Structural Characterization of Acid DES-Modified Alkaline Lignin and Evaluation of Antioxidant Properties

Abstract

Lignin is an abundant and environmentally friendly biopolymer that contains a large number of phenolic hydroxyl functional group. In this paper, alkaline lignin was modified using different acidic DES (choline chloride/p-toluenesulfonic acid and choline chloride/lactic acid) at 130 ℃ (TC-lignin and LC-lignin) and the conformational relationship between the modified products and the antioxidant activity was investigated. Lignin was characterized by ³¹P NMR, gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FT-IR), and its antioxidant activity was evaluated. The results showed that the alkaline lignin products modified by acidic DES formed relatively homogenous dispersions and were characterized by a relatively low molecular weight and a high content of phenolic hydroxyl groups (e.g., TC-lignin, aliphatic-OH: 3.52 mmol/g, G-OH: 4.18 mmol/g, M_w: 3726, M_n: 2053, PDI: 1.81). The antioxidant activity (free radical scavenging rate, 90.35%) of TC-lignin was somewhat higher than that of LC-lignin (free radical scavenging rate, 89.12%) and both were higher than that of the commercially available antioxidant BHT (free radical scavenging rate, 88.79%). More specifically, we discussed the possible mechanisms of antioxidant reactions of lignin model substances in DPPH solutions. In addition, LC-lignin has an excellent UV-blocking capacity due to the specific phenolic hydroxyl and phenyl propane structure. A simple method is proposed for the modification of industrial lignin to make it suitable for use as an antioxidant and UV-resistant product.

1. Introduction

Antioxidants are molecules that reduce or prevent oxidation of substances and the presence of these molecules prevents free radical chain reactions from occurring [1] Antioxidants are additives to numerous materials and are widely found in plastics [2], biopharmaceuticals [3], food and [4] beauty products [5]. With the development of society, the global market demand for antioxidants is continuing to grow [6]. From 1990 to 2019, the number of research articles and reviews indexed in Scopus for the keyword “phenolic compounds” has doubled compared to the literature available in the last decade. The library of congress catalog shows 534 books on this topic, 81 of which focus on antioxidants in food [7]. In comparison to synthesized antioxidants such as butylated hydroxyanisole (BHA), propyl gallate (PG) and butylated hydroxytoluene (BHT), which currently occupy a major market share, natural antioxidants have unique advantages, such as freedom from toxicity and high antioxidant capacity, and have received a wide range of interests from researchers [8]. It is believed that in the future market, antioxidants will tend to be renewable, low toxicity and easily biodegradable natural antioxidants. And natural lignin-derived antioxidants stand out for their low toxicity and biodegradability making them a potential alternative to synthetic analogues in food, daily chemical and biopharmaceutical applications [9].

Lignin, which accounts for between 12 and 30 per cent of the biomass in plants, is a promising raw material which is abundant in nature. Over 50 million tonnes of lignin accumulate each year as a by-product of cellulose production and kraft lignin is the most commonly used by-product of pulping technology [10]. Lignin mostly synthesized by radical coupling of three hydroxypropanoids: coumaryl alcohol, coniferyl alcohol and sinapyl alcohol [11]. Due to its rich phenolic structure, lignin can be used as a natural antioxidant instead of chemically synthesized antioxidants [12,13]. Lignins with more phenolic hydroxyl groups, fewer aliphatic hydroxyl groups, low molecular weight and narrow polydispersity exhibit higher antioxidant activity [14,15]. Phenolic hydroxyl groups as well as double bonds on the C_α position, i.e., the conjugated double bonds in lignin have a positive effect on the antioxidant activity, while aliphatic hydroxyl groups of lignin are negatively correlated with antioxidant activity [16]. Therefore, developing a simple and efficient lignin treatment method while increasing the phenolic fraction is the key to improve the antioxidant activity of lignin [17,18].

