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Xie, Y., Jiang, C., Chen, X., Wu, H., and Bi, S. (2020). "Preparation of oligomeric phenolic compounds (DHPs) from coniferin and syringin and characterization of their anticancer properties," BioRes. 15(1), 1791-1809.

Abstract

Lignin precursors of coniferin and syringin were synthesized and used to prepare guaiacyl-type and guaiacyl-syringyl-type oligomeric compounds (designated here as dehydrogenation polymers DHP-G, DHP-GS) via bulk method. The carbon 13 nuclear magnetic resonance spectroscopy (13C-NMR) spectra indicated that both DHPs contained typical lignin substructures. The DHPs were extracted sequentially with petroleum ether, ether, ethanol, and acetone to obtain eight fractions (F11 to F14 and F21 to F24). The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) experimental results showed that the growth of cervical cancer cells was inhibited by the two ether-soluble fractions F12 and F22, with semi-inhibitory concentration (IC50) values of 81.60 ± 9.30 and 103.24 ± 14.09 μg/mL, respectively. The bioactive compounds in F12 and F22 were separated by a preparative chromatography method. Ten bioactive compounds (G1 to G5 and GS1 to GS5) were obtained. Mass spectroscopy analysis revealed the following chemical structures: G1, β-5 G-type dimer; G2, (β-5)(β-5) G-type trimer; GS1, β-5 GS-type dimer; and GS2, (β-O-4)(β-5) GS-type trimer. The compounds had inhibitory effects on cervical cancer cells. The syringyl aromatic ring decreased the anticancer activity of DHP, and the β-O-4 linkages did not contribute to the anticancer activity. It was also found that the carboxyl groups contributed to the anticancer activity of DHP.


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Preparation of Oligomeric Phenolic Compounds (DHPs) from Coniferin and Syringin and Characterization of their Anticancer Properties

Yimin Xie,a,b,* Chen Jiang,a Xuekuan Chen,a Hongfei Wu,a and Shuying Bi a

Lignin precursors of coniferin and syringin were synthesized and used to prepare guaiacyl-type and guaiacyl-syringyl-type oligomeric compounds (designated here as dehydrogenation polymers DHP-G, DHP-GS) via bulk method. The carbon 13 nuclear magnetic resonance spectroscopy (13C-NMR) spectra indicated that both DHPs contained typical lignin substructures. The DHPs were extracted sequentially with petroleum ether, ether, ethanol, and acetone to obtain eight fractions (F11 to F14 and F21 to F24). The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) experimental results showed that the growth of cervical cancer cells was inhibited by the two ether-soluble fractions F12 and F22, with semi-inhibitory concentration (IC50) values of 81.60 ± 9.30 and 103.24 ± 14.09 μg/mL, respectively. The bioactive compounds in F12 and F22 were separated by a preparative chromatography method. Ten bioactive compounds (G1 to G5 and GS1 to GS5) were obtained. Mass spectroscopy analysis revealed the following chemical structures: G1, β-5 G-type dimer; G2, (β-5)(β-5) G-type trimer; GS1, β-5 GS-type dimer; and GS2, (β-O-4)(β-5) GS-type trimer. The compounds had inhibitory effects on cervical cancer cells. The syringyl aromatic ring decreased the anticancer activity of DHP, and the β-O-4 linkages did not contribute to the anticancer activity. It was also found that the carboxyl groups contributed to the anticancer activity of DHP.

Keywords: Lignin; Oligomer; DHP; Anticancer activity; Structure

Contact information: a: Research Institute of Pulp and Paper Engineering, Hubei University of Technology, 430068, Wuhan, China; b: Hubei Provincial Key Laboratory of Green Materials for Light Industry, Hubei University of Technology, 430068, Wuhan, China;

* Corresponding author: ppymxie@163.com

INTRODUCTION

Currently, considerable effort is being made to find new applications of lignins in order to add extra value (Vinardell and Mitjans 2017). Cancer incidence is increasing every year. Current treatments include surgery, chemotherapy, radiation therapy, and immunotherapy. Many natural plant extracts can be used as effective anticancer agents, such as vinblastine, etoposide, paclitaxel, bleomycin, and taxanes (Mathi et al. 2014). However, the amount of commercial anticancer components in plant extracts, such as paclitaxel and taxanes, is minimal, less than 0.1%. Therefore, the focus of recent research has shifted to the development of new natural anticancer drugs from rich lignin resources (Gomes et al. 2003). Plant polyphenols have drawn increasing attention due to their potent antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer. For instance, lignin, flavonoids, phenolic acids, and tannins are compounds possessing one or more aromatic rings with one or more hydroxyl groups. They are broadly distributed in the plant kingdom and are the most abundant aromatic secondary metabolites of plants (Dai and Mumper 2010).

