Lignin modification supported by DFT-based theoretical study as a way to produce competitive natural antioxidants

Article abstract of DOI:10.3390/molecules24091794

The valorization of lignins as renewable aromatic feedstock is of utmost importance in terms of the use of sustainable resources. This study provides a deductive approach towards market-oriented lignin-derived antioxidants by ascertaining the direct effect of different structural features of lignin on the reactivity of its phenolic OH groups in the radical scavenging reactions. The antioxidant activity of a series of compounds, modeling lignin structural units, was experimentally characterized and rationalized, using thermodynamic descriptors. The calculated O-H bond dissociation enthalpies (BDE) of characteristic lignin subunits were used to predict the modification pathways of technical lignins. The last ones were isolated by soda delignification from different biomass sources and their oligomeric fractions were studied as a raw material for modification and production of optimized antioxidants. These were characterized in terms of chemical structure, molecular weight distribution, content of the functional groups, and the antioxidant activity. The developed approach for the targeted modification of lignins allowed the products competitive with two commercial synthetic phenolic antioxidants in both free radical scavenging and stabilization of thermooxidative destruction of polyurethane films.

Full text of DOI:10.3390/molecules24091794

molecules
Article
Lignin Modification Supported by DFT-Based
Theoretical Study as a Way to Produce Competitive
Natural Antioxidants
1
2
1
1
Liga Lauberte , Gabin Fabre
, Jevgenija Ponomarenko , Tatiana Dizhbite ,
Dmitry V. Evtuguin 3 , Galina Telysheva * and Patrick Trouillas
1,
2,4,
*
1
Latvian State Institute of Wood Chemistry, Dzerbenes Str. 27, LV-1006 Riga, Latvia; roze@edi.lv (L.L.);
jevgenijaponomarenko@inbox.lv (J.P.); lcl@edi.lv (T.D.)
INSERM UMR 1248, Université de Limoges, Faculté de Pharmacie, 2 rue du Docteur Marcland,
2
8
7025 Limoges Cedex, France; gabin.fabre@gmail.com
3
4
CICECO/University of Aveiro, Campus Universit rio de Santiago, 3810-193 Aveiro, Portugal; dmitrye@ua.pt
Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of
Science, Palacký University, t rˇ . 17 listopadu 12, 771 46 Olomouc, Czech Republic
á
*
Correspondence: ligno@edi.lv (G.T.); patrick.trouillas@unilim.fr (P.T.); Tel.: +371-67555916 (G.T.);
+33-519-564-251 (P.T.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 9 April 2019; Accepted: 8 May 2019; Published: 9 May 2019
Abstract: The valorization of lignins as renewable aromatic feedstock is of utmost importance in terms
of the use of sustainable resources. This study provides a deductive approach towards market-oriented
lignin-derived antioxidants by ascertaining the direct effect of different structural features of lignin on
the reactivity of its phenolic OH groups in the radical scavenging reactions. The antioxidant activity
of a series of compounds, modeling lignin structural units, was experimentally characterized and
rationalized, using thermodynamic descriptors. The calculated O–H bond dissociation enthalpies
(
BDE) of characteristic lignin subunits were used to predict the modification pathways of technical
lignins. The last ones were isolated by soda delignification from different biomass sources and their
oligomeric fractions were studied as a raw material for modification and production of optimized
antioxidants. These were characterized in terms of chemical structure, molecular weight distribution,
content of the functional groups, and the antioxidant activity. The developed approach for the
targeted modification of lignins allowed the products competitive with two commercial synthetic
phenolic antioxidants in both free radical scavenging and stabilization of thermooxidative destruction
of polyurethane films.
Keywords: lignins; antioxidant activity; molecular rationalization; modification; stabilizers for polymers
1
. Introduction
The lignocellulosic plant biomass has appeared a promising alternative to the fossil resources
because of its abundance, renewability, versatility, and environmental safety. Lignin is one of the main
components of plant biomass. However, unlike the polysaccharides, only a little amount of lignin is
exploited to produce biobased chemicals so far [1–6].
Lignins are arranged as amorphous network polymers, as a result from the dehydrogenative
radical polymerization of monolignols (p-coumaryl, coniferyl, and sinapyl alcohols), containing divers
functional groups and reactive sites, e.g., phenolic, aliphatic, or carboxylic OH groups, as well as
substituted aromatic sites or C=C bonds [
can be valorized as prospective antioxidants. Low toxicity and biodegradability properties of natural
lignin-derived antioxidants make them a promising alternative to the synthetic analogs [10 13] in food,
7–9]. Due to stereo-hindered phenolic OH groups, lignins
–
Molecules 2019, 24, 1794; doi:10.3390/molecules24091794
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1794
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cosmetic, and pharmaceutical applications [10
,11
,14
,
15]. Applications of lignin-based antioxidants
in composite materials have also been recognized [12
radical scavenger effect to prevent autoxidation and depolymerization of cellulose in pulps and
papers [19 20]. The incorporation of lignins into synthetic polymer systems can prevent them from
,16–18]. Lignin functionalities exhibits effective
,
excessive photo- and thermal oxidation. For example, the introduction of 0.5–10% (w/w) of lignins,
isolated from the byproducts of the prehydrolysis of birch wood in polypropylene or recycled
polypropylene, significantly increases its stability, in a synergic way with the commercial Irganox 1010
antioxidant [21].
The main factors which limit the commercial use of lignins as antioxidants are their high
structural variability and variation in chemical composition, as well as the absence of clear guidance for
structure–activity relationships, which would allow the control of their antioxidant activity. Some recent
technological developments propose to separate lignin molecules into fractions with low polydispersity
and well-defined properties [22
is an effective method allowing to select lignin antioxidants by regulating molecular mass, polarity,
compatibility to a given substrate, and number of phenolic OH and other functionalities [22 23 26 32].
–25]. Among such processes, solvent fractionation of technical lignins
,
,
–
Despite the success of fractionation to enhance the antioxidant response, the lignin-derived antioxidants
still cannot compete with commercial phenolic antioxidants [33–35].
Due to the incredibly complicated and irregular structure of lignins, the measure of their
antioxidant capacity does not allow to evaluate the contribution of each structural fragment on this
activity [36–38]. The constitution of a library of lignin fragments has enabled experimental studies
of the structure–antioxidant activity relationship, which, for the first time, included some features of
the side-chain structure of the phenylpropane subunits revealing major structural descriptors [33,34].
To improve the predictive character of these structure–activity relationships, the experimental data
can also take advantage of theoretical chemistry data on various phenolic compounds [39–41]. A joint
experimental and theoretical approach sounds particularly adapted to solve the problems of lignin
chemistry and to develop the methods of lignins modification for enhancing of their antioxidant
activity. Based on the previous experimental studies made for a diversity of technical lignins, a series
of low-molecular weight phenolic fragments have been chosen, considered as being sufficiently
representative to rebuild the response of the “lignin puzzle.”
The aim of the present work was to obtain new quantitative information about the direct
contribution of the different substituents on the side-chains of lignin structural units and the linkages
between these units on the capacity of phenolic OH groups to scavenge free radicals and to develop
the approach for lignins modification to enhance their antioxidant potential. Lignin modification was
made based on data obtained by joining density functional theory (DFT) calculations and experimental
measurements. Such targeted modification supports promotion of market-oriented natural antioxidants
from renewable sources. The sulfur-free lignins isolated by the soda delignification from different
plant sources and their fractions were examined as prospective raw materials. The antioxidant activity
of lignin products was compared to that of commercially available phenolic antioxidants in terms
of stable free radical scavenging activity and the ability to reduce the thermooxidative destruction
of polyurethane films. Unlike commercial phenolic antioxidants, which significantly inhibit the
biodegradation of the composite materials, antioxidants derived from soda lignins are not only
biodegradable, but they can also catalyze the biological decomposition of the material into which they
are introduced [13].
2
. Results and Discussion
The antioxidant activity of lignins is mainly attributed to phenolic OH groups whose reactivity
in redox reactions is modulated by OCH groups [20,42,43]. This study has been focused on the
3
contribution to antioxidant activity of the guaiacyl phenylpropane unit with different substitution
patterns including side-chains and bonds between the structural units, which are insufficiently
characterized so far. Accordingly, the antioxidant activity of 19 lignin-derived structural units