Phenolic monomers originated from lignin and their derivatives have good antioxidant, anti-inflammatory and anti-hypertensive properties. Lignin itself has also been shown to have antioxidant, antibacterial and insecticidal properties [19]. The depolymerization modification of lignin is the simplest way to increase the phenolic hydroxyl content, which is also the most promising method to improve the antioxidant activity of lignin [20]. Zhao et al. [21] used formic acid as a hydrogen donor to accomplish depolymerization of alkaline lignin (AL) through hydrogenolysis. The products of hydrogenolysis of AL achieved 25.2 mg/L in terms of DPPH radical scavenging activity, which was superior to AL (63.5 mg/L) and the commercial antioxidant BHT (38.7 mg/L), indicating that the lower molecular weight of the modified lignin was beneficial in improving the antioxidant activity. Li et al. [22] treated hydrolyzed lignin with laccase and obtained a product with the highest phenolic hydroxyl content and lower molecular weight. The product obtained from laccase treatment had the highest phenolic hydroxyl content and lower molecular weight, and showed the exceptional antioxidant activity (IC₅₀ = 28.8 μg/mL), even compared to the commercially available antioxidants BHT (3,5-di- tert-butyl-4-hydroxytoluene, IC₅₀ = 38.2 μg/mL) and BHA (3-tert-butyl-4-hydroxyanisole, IC₅₀ = 56.3 μg/mL). Our group also studied the effect of deep eutectic solvents (DES) on the degradation of AL in the early stage and found that the acidic choline chloride-based DES system was effective in obtaining lignin monomers, with more phenolic hydroxyl groups, lower molecular weight and more homogeneity, which have good prospects for improving their antioxidant properties [23,24].

For this purpose, AL was used as a raw material to modify alkaline lignin using an acidic DES system. Different analytical techniques were used to study the structural characteristics and antioxidant activity of the modified lignin, to analyse the structural changes of AL during different acidic DES modification treatments, and to assess the antioxidant activity of the analyzed lignin samples by DPPH assay. More importantly, the lignin-based polyphenols with high phenolic hydroxyl content, low molecular weight, good antioxidant activity and reduced cytotoxicity prepared from AL by the DES-based one-step method proposed in this report are expected to be used in materials, daily chemicals, ecological agriculture and other fields of products, with potential tremendous economic value and broad application prospects.

2. Materials and Experimental Methods

2.1. Source of Materials

The AL obtained from Suzhou UPM Pulp and Paper Mill (Suzhou, China). Chemicals such as choline chloride (ChCl), lactic acid and p-toluenesulfonic acid monohydrate were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Deionized water prepared by our laboratory.

2.2. Modification of Alkaline Lignin

First, a 1:1 molar ratio of DES (ChCl as the hydrogen bond acceptor and p-toluenesulfonic acid/lactic acid as the hydrogen bonding donor variable) was prepared. The ChCl and p-toluenesulfonic acid system, noted as TC, and the ChCl and lactic acid system, noted as LC (for comparison purposes, the masses of ChCl were kept similar). The resulting mixture was stirred at 80 °C until clarified. Next, 2 g of AL is weighed and added to 40 g of DES and the mixed solution is put into a three-necked flask. The reaction apparatus was a magnetic oil bath stirrer and the reaction was modified by stirring at 130 °C for 5 h with the introduction of nitrogen gas (the optimum reaction temperature and time can be found in Figures S1 and S2). After time termination, 1 M hydrochloric acid was added to the reaction system and the pH was adjusted to 2. Centrifugation was then carried out and a solid-liquid separation interface could be observed, with the solid being the regenerated lignin and the aqueous phase being the DES-water mixture. Afterwards, distilled water was added several times to separate out the pure regenerated lignin and freeze-dried overnight to obtain solid products, noted as TC-lignin and LC-lignin (the solids residual rate of lignin after DES treatment can be found in Figure S3). The whole DES modification experimental procedure and product purification is shown in Scheme 1.

2.3. Characterization

Acetylation of lignin was completed prior to molecular weight testing. The molecular weight and dispersion of lignin samples were determined by gel permeation chromatography (LC-20A, Shimadzu, Japan). The mobile phase was approximately 5 mg/mL in tetrahydrofuran (THF) at a flow rate of 1 mL/min with a 50 μL injection volume and THF as the irrigation solution. The relative molecular mass of lignin was calculated from the standard curve of a monodisperse polystyrene standard. To increase the solubility of lignin in the solvent, the test sample was first acetylated.

The infrared absorption spectra of the different lignin samples were determined using a VERTEX 80V Fourier transform infrared spectrometer (Bruker, Berlin, Germany), wavelength analysis range: 4000–500 cm⁻¹, resolution: 4 cm⁻¹, number of scans: 32.

