Preparation and Anti-Lung Cancer Activity Analysis of Guaiacyl-Type Dehydrogenation Polymer

In this paper, guaiacyl dehydrogenated lignin polymer (G-DHP) was synthesized using coniferin as a substrate in the presence of β-glucosidase and laccase. Carbon-13 nuclear magnetic resonance (13C-NMR) determination revealed that the structure of G-DHP was relatively similar to that of ginkgo milled wood lignin (MWL), with both containing β-O-4, β-5, β-1, β-β, and 5-5 substructures. G-DHP fractions with different molecular weights were obtained by classification with different polar solvents. The bioactivity assay indicated that the ether-soluble fraction (DC2) showed the strongest inhibition of A549 lung cancer cells, with an IC50 of 181.46 ± 28.01 μg/mL. The DC2 fraction was further purified using medium-pressure liquid chromatography. Anti-cancer analysis revealed that the D4 and D5 compounds from DC2 had better anti-tumor activity, with IC50 values of 61.54 ± 17.10 μg/mL and 28.61 ± 8.52 μg/mL, respectively. Heating electrospray ionization tandem mass spectrometry (HESI-MS) results showed that both the D4 and D5 were β-5-linked dimers of coniferyl aldehyde, and the 13C-NMR and 1H-NMR analyses confirmed the structure of the D5. Together, these results indicate that the presence of an aldehyde group on the side chain of the phenylpropane unit of G-DHP enhances its anticancer activity.


Introduction
In 2020, there were approximately 19.3 million cancer patients worldwide, and 10 million people died of cancer. At present, lung cancer remains the most lethal cancer [1,2]. Chemotherapy is the most common therapeutic approach used to fight lung cancer. However, it has several limitations, including low bioavailability, poor water solubility, a low therapeutic index, high dose requirements, and poor targeting [3]. In addition, chemotherapy has severe adverse effects on the human body. Therefore, there is a critical need to identify natural drugs that can be applied in the treatment of lung cancer.
Lu et al. [4] found that alkali-treated lignin inhibited enzymatic and non-enzymatic lipid peroxidation, hindered the activity of glucose-6-phosphate dehydrogenase (G6PD), which is an enzyme implicated in the generation of superoxide anion radicals, and reduced the growth of HeLa S3 cervical cancer cells. Zhang et al. [5] showed that lignin macromolecules can promote cancer cell apoptosis and inhibit the expression of the transcription factor nuclear factor-k gene binding (NF-κB) in cancer cells. Lábaj et al. [6] separated lignin from the waste liquor of kraft pulping and determined the anti-tumor activity of lignin in mouse lymphocytes. As compared with the control group, the DNA strand damage induced by H 2 O 2 was significantly reduced, i.e., the resistance to H 2 O 2 -induced oxidative stress was enhanced. Geies et al. [7] used the in situ sodium hydroxide-sodium bisulphate method to separate lignin from three different agricultural and industrial wastes, i.e., sweet sorghum, rice straw, and bagasse. The results indicated that all lignin samples had high cytotoxicity potential on A549 lung cancer cells (IC 50 = 12~17 µg/mL).
Lignin also has good biocompatibility. Sakagami et al. [8,9] found that after oral administration of labelled natural lignin and synthetic lignin to mice, lignin was absorbed through the digestive system and excreted through the urine and feces, and did not remain in the body. Ugartondo et al. [10] found that bagasse lignin had a high antioxidant capacity within a certain concentration range and was harmless to normal human cells. Barapatre et al. [11] used pressurized solvent extraction (PSE) and continuous solvent extraction (SSE) to extract different lignin components from acacia wood. The authors found that these components had high cytotoxicity potential on MCF-7 human breast cancer cells (IC 50 : 2~15 µg/mL), but only had significant inhibitory effects on normal primary human hepatic stellate cells (HHSteCs) when the concentration was greater than 100 µg/mL. A low-molecular-weight lignin sample was found to have stronger anti-human immunodeficiency virus type 1 (HIV-1) performance than high-molecular-weight lignin [12]. Further, synthetic lignin dimer-like compounds with a β-5 structure displayed stronger inhibitory activities [12]. Together, findings in the literature demonstrate that lignin has anti-tumor, antibacterial, antiviral, antioxidant, and other physiological activities [8][9][10][11][12][13][14][15]. However, due to its complex structure, the source of lignin's anticancer activity has not yet been fully identified. Studies have examined the effects of lignin dehydrogenated polymer (DHP) on breast cancer cells (MCF7), normal fetal lung fibroblasts (MRC5) [16], and cervical cancer HeLa cells [17], and DHP has been found to contain β-5. Dimer and trimer DHP with a β-5 structure can inhibit the growth of cervical cancer cells; the β-O-4 ether bond does not participate in this anticancer activity, while the carboxyl group helps to increase the inhibition effect of DHP on cervical cancer cells [17]. Further, low-molecularweight DHP can inhibit the growth of MRC5 human embryonic lung cells [16]. Therefore, human malignant tumor cells are highly sensitive to DHP, which further demonstrates the potential value of DHP as an anticancer drug. However, there remains a paucity of studies on the anti-lung cancer activities of DHP, especially low-molecular-weight DHP with a guaiacyl substructure.
In this study, guaiacyl-type DHP was synthesized and extracted with different solvents. The phenolic compounds with good anti-lung cancer effects were separated by medium-pressure chromatography. Their anti-tumor activities against A549 lung cancer cells were analyzed. Furthermore, their chemical structures were analyzed by mass spectrometry (MS), carbon-13 nuclear magnetic resonance ( 13 C-NMR), and proton nuclear magnetic resonance ( 1 H-NMR), and the structure-activity relationship of lignin's biological activity of lignin was investigated. These findings offer novel ideas for exploring the commercial application of the DHP.