Over the years, botanists, plant physiologists, phytochemists, and biochemists, as well as organic chemists have studied polyphenols in more detail. Results of such studies have shown the significance of polyphenols not only as major and ubiquitous plant secondary metabolites, but also as compounds that express properties with numerous implications and potential exploitations in various domains of general public and commercial interests (Quideau et al. 2011). Many studies have demonstrated plant polyphenols to be a major source of anticancer drugs. This is a large class of compounds with high biological activity. For example, the anticancer activity of lignin, as well as its derivatives, has been verified. Barapatre et al. (2016) extracted four physiologically active lignin fractions from acacia wood by pressurized solvent extraction (PSE) and successive solvent extraction (SSE) and found that these extracts had inhibitory effects on breast cancer cells (MCF-7). Its semi-inhibitory concentration (IC50) was less than 15 μg/mL, but there was no obvious inhibitory effect on the growth of normal primary human hepatic stellate cells (HHSteCs). In another example, after ethanol defatting of pine fruit by Sakagami et al. (2005), the lignin-carbohydrate complex was collected through hot water and alkaline extraction. This complex had an obvious inhibitory effect on mouse ascitic cancer cells and also enhanced the immune response of mice. According to work of Niedzwiecki et al. (2016), polyphenols were able to penetrate human tissues, in particular the intestine and liver, where they were metabolized. Similar to the structure and physical properties of natural polyphenols, the oligomeric DHP may also penetrate the tissues of the human organ.

There are many lignin extraction methods (Obst and Kirk 1988; Jia et al. 2013). However, the extracted lignin typically has a large scale of molecular weight and contains a small amount of carbohydrate impurities. An artificial lignin with structure most similar to protolignin in plant can be synthesized by dehydrogenation reaction combined with free radical polymerization catalyzed by laccase or lignin peroxidase. Freudenberg (1952) found that coniferyl alcohol could be polymerized to form a dehydrogenation polymer (DHP) under enzymatic catalysis, with a structure that was similar to natural lignin (Guan et al. 1997; McCarthy and Islam 1999). To maintain consistent terminology with other publications, the letters DHP will be used in this paper to mean products of such reactions; however, readers are urged to keep in mind that the reaction products may include dimers and trimers rather than polymers. The degree of polymerization of DHP can be regulated by controlling the concentration of the substrate, the ratio of enzyme, temperature, pH, and reaction time. Therefore, DHP with a low degree of polymerization, simple structure and connection mode, and more functional groups can be obtained, resulting in higher biological activity than that of naturally extracted lignin.

The synthesis of oligolignin has been reported (Ye et al. 2016; Chen et al. 2018; Xie et al. 2019). Chen et al. (2018) synthesized DHP with isoeugenol as the precursor, and found that the ether-soluble component had strong antioxidant activity with an IC50 of 0.12 g/L. However, most of this work focused on the chemical structure of oligolignin rather than its biological activity and anticancer properties. Therefore, research into the relationship between the oligolignin structure and biological activity is still required.

In this study, in order to synthesize the oligomeric artificial lignin, G-type lignin dehydrogenation polymer (DHP-G) and GS-type lignin dehydrogenation polymer (DHP-GS) were synthesized via a bulk method starting with coniferin, or a mixture of coniferin and syringin (1:1, molar ratio) under laccase and β-glucosidase catalysis. The two types of DHP were extracted by solvents. The inhibitory effect of DHP fractions on the growth of human cervical cancer Hela cells was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The fractions with higher biological activity were further purified and screened to obtain bioactive compounds, which were structurally identified by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The anticancer activity of these compounds was determined and the source of biological activity discussed from the analysis of the structure-effect relationship.

EXPERIMENTAL

Materials

Coniferin and syringin (Fig. 1) were synthesized in the laboratory from starting materials vanillin and syringaldehyde (Aladdin, Shanghai, China), respectively, according to the previous methods of Xie Yimin(1991) and Xie et al. (1994a). β-Glucosidase was purchased from Sigma Co., Ltd. (Shanghai, China) and laccase (No. 51003) from Novazyme Co., Ltd. (Tianjing, China). All other chemicals were of an analytical grade.

