignins,whichwere 6, 15, 30, 75, 150, 300, 600, 900 and 1200 μL.Measurementswere carried out in the spectrum range from 300 to 600 nmwith spec-tral resolution of 0.5 nm. The antioxidant activity is expressed in termsof IC50 values in μgmL−1, whichwasdetermined using the linear regres-sion of inhibition versus antioxidant concentration data, using λ =517 nmas the standardwavelength for analysis. The analyseswere per-formed in duplicate. The percentage of inhibition was calculated basedon the Eq. (2):

Inhibition I;%ð Þ ¼ AbsS−AbsBAbsB

� 100 ð2Þ

where AbsS is the absorbance of DPPH at 517 nm in the presence of theantioxidant compound and AbsB is the absorbance of DPPH at 517 nmwithout the addition of the antioxidant compound.

Based on the results obtained in the assays described, a kinetic studywas performed in order to obtain complementary data about the anti-oxidant activity. For this study, an aliquot from the lignins, BHT andIrganox 1010 solution (75 μL)was added to 1350 μL of working solutionof DPPH in 90% (v/v) dioxane-water before the sample was brought tofinal volume of 3000 μL with 90% (v/v) dioxane-water. After this addi-tion, the sample was incubated in a dark place at room temperaturefor 10 min before each measurement. The parameters of analysis wereequal to those described aforementioned. The analyses were performedin duplicate.

3. Results and discussion

3.1. Lignin extraction

According to the results, PPOMF submitted to acetosolv process pro-vided a lignin yield around 48.5%with insoluble-lignin content of 92.1%and acid-soluble lignin content of 2.2%, totalizing a purity of 94.3%. Thelignin yield was relatively high, considering the extraction was carriedout undermild conditions. In addition, the high purity of the isolated lig-nin also shows the efficiency of the acetosolv process to promote thecleavage of the lignin-carbohydrate complexes (LCC), yielding a ligninwith low residual carbohydrates content.

It is worth mentioning that the greatest advantage of the acetosolvprocess used in the present work in comparison with those already ex-istent in the literature is the successful lignin isolation with higher pu-rity and yields under mild conditions. There are some authors thatreported palm oil lignin yields ranging from 8 to 14% by usingethanosolv process under high severity condition, generally at 190 °Cduring 60 min [18,27–29].

The works in the literature about acetosolv palm oil lignin are verylimited [30,39], but they report higher lignin yields and purities thanthose obtained by using ethanosolv process. Souza et al. [39] andOliveira et al. [30] reported lignin yields of 63% and 57% and purities of83.4% and 91.6%, respectively, for the acetosolv pulping of PPOMFs, inwhich similar extraction and purification conditions in relation tothose used in the current work were employed.

These results emphasize that even though a high severity conditionis used, which theoretically would promote a great extension cleavageof lignin inter-units, is not a guarantee of high lignin yields. This occurs

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because the efficiency of the lignin extraction process depends on thetemperature, reaction time, pressure, solvent and catalyst.

However, based on the works aforementioned, one can assume thatthemain difference between the currentwork and others is the solvent.Acetic acid is a source of protons, which combinedwith the acid catalystwill protonate a higher amount of active sites, favoring their hydrolysis.In addition, it is also important to considerer the lignin solubility in bothsolvents (acetic acid and ethanol), which will affect directly in the pro-cess yield as well.

The isolation of high quality lignin with elevated yield atmild condi-tions using a recyclable system can be considered an alternative and ef-ficient tool for the biorefinery and lignin platform. This process allowscarry out successive extractions per day in lab, as well as in benchscale, in order to produce high amount of lignin at once to use it as feed-stock for several technological applications.

3.2. FT-IR spectral analysis

The FT-IR spectra of DPOAL and its fractions are illustrated in Fig. 1(A). The peaks were assigned according to previous published data[13,31]. The main bands assigned in the spectra were compared by theratio between their areas (AX) and that of the band at 1515 cm−1

(A1515). The results of relative contents of each band are shown inFig. 1(B).