13 C-NMR Spectral Analysis of DHP
The 13 C-NMR spectrum of G-DHP is shown in Figure 1. A weak γ-CHO signal of cinnamaldehyde can be observed at 194.2 ppm (No. 1), indicating that a small amount of oxidation of G-DHP occurs during the polymerization process [18]. At 172.1 ppm (No. 2), the signal of ferulic acid at the γ-position of ferulic acid is observed, indicating that the hydroxymethyl group at the γ-position is oxidized to form cinnamic acid [19,20] 29) is of C-β in the β-β structure [22]. These results are in general agreement with those of previous studies [17,25]. Together, these findings suggest that the synthesized G-DHP contained mainly β-O-4 and β-5 structures, but also β-β, β-1 and 5-5 structures and a small amount of cinnamic aldehyde and ferulic acid.

Analysis of Molecular Weights of the DHP Fractions
The molecular weights of each classified fraction of G-DHP are shown in Table 1. From the Table. It can be found that the molecular weights of the classified fractions of G-DHP increased with the increasing polarity and solubility of the solvent. The molecular weights of G-DHP ranged from 250 Da to 3500 Da.

Analysis of the Total Phenolic Content (TPC) of DHP-Classified Fractions
The phenolic hydroxyl groups in phenolic compounds under alkaline conditions will change the Folin-Phenol reagent from yellow to blue. By measuring the absorbance of the standard sample at different concentrations, a standard curve was established, and the TPC of the experimental samples relative to the standard was calculated by substituting the absorbance of each sample into the standard curve equation.
The relationship between the concentration of gallic acid and absorbance at 760 nm is shown in Figure 2. The TPC is expressed as mg of gallic acid per g of sample. The concentration of gallic acid was linearly related to the absorbance, and the equation of the standard curve was as follows: y = 0.9581x + 0.07432, R 2 = 0.98106. The absorbance of each classified component of G-DHP was substituted into the equation to obtain the concentration. The TPC of each classified component relative to gallic acid is presented in Table 2. The TPC of each classified component decreased with increasing molecular weight.

Analysis of Molecular Weights of the DHP Fractions
The molecular weights of each classified fraction of G-DHP are shown in Table 1. It can be found that the molecular weights of the classified fractions of the G-DHP increased with the increasing polarity and solubility of the solvent. The molecular weights of the G-DHP ranged from 250 Da to 3500 Da.

Analysis of the Total Phenolic Content (TPC) of DHP-Classified Fractions
The phenolic hydroxyl groups in phenolic compounds under alkaline conditions will change the Folin-Phenol reagent from yellow to blue. By measuring the absorbance of the standard sample at different concentrations, a standard curve was established, and the TPC of the experimental samples relative to the standard was calculated by substituting the absorbance of each sample into the standard curve equation.
The relationship between the concentration of gallic acid and absorbance at 760 nm is shown in Figure 2. The TPC is expressed as mg of gallic acid per g of sample. The concentration of gallic acid was linearly related to the absorbance, and the equation of the standard curve was as follows: y = 0.9581x + 0.07432, R 2 = 0.98106. The absorbance of each classified component of the G-DHP was substituted into the equation to obtain the concentration. The TPC of each classified component relative to gallic acid is presented in Table 2. The TPC of each classified component decreased with increasing molecular weight.

Analysis of the Anti-tumor Activities of the G-DHP Classified Fractions
The effects of the G-DHP classified fractions and coniferin, at a concentration of 800 µg/mL, on the growth of A549 lung cancer cells are shown in Figure 3. It can be found that the lignin precursor coniferin did not have any obvious inhibitory effect on lung cancer cells. Meanwhile, the cell growth status of the control group, using the solvent only, was also normal, indicating that the solvent had no effect on the growth of A549 lung cancer cells. The inhibitory effects of the classified fractions of G-DHP on A549 lung cancer cells were significant, especially for the ether-soluble fraction DC2. Specifically, the cells became round and contracted and no longer adhered to the wall, and some even broke into small fragments. Further, many apoptotic cells died.