Molecules 2019, 24, 1794
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was evaluated (Figure 1), all bearing a guaiacyl moiety with various structural modulations, being
considered as representative of lignin structural fragments [34]. A contribution of each of these
structures to the antioxidant activity was examined to develop the most appropriate method for
modifying lignin to enhance its antioxidant activity.
Figure 1. Chemical structures of the lignin model compounds.
To establish a reliable structure–activity relationship (SAR), the related mechanism of free radical
scavenging should be considered. The basic antioxidant mechanism of polyphenolics is the direct ROS
(reactive oxygen species) scavenging by hydrogen atom transfer (HAT) mainly from phenolic OH
groups (Figure 2). HAT may proceed either via (i) proton-coupled electron transfer (PCET), which is
the concerted electron and proton transfer from the polyphenol to the free radical, (ii) or by sequential
proton loss–electron transfer (SPLET), which is electron transfer from the deprotonated form (mainly
phenolate or carboxylate in the presence of carboxylic acid moieties). Electron transfer directly from the
polyphenol (electron transfer then proton transfer, ET-PT) is unlikely [41], being much more favored
from the deprotonated forms (SPLET).
Figure 2. Different mechanisms for hydrogen atom transfer from polyphenols (ArO–H).

Molecules 2019, 24, 1794
4 of 21
Disregarding the mechanism involved, the free radical scavenging by polyphenols is mainly driven
by bond dissociation enthalpies (BDEs) of phenolic O–H. This descriptor is indeed directly related
to the thermodynamic balance of the scavenging process, which is similar for both PCET and SPLET
mechanisms. Therefore, BDE is a global descriptor of the antioxidant activity. In turn, ETE is a descriptor
more specific for SPLET: it has no relevance under apolar and acidic medium conditions, but it may be
seen as a minor descriptor under polar and neutral to alkaline conditions i.e., when SPLET may proceeds.
−
1
The calculated BDE values varied from 77.6 kcal.mol (typical for highly-reactive phenolic antioxidants)
−
1
to 86.9 kcal.mol (characteristic of low antioxidant activity), as shown in Table 1. The scavenging of the
•
•+
DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) free radicals often correlates with these thermodynamic descriptors [44 46] Conversely to BDE,
–
the higher the radical deactivation index (RDI) value, the higher the antioxidant activity. Wide
•
variations of RDI values were observed, ranging from 0.02 to 1.72 and from 0.16 to 2.39 in DPPH and
•
+
ABTS assays, respectively (Table 1). Assuming the RDI = 1.00 for guaiacol (1) as a reference (Table 1),
RDI values lower or higher than 1.00 indicated a decrease or increase, respectively, in reactivity of the
guaiacyl phenolic OH group.
Table 1. Calculated O–H bond dissociation enthalpy (BDE), electron transfer enthalpy (ETE), and
•
•+
radical deactivation indexes (RDI) in DPPH and ABTS assays of the lignin modeling compounds.
ABTS +
•
Number of the
Compound
Lignin Modeling
Compound
BDE (kcal
ETE (kcal
pKa inb
Water
•
DPPH RDI
−
1
−1
mol )
mol
)
RDI
1
2
3
4
5
6
7
8
9
guaiacol
methylguaiacol
ethylguaiacol
propylguaiacol
isoeugenol
eugenol
vanillin
acetovanillone
propiovanillone
82.4
80.3
80.5
80.4
77.7
81.0
85.3
85.2
85.0
1.00 ± 0.03
1.38 ± 0.03
1.35 ± 0.04
1.53 ± 0.02
0.90 ± 0.09
1.72 ± 0.07
0.02 ± 0.01
0.03 ± 0.01
0.05 ± 0.01
1.18 ± 0.03
0.98 ± 0.02
0.08 ± 0.01
109.0
105.7
106.0
106.0
105.7
107.3
121.4
119.5
118.9
-
9.93
10.27
-
9.85
9.89
10.15
7.40
7.81
7.98
0.92 ± 0.03
-
1.58 ± 0.07
0.67 ± 0.05
0.96 ± 0.04
0.57 ± 0.03
0.54 ± 0.03
0.45 ± 0.04
1.67 ± 0.02
0.92 ± 0.02
0.26 ± 0.01
2.39 ± 0.07
4
.41/
0.52
.43/
10
11
12
13
homovanillic acid
vanillylmandelic acid
vanilglycolic acid
ferulic acid
82.0/78.7
82.5/79.0
86.9/83.7
81.8/77.6
109.1/102.0
111.3/103.3
125.7/116.7
116.8/106.0
1
3
9
.93
1
7
.60/
.54
4
9
.56/
.39
1.23 ± 0.03
1.23 ± 0.06
14
15
16
dihydroferulic acid
dehydrodiisoeugenol
dehydrodieugenol
81.2/79.5
81.9
80.1
107.4/103.5
109.5
106.3
1.39 ± 0.04
-
-
-
a
0.67 ± 0.02
-
-
a
2.72 ± 0.03
6
.16/
17
divanillin
84.8
n.d. c
121.6
0.16 ± 0.03
1
0.07
-
1
1
8
9
dipropiovanillone
acetovanillonylvanillic acid
86.0
86.5/85.6
0.014 ± 0.01
0.02 ± 0.01
122.0
121.5/120.0
-
0.26
-
a
imported from Bortolomeazzi, et al. [47]; b imported from Ragnar, et al. [48]; c the compounds insoluble in the
reaction medium.
•
•+
BDE values followed a trend similar to the RDI values of DPPH and ABTS , as evidenced by
2
regression coefficients (R ) of 0.63 and 0.48, respectively. The regression analysis by excluding extreme
2
cases (i.e., compounds
5
and 16) nearly reached an acceptable linear correlation (R = 0.89 and 0.72,
respectively). This confirms the relevance of BDE as the major descriptor of the antioxidant activity, as
•
already seen for DPPH scavenging activity of flavonoids [44–46].
2
.1. Role of the Alkyl Substituents
−
1
•
1) is an efficient DPPH scavenger
With a BDE of 82.4 kcal mol , the unsubstituted guaiacol (
Table 1). Compounds , and (creosol, ethylguaiacol, propylguaiacol, and eugenol, respectively)
(
2,
3
,
4
6
•
•+
exhibited higher antioxidant activity in both DPPH and ABTS assays, as seen from the RDI value
higher than 1.00 (Table 1). This result agrees with the corresponding BDE values, which are lower