Fig. 1. Chemical structure of the coniferin (I) and syringin (II)

Fig. 2. Fractionation of the DHP-G and DHP-GS by solvents

Synthesis of the DHP-G and DHP-GS

Coniferin (Xie et al. 1994b) (9.62 mmol) was dissolved in an acetic acid/sodium acetate buffer solution (pH 4.6, 100 mL). β-Glucosidase (30 mg, 6.4 U/mg) and laccase (2 mL, 1093 IU/mL) were added and the solution mixed with bubbling of sterilized air at 30 °C. The reaction was stopped by addition of hot distilled water (80 °C, 100 mL) after 30 min reaction. After centrifugation (8000 r/min, 8 min), the supernatant was removed and the residue washed thoroughly with water to remove the enzyme and residual lignin precursor, then freeze-dried. The precipitation was extracted with dichloroethane/ethanol (2:1 v/v) mixture, centrifuged at 8000 r/min for 8 min, and the supernatant collected. The solvent was removed in vacuo to give the purified DHP-G with 88.3% yield.

The above procedure was also conducted for a mixture of syringin (4.0 mmol) and coniferin (4.0 mmol) as starting material of polymerization, resulting in DHP-GS in 80.9% yield.

Classification of the DHP-G and DHP-GS

As shown in Fig. 2, the DHP-G and DHP-GS were fractionated by the solvent method (Wang et al. 2010). The DHPs were extracted with petroleum ether (boiling point 30 to 60 °C), diethyl ether, absolute ethanol, and acetone, sequentially, according to their solubility in the organic solvent. From DHP-G, a petroleum ethersoluble fraction (F11), ether soluble fraction (F12), ethanol soluble fraction (F13), and acetone soluble fraction (F14) were obtained with yields of 2.1%, 26.2%, 31.3%, and 7.8%, respectively. From the DHP-GS, a petroleum ether soluble fraction (F21), ether soluble fraction (F22), ethanol soluble fraction (F23), and acetone soluble fraction (F24) were obtained with yields of 2.5%, 24.3%, 40.8% and 9.7%, respectively.

Methods

13C NMR spectroscopy of DHPs

The DHP-G or DHP-GS (80 mg) was dissolved in dimethyl sulfoxide (DMSO)-d6 (0.6 mL) and put into a Φ 5 mm NMR tube. The solutions were scanned with a 600-DD2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) at 150.83 MHz to obtain the corresponding 13C-NMR spectrum. The parameters of the instrument were: pulse delay: 2.5000 s, acquisition time: 0.9437 s, and number of scanning time: 6,000.

Molecular weight determination of the DHP fractions

The relative molecular mass was determined by size exclusion chromatography. Each DHP fraction (2 mg) was dissolved in N,N-dimethylformamide (DMF) (2 mL), filtered through a 0.22-μm membrane and injected into a Shimadzu LC 20A gel permeation chromatograph (GPC) (Shimadzu, Kyoto, Japan). The separation column was Shim-pack GPC-803D (300 mm × 8 mm) (Shimadzu, Kyoto, Japan), the mobile phase was DMF, and the flow rate was 0.6 mL/min with a column temperature of 35 °C. The injection volume was 25 μL. Polystyrene was used as standard.

Preparative column chromatography purification of fractions

The fractions with higher anticancer activity were further purified by preparative column chromatography (Büchi C-615, Büchi Lab Equipment, Flawil, Switzerland) according to the following procedure (Tan et al. 2011; Xiang 2015). The F12 and F22 fractions were eluted with acetone/n-hexane (2:3, v/v), acetone/n-hexane (3:2, v/v), and methanol/chloroform (1:18, v/v), respectively, as shown in Fig. 3. The yields of purified compounds Gto G5 were 39.2%, 28.7%, 15.4%, 8.9%, and 7.8%, respectively. The yields of purified compounds GSto GS5 were 43.5%, 26.9%, 12.0%, 10.4%, and 7.2%, respectively.

Fig. 3. Column chromatography fractionations of the ether-soluble fractions F12 and F22

Determination of anticancer activity of the DHP fractions and purified compounds

Cervical cancer Hela cells (primary cell) in the logarithmic growth phase were adjusted to a concentration of 1 × 104 cells/well in a 96-well flat bottom plate (Mathi et al. 2015). The cells were incubated for 24 to 48 h until the cells spread over the bottom. The DHP fractions (100 µL) across a concentration gradient of 10 to 1000 μg/mL were added to each well. After 24 h incubation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetr-azolium bromide (MTT) solution (5 mg/mL, 20 μL) was added to each well (Van et al. 2011). After 4 h, 150 μL of DMSO was added to each well and the plate was shaken for 10 min on a shaker to fully dissolve the crystals. Finally, the absorbance of each well at 490 nm was measured on a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The relationship between the concentration of each sample and the inhibition rate was plotted and the IC50 calculated.

