The following reagents were used: l-cysteine (99%), NaOH (97%), 1,4-dioxane (98%), cellulase (hydrolyzing activity is >1000 U/g), viscozyme L (a cellulase with enzymatic activity value of 100 FBG/g), γ-valerolactone (98%), methanol (≥99.9%, HPLC level), and acetonitrile (≥99.9%, HPLC level). All chemicals used in this study were purchased from Macklin except the cellulase was from Sigma and viscozyme L from Novozymes.

The wheat straw collected in Jiaxiang county, Shandong province, was physically cut, crushed, and sieved to obtain the 40–60 mesh fraction. The crushed wheat straw was dewaxed in 80% ethanol (v/v) at 80°C for 8 h. This step was repeated twice and then followed by extraction in dichloromethane at 40°C for 8 h (Li et al., 2019). The final dewaxed sample was air-dried in the fume hood and then stored in a desiccator for later use.

The lignin content of wheat straw was determined by the l-cysteine assisted dissolution method as reported previously (Lu et al., 2021). A 0.1 g/mL l-cysteine stock solution was prepared by dissolving 1.0 g of l-cysteine in 10 ml 72% H₂SO₄. 0.3 g of the absolutely dried lignocellulosic sample was added to 10 ml of l-cysteine solution at 60°C in a sand bath with continuous stirring for 30 min to dissolve lignin. 105 ml of deionized water was subsequently added to the reaction to dilute the solution. The content of lignin was calculated using Bill’s formula (1): S L ( % ) = A b s ′ × V f × δ ε × m s × P × 100 % (1) where Abs’ is the UV absorption values at 283 nm; V_f (ml) is the volume after dilution, V_f = 115 ml; ɛ (L/gcm) is the UV absorptivity, ɛ = 15.0; m_s (mg) is the mass of absolutely dried lignin; p (cm) is the optical path length, p = 1 cm; δ is the absolute dryness, δ = 1.

The lignin content of wheat straw was 16.46% ± 0.31 according to our determination.

The above-extracted lignin samples were purified by the following method. 150 mg of the crude lignin was added to 15 ml 96% (v/v) 1,4-dioxane/water mixture and stirred at room temperature for 4 h, repeated three times. For the AL, the extraction liquid was evaporated to obtain pure lignin after centrifugation. As for the acid lignins, the 1,4-dioxane solutions were concentrated to ∼3 ml, transferred into a centrifuge tube filled with 40 ml cold deionized water by drops, then centrifuged to remove the supernatant, and lyophilized to get the purified lignins.

Wheat straw contains 15.9 mg tricin per g of dry matter biomass according to our determination, which is higher than many other plant species. Wheat, as one of the most common staple cereals, is widespread in Asia, especially in China. Accordingly, wheat straw lignin could be a potential source of natural tricin with high value for human health. In this study, we investigated the influence of fractionation methods on the tricin content of the lignin from wheat straw under different conditions, including those performed under acid, alkaline, and neutral conditions. For instance, we used alkaline extraction, mild acidolysis, cellulosic enzymatic hydrolysis, GVL pretreatment, and organosolv pretreatment to extract the wheat straw lignin and six different lignin samples were obtained (Table 1). As report previously, tricin was almost completely depleted under the alkaline condition at high temperature (Zikeli et al., 2016). Aiming to reserve the tricin component, we, therefore, performed the alkaline extraction under low temperature and obtained 13.1% crude AL on the base of wheat straw. The crude lignin was purified by dissolving in dioxane/H₂O solution and gave 8.9% yield, accounting for 54.2% of the lignin in wheat straw. A controlled experiment using ball-milled wheat straw samples was also performed hoping to improve the lignin yield. The crude lignin yield increased to 21.2%. But the increment turned out to be from the carbohydrate fraction, which was eliminated during purification and leading to a similar purified lignin yield (55.1%). The mild acidolysis, organic acid, and cellulase enzymatic hydrolysis methods all produced about 50% lignin. But the GVL pretreatment yielded only 22.5% lignin, which might be because of the incomplete precipitation of lignin from the GVL solution.