Molecules 2019, 24, 1794
5 of 21
than 82.4 kcal mol 1 for these three compounds (Table 1). This effect can be attributed to a positive
−
inductive effect of the alkyl chain thus increasing the electrophilicity of this lignin structural unit. The
•
length of saturated alkyl chains (methyl, ethyl or propyl) had negligible effect on BDEs and DPPH
scavenging activity. Compounds 1–4 exhibited RDI values higher than 1, meaning that more than one
radical can be scavenged per OH group. This also means that these guaiacol derivatives may form
oxidative products (e.g., dimers) also behaving as efficient scavengers. The spin density distribution of
the aryloxy radicals formed after HAT from the guaiacyl moieties highlighted multiple reactive sites
for the further dimerization reactions in solution (Figure 3).
Figure 3. The spin density distribution of the aryloxyl radicals formed from the OH groups of the
lignin-modeling structures.
2
.2. Role of π-Conjugation in the Side-Chain
Compounds and are special cases because of possessing delocalized
at the para position to phenolic OH group. Both compounds differ from the location of the double bond
Figure 1). In , the double bond is conjugated with the phenyl ring, allowing the radical delocalization
after HAT over the phenyl ring and the side-chain. The longer the delocalization, the more stable
5
6
π electrons in the side-chain
(
5
−1
the radical, and the lower the BDE. Compound
Table 1). This makes efficient at scavenging both DPPH and ABTS free radicals. Bortolomeazzi and
coauthors evidenced that may form oxidatively-induced biphenyl dimers, following fast kinetics [47].
These dimers appeared 90 times less active than in terms of antioxidant efficacy, most probably
because of the lowering of electrophilicity by acquiring the electron-accepting substituent in the
th position of the guaiacyl unit (i.e., increasing BDE values). This is confirmed by the O–H BDE
5 indeed exhibits a low BDE value of 77.7 kcal mol
•
•+
(
5
5
5
5

Molecules 2019, 24, 1794
6 of 21
of 81.9 kcal mol 1 as obtained for the most abundant dimer dehydrodiisoeugenol (15), as seen in
−
Table 1. Some contradiction between a very low BDE and the RDI value lower than of isoeugenol is
1
therefore rationalized by the quick formation of less active isoeugenol dimers. The high rate constant
of dimerization may also cause quick self-termination between two isoeugenol radicals, explaining
•
the fact that its stoichiometric factor is lower than
1
against DPPH [20]. The double bond in
6
is not
conjugated with the phenyl ring, therefore decreasing the
direct consequence, the BDE of the former is higher than that of the latter (81.0 and 77.7 kcal mol ,
π
-conjugated path compared to
5
. As a
−
1
•
respectively). However, surprisingly,
6
is more active than
2
–
4
at scavenging DPPH (Table 1). Dimers
can also be formed by two eugenolyl radicals [47]. The most abundant dimer, dehydrodieugenol (16),
is approximately twice as active as the monomer ( ) in terms of free radical scavenging. The BDE of
is 80.1 kcal mol , confirming its better activity. As steric hindrance may prevent planarity of the
phenyl rings and extension of the -conjugated path in the dimer, the increase in activity is mainly due
5
−
1
1
6
π
to the presence of two phenolic OH groups, each one being prone to HAT and to efficient scavenge
•
more than one free radical per one phenolic group. Additionally, 16 reacts quicker with DPPH than
6
[
47]. The
6
is therefore able to scavenge free radicals multiple times due to the formation of dimers,
•
thus explaining the difference between its high BDE and the stoichiometric factors against DPPH .
•
+
Surprisingly, both
values 1.45 and 1.44 for
SPLET mechanism in water. Both compounds exhibit low ETE values (105.7 and 107.3 kcal mol ,
5
and
6
showed a similar and efficient antioxidant activity against ABTS (RDI
5
and
6, respectively). This can be rationalized by the kinetically preferential
−1
•
+
respectively), which correlate with their efficient antioxidation activity in water against ABTS
.
2
.3. Role of the Carbonyl Group in the α-Position of Guaiacyl Unit
Vanillin, acetovanillone, and propiovanillone (
7
–
9
) are guaiacol derivatives substituted with a
•
chain bearing a carbonyl group in the
α
-position. These three compounds almost entirely lost DPPH
−1
scavenging activity (Table 1) and exhibited high BDE values ranging from 85.0 to 85.3 kcal mol .
The carbonyl group, being highly electronegative, attracts the density of electrons, thus reducing the
π-conjugation and the stability of the aroxyl radical (Figure 3).
The length of the side-chain (one to three carbon atoms for
and antioxidant activities. Interestingly, these three compounds still exhibited ABTS scavenging
activities (0.57, 0.54, and 0.45 for , and , respectively). As expected from the high BDE values, these
activities were rather low, much lower than that of compounds
7 to 9) had no influence on BDEs
•
+
7
,
8
9
1
–
6
. The discrepancy between both
•
assays may come from the thermodynamic balance of scavenging reactions. In the case of DPPH ,
DPPH-H exhibits a BDE of 80.4 kcal mol in polar solvent [42], hence HAT from compounds
DPPH is thermodynamically unlikely, therefore forbidding almost all primary scavenging action.
−1
7–9
to
•
Moreover, compounds 7, 8, and 9 have pKa of 7.40, 7.81, and 7.98 (Table 1), reflecting acidity of the
phenolic OH group. In buffered solution (pH = 7.35, see Experimental section), this corresponds to the
presence of 44%, 11%, and 5% phenolates (deprotonated forms of the phenolic compounds), for the
three compounds, respectively. Therefore, the SPLET mechanism may also proceed, as reflected by the
•
+
−1
ETE descriptors. Indeed, ABTS electron affinity is 131 kcal mol and the ETE values of 7, 8, and 9
−
−1
are 121.4, 119.5, and 118.9 kcal mol , respectively.
2
.4. Role of the Carboxylic Group in the β-Position of Guaiacyl Unit
Homovanillic acid (10) and vanillylmandelic acid (11) are guaiacyl derivatives substituted
with a chain containing a carboxylic acid in -position. The pK of both compounds are 4.41 and
.43, respectively, in water solution corresponding to the carboxylic acid group [48]. Thus, in all
β
a
3
•
+
assays assessed in water, such as the ABTS assay, the monoanionic form must be considered for
interpretation. In pure methanol, the carboxylic acid pK
Therefore, concerning the DPPH assay performed in methanol, the protonated form is predominant.
a
of both compounds is higher than
9
[
49].
•
The antioxidant activity of 10 and 11 is similar to that of
respectively, as depicted in Table 1). Compound 10 appears slightly more active than
1
(RDI of 1.18 and 0.98 for both compounds,
and 11
1
,