Mass spectroscopic analysis of the structure of purified compounds

The molecular weights of the purified compounds were determined using a high performance benchtop quadrupole trap atmospheric pressure chemical ionization mass spectrometry (APCI-MS; Thermo Fisher Scientific, MA, USA). The ion source was with a scan range of m/z 70 to 1050 and spray voltage of 5000 V, using nitrogen as the dry gas with a flow rate of 45 L/min.

RESULTS AND DISCUSSIONS

13C-NMR Spectral Analysis for the DHP-G and DHP-GS

The 13C-NMR spectra of the DHP-G and DHP-GS are shown in Figs. 4 and 5, respectively. The possible substructures are shown in Fig. 6. In Fig. 4, a weak α-CHO signal peak at 190.9 ppm (No. 1) was observed, indicating that a small amount of oxidation occurred during the DHP-G polymerization (Xie et al. 2000). The aliphatic carboxylic acid signal at 172.2 ppm (No. 2) showed that the γ-position had been oxidized to form cinnamic acid. Signals from 149.8 to 147.0 ppm (No. 4 to 8) were assigned to aromatic carbons of the guaiacyl ring. The signal at 143.6 ppm (No. 9) was from C4 of the 5-5 structure. The signals near 130 ppm (No. 10 to 14) were mainly the Cα/Cβ signals from C=C, indicating that some double bonds in the side chain were not involved in the dehydrogenation reaction. Signals from 76.7 to 74.8 ppm (No. 22 to 23) and at 67.2 ppm (No. 27) were mainly from Cα and Cγ in the β-5 structure. This finding was similar to those of previous studies (Lüdemann and Nimz 1973; McElroy and Lai 1988). Peaks at 85.1 ppm (No. 21), 70.2 ppm (No. 26), and 62.0 ppm (No. 29) were mainly from Cβ, Cα, and Cγ of the β-O-4 structure, respectively (Harman-Ware et al. 2017). The signal at 63.5 ppm (No. 28) was from Cα of the β-1 structure. The peak at 53.5 ppm (No. 31) was assigned to Cβ of the β-β structure. The peak intensities indicated the content of β-5 and β-O-4 in the DHP-G was considerably higher than that of other substructures.

The 13C-NMR spectra of the DHP-GS (Fig. 5) was similar to that of the DHP-G (Fig. 4), except for the signals that arose from the syringyl units. At low field chemical shifts of 191.3 ppm (No. 1′) and 172.3 ppm (No. 2′), there were peaks corresponding to the C=O groups of the aromatic aldehyde and γ-position of ferulic acid, respectively (Holtman et al. 2004; Hage et al. 2009). The signal at 152.8 ppm (No. 4′) was assigned to C3 and C5 on the etherified syringyl unit (Choi and Faix 2011). The resonances at 143.8 ppm (No. 7′) and 129.1 ppm (No. 8′) corresponded to C4 and C1 of the 5-5 structure, respectively. The peaks at 76.9 ppm (No. 16′) and 67.3 ppm (No. 20′) corresponded to Cα and Cγ of the β-5 structure, respectively. The peaks at 85.3 ppm (No. 15′), 71.0 ppm (No. 18′), and 61.8 ppm (No. 22′) arose from the Cβ, Cα, and Cγ signals of the β-O-4 structure, respectively. The weak signal at 63.0 ppm (No. 21′) corresponded to Cα of the β-1 structure. The peak at 45.2 ppm (No. 26′) illustrated that some β-β structures were present. These results indicated that the DHP-GS also contained substructures, such as 5-5, β-O-4, β-5, β-1, and β-β, of which β-O-4 and β-5 were dominant.

Fig. 4.13C-NMR spectrum of the DHP-G

Fig. 5.13C-NMR spectrum of the DHP-GS

Molecular Weight Analyses of the DHP Fractions

To understand the degree of polymerization of the DHP fractions, the weight of average molecular weight (Mw) and number of average molecular weight (Mn) of each DHP fraction were determined. As shown in Table 1, the molecular weight of the four DHP-G fractions was much lower than that of naturally extracted lignin as milled wood lignin (MWL) according to the result of Huang et al. (2011).According to the molecular weight of coniferyl alcohol, i.e. 180 g/ mol, it was speculated that the dimer structure was dominant in F12 and F22.