The FT-IR spectra of DPOAL and the fractionswere quite similar,withonly few changes in the relative intensity of some peaks,which suggeststhat the process of solvent fractionation did not promote significantchanges in the lignin-bonding pattern. In addition, their FTIR spectraalso presented characteristics bands related to S (at 1328 and1119 cm−1) and G units (at 1515, 1269 and 1035 cm−1). Amongthese bands, those related to G units presented higher intensities, sug-gesting that DPOAL was mostly composed by G units.

The broad band at 3400 cm−1 is assigned to the hydroxyl group(O\\H) stretching, while bands at 2935 and 2855 cm−1 were attributedto the C\\H asymmetric and symmetrical vibrations of methyl (\\CH3)and methylene (\\CH2) groups, respectively. The band at 1710 cm−1 isassigned to carbonyl stretching in unconjugated ketones and conju-gated carbonyl groups. The bands at 1603, 1515, 1453 and 1425 cm−1

arise from the aromatic skeletal vibrations. Derived from the asymmet-ric C\\O stretch of ester groups, the band at 1164 cm−1was indicative ofthe presence of p‑coumarate (pCE) units, which were originated fromthe oxidation of H units. Therefore, based on the aforementioned spec-tral features, DPOAL and its soluble fractions can be classified as a HGSlignin.

Fig. 1. (A) FTIR spectra of DPOAL and its soluble fractions and (B)

It is worth mentioning that in all soluble fractions, especially in theethanol fraction (EtOH-F), a significant increase in the intensity of thebands attributed to the stretching of the C\\H and C_O bonds was ob-served. This fact suggests the enrichment of the soluble fractions withLCC or remaining fatty acids presents in DPOAL. A possible explanationfor this behavior is the high ability of ethanol to solubilize fragmentswith LCC and fatty acids.

3.3. 1H\\13C HSQC NMR spectroscopy

DPOAL and its soluble fractions were analyzed by 1H\\13C HSQCNMR in order to obtain complementary information on their structure,such as the monomeric composition and the relative proportion ofmonomers (RPM). Moreover, it is possible to evaluate the effects thatthe extraction process caused on their structure. Fig. 2 shows the aro-matic region (δC/δH 100–150/6.0–8.0) of the 1H\\13C HSQC NMR spec-tra of lignins, where the most important cross-peaks were assignedaccording to Hussin et al. [29,32] and are summarized in Table 1. In ad-dition, the main substructures identified in the spectra are depicted inFig. 2.

Fig. 2 shows that all lignins were mainly composed by syringyl (S),guaiacyl (G), p‑hydroxyphenyl (H) and p‑coumarate (pCE) units,which is consistent with the previous results of palm oil lignin obtainedby Sa'don et al. [22] and Hussin et al. [29,32].

pCE units were originated from H units through the oxidation oftheir phenylpropanoid chains, which suggests that the H units were se-verely modified during the organosolv process, corroborating with theprevious results described by Constant et al. [33].

The data presented in Table 2 show that themajor constituent of lig-nins was G followed by S and H units, which corroborates with theamount and intensities of the absorption bands related to these mono-meric units in their FTIR spectra.

It was also observed from the data presented in Table 2 that the frac-tionation process generated lignins with higher syringyl contents thanthat of DPOAL, which can be easily seen by the increase in the S/Gratio values. This behavior occurred due to the preferential interactionof the solvents with the fragments rich in polar functional groups,such as methoxyls that are present in abundance in syringyl units, fa-voring their solvation and dissolution in detriment of the otherconstituents.

3.4. Gel permeation chromatography (GPC)

Fig. 3 shows the differentialmolecularweight distributions curves ofDPOAL and its soluble fractions (ACT-F, EtOH-F and MeOH-F) obtained

Relative content of the main assigned bands in their spectra.

Fig. 2. Aromatic region in the 1H\\13C HSQC NMR spectra of (A) DPOAL; (B) ACT-F; (C) EtOH-F and (D) MeOH-F and their main substructures.