Molecules 2019, 24, 1794
7 of 21
in agreement with slightly lower BDE values (BDE of 82.0, 82.4, and 82.5 kcal.mol 1 for the three
compounds, respectively); however, both from theoretical and experimental points of view these
differences are almost insignificant. In 10 and 11, the antioxidant constructive effect of the alkyl chain
appears counterbalanced by the antioxidant destructive effect of the carboxylic acid moiety. It is worth
−
noting that the hydroxyl group in the
α-position of 11 has a slight detrimental effect. Low phenolic
−
1
BDEs for the monoanionic forms (78.7 and 79.0 kcal mol for 10 and 11, respectively) rationalized
•
+
the rather high ABTS scavenging (RDI of 1.67 and 0.92, respectively). The slightly lower BDE of
may explain its somewhat better activity with respect to 11, however this difference is not very
1
0
significant. A better explanation is given by ETE, which appears slightly lower for 10 than for 11. This
confirms, at least partially, the contribution of SPLET mechanism, i.e., facilitated electron transfer
•
+
from the carboxylated form to ABTS . Vanilglycolic acid (12) bears both the properties of a carbonyl
group in
α
-position and a carboxylic acid in the
β
-position. This compound has no scavenging activity
•
−1
against DPPH , which is fully supported by the high O–H BDE value (86.9 kcal mol ). In water, 12 has
two low pK
•
+
a
(pK
a
1 = 1.60 and pK
a
2 = 7.54 [43]), meaning that in the ABTS assay, this compound is
mainly mono-deprotonated (carboxylate) and slightly bi-deprotonated (carboxylate and phenolate).
The first ETE value (energy required to remove an electron from the carboxylate) is high thus making
SPLET inefficient. The second ETE (energy required to remove an electron from the phenolate) is
•
+
•+
significantly lower, allowing SPLET towards ABTS . Therefore, the remaining ABTS scavenging
activity is most probably due to the small percentage of bi-deprotonated form of 12.
2
.5. Role of the Carboxylic Group in the γ-Position of Guaiacyl Unit
Ferulic acid (13) is a very well-known antioxidant polyphenol. Experimentally, it showed very
•
good DPPH scavenging activity (RDI = 1.23), in agreement with previously reported values [50
,51].
•
Its dihydrogenated analog dihydroferulic acid (14) exhibits the same DPPH scavenging activity. As for
compounds with carboxylic acid moieties in
water (4.56 for 13) but their pK values in methanol are expected to be much higher [49]. Therefore,
and 14 are most likely present in their protonated form in methanol. Hence, the similar BDEs of
a
β-position, these compounds exhibit low pK values in
a
1
3
−
1
•
the protonated forms (81.8 and 81.1 kcal mol , respectively) rationalize similar DPPH scavenging
activities for both compounds behaving by PCET. In water, both 13 and 14 are deprotonated from
the carboxylic acid moieties (first pK
a
value is 4.56 for 13
[48]). Therefore, the higher antioxidant
•
+
activity of 13 against ABTS can be explained by the BDE of its deprotonated form (ferulate, BDE of
−1
−1
7
π
7.6 kcal mol ) compared to 14 (dihydroferulate, BDE of 79.5 kcal mol ). Additionally, the larger
-conjugated path (delocalization of electron density) in ferulate with respect to dihydroferulate also
accounts for the higher activity of ferulate. In their respective radical forms, the spin density of the
radical is only located on the phenyl ring for dihydroferulatoxyl radical, whereas it also spans the
conjugated side-chain for ferulatoxyl radical (Figure 3). A higher delocalization of the spin density
induces higher stability of the radical, thus in agreement with lower BDE and higher antioxidant
activity. It should be noted that the effect of the
π-conjugation is different in 13 and 14, compared to 5
and 6, due to the presence of the carboxylic moiety.
2
.6. Effect of Dimerization
Compound 17 (divanillin) is the C5–C5 dimer formed from two vanillin molecules (Figure 1).
It possesses two active symmetrical OH groups. Compounds 18 and 19 are arylether guaiacol dimers
found in lignins. In both cases, the two guaiacyl units are -O-4’linked. Thus, both compounds have
only one active phenolic OH group. These three dimers exhibit carbonyl moieties in -position on their
para side-chains, so they share the same properties as their corresponding monomers (compounds
and ). Moreover, no additional -conjugation arises from the dimerization process, even for divanillin
in which steric hindrance prevents coplanarity of both phenyl rings. Therefore, these compounds show
β
α
7
8
π
•
•+
the absence or just weak activity against DPPH and ABTS , respectively, as confirmed by the high
O–H BDE values (84.8, 86.0, and 86.5 kcal mol for 17, 18, and 19, respectively). It is worth noting that
−
1

Molecules 2019, 24, 1794
8 of 21
even though 19 has a carboxylic acid moiety that can be deprotonated in solution, it is located on the
guaiacyl moiety without the active OH group so with almost no influence on the BDE (Table 1).
2
.7. Quantification of the Direct Influence of the Each Structural Descriptor on the Antioxidant Activity
of Lignins
Since BDE allows direct describing of the structure–antioxidant activity relationship, this parameter
was chosen to quantify the contribution of each structural descriptor. For this purpose BDE values
∆
were calculated among derivatives with or without of the corresponding chemical pattern (Table 2).
The ∆BDE was calculated for each pair of compounds shown in Table 2 and the average ∆BDE value
of the involved pairs of compounds was taken for each structural descriptor (Table 2).
Table 2. Quantitative evaluation of the impact of different structural descriptors (chemical substituents)
on the reactivity of the guaiacyl OH group.
Average ∆BDE of Phenolic
Normalized
Negative Impact
of the Structural
Descriptor, %
Normalized
Positive Impact of
the Structural
Compounds with and
without the Indicated
Chemical Feature *
OH Group between
Compounds Bearing or not
the Corresponding
Structural
Descriptor
Descriptor, %
Chemical Substituent
α-CH2
α-CH2-CH3
α-CH2-CH2-CH3
α-C=C
2 (1)
3 (1)
4 (1)
−2.1
−1.9
−2.0
−2.3
+0.6
+4.8
+1.6
+0.8
+0.5
+1.5
+1.4
−0.7
-
-
-
-
12
98
33
16
10
31
29
-
43
39
41
47
-
-
-
-
-
5(4), 13(14)
6(4)
7(2), 8 (3), 9(4), 12(10)
10 (3), 12 (8)
14 (4)
β-C=C
α-C=O
β-COOH
γ-COOH
α-OH
11 (10)
15 (4)
18 (9), 19 (8)
16 (6), 17 (7)
α-O-4/β-5
β-O-4 ether linked
biphenyl (5-5)
-
-
14
*
model structures are depicted in Figure 1. Parent basic lignin structures are presented in parentheses.
The presence of
α-carbonyl group affects the reactivity of phenolic OH group much stronger in
comparison to other structural descriptors, as can be seen by the biggest increase of BDE (highest
∆
BDE value) (Table 2). This rationalizes that compounds containing
α
-carbonyl groups are almost
•
inactive at scavenging DPPH radicals (e.g., RDI of compound
7 was 0.02, see Table 1). Because of this
on/off effect, the
∆BDE of α-carbonyl groups was taken as a reference to normalize the impact of the
other descriptors (Table 2).
2
.8. Experimental Evaluation of Lignin Antioxidant Activity with Respect to Impact of Different
Structural Descriptors
The quantitative impact of different structural descriptors (chemical substituents) on the reactivity
of the guaiacyl OH group (Table 2) was used to rationalize the antioxidant activity of the oligomeric and
polymeric lignin products and to find the most appropriate way for modification of technical lignins to
obtain highly efficient phenolic antioxidants. Soda lignins used as a raw material in this study are
heterogeneous both from point view of their chemical composition and the structure with a rather broad
molecular weight distribution. Regarding the practical application of these antioxidants, numerous
studies have highlighted the advantage of isolation of relatively uniform lignin fractions for these
purposes, e.g., obtained by solvent fractionation. The most prospective approach is reported to obtain
oligomeric fractions isolated from technical lignins by dichloromethane (CH Cl ) [22
,
23,26–29,31,32,35].
2
2
Here, different soda lignins and their CH Cl fractions were characterized in terms of the chemical
2
2
composition, molecular weight distribution, content of functional groups, and other structural
properties (Table 3).