Analysis of the Anti-Tumor Activities of the G-DHP Classified Fractions
The effects of the G-DHP classified fractions and coniferin, at a concentration of 800 µg/mL, on the growth of A549 lung cancer cells are shown in Figure 3. It can be found that the lignin precursor coniferin did not have any obvious inhibitory effect on lung cancer cells. Meanwhile, the cell growth status of the control group, using the solvent only, was also normal, indicating that the solvent had no effect on the growth of A549 lung cancer cells. The inhibitory effects of the classified fractions of the G-DHP on A549 lung cancer cells were significant, especially for the ether-soluble fraction DC 2 . Specifically, the cells became round and contracted and no longer adhered to the wall, and some even broke into small fragments. Furthermore, many apoptotic cells died.

Analysis of the Anti-tumor Activities of the G-DHP Classified Fractions
The effects of the G-DHP classified fractions and coniferin, at a concentration of 800 µg/mL, on the growth of A549 lung cancer cells are shown in Figure 3. It can be found that the lignin precursor coniferin did not have any obvious inhibitory effect on lung cancer cells. Meanwhile, the cell growth status of the control group, using the solvent only, was also normal, indicating that the solvent had no effect on the growth of A549 lung cancer cells. The inhibitory effects of the classified fractions of G-DHP on A549 lung cancer cells were significant, especially for the ether-soluble fraction DC2. Specifically, the cells became round and contracted and no longer adhered to the wall, and some even broke into small fragments. Further, many apoptotic cells died.  The relationships between the concentration of the classified fractions and the inhibition rate on A549 lung cancer cells are shown in Figure 4. The IC 50 values are shown in Table 3. It can be found that all classified fractions inhibited A549 lung cancer cells. Furthermore, the relationships between the concentration and the inhibition rate were approximately logarithmic. The ether-soluble fraction (DC 2 ) showed the strongest inhibitory effect on A549 lung cancer cells, with an IC 50 of 181.46 ± 28.01 µg/mL. According to the standards of the National Cancer Institute. IC 50 ≤ 20 µg/mL is highly cytotoxic; ranging from 21 to 200 µg/mL is moderately cytotoxic; ranging from 201 to 500 µg/mL is weakly cytotoxic; and an IC 50 > 501 µg/mL is noncytotoxic [26][27][28].
Molecules 2023, 28, x FOR PEER REVIEW 5 of 17 The relationships between the concentration of the classified fractions and the inhibition rate on A549 lung cancer cells are shown in Figure 4. The IC50 values are shown in Table 3. It can be found that all classified fractions inhibited A549 lung cancer cells. Further, the relationships between the concentration and the inhibition rate were approximately logarithmic. The ether-soluble fraction (DC2) showed the strongest inhibitory effect on A549 lung cancer cells, with an IC50 of 181.46 ± 28.01 µg/mL. According to the standards of the National Cancer Institute. IC50 ≤ 20 µg/mL is highly cytotoxic, ranging from 21 to 200 µg/mL is moderately cytotoxic, ranging from 201 to 500 µg/mL is weakly cytotoxic, and an IC50 > 501 µg/mL is noncytotoxic [26][27][28].

Analysis of the Anti-Tumor Activity of the Purified Substances of G-DHP Ether-Soluble Fraction
The effects of the purified substances from the DC2 fraction, at a concentration of 800 µg/mL, on the growth state of A549 lung cancer cells are shown in Figure 5. It can be found that the A549 lung cancer cells were apoptotic in the experimental groups with the addition of each purified substance. The cells became smaller and rounder, the space between the cells became larger, and the cells no longer adhered to the wall; some of the apoptotic cells broke off into small fragments. The purified substances exhibited different degrees of inhibitory effects on A549 lung cancer cells, with compound D5 exhibiting the best inhibitory effect.