For ³¹P NMR characterization, 40 mg of lignin of different raw materials were dispersed in anhydrous pyridine/CDCl₃ mixture (0.5 mL, 1.6:1, v/v), 0.2 mL of internal standard reagent (e-NHI, 9.23 mg/mL) and 0.05 mL of relaxant reagent (chromium (III) acetylacetonate, 5.6 mg/mL) were added, and then 0.1 mL of phosphitylating reagent (TMDP, 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphosphplane) was phosphorylated, and the samples were signalized after 15 min using a ³¹P NMR spectral counterpart.

2.4. Evaluation of Antioxidant Properties

The DPPH radical scavenging assay for lignin fractions was based on a previous method represented in the literature [8]. DPPH· is a stable radical with maximum UV absorption at 517 nm and is widely used for antioxidant measurements in plant extracts and food supplements, additives [25]. A 6 mL sample of lignin was mixed with 5 different concentrations (0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, 0.1 mg/mL) of methanol solution with 6 mL of 0.15 mM DPPH-CH₃OH solution in the dark for 45 min at room temperature. The absorbance of the products was measured at 517 nm using UV-Vis light. Each experiment was repeated three times. The common antioxidant BHT was used as a positive control. Sample DPPH radical scavenging activity (RSA) was calculated using the Formula (1) [8]:

$$\mathrm{RSA} (\%)=\left(1-\frac{A_s-A_b}{A_c}\right)$$

(1)

where A_s is the absorbance of mixture with regenerated lignin; A_c is the absorbance of mixture without regenerated lignin instead of 6 mL methanol; A_b is the absorbance of mixture with only regenerated lignin and methanol. The IC₅₀ value is the concentration of antioxidant required for 50% scavenging of DPPH·, which is calculated from the curve of free radical scavenging rate with concentration.

2.5. Evaluation of UV Absorption Properties

The alkaline lignin and its regenerated lignin were dissolved in 1,4-dioxane and sample solutions of different concentrations were prepared and UV-Vis was used to measure the UV absorption at 250–700 nm.

3. Results & Discussion

3.1. Molecular Weight Determination

Gel permeation chromatography (GPC) analysis was used to obtain molecular weights and their distributions. The mean molecular weight (M_w), mean molecular number (M_n), and polydispersity values (M_w/M_n) are shown in Figure 1a. According to Figure 1a, degradation-modified alkaline lignin would result in lower mean molecular weight (M_w) and polydispersity values. Degradation-modified lignin (TC-lignin M_w: 3726 g/mol, LC-lignin M_w: 4356 g/mol) showed significantly lower M_w than unmodified lignin (AL M_w: 7308 g/mol). In addition, TC-lignin had the lowest polydispersity value (M_w/M_n: 1.81) compared to unmodified lignin AL (M_w/M_n: 3.30). Alkaline lignin modified by acidic DES can be found to have smaller fragments of lignin, while being more soluble in certain organic solvents [26]. Figure 1a clearly shows the high depolymerisation of alkaline lignin by acid DES, which could be due to the extensive breakdown of the aryl ether bonds. The distribution of the lower molecular weight lignin oligomers dissolved by the degradation of lignin macromolecules by acid DES is also more uniform [27].

3.2. FT-IR Analysis

FTIR is a simple and versatile technique for the determination of different functional groups. As shown in Figure 1b, typical absorption bands are observed at 1602, 1510 cm⁻¹ and these distinct peaks are the result of vibrations of the aromatic ring of the phenyl propane unit in the lignin. The DES modification did not affect the structure of the benzene ring, indicating that the DES treatment was relatively mild. The characteristic peak 1260 cm⁻¹ (guaiacol unit) is observed. Two absorption peaks at 815 cm⁻¹ and 855 cm⁻¹ can be clearly observed. This two peak clearly indicates that the alkaline lignin is derived from coniferous material (this is consistent with our raw material kraft softwood lignin). The band at 1120 cm⁻¹ is due to bending deformation within the aromatic C-H plane [28]. The intense absorption band at 3405 cm⁻¹ is caused by the O-H stretching vibration of the OH group in the product, which is where we need to focus our attention, and the unique peak signal of the symmetric and asymmetric vibration of the different peaks C-H at 2930 cm⁻¹. The spectrum exhibits a weaker peak at 1705 cm⁻¹, suggesting that the DES treatment process may have resulted in more β-O-4′ bond cleavage [23,24]. Consequently, the relatively large yield of conjugated carbon groups in lc-lignin may have resulted in reduced antioxidant activity in this specimen. At 1045 cm⁻¹ related with deformation vibrations within the aromatic C-H plane, the absorption band is assigned to the antisymmetric C-O stretching of the ester group at 1160 cm⁻¹. By the reduction of this peak we can find that the ether bond is reduced and that the DES modification of lignin is based on degradation, which breaks most of the β-O-4′ bonds. The absorption wavelength at 1120 cm⁻¹ relates to the C-H outer plane vibrations of the benzene ring in the sample [29].