Molecules 2019, 24, 1794
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Table 3. Characterization of the lignin samples in terms of their composition, molecular weight
distribution, functionality, and antioxidant activity.
Phenolic
OH/100 Phenyl
Propane Units
Relative Content of
α-Carbonyl group in
the Phenyl-Propane
Units, % **
Number of
Scavenged DPPH
Radicals Per OHphen
(RDI)
•
Lignin
Content, % *
Sample
OCH3, %
Mw, Da
(PPU)
Flax soda lignin
Flax soda lignin CH2Cl2 fraction
Black alder soda lignin
Black alder soda lignin, CH2Cl2
fraction
Ash-tree soda lignin
Ash-tree soda lignin CH2Cl2
fraction
94.5 ± 0.5
95.4 ± 0.4
74.0 ± 0.7
8.1 ± 0.1
6.5 ± 0.1
11.2 ± 0.1
24.0 ± 0.2
16.3 ± 0.1
13.3 ± 0.1
8358
847
7617
26
33
48
9.1 ± 0.1
22.3 ± 0.1
32.5 ± 0.1
49.5 ± 0.1
21.7 ± 0.1
39.9 ± 0.1
1.33 ± 0.07
0.71 ± 0.04
0.77 ± 0.03
0.69 ± 0.03
1.01 ± 0.05
0.72 ± 0.04
9
9.2 ± 0.7
638
4505
818
88
47
85
92.0 ± 0.9
9
8.2 ± 0.5
TBHQ
Irganox
-
-
-
-
-
-
200
100
-
-
1.31 ± 0.05
1.31 ± 0.07
*
based on the results of Py-GC/MS analysis of lignin-derived and carbohydrate-derived products. ** Relative
content (%) of structures containing α-carbonyl groups in all lignin-derived products, according to results of the
Py-GC/MS analysis.
Their antioxidant activity was examined and compared to that of the synthetic antioxidants
TBHQ and Irganox 1010 (Figure 4), which are highly effective and widely used commercial phenolic
antioxidants.
Figure 4. Chemical structures of the commercial antioxidants TBHQ and Irganox 1010, used as references.
Despite numerous advantages of CH Cl -fractioning, including a significantly higher content
2
2
of phenolic OH moieties, lower heterogeneity in terms of composition, and the molecular weight
distribution in comparison to the parent lignins, a noticeable part of phenolic OH groups in CH Cl
2
2
fractions is inactive in radical scavenging reactions (i.e., RDI < 1, see Table 3). The antioxidant activity
assay of the oligomeric lignin CH Cl fractions shows that they are not competitive in comparison to
2
2
the commercial phenolic antioxidants TBHQ and Irganox (Table 3). This behavior could be explained
by the negative effect of the structural descriptors mentioned in Table 2 within the lignin structure,
being the
α-C=O the most suspicious. Hence, this proposition was verified by more detailed analysis
of lignins and the corresponding CH Cl fractions.
2
2
2
.9. Rationalization and Enhancement of the Lignin Antioxidant Activity
The analysis of lignins in strongly basic aprotic solvents allow to discriminate and quantify the
phenolic hydroxyls in noncondensed and condensed structures [52
,53]. As an example, the analysis of
black alder lignin and its corresponding CH Cl fraction is envisaged, but very similar features were
2
2
1
find for other lignins in study. The H-NMR spectra in highly basic solution (HMPT-d ) show the
18
difference between the black alder parent lignin and the corresponding CH Cl fraction (Figure 5).
2
2

Molecules 2019, 24, 1794
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Figure 5. 1H-NMR spectra (HMPT-d18, 298K) of parent black alder soda lignin and its fraction,
extracted by CH Cl . Solvent contaminants peaks are marked by asterisk.
2
2
The phenylpropane units containing phenolic OH revealed the characteristic resonances in the
spectra range of 8.6 to 11.0 ppm [52]. CH Cl fraction showed an enhanced content of -carbonyl
CO) (signal at 10.5–10.7 ppm [52]) and aldehyde groups (CHO) (signals at 10.2–10.4 ppm [52]) in
α
2
2
(α
the units containing the phenolic groups (Figure 5). They also have an increased content of carboxyl
groups (signal at 11.46 ppm [52]) in comparison to parent lignin. According to the data, obtained
by density functional theory (DFT) calculations (Table 2), the presence of these oxygen-containing
groups in the side-chain, especially carbonyl groups, is the major reason for the inactivation of lignin
1
CH Cl fractions in radical scavenging reactions. The results of H-NMR analysis were confirmed by
2
2
the Py-GC/MS, which shows the significantly higher yield of
α-carbonyl group-containing compounds
in the lignin derived products for CH Cl fractions in comparison to the parent lignins (Table 3).
2
2
Due to the aforementioned structural features, the elimination of
drastically the antioxidant activity of lignin fractions. To tackle this hypothesis, the chemical reduction
of -carbonyl moieties was performed. The most commonly used reaction for the reduction of carbonyl
α-carbonyl is prone to increase
α
groups of lignins is by using NaBH ; however, in such case the carbonyl groups are reduced to aliphatic
4
OH groups which also have slightly negative effect on the antioxidant activity (Table 2). Instead, the
chemical transformation of carbonyl groups into CH fragments would not only avoid the negative
2
effect of the former but also bring the positive effect of the latter, see Table 2. Therefore, this modification
of lignin products was performed according to the reaction sequence shown in Figure 6.
The elimination of the carbonyl groups was also controlled by FTIR spectroscopy (Figure 7).
The variation of the intensity of the band assigned to the carbonyl groups vibration in the normalized
spectra of flax, black alder, and ash-tree soda lignin CH Cl fractions after their modification decreased
2
2
from 76.2 to 19.5%, from 29.7 to 11.3%, and from 28.0 to 13.0%, respectively (Figure 7).
The elimination of the carbonyl groups was also confirmed by Py-GC/MS showing a significant
decrease in the yield of the lignin-derived compounds containing the carbonyl group with a
simultaneous increase in the yield of the compounds containing α-CH fragments (Figure 8).
2