Analysis of the Anti-Tumor Activity of the Purified Substances of G-DHP Ether-Soluble Fraction
The effects of the purified substances from the DC 2 fraction, at a concentration of 800 µg/mL, the growth state of A549 lung cancer cells are shown in Figure 5. It can be found that the A549 lung cancer cells were apoptotic in the experimental groups with the addition of each purified substance. The cells became smaller and rounder, the space between the cells became larger, and the cells no longer adhered to the wall. Some of the apoptotic cells broke off into small fragments. The purified substances exhibited different degrees of inhibitory effects on A549 lung cancer cells, with compound D 5 exhibiting the best inhibitory effect. The relationships between the different concentrations of purified compounds from the DC2 fraction and the inhibition rate on A549 lung cancer cells are shown in Figure 6.
The IC50 values are shown in Table 4. The results revealed that all purified compounds had inhibitory effects on A549 lung cancer cells, while the concentrations and inhibition rates exhibited roughly logarithmic relationships. Obvious inhibitory effects of D4 and D5 on A549 lung cancer cells were observed, with IC50 values of 61.54 ± 17.10 µg/mL and 28.61 ± 8.52 µg/mL, respectively. As compared with the original DC2 fraction, the purified substances showed significantly enhanced inhibitory effects on A549 lung cancer cells, indicating that the purification by medium-pressure liquid preparative chromatography was successful for the isolation of the bioactive substances. In our previous research, some compounds isolated from DHP had a certain inhibitory effect on the HeLa cervical cancer cells. The inhibitory property of a compound, i.e., 4-[3-Hydroxymethyl-5-(3-hydroxy-propenyl)-7-methoxy-2,3-dihydro-benzofuran-2-yl]-2-methoxy-phenol from ether-soluble fractions, were relatively strong, with IC50 values of 15.1 ± 2.3 µg/mL. This compound was a typical (β-5) G-type dimer, and very similar to compounds D4 and D5 in structure in the present work. This also meant that the D4 and D5 compounds are relatively effective in anti-cancer treatments [17]. We have conducted a large number of experiments on the metabolic activity of hepatocytes cultured in vitro in the lignin and lignin-carbohydrate complexes. The growth of normal hepatocytes cells was enhanced by the addition of lignin and linin-carbohydrate complexes, and the inhibition effect could be ignored [29,30]. 28.61 ± 8.52 The relationships between the different concentrations of purified compounds from the DC 2 fraction and the inhibition rate on A549 lung cancer cells are shown in Figure 6. The IC 50 values are shown in Table 4. The results revealed that all purified compounds had inhibitory effects on A549 lung cancer cells, while the concentrations and inhibition rates exhibited roughly logarithmic relationships. Obvious inhibitory effects of D 4 and D 5 on A549 lung cancer cells were observed, with IC 50 values of 61.54 ± 17.10 µg/mL and 28.61 ± 8.52 µg/mL, respectively. As compared with the original DC 2 fraction, the purified substances showed significantly enhanced inhibitory effects on A549 lung cancer cells, indicating that the purification by medium-pressure liquid preparative chromatography was successful for the isolation of the bioactive substances. In our previous research, some compounds isolated from DHP had a certain inhibitory effect on the HeLa cervical cancer cells. The inhibitory property of a compound, i.e., 4-[3-Hydroxymethyl-5-(3-hydroxypropenyl)-7-methoxy-2,3-dihydro-benzofuran-2-yl]-2-methoxy-phenol from ether-soluble fractions, were relatively strong, with IC 50 values of 15.1 ± 2.3 µg/mL. This compound was a typical (β-5) G-type dimer, and very similar to compounds D4 and D5 in structure in the present work. This also meant that the D4 and D5 compounds are relatively effective in anti-cancer treatments [17]. We have conducted a large number of experiments on the metabolic activity of hepatocytes cultured in vitro in the lignin and lignin-carbohydrate complexes. The growth of normal hepatocytes cells was enhanced by the addition of lignin and linin-carbohydrate complexes, and the inhibition effect could be ignored [29,30].

HESI-MS Mass Spectrometry Analysis of the Purified Substances
Electrospray ionization mass spectrometry (ESI-MS) is appropriate for investigating the primary and fragment structure of biopolymers. Several previous studies on lignin that used ESI-MS have shown the utility of this analytical method for estimating the average molecular mass of lignin macromolecules, distinguishing their biological origins, and exploring the reactions occurring in solutions [31,32]. Given the high contents of hydroxyl and carboxyl groups in lignin, mass spectrometric information in the negative ion mode (M − H + ) will produce better signals than in the positive ion mode (M + H + ). Therefore, ESI under negative ionization conditions is the most widely used method for determination of lignin structure [33]. Coniferin was enzymatically hydrolyzed to coniferyl alcohol before polymerization to DHP. The molecular weight of the monomer coniferyl alcohol was about 180 g/mol. In addition, functional groups such as carboxyl and aldehyde groups may be generated under the catalytic action of laccase, thus, the molecular weight distributions of DHP with different degrees of polymerization were as follows: monomer (130-190 g/mol), dimer (190-380 g/mol), trimer (380-570 g/mol) [34][35][36].