3.3. ³¹P NMR Spectral Analysis

The phenolic hydroxyl group is an important factor affecting the antioxidant activity of lignin, and LC-lignin and TC-lignin were further determined by quantitative ³¹P NMR spectroscopy after phosphorylation to determine the amount of phenolic hydroxyl groups, as shown in Figure 1c. The phenolic hydroxyl groups were first phosphorylated using 1,3,2- dioxaphosphorane chloride and then ³¹P-NMR was used to determine the amount of different hydroxyl groups (aliphatic OH and phenolic OH) in lignin, as shown in Figure 1d. Based on previous reports, the signal peaks of the different hydroxyl groups were assigned [30]. The carboxyl (-COOH) and aliphatic hydroxyl (-OH) groups in alkaline lignin were 133.6–136.0 ppm and 145.4–149.0 ppm, respectively [30]. Phenolic hydroxyl groups in the G unit were observed at 138.0–140.5 ppm [31], indicating that DES treatment of alkaline lignin at this temperature is not prone to condensation. As can be seen from Figure 1d, the aliphatic-OH of TC-lignin was 3.52 mmol/g and the G-OH was 4.18 mmol/g, which was much higher than the phenolic hydroxyl content of alkaline lignin (ph-OH: 2.32 mmol/g, Figure S4), demonstrating that this is an efficient and convenient way to increase the phenolic hydroxyl content. Nevertheless, this modification of lignin with DES is based on the principle of breaking the ether bond [23,24] and the phenolic monomers content of the treated lignin is the same as that of other phenolization methods without obtaining a higher phenolic hydroxyl content [32].

3.4. Antioxidant Activities against DPPH Radical

On the basis of the above chemical and structural characterisation, the antioxidant activity of the depolymerisation products was further evaluated using DPPH radical scavenging assays. As shown in Figure 2a, the scavenging of DPPH radicals gradually increased over the test concentration of 0.02–0.1 mg/mL for each group of specimens, and at the concentration of 0.1 mg/mL, the scavenging rates of TC-lignin, LC-lignin and BHT were 90.35%, 89.12%, and 88.79% respectively, which were all higher than the scavenging rate of AL on DPPH·. As shown in Figure 2b, the IC₅₀ values for LC-lignin and TC-lignin were 25.53 μg/mL and 12.8 μg/mL respectively, which were significantly lower compared to the IC₅₀ values for untreated AL (38.41 μg/mL). These results suggest that modifications of lignin introduced through the DES treatment also improved the free radical scavenging capacity of lignin and that the antioxidant capacity was further enhanced with increasing acidity of the acidic system DES. Notably, the free radical scavenging capacity of TC-lignin (IC₅₀, 12.8 μg/mL) was similar to that of the common antioxidant BHT (IC₅₀, 14.54 μg/mL), indicating the excellent antioxidant activity of TC-lignin and its potential application as a biomass-based antioxidant [33]. Lignin with higher levels of G-OH exhibited stronger antioxidant activity. Previous studies have shown that an increase in the content of phenolic OH groups in depolymerised lignin products would enhance the antioxidant activity of lignin [28,34]. Although LC-lignin and TC-lignin have similar molecular weights, there is a significant difference in the DPPH radical scavenging ability of LC-lignin and TC-lignin, which may be due to the molecular weight of TC-lignin lower polydispersity and higher solubility, both of which contribute positively to the antioxidant activity [34]. Promising applications for lignin in cosmetics exist due to the role of acid-catalyzed depolymerization in enhancing the antioxidant activity of lignin, the ability to reduce free radical production, and the ability to stabilize reactions induced by oxygen and its free radical species.