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Figure 6. Reaction pathways used for the elimination of lignin carbonyl groups.
The decrease of the relative content of phenolic OH groups conjugated with carbonyl groups (with
respect to the total OH phenolic) was also confirmed using differential UV/Vis spectroscopy (Table 4).
As it was predicted, the elimination of carbonyl groups drastically increases the reactivity of lignins
phenolic OH groups making them competitive with respect to the commercial phenolic antioxidants
TBHQ and Irganox 1010 (Table 4).
Table 4. Relative content of the phenolic units conjugated with α-carbonyl group given with respect to
the total OH phenolic and antioxidant activity of lignins.
Relative Content of the Phenolic
Units Conjugated with α-Carbonyl
Group, vs. Total Phenolic Groups, %
•
Number of Scavenged DPPH Radicals
Sample
Per One Phenolic OH Group
Flax soda lignin CH Cl fraction
14.0
0.71 ± 0.04
2.05 ± 0.8
2
2
Flax soda lignin CH Cl fraction
2
2
6
.0
modified
Black alder soda lignin
CH Cl fraction
3
1
2
1
6.5
3.9
5.6
1.3
0.69 ± 0.03
1.80 ± 0.02
0.72 ± 0.04
1.26 ± 0.07
2
2
Black alder soda lignin
CH Cl fraction, modified
2
2
Ash-tree soda lignin CH Cl
2
2
fraction
Ash-tree soda lignin
CH Cl fraction, modified
2
2
TBHQ
Irganox 1010
-
-
1.31 ± 0.05
1.31 ± 0.07

Molecules 2019, 24, 1794
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Figure 7. FTIR spectra of fractions obtained from soda lignins under study (
chemical modification (elimination of carbonyl groups).
1) before and (2) after their

Molecules 2019, 24, 1794
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Figure 8. Change of the relative content of the compounds containing carbonyl groups (C=O) and
CH fragment at the -position of side-chain ( -CH ) in the lignin-derived compounds (Py-GC/MS
data), caused by the modification of the CH Cl fractions of soda lignins.
–
α
α
2
2
2
2
2
.10. Modified Lignin Fractions as Alternative Antioxidants in Polyurethane Films
Commercially available synthetic antioxidants inhibit significantly the biodegradation of polymeric
materials. Conversely, lignins can stimulate their decomposition under natural environment after the
end of their service life. Therefore, replacing synthetic antioxidants with natural ones would promote
the production of environmentally friendly materials. For example, it was shown that the addition of
lignin (4.2% to 9.3% in weight) in the poly(ethylene adipate) urethane films significantly improved
their degradability by fungi enzymes (e.g., by laccase isolated from the Aspergillus sp.) [13].
In this work, the influence of different antioxidants on thermooxidative destruction of polyurethane
◦
(
PU) films at 310 C in air was studied for the demonstration purposes. Accordingly, flax soda
lignin modification products were compared to Irganox 1010, a highly effective antioxidant against
thermooxidative degradation of polyurethanes [54]. The kinetic curves of the thermooxidative
degradation of the polyurethane films show that the addition of the antioxidant decreases the weight
loss rate, especially in the first stage of thermooxidation destruction (Figure 9), which proceeds by
radical mechanism [55].

Molecules 2019, 24, 1794
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Figure 9. Kinetics of the thermooxidative degradation of polyurethane (PU) films (with and without
addition of antioxidant).
Without antioxidant, 50% of the weight loss of the material is reached after 32.5 min of incubation.
With 2% of flax soda lignin CH Cl fraction 50% of the weight loss of the material is reached after
2
2
7
4.5 min of incubation. Adding 2% of Irganox or modified flax soda lignin CH Cl fraction increases
2 2
this time to 100 or 111.5 min, respectively (Figure 9).
The antioxidant activity was also assessed as integrals of the kinetic curves (Figure 9). The integral
antioxidant activity as portion from the activity of Irganox was 0.6 for flax soda lignin CH Cl fraction
2
2
and 1.3 for modified flax soda lignin CH Cl fraction. This indicated that the modified flax soda
2
2
lignin CH Cl fraction performed 30% better inhibition effect than Irganox. This confirms a highly
2
2
efficient antioxidant activity of this lignin derivative, and outlines the promising application of lignins
in environmentally friendly materials.
3
. Materials and Methods
3
.1. Compounds, Modeling Lignin Structural Units
Model compounds 1–11, 13, 14, and 17 (Figure 1) used in this study are commercially available:
1
2
3
4
5
6
7
8
9
.: 2-methoxyphenol (guaiacol), ≥ 98%, Sigma Aldrich, CAS: 90-05-1
.: 2-methoxy-4-methylphenol (4-methylguaiacol), ≥ 99%, Acros organics, CAS 93-51-6
.: 2-methoxy-4-ethylphenol (p-ethylguaiacol), ≥ 98%, SAFC, CAS 2785-89-9
.: 2-metoxy-4-propylphenol (4-propylguaiacol), ≥ 99%, Sigma Aldrich, CAS 2785-87-7
.: 2-methoxy-4-propenylphenol (isoeugenol), 99%, Sigma Aldrich, CAS 97-54-1
.: 4-allyl-2-methoxyphenol (eugenol), ≥ 98%, Sigma Aldrich CAS 97-53-0
.: 4-hydroxy-3-methoxybenzaldehyde (vanillin), 99%, Sigma Aldrich, CAS 121-33-5
.: 4’-hydroxy-3’-methoxyacetophenone (acetovanillone), 98%, Acros organics, CAS498-01-2
.:
1-(4-hydroxy-3-methoxyphenyl)-1-propanone (propiovanillone),
≥
95%, Bide Pharmatech,
CAS 1835-14-9
1
1
0.: 4-hydroxy-3-methoxyphenylacetic acid (homovanilic acid), Acros organics, CAS 306-08-1
1. (RS)-hydroxy(4-hydroxy-3-methoxy-phenyl)acetic acid (vanillylmandelic acid), 99%, Acros
:
organics, CAS 55-10-7