HESI-MS Mass Spectrometry Analysis of the Purified Substances
Electrospray ionization mass spectrometry (ESI-MS) is appropriate for investigating the primary and fragment structure of biopolymers. Several previous studies of lignin that have used ESI-MS have shown the utility of this analytical method for estimating the average molecular mass of lignin macromolecules, distinguishing their biological origins, and exploring the reactions occurring in solutions [31,32]. Given the high contents of hydroxyl and carboxyl groups in lignin, mass spectrometric information in the negative ion mode (M − H + ) will produce better signals than in the positive ion mode (M + H + ). Therefore, ESI under negative ionization conditions is the most widely used method for determination of lignin structure [33]. Coniferin was enzymatically hydrolyzed to coniferyl alcohol before polymerization to DHP. The molecular weight of the monomer coniferyl alcohol was about 180 g/mol. In addition, functional groups such as carboxyl and aldehyde groups may be generated under the catalytic action of laccase; thus, the molecular weight distributions of DHP with different degrees of polymerization were as follows: monomer (130-190 g/mol), dimer (190-380 g/mol), trimer (380-570 g/mol) [34][35][36].
The mass spectral information of the purified substance D5 from the DC2 fraction of G-DHP is shown in Figure 7. The signal at m/z 353.103 is of the molecule D5; the molecular structure formula of D5 was calculated to be C20H18O6 based on the other fragments The most abundant ion fragment is at m/z 149.060, which is the ion peak of 2-methoxy-4-vinyl coniferyl alcohol. This ionic peak originates from the cleavage of the coumarin structure, indicating that the Cγ on the side chain of the phenyl propane monomer in guaiacyl lignin is unstable as compared to other positions. The signal at m/z 137.0 is of the fragment peak of 2-methoxy-4-methylphenol formed by the elimination of Cβ on the side chain of the fragment at m/z 149.060. This is consistent with previous findings, where m/z 177.0 was found to be the fragment peak of 2-methoxy-4-methylphenol [37]. The ionic peak of the coniferyl alcohol monomer at m/z 177.0 is also consistent with previous findings [38]. The peak at m/z 353.1 is of the β-5-linked dimer of coniferyl aldehyde (β-5, γ-CHO, γ'-CHO),  61.54 ± 17.10 D 5 28.61 ± 8.52 The mass spectral information of the purified substance D 5 from the DC 2 fraction of G-DHP is shown in Figure 7. The signal at m/z 353.103 is of the molecule D 5 . The molecular structure formula of D 5 was calculated to be C 20 H 18 O 6 based on the other fragments The most abundant ion fragment is at m/z 149.060, which is the ion peak of 2-methoxy-4-vinyl coniferyl alcohol. This ionic peak originates from the cleavage of the coumarin structure, indicating that the Cγ on the side chain of the phenyl propane monomer in guaiacyl lignin is unstable as compared to other positions. The signal at m/z 137.0 is of the fragment peak of 2-methoxy-4-methylphenol formed by the elimination of Cβ on the side chain of the fragment at m/z 149.060. This is consistent with previous findings, where m/z 177.0 was found to be the fragment peak of 2-methoxy-4-methylphenol [37]. The ionic peak of the coniferyl alcohol monomer at m/z 177.0 is also consistent with previous findings [38]. The peak at m/z 353.1 is of the β-5-linked dimer of coniferyl aldehyde (β-5, γ-CHO, γ'-CHO), while the peak at m/z 325.1 differs from m/z 353.1 by 28, indicating that m/z 325.1 is a fragment peak formed by the elimination of the aldehyde group on Cγ of the molecular ion at m/z 353.1. These results indicate that the oxidation of coniferin catalyzed by laccase and β-glucosidase led to the formation of the β-5 structure of the coniferyl aldehyde dimer. Similar findings have been reported in previous studies that have synthesized DHP [39].
while the peak at m/z 325.1 differs from m/z 353.1 by 28, indicating that m/z 325. fragment peak formed by the elimination of the aldehyde group on Cγ of the molecul at m/z 353.1. These results indicate that the oxidation of coniferin catalyzed by laccas β-glucosidase led to the formation of the β-5 structure of the coniferyl aldehyde dimer ilar findings have been reported in previous studies that have synthesized DHP [39]. The mass spectrum of the purified substance D4 from the DC2 fraction of G-D shown in Figure 8. Similarly to D5, the molecular ion of D4 is also found at m/z 35 Considering the other fragments, the molecular structure formula of D4 should al C20H18O6. All of the fragment ion peaks in D5 can be found in the spectrum of comp D4, while D4 has additional peaks at m/z 129.018, m/z 167.034, m/z 187.061, m/z 203.056 m/z 301.072, indicating that D4 contains some impurities. These results indicate th and D5 are generally the same substance, but D4 contains more impurities. This asse is also confirmed by the slightly lower inhibitory effect of the compound D4 on A549 as compared to the compound D5. The mass spectrum of the purified substance D 4 from the DC 2 fraction of G-DHP is shown in Figure 8. Similarly to D 5 , the molecular ion of D 4 is also found at m/z 353.103. Considering the other fragments, the molecular structure formula of D 4 should also be C 20 H 18 O 6 . All of the fragment ion peaks in D 5 can be found in the spectrum of compound D 4 , while D 4 has additional peaks at m/z 129.018, m/z 167.034, m/z 187.061, m/z 203.056, and m/z 301.072, indicating that D 4 contains some impurities. These results indicate that D 4 and D 5 are generally the same substance, but D 4 contains more impurities. This analysis is also confirmed by the slightly lower inhibitory effect of the compound D 4 on A549 cells, as compared to the compound D 5 . The mass spectrum of the purified substance D4 from the DC2 fraction of G-DHP is shown in Figure 8. Similar to that of D5, the molecular ion of D4 is also found at m/z 353.103. Considering the other fragments, the molecular structure formula of D4 should also be C20H18O6, and all of the fragment ion peaks in D5 can be found in the spectrum of compound D4, while D4 has additional peaks at m/z 129.018, m/z 167.034, m/z 187.061, m/z The mass spectrum of the purified substance D 4 from the DC 2 fraction of G-DHP is shown in Figure 8. Similar to that of D 5 , the molecular ion of D 4 is also found at m/z 353.103. Considering the other fragments, the molecular structure formula of D 4 should also be C 20 H 18 O 6 , and all of the fragment ion peaks in D 5 can be found in the spectrum of compound D 4 , while D 4 has additional peaks at m/z 129.018, m/z 167.034, m/z 187.061, m/z 203.056, and m/z 301.072, indicating that D4 contains some impurities.
The results indicate that D 4 and D 5 are mainly the same substance, but D 4 contains more impurities. This is also confirmed by the slightly less inhibitory effect of D 4 on A549 cells than D 5 .