The extracted lignin samples were characterized by HSQC NMR spectra to determine their structure (Figure 2 and Table 2). In general, all the methods applied in this study were able to extract the lignin from the wheat straw without serious alternation. The characteristic signals of guaiacyl, syringyl, and p-hydroxyphenyl units were clearly identified on the HSQC spectra, as well as the peaks of tricin and p-coumarate (Zheng et al., 2021). Unlike most of the AL in other studies (Lan et al., 2019; Zikeli et al., 2016), a certain amount of p-coumarate remained on the lignin after alkaline treatment in this study, which should be due to the mild condition (30°C) that we applied. The relative content of tricin was calculated by integrals of the corresponding peaks. The percentages of tricin in GVL-pretreated lignin, CEL, and MAL were 8.6, 6.7, and 6.3, respectively, higher than that in AL and organic acid lignin, which was about 1.1–1.7 (Table 3). A close inspection on the spectrum of the GVL lignin revealed that a certain amount of free tricin was presented in this sample, as indicated by the characteristic peak of C₃/H₃ in free tricin at δ _C/δ _H 103/7.0. The ratio of linked tricin to free tricin was about 6:4 according to the contour area of the corresponding signals. This result suggests that the dilute H₂SO₄ treatment can selectively cleave the β–O–4-tricin ether bonds in GVL at high temperature.

In order to determine the absolute content of tricin in the lignin samples, we applied the thioacidolysis degradation method followed by measurement using HPLC-MS under multiple reaction monitoring mode (MRM), as reported previously (Lan et al., 2016; Chen et al., 2021). The GVL lignin contained 89.3 mg/g of tricin. This was the highest tricin level among the six lignin samples, even higher than the tricin content in CEL (67.3 mg/g). The previous study also showed that the tricin level in the GVL lignin sample was higher than that in the CEL sample, according to the HSQC characterization. One of the possible reasons is that some of the tricin-(β–O–4)-lignin linkages were cleaved during GVL lignin extraction and the clipped-off tricin was isolated along with lignin, leading to a bit higher tricin level in the GVL lignin sample. The OL contained only 35.7 mg/g lignin, which might imply that tricin was degrade under such high concentration of acid solution at high temperature. The AL displayed the lowest tricin content with only 23.1 mg/g, although a mild condition was applied for the extraction. These results are generally in accordance with the semiquantitative result from HSQC. We further evaluated the stability of tricin under similar conditions using a pure tricin compound. Results showed that more than 98% of tricin remained after 9 h reaction at 30°C in 1.0 M NaOH solution, indicating that the tricin moiety was stable under this condition. The reason for the poor yield of tricin under alkaline conditions is still unclear. Further study using the dimeric tricin-monolignol model compound would be required to figure out the underlying mechanism.

Tricin is beneficial to human beings because of its various biological activities. The flavonoid backbone structure containing phenyl, carbonyl, and C=C double bonds endows strong UV absorption and radical scavenging ability to tricin. To explore the potential application of tricin-lignin, we further evaluated the SPF value of lignin-based sunscreens and radical scavenging ability of the extracted lignin samples, Table 4, to figure out how does the tricin level affect these properties.

The SPF of tricin-based sunscreen was 5.65, which was significantly higher than that of other dimeric model compounds (β–O–4, β–β, β–1, and β–5) and their derivatives formed by conventional monolignol (Lin M. et al., 2021). The SPF of AL was 4.30, similar to that of CEL, even though its tricin content was much lower than CEL. On the other hand, GVL lignin-based sunscreen displayed the highest SPF value, even slightly higher than that of pure tricin. The free radical scavenging activity showed a similar trend as the SPF value. The EC₅₀ of the free radical towards tricin was 0.26 mg/ml (0.00079 mmol/ml). The AL and CEL presented a similar EC₅₀ value (1.63 and 1.80 mg/ml), while the GVL lignin was the most active lignin towards radical with EC₅₀ of 1.02 mg/ml. In our previous study, we revealed that the β–O–4 and β–β interlinkages were not favorable to UV absorption and radical scavenging. Moreover, the CEL showed lower SPF and radical scavenging ability compared with OL and kraft extracted from Pinus kesiya and Eucalyptus. This might explain why even the CEL contains a much higher tricin level but showed similar SPF and EC₅₀ values with AL. The SPF value of tricin itself was remarkable, and the lignins containing tricin extracted from wheat straw displayed better SPF than those from softwood and hardwood without tricin. This suggests that retaining tricin is beneficial to the anti-UV property of the lignin samples. However, tricin does not impact the radical scavenging ability that much, as the kraft lignin of eucalyptus exhibited very low EC₅₀ as well (Lin M. et al., 2021).