Molecules 2019, 24, 1794
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1
3.: (E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid (trans-ferulic acid), 99%, Sigma Aldrich,
CAS 537-98-4
4. 3-(4-hydroxy-3-methoxyphenyl) propanoic acid (dihydroferulic acid), 96%, Sigma Aldrich,
1
:
≥
CAS 1135-23-5
0
0
0
0
17.
:
6,6 -dihydroxy-5,5 -dimethoxy-[1,1 -biphenyl]-3,3 -dicarboxaldehyde (divanillin), Fluorochem,
CAS 2092-49-1
Compound 12 (4-hydroxy-3-methoxyphenyl)(oxo) acetic acid (or vanilglycolic acid) was
synthesized at the Latvian State Institute of Wood Chemistry by the oxidation of compound
by nitrobenzene in alkaline medium at 100 C, using the method reported by Mottern [56] with minor
8
◦
modifications. Compound
at 100 C for 24 h. Aniline and small amount of nitrobenzene were distilled with water vapor; the
cooled solution was acidified, treated with active oxygen and filtered. The solution was cooled and
8 (35 g), nitrobenzene (27 g), and NaOH (25 g) were mixed in 75 mL water
◦
◦
compound 12 was crystallized. The amount of compound 12 was 34 g (85%) (Tmelting = 133 C) and its
purity was 85%, as checked chromatographically (GC, gas chromatography).
Compounds 18 and 19 (Figure 1) were synthesized by the methods described previously [57–59].
Their purity was 88 and 90%, respectively, as checked by GC.
3
.2. High-Molecular Weight Lignins
The soda sulfur-free lignins were produced on industrial scale from annual (nonwood) plants
using the patented lignin precipitation process applied to black liquor from soda pulping [60]. Soda
lignin from flax (Linum usitatissimum) was kindly supplied by the Granit SA (Lausanne, Switzerland).
The wood based soda lignins were obtained as a by-product from the black liquor of soda pulping
(
the IWC pilot equipment). The wood chips of black alder (Alnus glutinosa) and the ash-tree (Fraxinus
excelsior) were pre-extracted with acetone to remove the extractives according to TAPPI standard T
80 pm-99. The residual content of the extractives was 0.15% on the oven-dried wood. The 280 g of
2
chips were cooked in the digester with 2240 mL of 5% sodium hydroxide (Sigma Aldrich, Steinheim,
Germany) which corresponds to an alkali charge of 42% based on the dry wood. Once the reactor
◦
reached the operating temperature of 160 C, it was maintained for 3 h. The pulp was filtered, and the
filtrate was acidified to pH 2 to precipitate the lignin part. Lignin was filtered off and washed with a
◦
hot water until neutral reaction. The obtained lignin was dried under vacuum at 40 C. The yields of
soda lignins from black alder and ash tree were ~20%.
Both industrial and laboratory lignins were used as raw materials for modification to obtain
the highly effective lignin-derived antioxidants. The oligomeric fraction of lignin was obtained by
fractionation with CH Cl as described previously [61].
2
2
3
.3. Computational Methods
The O–H bond dissociation enthalpy (BDE) of each phenol moiety (ArO-H) was calculated based
on the enthalpies balance as follows.
0
•
0
•
0(
BDE(ArO-H) = H (ArO ) + H (H ) − H ArOH)
(1)
It is noteworthy that the lower the BDE, the easier the cleavage of the O–H bond, and the higher
the antioxidant activity. Similarly, the electron transfer enthalpy (ETE) was calculated as follows.
−
0
•
0
−
ETE(ArO ) = H (ArO ) − H (ArO )
DFT calculations were performed with the B3P86 functional in Gaussian program as it was
shown to accurately describe polyphenol BDEs [39 40 62 63]. The 6-31+G(d,p) basis set was used
as providing very similar results compared to the larger and more computationally demanding
-311+G(2d,3pd) basis set. Ground-state geometries were confirmed by a vibrational frequency
analysis that indicated the absence of imaginary frequency. Enthalpies were calculated at 298 K
(2)
,
, ,
6

Molecules 2019, 24, 1794
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and 1 atm. As many lignin fragments possess carboxylic acid moieties that can be deprotonated in
aqueous media, all protonation states were evaluated. The solvent effect was considered using the
integral-equation-formalism polarizable continuum model (IEF-PCM). Continuum models consider
the molecular system embedded in a shape-adapted cavity surrounded by a dielectric continuum
characterized by its permittivity (
conditions of the DPPH assay. All calculations were performed with the Gaussian09 software from
Gauss Inc, USA [64].
ε
= 32.61 for methanol). Calculations in methanol reproduced the
•
®
•
•+
)
3
.4. Antioxidant Activity Assays (DPPH , ABTS
•
•+
Chemicals used for the antioxidant evaluation (DPPH , ABTS and solvents) were of analytical
grade (Sigma–Aldrich). All solutions were prepared freshly before the measurements.
•
DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in methanol at a final concentration of
−1
~
10–4 mol L and incubated in the dark at room temperature for 16 h. The exact concentration of
•
•
DPPH in terms of absorbency at 515 nm was calculated from a calibration curve (A5 = 8.3
×
CDPPH
15
+
0.001).
A stock solution of ABTS (2 mM) was prepared in 50 mM phosphate buffered saline (PBS) made
of 8.18 g NaCl, 0.27 g KH PO , 3.58 g NaHPO x11 H O, and 0.15 g KCl in 1 L of distilled water. When
2
4
4
2
•
+
necessary, the pH of the solutions was adjusted at 7.4 with 0.1 M NaOH. The ABTS solution was
produced by mixing 50 mL of ABTS stock solution with 0.2 mL of K S O (70 mM) aqueous solution.
2
2
8
The mixture was kept in the dark at room temperature during hours 15 to 16 before use. To evaluate
•
+
the antioxidant capacity, the ABTS solution was diluted with PBS to reach the absorbance at 734 nm
•
+
of 0.800
±
0.030 [65]. The exact concentration of ABTS was calculated from the calibration curve
•
+
(A734 = 23.85 × C
− 0.007).
ABTS
A 0.03 mL DMSO solution of the lignin or relative phenolic compounds was added to 3 mL
of the prepared the DPPH or the ABTS solution. Five different concentrations were used for
each derivative. The decrease in DPPH or ABTS concentration was followed at a 515 or 734 nm
•
•+
•
•+
respectively until the reaction’s completion using Perkin Elmer UV/VIS spectrometer Lambda 650
◦
•
equipped with a thermally controlled cell at 22 C. All experiments were done in triplicate. The DPPH
•
+
or ABTS concentrations at steady state (i.e., primary reaction completed and no change in solution
for most of compounds) were plotted as a function of the molar concentrations of the lignins or relative
phenolic compounds. The antioxidant activity was expressed as a radical deactivation index (RDI)
which corresponds to the number of deactivated free radicals per molecule of tested compounds in the
case of lignin modeling compounds, or as the number of deactivated free radicals per phenolic OH
group in the case of technical lignins and their fractions. The obtained results are shown as the average
with a confidence interval (α = 0.05 level of significance). The RDI index is independent of the free
radical concentration. The higher the RDI, the higher the free radical scavenging activity.
3
.5. Characterization of the Polymeric and Oligomeric Lignin Products
3
.5.1. Molecular Weight Distribution
The molecular weight distribution (MWD) of the parent lignins and the fractions obtained was
analyzed by size exclusion chromatography (SEC) described in [66] using the Agilent 1100 system
−
1
(
PaloAlto, CA, USA). Lignin samples dissolved (1 mg mL ) in the eluent—DMSO—were injected into
−1
the PolarGeL-L 300
×
7.5 mm column (Polymer Laboratories). The flow rate was 0.8 mL min , column
was thermostated at 60 C. A differential refractometer (RI) and a UV photometer (at
◦
λ = 254 nm) were
used as detectors. For calibration, polystyrene standards were used in the range of 600 to 60,000 Da.
3
.5.2. Analytical Pyrolysis
The analytical pyrolysis (Py-GC/MS) analysis was performed using a Frontier Lab (Japan) Micro
◦
◦
−1
Double-shot Pyrolyser Py-2020iD (500 C pyrolysis temperature, 600 C s heating rate) directly