Discussion of the Structure-Effect Relationships of the Purified Substances
The HESI-MS, 13 C-NMR, and 1 H-NMR results indicated that compound D5 is a coniferyl aldehyde dimer with a β-5 linkage. The compounds D4 and D5 are the same substance, although the compound D4 contains more impurities, leading to a reduction in its anti-tumor activity. Zemek et al. [40] studied the biological activity of lignin model substances and found that those containing Cα=Cβ on the side chain of the benzene ring and

Discussion of the Structure-Effect Relationships of the Purified Substances
The HESI-MS, 13 C-NMR, and 1 H-NMR results indicated that compound D 5 is a coniferyl aldehyde dimer with a β-5 linkage. The compounds D 4 and D 5 are the same substance, although the compound D 4 contains more impurities, leading to a reduction in its anti-tumor activity. Zemek et al. [40] studied the biological activity of lignin model substances and found that those containing Cα=Cβ on the side chain of the benzene ring and with methyl on Cγ were the most effective. In addition, increases in hydroxyl, carbonyl, and carboxyl groups on the side chain reduced the biological activity of the compound. Xie et al. [17,41] synthesized DHP from isoeugenol and found that the presence of an aldehyde group on the side chain of the phenylpropane structure weakened the antioxidant and antibacterial properties of DHP. However, the results of the present study indicated that compound D 5 contains aldehyde groups on the side chain, yet it had strong anti-tumor activity. Gładkowski et al. [42] used aromatic aldehydes as raw materials to synthesize 20 aromatic aldehydes. The authors found that some of the obtained compounds had significant cytotoxic effects on two cancer cells: human leukemia cells (Jurka cells) and canine osteosarcoma cells (D 17 cells). These results suggest that aldehydes have potential anti-tumor applications.
Previous studies have shown that β-5-linked oligomeric lignins have good inhibitory effects on cervical cancer HeLa cells, with IC 50 values as low as 15.1 µg/mL [17], while the present study found that β-5-linked oligomeric lignin had an inhibitory effect on A549 lung cancer cells, indicating that β-5 structured oligomeric lignins have broad anticancer activity.

Synthesis of DHP
The DHP synthesis mechanism is shown in Figure 12. In 150 mL of 0.2 mol/L sterile acetic acid/sodium acetate buffer solution with pH 4.83, 5 g of coniferin, 50 mg of β-glucosidase (6.4 U/mL), and 3 mL of laccase (648 U/mL) were added with the continuous blowing of sterile air filtered by activated carbon with an air pump. Then, the mixture was allowed to continue reacting in a water bath at 30 °C for 25 min. After the reaction was completed, 150 mL of distilled water was added and then heated to 70 °C to stop the re-

Synthesis of DHP
The DHP synthesis mechanism is shown in Figure 12. In 150 mL of 0.2 mol/L sterile acetic acid/sodium acetate buffer solution with pH 4.83, 5 g of coniferin, 50 mg of β-glucosidase (6.4 U/mL), and 3 mL of laccase (648 U/mL) were added with the continuous blowing of sterile air filtered by activated carbon with an air pump. Then, the mixture was allowed to continue reacting in a water bath at 30 • C for 25 min. After the reaction was completed, 150 mL of distilled water was added and then heated to 70 • C to stop the reaction. The mixture was then centrifuged, the precipitate collected and washed with distilled water more than 5 times to remove the unreacted coniferin, and some enzymes freeze-dried to obtain crude DHP. The mixture was dissolved in dichloroethane/ethanol (v/v = 2:1), and the precipitate of the residual enzyme was removed by centrifugation. The solvent was then removed in vacuo. The DHP was obtained by vacuum drying with a yield of 38.36%.
Molecules 2023, 28, x FOR PEER REVIEW 12 of 17 action. The mixture was then centrifuged, the precipitate collected and washed with distilled water more than 5 times to remove the unreacted coniferin, and some enzymes freeze-dried to obtain crude DHP. The mixture was dissolved in dichloroethane/ethanol (v/v = 2:1), and the precipitate of the residual enzyme was removed by centrifugation. The solvent was then removed in vacuo. The DHP was obtained by vacuum drying with a yield of 38.36%.

13 C-NMR Measurement of G-DHP
An 80 mg DHP sample was placed into φ5 mm NMR tubes and dissolved with 0.6 mL of DMSO-d6. A 600-dd2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to scan the solution at 150.90 MHz to obtain the corresponding 13 C-NMR spectrum. The parameters of the instrument were as follows: test temperature: 25 °C; pulse delay: 2.0000 s; acquisition time: 0.6900 s; and number of scans: 3000.