Table 1. Molecular Weight of the DHP Fractions

Fig. 6. Main substructures in the DHP-G and DHP-GS

Determination of Anticancer Activities of the DHP Fractions

The anticancer activities of the DHP fractions and the two lignin precursors are shown in Figs.7 and 8, and Table 2. The relationship between the sample concentration and inhibitory rates were shown in log-fitting curves. Every fraction of DHP had a certain inhibitory effect on the Hela cervical cancer cells.

The inhibitory properties of two kinds of ether-soluble fractions, F12 and F22, were relatively strong, with IC50 values of 81.60±9.30μg/mL and 103.24±14.09μg/mL, respectively. This also meant that the ether-soluble fraction from the DHP-G was more active than that from the DHP-GS. However, the IC50 values of coniferin and syringin were as high as 3844.75±86.51 μg/mL and 4410.96±106.94μg/mL, respectively, indicating that the activities of the two lignin precursors were low. As compared with the commercial anticancer drugs from plant as paclitaxel with IC50 of 7.08μM (6.04μg/mL) to cervical cancer (Yilmaz et al. 2016), the IC50 of the coniferin and the syringin was too high to be applied as drugs in anticancer purpose.

Fig. 7. The relationship between the concentrations of F11, F12, F13, F14, and coniferin and the inhibitory rates on cervical cancer cells

Fig. 8. The relationship between the concentrations of F21, F22, F23, F24, and syringin and the inhibitory rates on cervical cancer cells

Table 2. Inhibitory Effect of the DHP Fractions on the Hela Cervical Cancer Cells

Anticancer Activity Analyses of the Purified DHP Compounds

Results from the anticancer experiment with purified compounds G1 to G5 and GS1 to GS5 are shown in Figs.9 and 10, and Table 3. It was found that only compounds G1, G2, GS1, and GS2 had low IC50 values on the Hela cervical cancer cells of 15.1±2.3, 40.5±7.6, 30.8±5.4, and 87.8±9.2 μg/mL, respectively. Of these, G1 had the greatest inhibitory effect on growth, although its IC50 value on Hela cervical cancer cells was a little higher than that of paclitaxel (Yilmaz et al. 2016). However, because the IC50 value of the original F12 fraction was only 81.60±9.30μg/mL, these results further indicated G1 and G2 can be the most active compounds and play an important role in the efficacy of F12. Similarly, the active anticancer substances in F22 were mainly GS1 and GS2, with IC50 values of 30.8±5.4 and 87.8±9.2μg/mL, respectively.

Fig. 9. Relationship between inhibitory rate and concentration of the purified compounds G1 to G5 on the Hela cervical cancer cells

Fig. 10. Relationship between inhibitory rate and concentration of the purified compounds GS1 to GS5 on the Hela cells

Table 3. Inhibitory Effects of the Purified DHP Compounds on the Hela Cervical Cancer Cells

Structural Analyses of the Active Anticancer Compounds Isolated from the DHP-G and DHP-GS

Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is widely used to analyze weakly polar small-molecule polymers, with remarkable advantages in speed, specificity, and sensitivity over other methods. Mass spectrometry has now become one of the most important instruments in the analysis of lignin structures (Evtuguin and Amado 2003; Reale et al. 2004; Yang et al. 2010).

Molecular weight information for G1 is shown in Fig.11. It was found that the molecular ion signal of G1 appeared at m/z357.133. The fragment peak at m/z149.023 was formed by the γ-position carbon ionization in the side chain of coniferyl monomer. There was a structural signal peak formed by the cleavage of the γ-hydroxyl group at m/z 163.039. The ion signal peak at m/z 279.159 was a dimer of the β-5 structure. The fragment peak at m/z341.138 was from β-5, γ-CH2+. From the analyses of fragment peaks, it was proposed that the Gmolecular ion at m/z357.133 was a G-type dimer (β-5, γ-CH2OH, γ’-CH2OH) (Fig.15, G1), i.e.m/z 4-[3-hydroxymethyl-5-(3-hydroxy-propenyl)-7-methoxy-2,3-dihydro-benzofuran-2-yl]-2-methoxy-phenol. This indicated that a coniferyl monomer with double bond in the side chain combined with the phenolic hydroxyl group of another monomer under laccase catalysis to form a β-5 structure.