Molecules 2019, 24, 1794
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coupled with the Shimadzu GC/MS–QP 2010 apparatus (Tokyo, Japan) with capillary column RTX-1701
Restek, Bellefonte, PA, USA), 60 m 0.25 mm 0.25 m film (injector temperature 523 K, ion source
23 K with EI of 70 eV, MS scan range m/z 15 to 350, carrier gas helium at the flow rate of 1 mL min
and split ratio of 1:30. The mass of a sample was 1.00–2.00 mg. The oven program was 1 min isothermal
(
5
×
×
µ
−1
,
−1
at 333 K, then 6 K min to 543, and finally held at 543 K for 10 min. The apparatus was modified by
installation of the splitter of gas carrier flow Vitreous Silica Outlet Splitter VSOS (SGE, Australia) to
operate FID and MS detectors simultaneously. The identification of the individual compounds was
performed on the basis of GC/MS chromatogram using the Library MS NIST 147.LI13; whereas, the
relative area of the peak of individual compounds was calculated using the Shimadzu software on
the basis of GC/FID data. The summed molar areas of the relevant peaks were normalized to 100%.
Relative peak areas calculated for pyrolysis products of different origin (lignins, carbohydrates, and
lipophilic extractives) were used for assessment of the composition of the lignin samples.
3
.5.3. Spectroscopic Analyses
Fourier transform infrared (FTIR) spectra of lignins were recorded in KBr pellets by Spectrum One
−
1
1
apparatus (Perkin Elmer) with 4 cm resolution and 64 scans. H-NMR spectra of the non-acetylated
lignin samples were recorded on a BrukerAvance 300 spectrometer (Bruker, Wissembourg, France)
operating at 300.1 MHz and at 298K. The spectra were registered in the hexamethylphosphoroustriamide
(HMPT-d18) using a sample concentration of 2.5% (w/v). Tetramethylsylane (TMS) was used as an
internal standard (
δ
= 0.00). The relaxation delay was 3.0 s and about 300 scans were collected
◦
(90 pulse).
3
.5.4. Functional Analysis
The content of OCH groups in lignin samples was determined by the Viebock–Schwappach
3
method [45]. The results were expressed on a dry weight and ash-free basis. The number of total and
1
phenolic OH groups was determined, using H-NMR spectroscopy for the acetylated lignin samples [67].
1
H-NMR spectra were recorded on a Bruker Avance 300 spectrometer (Bruker, Wissembourg, France)
operating at 300.1 MHz and at 298K. The spectra were registered in CDCl using typical sample
3
concentrations of 2.5% (w/v). Tetramethylsylane (TMS) was used as an internal standard (
δ
= 0.00).
◦
The relaxation delay was 3.0 s and about 300 scans were collected (90 pulse). The number of phenolic
OH groups with conjugated carbonyl groups was determined by differential UV/VIS spectroscopy.
The lignin samples (10–15 mg) were dissolved in 10 mL dioxane. Two milliliters of the prepared
solution was transferred in three 50-mL volumetric flasks. One of the flasks was filled with the buffer
solution at pH 6, the second flask was filled with the buffer solution at pH 12, and the third flask was
filled with 0.2 M NaOH. The optical density of the prepared alkaline solutions was measured against
the optical density of the neutral solution (pH 6) at 300 nm and 360 nm, using Perkin Elmer UV/Vis
spectrometer Lambda 650. (Waltham, MA, USA). Calculations of the number of phenolic OH groups
were made as described elsewhere [59].
3
.6. Modification of the Lignins by Reduction of Carbonyl Groups
The procedure describing the reduction of aldehyde and ketone functions to methylene groups,
using the tosylhydrazone derivatives under very mild conditions [68] was adapted for lignin. The yield
of the modified lignin was ~75%. Recrystallization from the aqueous methanol provides analytically
pure product with the yield ~64%.
3
.7. The Effect of Lignin Products on Thermooxidative Destruction of Polyurethane Films
Thermooxidative destruction of the model polyurethane (PU) elastomer (control and containing 2%
◦
of lignin sample films) was studied at 310 C under isothermal regime by TGA in air atmosphere (flow
−1
rate 50 mL min ) using the Metler Toledo Star System TGA/ADTA 851e device. A 8–10 mg sample was
used. The PU elastomer films were obtained as described previously [33].

Molecules 2019, 24, 1794
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4
. Conclusions
In this work, an integrated and multidisciplinary approach for the better understanding the
antioxidant activity of lignins has been applied. By combining molecular modeling and antioxidant
assays, the antioxidant properties of lignin phenylpropane units were rationalized. The calculation
of O–H bond dissociation enthalpies (BDE) of characteristic lignin subunits allowed us to quantify
the direct influence of key structural features on antioxidant activity of lignin. Importantly, the
presence of carbonyl groups conjugated with phenolic units almost completely (98%) inactivated the
antioxidant activity of phenylpropane units. Other structural features, such as aryl ether linkages
or carboxyl groups in phenolic units, contributed to a reduction of the redox reactivity to ~30% or
1
6–33% (calculated as normalized
On the other hand, the presence of –
units resulted in an increase of the redox reactivity of their phenolic groups by approximately 40%
∆
α
BDE), respectively, when compared to an unsubstituted analog.
CH – or – CH fragments in the side-chains of phenylpropane
α
2
3
(calculated as normalized ∆BDE).
The antioxidant activity of soda lignins from flax, black alder and ash-tree was also examined.
It was suggested that the decrease in heterogeneity and polydispersity, as well as a high content of
phenolic OH groups in the oligomeric fractions of lignins make them good candidates in antioxidation
applications. However, such undesirable structural features as high content of carbonyl or carboxyl
groups at the alpha or beta positions of the propane chain, drastically decrease the antioxidant capacity
of lignins over commercial phenolic type antioxidants. However, the proposed targeted modification of
lignins fractions allowed a significant decrease in carbonyl groups conjugated with phenolic structural
units, with a simultaneous increase in CH groups at α-position of the side-chains. This resulted in
2
a drastic increase of the phenolic reactivity making their antioxidant activity competitive with the
synthetic commercial antioxidants produced from fossil resources. These modified lignins were more
effective in preventing the thermo-oxidation of PU films compare to the reference synthetic antioxidants,
therefore appearing as very promising innovative environmentally friendly and biodegradable
antioxidation agents.
Author Contributions: Devising of the project, G.T. and P.T.; Conceptualization, G.T. and P.T.; Methodology,
T.D., J.P., and D.V.E.; Data Curation, J.P., G.F., and D.V.E.; Visualization, G.F. and D.V.E.; Writing—Original
Draft Preparation, J.P., G.F., and P.T.; Writing—Review and Editing, P.T., G.T., and L.L.; DFT calculations and
interpretation of the obtained results, G.F. and P.T.; Chemical analysis, synthesis, and characterization of the
obtained products, L.L. and T.D.
Funding: Financial support from the Czech Science Foundation (P208/12/G016), the Ministry of Education,
Youth and Sports of the Czech Republic (project LO1305), and the student project IGA_PrF_2017_028 of Palacky
University is gratefully acknowledged. This study was supported also by the Latvian-France collaboration
programme “OSMOZE” and by the Latvian State Institute of Wood Chemistry Bioeconomy grant “LigProBK”.
This work was supported by CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref.
UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate cofinanced by
FEDER under the PT2020 Partnership Agreement.
Acknowledgments: The authors thank Cali (Calcul en Limousin) for computer facilities. The authors are grateful
to Maris Lauberts for the help in antioxidant activity tests.
Conflicts of Interest: The authors declare no conflicts of interest.
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