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by GPC. In addition, Table 3 summarizes the molecular weight values(Mw and Mn), polydispersity index (Mw/Mn) and the functional groupscontent, which will be discussed latter.

Table 3 shows that DPOAL and its soluble fractions had a narrowmo-lecular weight distribution, which is confirmed by the low polydisper-sity values, showing that the fragments composing these lignins hadlow heterogeneity in terms ofmolecularweight. Moreover, it is possibleto observe that the fractionation process produced ligninswith differentMw values, since the change in the solvent polarity caused a displace-ment of the differential molecular weight distributions curves to lowerlog MW values.

The solvent polarity had influence in both the fractions Mw andyields. Both parameters decreased following the order ACT-F N EtOH-FNMeOH-F. This can be explained by the lower polarity of acetone in re-lation to EtOH andMeOH, resulting in the dissolution of highermolecu-lar weight fragments, yielding a higher amount of soluble material as

Table 1Assignments of theDPOAL and its soluble fractions 1H\\13C correlation signals in theHSQCNMR spectra.

Label δC/δH (ppm) Assignment

S2,6 6.71/104.1 C2\\H2 and C6\\H6 in S unitsG2 6.97/112.0 C2\\H2 in G unitsG5 6.80/116.0 C5\\H5 in G unitsG6 6.79/120.0 C6\\H6 in G unitsH2,6 7.25/129.6 C2,6\\H2,6 in H unitspCE2,6 7.86/132.9 C2,6\\H2,6 in p‑coumarate (pCE)

well. This behavior can also be seen by the slightly displacement ofACT-F curve to higher log MW values.

However, the use of higher polarity solvents, such as EtOH andMeOH, caused a decrease in the yields of their soluble fractions, aswell as in their Mw values. This is due to the ability of these solvents topromote the dissolution of lower molecular weight fragments that gen-erally havemore polar groups to interact with them. Comparing the dif-ferential molecular weight distribution curves of EtOH-F and MeOH-Fwith those of DPOAL and ACT-F is observed that the higher the solventpolarity, the displacement of the curves will be to lower logMW values.This shows the great potential of this kind of solvent to dissolve selec-tively lower molecular weight fragments.

3.5. 31P NMR analysis

Quantitative 31P NMR spectrawere obtained to assess the amount ofaliphatic, phenolic (5‑substituted‑OH, guaiacyl‑OH andp‑hydroxyphenyl‑OH) and carboxylic acids hydroxyl groups. The

Table 2Relative proportion of monomers for DPOAL and its soluble fractions obtained from their1H\\13C HSQC spectra.

Lignin % S % G % H S/G ratio H/G ratio

DPOAL 9.7 72.3 18.0 0.13 0.25ACT-F 12.2 75.0 12.8 0.16 0.17EtOH-F 12.4 74.5 13.2 0.17 0.18MeOH-F 20.1 61.1 18.8 0.33 0.31

Fig. 3.Differential molecular weight distribution curves of DPOAL and its soluble fractions.

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obtained 31P NMR spectra for DPOAL, ACT-F, EtOH-F and MeOH-F withthe main identified structures are shown and depicted in Fig. 4 andthe data of quantification are summarized in Table 3.

Fig. 4 shows that the aromatic components of DPOAL, ACT-F, EtOH-Fand MeOH-F were syringyl/G-condensed (144.5–141.2 ppm), guaiacyl(141–138.5 ppm) and p‑hydroxyphenyl (138.4–137.2 ppm), which isconsistent with the 1H\\13C HSQC and FTIR data.

The measured amounts of hydroxyl groups for DPOAL and its frac-tions are in agreement with the values reported in the literature fororganosolv lignins [33,34]. Table 2 shows that the ACT-F had a reductionof 35% in the amount of aliphatic hydroxyls compared with the otherlignins. A possible explanation for this behavior is the low ability of ace-tone to solubilize fragments with LCC, since this solvent has a lower po-larity than EtOH and MeOH to interact with hydroxyl groups ofcarbohydrates.