Classification of G-DHP
G-DHP was classified according to the polarity and solubility of different organic solvents, with reference to the method of Li et al. [43]. G-DHP extraction was graded using n-hexane, ether, ethyl acetate, and dioxane. The classification process is shown in Figure  13. G-DHP was placed into a Soxhlet extractor together with n-hexane and heated at boiling point to reflux for 6 h. The extracted solution was distilled in vacuo to remove the

13 C-NMR Measurement of G-DHP
An 80 mg DHP sample was placed into ϕ 5 mm NMR tubes and dissolved with 0.6 mL of DMSO-d 6 . A 600-dd2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to scan the solution at 150.90 MHz to obtain the corresponding 13 C-NMR spectrum. The parameters of the instrument were as follows: test temperature: 25 • C; pulse delay: 2.0000 s; acquisition time: 0.6900 s; and number of scans: 3000.

Classification of G-DHP
G-DHP was classified according to the polarity and solubility of different organic solvents, with reference to the method of Li et al. [43]. G-DHP was graded using n-hexane, ether, ethyl acetate, and dioxane. The classification process is shown in Figure 13. G-DHP was placed into a Soxhlet extractor together with n-hexane and heated at boiling point to reflux for 6 h. The extracted solution was distilled in vacuo to remove the solvent and then dried under a vacuum to obtain the n-hexane-soluble component (DC 1

Determination of Molecular Weight of the G-DHP Fractions
The relative molecular weights of individual fractions were determined by gel permeation chromatography (GPC). The molecular weight standard curves were prepared with EasiVial Polystyrene PS-M as the standard. Each DHP fraction (2 mg) was dissolved in 2 mL N,N-dimethylformamide (DMF) filtered through a 0.22 µm microporous membrane, and then injected into the GPC system (20AT, Shimadzu, Osaka, Japan) for molecular weight measurement. The instrument parameters were as follows: column: Stirrage HR 4E DMF (7.8 × 300 mm) with a guide column of Styrage DMF (4.6 × 30 mm); detector: RID-10A differential refraction detector; mobile phase: DMF (containing 0.1 mol/L LiCl); flow rate: 1.0 mL/min; column temperature: 50 °C; injection volume: 20 µL.

Determination of Total Phenol Content (TPC)
The Folin-Ciocalteu colorimetric method [44] was used to determine the TPC of the various fractions. For the quantification of these extracts, a standard curve was constructed from the analysis of different concentrations of gallic acid varying from 125-1000 µg/mL. Then, 100 µL methanol extract was added to test tubes with 500 µL Folin-Ciocalteu reagent. Six milliliters of distilled water was then added. After shaking for 1 min, 2 mL of 15% Na2CO3 solution was transferred to the test tubes within 0.5~8 min. Finally, distilled water was added to the tubes to make a total volume of 10 mL. After being kept in the dark at room temperature for 2 h, the test tubes were homogenized with ultrasonic agitation. The spectrophotometer (Nakagyo-ku, Kyoto, Japan) was set at a wavelength of

Determination of Molecular Weight of the G-DHP Fractions
The relative molecular weights of individual fractions were determined by gel permeation chromatography (GPC). The molecular weight standard curves were prepared with EasiVial Polystyrene PS-M. Each DHP fraction (2 mg) was dissolved in 2 mL N,Ndimethylformamide (DMF) filtered through a 0.22 µm microporous membrane, and then injected into the GPC system (20AT, Shimadzu, Osaka, Japan) for molecular weight measurement. The instrument parameters were as follows: column: Stirrage HR 4E DMF (7.8 × 300 mm) with a guide column of Styrage DMF (4.6 × 30 mm); detector: RID-10A differential refraction detector; mobile phase: DMF (containing 0.1 mol/L LiCl); flow rate: 1.0 mL/min; column temperature: 50 • C; injection volume: 20 µL.

Determination of Total Phenol Content (TPC)
The Folin-Ciocalteu colorimetric method [44] was used to determine the TPC of the various fractions. For the quantification of these extractives, a standard curve was established from the analysis of different concentrations of gallic acid varying from 125-1000 µg/mL. Then, 100 µL methanol extractive was added to test tubes with 500 µL Folin-Ciocalteu reagent. Six milliliters of distilled water was then added. After shaking for 1 min, 2 mL of 15% Na 2 CO 3 solution was transferred to the test tubes within 0.5~8 min. Finally, distilled water was added to the tubes to make a total volume of 10 mL. After being kept in the dark at room temperature for 2 h, the test tubes were homogenized with ultrasonic agitation. The spectrophotometer (Shimadzu 2550, Nakagyo-ku, Kyoto, Japan) was set at a wavelength of 760 nm, and the absorbance reading was measured. The TPC of each sample was calculated according to the following formula: TPC [mg (gallic acid)/g(sample)] = GAE × FV × DF/SW Legend: GAE is the concentration of gallic acid calculated from the standard curve (mg/mL); FV is the final volume of the sample extract (mL); DF is the dilution multiple; SW is the sample mass (g).