3.5. Ultraviolet Absorption Performance

The sun transmits electromagnetic emissions in three ultraviolet (UV) wavelength areas. The shortest wavelengths (UVC, 100–290 nm) are trapped by the ambient atmosphere, and UV-A radiation at medium wavelengths (UVA, 290–320 nm) and longer wavelengths (UVB, 320–400 nm) reach the earth’s surface. Overexposure to UVB can lead to sunburn, while UVA penetrates more deeply into the skin. Lignin is known to have UV-blocking ability. Lignin depending on its origin has an absorption maximum in average in the 270–280 nm range, while softwood lignins exhibit maxima at higher wavelengths than hardwood lignins. To assess the UV-blocking ability of the samples, the light transmission of all samples was measured in the wavelength range of 250–700 nm and the results are shown in Figure 3a. The results show that LC-lignin has a high transmittance in the UV spectrum. It has a total of 87% transparency at 550 nm, and 1.4% transparency at 250 nm for AL indicates that neither AL has an effective UV-blocking ability in the UV-A range. TC-lignin exhibits low transparency, blocking almost 100% of the UVC and UVB spectra and most of the UVA spectrum As shown in Figure 3b, the UV transmittance of different concentrations of LC-lignin in the same system, it can be seen that the UV absorption capacity increases with increasing concentrations, where the UV light below 300 nm can be blocked at 64 ppm, the UV light below 325 nm can be blocked by an increase to 150 ppm, and all UV light below 350 nm can be achieved at a concentration of 200 ppm. The superior UV-blocking properties were attributed to the specific phenolic hydroxyl and phenyl propane structures in the lignin molecule [35]. Furthermore, the charge-transfer complex between the phenolic groups that give electrons and the electron-accepting o-quinone portion could further enhance the UV-absorbing capacity of the modified lignin [36].

3.6. Possible Antioxidant Reaction Mechanism of Lignin Oligomers

A variety of phenolic monomers can be obtained from alkaline lignin by DES treatment [37]. The most abundant monomer obtained is 2-methoxy-4-propylphenol [23,24]. Therefore, we chose 2-methoxy-4-propylphenol as a lignin model species to model the reaction mechanism of lignin degradation products with DPPH. The possible antioxidant reaction pathway of lignin with DPPH is shown in Figure 4. The lignin model species first donates a hydrogen from the hydroxyl group to scavenge a partial head-site DPPH molecule (reaction [a] in Figure 4). The phase of reaction [b] methoxy stabilises the hydroxyl radical, which will enhance the antioxidant activity of the lignin. Reaction [c] is where the lignin aryl part of the head site can bind to the DPPH molecule to form an electron pair. Another hypothesis involves dimerisation between two phenoxy radicals, forming a complex that could provide another hydrogen via a hydroxyl group (reactions [d, e] of Figure 4) [8].

4. Conclusions

In this experiment, alkaline lignin was used as a raw material and modified using DES systems with different acidity. The degradation reaction of alkaline lignin occurred by the breaking of a large number of ether bonds in DES, resulting in a modified product with good solubility, low molecular weight, and high phenolic hydroxyl content (e.g., TC-lignin, aliphatic-OH: 3.52 mmol/g, G-OH: 4.18 mmol/g, M_w: 3726, M_n: 2053), which is the key to increased antioxidant activity. The acidic DES-modified alkaline lignin has stronger antioxidant activity than the commercially available antioxidant BHT (IC₅₀, 14.54 μg/mL), and TC-lignin has good DPPH radical scavenging ability (IC₅₀, 12.8 μg/mL) as a natural antioxidant, providing a new source of raw material for the preparation of bio-based antioxidant functional materials at a later stage. In addition, 2-methoxy-4-propylphenol was chosen as a lignin model substance to simulate the reaction mechanism of lignin degradation products with DPPH. TC-lignin and LC-lignin not only have high antioxidant properties, but also have good UV-screening, which makes the development of lignin-based products, especially sunscreens and UV shielding materials, even more beneficial.