Another important data presented in Table 3 that deserve a specialattention is the amount of hydroxyl groups from carboxylic acids inDPOAL and its fractions. This type of hydroxyl group can be originatedfrom fatty acids that remained in the lignins after their purification, aswell as from lignin fragments that were oxidized during the pulpingprocess. Table 3 shows that all soluble fractions presented higherCOOH content than DPOAL, suggesting that the fractionation process fa-vored the solubilization of fragments with COOH groups.

An interesting result can be obtained by the FTIR spectra of lignins, inwhich the absorption bands related to C\\H and C_O stretching in-creased for all fractions in relation to those of DPOAL, which possiblycan be attributed to the fatty acids or LCC fragments presents in the sol-uble fractions. This result combined with the COOH content calculated

Table 3Molecular weights, hydroxyl groups' content and Tg values of DPOAL and its solublefractions.

Index DPOAL ACT-F EtOH-F MeOH-F

Yield (%) 48.5 44.3 33.9 23.6Mw (g mol−1) 1394 1431 1149 1110Mn (g mol−1) 739 751 760 629Mw/Mn 1.88 1.91 1.51 1.77Tg (°C) 163 131 130 133OH aliphatic content (mmol g−1) 0.39 0.26 0.39 0.40Syringyl‑OH 0.67 0.81 0.62 0.82Guaiacyl‑OH 0.59 0.73 0.58 0.70p‑Hydroxyphenyl‑OH 0.34 0.41 0.33 0.37OH phenolic content (mmol g−1) 1.59 1.95 1.52 1.88COOH content (mmol g−1) 0.26 0.52 0.33 0.32Total OH groups' content (mmol g−1) 2.25 2.74 2.24 2.62OHphen/OHtotal 0.71 0.71 0.68 0.72

by 31P NMR suggests that the fractionation favored the solubilizationof residual fatty acids present in DPOAL in the following crescentorder: DPOAL b MeOH-F b EtOH-F b ACT-F, which is consistent withthe results described by Lange et al. [24] for the acetone soluble fractionof wheat straw lignin.

In addition, it is important to highlight that although the DPOAL andits soluble fractions had different amount of phenolic hydroxyls, theypresented almost equal OHphen/OHtotal ratio values, which means thatall lignins had very similar values of concentration of phenolic hydroxylgroups. This feature is very important to certain applications, such as theuse of lignin for antioxidant purposes, since it is well known that thephenolic hydroxyls play a key role in the mechanism of antioxidantaction.

3.6. Differential scanning calorimetry (DSC)

DSCmeasurements were carried out in order to determine the glasstransition temperature (Tg) of lignins. Theoretically, the Tg is reachedwhen the polymeric chain has enough energy to rotate. At first, thechains in lignin vibrate when it received energy and the rotation ofthe lignin creates a free volume. Smaller fragments with low molecularweight resulting in high speedmobility require less energy to rotate thechain [27]. In Fig. 5, the Tg of DPOAL and its fractions are indicated by ar-rows and their values are summarized in Table 3.

According to Fig. 5 and the Tg values in Table 3, it is possible to ob-serve that all soluble fractions had lower Tg values than that of DPOAL.This behavior ismainly linked to their molecular weights and branchingdegrees. Probably, DPOAL has a higher branching degree than its frac-tions, which requires a higher energy to provide the mobility of thepolymeric chains.

The fractionation process provided the selection of certain fragmentsby changing the solvent polarity, which had lower molecular weightsand probably branching degrees than DPOAL. These structural featuresincreased the degree of freedom of the polymeric chains of ACT-F,EtOH-F and MeOH-F and consequently their mobility as well, decreas-ing their Tg values.

It is worth mentioning that all soluble fractions had very similar Tgvalues, which suggests that they had similar structural features aswell, in agreement with the aforementioned FTIR, 1H\\13C HSQC and31P NMR and GPC results.