Determination of the Anti-Tumor Activities of DHP Fractions and Purified Compounds
According to the method of Ahamed, Le et al. [45,46], the antitumor activities of the G-DHP fractions and purified compounds were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. First, samples were dissolved in acetone: CM2-1 medium (0.99:99.1 v/v, 0.1% polysorbate 80 as dispersant) to obtain a series of solutions with concentration in the range from 12.5 µg/mL to 800 µg/mL. Second, 5 × 10 3 exponentially growing tumor cells were suspended in 100 µL of the CM2-1 medium and plated per experimental well on 96-well plates. Blank wells were also filled with 200 µL of complete medium. Third, the cells were cultured in a carbon dioxide cell incubator (37 • C, 5% CO 2 ). After 16-48 h, 100-µL sample solutions with different concentrations (12.5~800 µg/mL) were transferred to every experimental well, which was different from the sample solvent added to the control wells. After incubation for 48 h, 20 µL MTT solution (5 mg/mL) was added to each well. Four hours later, the liquid in each well was discharged with a sterile syringe, and then 150 µL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the plate was placed into a microplate reader after shaking at a low speed for 10 min. The absorbance of each well was measured at 490 nm. The inhibition rate of each sample was calculated using the following equation: Then, SPSS 26 software was used to calculate the semi-inhibition concentration (IC 50 ) of each sample [47].

Purification of the Ether-Soluble Fraction of DHP with Preparative Column Chromatography
The ether-soluble component DC 2 of DHP was gradient eluted and repeatedly purified using a medium-pressure liquid preparation chromatograph (Buchi C-615, Zurich, Switzerland) equipped with an ultraviolet (UV) detector at a wavelength of 280 nm. The stationary phase was Sephadex LH-20 gel (100~200 mesh), and the mobile phase was a chloroform-methanol mixed eluent (the concentration gradient of methanol was in the range of 0% to 50%), with a flow rate of 2.5 mL/min.
First, the samples were dissolved in a 50% chloroform-methanol solution, and then the sample solution was injected into the dosing ring using a glass syringe. After eluting the separation column with 50% chloroform-methanol, the concentration of methanol was gradually increased, and finally, the column was completely washed with methanol. During the separation process, a UV 230 II ultraviolet-visible detector was applied. The ether-soluble component DC 2 of DHP was purified to obtain compounds D 1 , D 2 , D 3 , D 4, and D 5 , with yields of 7.47%, 41.67%, 29.91%, 10.83%, and 10.12%, respectively.

Mass Spectrometry Analysis of the Structure of the DHP Purified Compound
The mass spectrum information of D 5 was obtained using electrostatic field orbital hydrazine mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). The ion source was HESI. The determination conditions were as follows: dry gas: N 2 ; spray voltage: 3200 V; temperature of the ion transmission capillary: 300 • C; auxiliary gas temperature: 280 • C; scanning mode: full MS/dd-MS2; scanning range m/z: 50-750.
3.2.9. 13 C-NMR and 1 H-NMR Measurement of the Purified Substance D 5 13 C-NMR Measurement of the Purified Substance D 5 An 80 mg purified substance D 5 sample was placed into ϕ 5 mm NMR tubes and dissolved with 0.6 mL of DMSO-d 6 . A 600-dd2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to scan the solution at 150.90 MHz to obtain the corresponding 13 C-NMR spectrum. The parameters of the instrument were as follows: test temperature: 25 • C; pulse delay: 2.0000 s; acquisition time: 0.6900 s; and number of scans: 3000. 1 H-NMR Measurement of the Purified Substance D 5 A 15 mg volume of the purified substance D 5 sample was placed into ϕ 5 mm NMR tubes and dissolved with 0.6 mL of DMSO-d 6 . A 600-dd2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to scan the above sample at 600 MHz to obtain the corresponding 1 H-NMR spectrum. The parameters of the instrument were as follows: test temperature: 25 • C; pulse delay: 5.0000 s; acquisition time: 1.4500 s; and number of scans: 256.

Conclusions
(1) 13 C-NMR determination revealed that the structure of G-DHP was relatively similar to that of the gymnosperm ginkgo MWL, which was dominated by β-O-4, β-5, β-1, β-β, and 5-5 substructures. (2) G-DHP was classified with different polar solvents to obtain a hexane-soluble fraction (DC 1 ), ether-soluble fraction (DC 2 ), ethyl acetate-soluble fraction (DC 3 ), and dioxane-soluble fraction (DC 4 ). The GPC results and total phenolic content measurements showed that the total phenolic content of fractions decreased with an increase in molecular weight. The bioactivity assay showed that the ether-soluble fraction (DC 2 ) had the strongest inhibitory effect on lung cancer A549 with an IC 50 of 181.46 ± 28.01 µg/mL. (3) Further purification of the DC 2 fraction revealed that the obtained compounds D 4 and D 5 had significant anti-tumor activities, with IC 50 values of 61.54 ± 17.10 µg/mL and 28.61 ± 8.52 µg/mL against lung cancer A549, respectively. HESI-MS measurements indicated that D 5 was a coniferyl aldehyde dimer linked by β-5. The compounds D 4 and D 5 were similar substance, but D 4 contained more impurities. 13 C-NMR and 1 H-NMR results also confirmed the chemical structure of D 5 .
In combination with the results of the anti-tumor assay, for the small molecule compound of guaiacyl type DHP, it was found that the presence of an aldehyde group on the side chain of the phenylpropane subunit can enhance the anti-cancer activity.