Fig. 11. Mass spectrum of the compound G1

Legend: m/z137.059:2-Methoxy-4-methyl-phenol;m/z149.023: 2-Methoxy-4-vinylphenol;m/z163.039:2-Methoxy-4-propenyl-phenol;m/z205.086:4-(3-Hydroxy-propenyl)-2-methoxy-phenol[Na+];m/z341.138: 4-(3-Hydroxymethyl-7-methoxy-5-propenyl-2,3-dihydro-benzo-furan-2-yl)-2-methoxy-phenol;m/z357.133:4-[3-Hydroxymethyl-5-(3-hydroxy-propenyl)-7-methoxy-2,3-dihydro-benzofuran-2-yl]-2-methoxy-phenol

The mass spectrum of G2 is shown in Fig. 12. The signal peak at m/z 219.065 was formed by the breaking of the ether bond and carbon-carbon bond on the coumaran ring in the structure of phenylcoumaran. The signal at m/z314.177 was generated by the cleavage of the carbon-carbon double bond in the side chain of the β-5 dimer. The signal of m/z341.138 (β-5, γ-COOH) was strong, indicating that many monomers were polymerized by β-5 linkage, and the side chain was easily oxidized to a carboxylic acid structure. The parent ion of the fragment at m/z219.065 appeared at m/z357.133 and its structure was β-5, γ’-COOH. The signal of m/z392.287 was produced by β-5 (γ-CH2OH, γ’-COOH) dimer capturing Na+. The molecular ion peak of G2 appeared at m/z564.221. According to the analysis of fragment peaks, it was assigned to 5-(2-Carboxy-vinyl)-2′-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7,7′-dimethoxy-2,3,2′,3′-tetrahydro-[2,5′]bibenzofur-anyl-3′-carboxylic acid, which indicated the structure of G2 to be a G-type trimer [(β-5)(β-5), γ-COOH, γ’-CH2OH, γ”-COOH] (Fig. 15, G2). The fragment structure trimer [(β-5)(β-5), γ-COOH, γ’-CH2OH, β”-CH+] at m/z519.201 also confirmed the G2 structure.

Molecular weight information for GS1 is shown in Fig.13. The signal of m/z 205.086 was a typical fragment of the β-5 type dimer after coumaran ring breaking. The signal at m/z233.080 was formed by the capture of Na+ by the sinapyl alcohol monomer. The ion peak at m/z387.143 revealed the structure of 4-[3-hydroxymethyl-5-(3-hydroxy-propenyl)-7-methoxy-2,3-dihydro-benzofuran-2-yl]-2,6-dimethoxy-phenol, which was a β-5 GS-type dimer (Fig. 15, GS1). Further analyses of fragment ion peaks at m/z149.023, 187.075, and 205.086 confirmed signal at m/z387.143 to be a dimer [(β-5), γ-CH2OH, γ’-CH2OH] formed by G-type and S-type monomers. The molecular weight difference between m/z357.132 and m/z387.143 was 30, exactly the molecular weight of -CH2OH, indicating that the γ position was relatively easily to be eliminated, and reassured the structure of GS1.

Fig. 12. Mass spectrum of the compound G2

Legend: m/z 219.065: 3-(3-Ethyl-4-hydroxy-5-methoxy-phenyl)-acrylic acid; m/z314.127:4-(3-Hydroxymethyl-7-methoxy-5-methyl-2,3-dihydro-benzofuran-2-yl)-2-methoxy-phenol, (β-5, γ-OH);m/z 341.138: 2-(4-Hydroxy-3-methoxy-phenyl)-7-methoxy-5-vinyl-2,3-dihydro-benzofuran-3-carboxylic acid;357.m/z 133:3-[2-(4-Hydroxy-3-methoxy-phenyl)-7-methoxy-3-methyl-2,3-dihydro-benzofuran-5-yl]-acrylic acid; m/z 392.287:3-[2-(4-Hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-2,3-dihydro-benzofuran-5-yl]-acrylic acid[Na+]; m/z 519.201:2′-(4-Hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7,7′-dimethoxy-5-vinyl-2,3,2′,3′-tetrahydro-[2,5′]bibenzofuranyl-3′-carboxylic acid; m/z 564.211:5-(2-Carboxy-vinyl)-2′-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7,7′-dimethoxy-2,3,2′,3′-tetrahydro-[2,5′]bibenzofuranyl-3′-carboxylic acid.

The GS2 was a compound eluted from the preparative column chromatography with acetone/n-hexane mixture (3:2 v/v) as the mobile phase. The GS2