3.7. Antioxidant activity

Lignin is considered a promising natural antioxidant because of itspolyphenolic nature. It is widely recognized that the hydrogen-donating ability of phenolic compounds results in the ability to elimi-nate DPPH free radicals [35]. However, the radical scavenging abilityof phenolic compounds depends not only on the ability to form aphenoxy radical. The presence of groups that can stabilize these radicalsby resonance has a positive effect in the antioxidant capacity of com-pounds [36]. However, other factors, like the presence of conjugatedcarbonyl groups in the side chain or the presence of LCC or fatty acids,has an negative effect in the antioxidant activity of lignin [16].

The antioxidant capacity of DPOAL and its soluble fractions wereevaluated and compared to that of two commercial antioxidants (BHTand Irganox 1010). The DPPH• scavenging curves are depicted inFig. 6. The results are expressed in terms of percent inhibition of theDPPH radical versus the antioxidant concentration. In addition, Table 4summarizes the calculated IC50 values for the lignin samples, as wellas for BHT and Irganox 1010.

The results in Fig. 6 showed that all lignin samples had higher inhi-bition values compared to commercial antioxidants. Among the ligninsamples, only a slight increase in antioxidant activity was observed forMeOH-F and ACT-F in relation to DPOAL, while for EtOH-F it was ob-served a small decrease in the antioxidant activity in relation toDPOAL, as shown in Table 4.

Fig. 4. 31P NMR spectra of DPOAL, ACT-F, EtOH-F and MeOH-F and the main identified substructures.

Fig. 5. DSC curves of DPOAL, ACT-F, EtOH-F and MeOH-F.Fig. 6. DPPH free radical scavenging capacity of DPOAL, ACT-F, EtOH-F, MeOH-F andcommercial antioxidants BHT and Irganox 1010.

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Table 4DPPH free radical scavenging capacity (IC50 value) of DPOAL,MeOH-F, EtOH-F, ACT-F, BHT and Irganox 1010.

Sample IC50a (μg/mL)

DPOAL 45.0 ± 0.67ACT-F 43.4 ± 0.81EtOH-F 46.5 ± 0.59MeOH-F 42.5 ± 0.83BHT 637.7 ± 18.8Irganox 1010 1572.7 ± 9.85

a IC50 value is defined as the antioxidant concentration nec-essary to decrease the initial DPPH• concentration by 50%inhibition.

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Themost efficient antioxidant activity of lignin samples compared tocommercial antioxidants can be explained due to the greater number ofphenolic hydroxyl groups permass unit present in lignin.Moreover, thepresence of methoxyl groups (\\OCH3) in the S and G units of ligninhelps to stabilize the phenoxy radicals, favoring the process of DPPH•scavenging. In addition, conjugated double bonds in the side chain oflignin can provide additional stabilization of the phenoxy radicalsthrough extended delocalization. Similar results were obtained byGregorova, Košíková and Staško [37], where it was shown that ligninisolated from beechwood exhibited an antiradical power almost tentimes higher than Irganox 1010.

Fig. 7 shows possible pathways for the reaction of ligninwith DPPH•.Firstly, lignin donates a proton from the hydroxyl group to scavenge oneDPPH molecule, forming a phenoxy radical (path a, Fig. 7). This radicalcan then be stabilized by resonance with the methoxyl group (path b,Fig. 7) or with the conjugated double bond present in the side chain(path c, Fig. 7).

Since the antioxidant activity of lignin is attributed mainly to thephenolic hydroxyl groups present in its structure, it was expected that

Fig. 7. Possible pathways for reaction

the higher the amount of these groups, the greater its antioxidant activ-ity. This correlation, however, was not clearly observed when compar-ing the antioxidant activity of ACT-F and MeOH-F, since themethanolic fraction exhibited higher antioxidant activity but has alower amount of phenolic hydroxyl compared to the acetone fraction(Table 3). Thus, other factors may be acting more decisively in the im-proving of the antioxidant activity of this fraction.

The results in Tables 2 and 3 showed that the fractionation processwas able to select fragments with higher syringyl content (methoxylgroups) and syringyl hydroxyl groups (Syringyl‑OH), as evaluated by1H\\13C HSQC and 31P NMR, respectively. Among the fractions,MeOH-F had a syringyl content significantly higher than the other frac-tions. This fact may explain the better antioxidant activity of MeOH-F,since syringyl contains two methoxyl groups, which help to stabilizethe phenoxy radical formed in the reaction between lignin and DPPHby resonance and also to increase the steric hindrance around thephenoxy radical formed during the antioxidant mechanism.

Based on that, it is worthmentioning that the higher antioxidant ca-pacity (low IC50 values) of MeOH-F and ACT-F can be attributed to theirhigh syringyl (methoxyls), Syringyl‑OH and phenolic hydroxyl contentin relation to those of DPOAL. This shows that the fractionation processcan select very useful functional groups, in which their amount can alsobe selected by changing the solvent polarity.

The lower antioxidant activity of EtOH-F may be related to the factthat this fraction probably presented higher contents of LCC fragmentsand/or fatty acids, as previously suggested in Sections 3.2 and 3.5. It isimportant to note that studies have shown that the type of solventused in the antioxidant assay may have a great influence on the results.Bondet and coworkers [38], for example, have demonstrated that diox-ane is able to significantly decrease the kinetics of the DPPH/BHTreaction.

In order to evaluate if this solvent effect would be responsible for thebest results of the lignin samples in comparison to the commercial

of lignin with DPPH free radical.

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antioxidants, a kinetic study of the DPPH• scavenging was carried out.Fig. 8 shows the DPPH free radical inhibition curves versus time.

The results showed that even after a considerable period of time,commercial antioxidants continued to exhibit significantly lower inhibi-tion values than the lignin samples, proving the potential of DPOAL andits fractions to act as antioxidant agents even in chemical environmentsthat tend to inhibit the antioxidant/DPPH reactions. Similarly,Gregorova, Košíková and Staško [37] have demonstrated in their workthat the lignin obtained from beechwood is capable of inhibiting theDPPH radical much faster than Irganox 1010, in the same solvent (diox-ane) used in this work.

Thus, the higher antioxidant activity of DPOAL and its fractions,when compared to the commercial antioxidants BHT and Irganox1010 suggests that these compounds can be considered as potential al-ternative natural antioxidants to beused in food,medicine and chemicalindustry.

Moreover, lignins have a great advantage compared to BHT andIrganox 1010, which is the possibility of use in some applications thatrequires elevated temperatures.

However, the low solubility of lignin in other systems is a drawbackwhen considering its potential application as an antioxidant. In thissense, the use of organic soluble fractions with a similar or even betterantioxidant capacity than that of the non-fractionated lignin representa way to overcome this limitation.

Therefore, the combination of high antioxidant activity and thermalstability and selective solubility in organic solvents, makes the solublefractions of DPOAL a promising bio-based antioxidant compound withseveral technological applications.

4. Conclusions

High purity lignin can be obtained from pressed palm oil mesocarpfibers by using the acetosolv process under mild conditions with highyield. The fractionation process with different organic solvents pro-duced soluble fractionswith different structural properties, such asmo-lecular weight distribution, polydispersity and different amount ofhydroxyl groups. The antioxidant activity assays showed that DPOALand its soluble fractions had higher inhibition values compared to thecommercial antioxidants. These results show that lignin has a great an-tioxidant capacity with the potential for replace the commercial antiox-idants in different fields of industry. Moreover, the fractionation processis presented as a simple and time saving method for obtaining ligninfractions with different structural properties by changing the organicsolvent.

Fig. 8. Inhibition curves of lignins, BHT and Irganox 1010 obtained in the kinetic study ofthe DPPH• scavenging.

Acknowledgments

The authors gratefully acknowledge the National Council forScientific and Technological Development – CNPq (409814/2016-4)and CAPES for the financial support, as also the CENAUREMN (CentroNordestino de Uso e Aplicação da Ressonância Magnética Nuclear atUniversidade Federal do Ceará, Fortaleza, Brazil) for the NMR analyses.

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