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Determination of Molecular Structures of Condensed Tannins from Plant Tissues Using HPLC-UV Combined with Thiolysis and MALDI-TOF Mass Spectrometry
采用HPLC-UV 结合硫解和MALDI-TOF质谱的方法测定植物组织中缩合单宁的分子结构   

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Abstract

Condensed tannins extracted from plant tissues are suitable substitutes for phenolic resins. Their molecular structure, which might influence their chemical reactivity, can be assessed by the use of both HPLC-UV after acid thiolysis and MALDI-TOF mass spectrometry. Thiolysis of plant extracts in acidic methanol with cysteamine hydrochloride results in the release of the monomeric units of the condensed tannin oligomers that can be further quantified by reversed-phase HPLC-UV by comparison with analytical standards. MALDI-TOF mass spectrometry using 2,5-dihydroxybenzoic acid as matrix and K+ as cationization agent highlights the molecular structural characteristics (e.g., monomeric unit sequence) of the tannin oligomers. The methodologies permit the estimation of the mean and the maximum (observable) degree of polymerization, the type of monomeric units and the presence of glycosylation and/or esterification of the tannin oligomers.

Keywords: Condensed tannins(缩合单宁), Thiolysis(硫解), HPLC-UV(高效液相色谱), MALDI-TOF(MALDI-TOF), Plant extracts(植物提取物)

Background

Condensed tannins are polyphenolic oligomers made of flavan-3-ol monomeric units that could be extracted from several plant tissues (e.g., softwood barks). They have been recognized as suitable alternatives to synthetic phenolics in resin formulations such as wood adhesives and foamed materials. The most common flavan-3-ol monomers detected in condensed tannins, which differ in the hydroxylation pattern and stereochemistry, are shown in Figure 1.


Figure 1. Most common monomers identified in the structure of condensed tannins

The specific structure of monomeric units in the oligomer and the polymerization degree strongly influence the chemical reactivity and physical properties of the tannins, e.g., condensation reaction rate with aldehydes, heavy metal chelation ability, and viscosity of aqueous solutions (Pizzi and Stephanou, 1994; Yoneda and Nakatsubo, 1998; Garnier et al., 2001). Identification of the molecular structure of the tannins is therefore important to better determine their possible exploitation.

The analysis of the structure of condensed tannins monomers has been performed with different methodologies, e.g., size exclusion chromatography (SEC), normal and reversed-phase high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) (Hümmer and Schreier, 2008).

Depolymerisation of tannins in acidic methanol and in the presence of toluene-α-thiol or cysteamine hydrochloride (thiolysis) followed by reversed-phase HPLC-UV was recognised as a suitable method for the estimation of the mean degree of polymerization of condensed tannins and the configuration of their building units (Matthews et al., 1997; Cheynier et al., 2001; Jerez et al., 2007; Bianchi et al., 2015). As well, MALDI-TOF MS has been shown to be successful in the determination of the structure of condensed tannins (Pasch et al., 2001; Monagas et al., 2010; Bianchi et al., 2014).

The proposed method for the analysis of condensed tannins extracted from plant tissues represents an optimization of the previously published procedures for HPLC-UV after acid thiolysis and MALDI-TOF MS. A careful tuning of the mobile phase gradient in the HPLC analysis was performed in order to reach a sufficient separation between the released flavanols and their corresponding thioethers. The choice of the cationization agent in the preparation of the sample for MALDI-TOF MS was also carefully gauged, especially in consideration of samples containing a relatively high amount of inorganic compounds like bark extracts.

The method was successfully used in the characterization of condensed tannins extracted from the bark of softwood species, e.g., Silver fir, European larch, Norway spruce, Douglas fir and Scots pine (Bianchi et al., 2015).

Materials and Reagents

  1. 1 ml Eppendorf tubes 
  2. 2 ml Eppendorf tubes 
  3. HPLC-filters 17 mm, PTFE, 0.45 µm (infochroma, catalog number: 8817-P-4 )
  4. Cosmosil Protein-R ø4.6 x 250 mm HPLC column (NACALAI TESQUE, catalog number: 06527-11 )
  5. Dry extracts from plant tissues (e.g., softwood bark)
    The dry plant extracts could be obtained through different processes. For the extraction of tannins, the maceration of fine milled tissue (< 1 mm in size) in solvents like methanol, acetone or water is suggested. The extraction temperature should be between 30 and 90 °C, and the extraction time between 10 and 60 min. The drying of the extract has to be performed avoiding as much as possible post-modification (e.g., oxidation) of the extracts. Vacuum drying and/or freeze-drying are therefore recommended. After drying the extracts should be stored in a refrigerator (3-8 °C), protected from light and air.
  6. Deionized water (18.2 MΩ-cm)
  7. 2,5-dihydroxybenzoic acid (matrix substance for MALDI MS, HPLC grade) (Sigma-Aldrich, catalog number: 85707 )
  8. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  9. Cysteamine hydrochloride (HPLC grade) (Sigma-Aldrich, catalog number: 49705 )
  10. Hydrochloric acid (HCl) (37%) (Sigma-Aldrich, catalog number: 320331 )
  11. Methanol (for HPLC) (Sigma-Aldrich, catalog number: 34860 )
  12. Acetone (for HPLC) (Sigma-Aldrich, catalog number: 270725 )
  13. Trifluoracetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 )
  14. Acetonitrile (ACN) (Sigma-Aldrich, catalog number: 34998 )
  15. Flavan-3-ol analytical standards:
    (+)-Catechin (Sigma-Aldrich, catalog number: 43412 )
    (-)-Epicatechin (Sigma-Aldrich, catalog number: 68097 )
    (-)-Gallocatechin (Sigma-Aldrich, catalog number: 01338 )
    (-)-Epigallocatechin (Sigma-Aldrich, catalog number: 08108 )
  16. Mass spectra calibration standard covering a range from about 750 to 4000 m/z (e.g., Peptide calibration standard II, Bruker Daltonics, Germany)
  17. Thiolysis media (see Recipes)
  18. HPLC mobile phases (see Recipes)

Equipment

  1. Eppendorf pipette 1-10 µl
  2. Eppendorf pipette 100-1,000 µl
  3. HPLC system equipped with a UV-photodiode array detector. In our study an Agilent 1100 LC system was used (Agilent, Waldbronn).
  4. Water bath (Polyscience, UK)
  5. Ultrasonic bath (BANDELIN electronic, model: Sonorex RK510 )
  6. MALDI-TOF mass spectrometer equipped with laser emitting in UV wavelength. In our study a MALDI-TOF mass spectrometer Reflex III was used (Bruker Daltonics, Germany).
  7. Analytical balance
  8. Refrigerator
  9. HPLC vials (1 ml)

Procedure

  1. HPLC-UV after thiolysis
    1. Sample and standard preparation
      1. Dissolve the dried plant extracts (5 mg) in methanol (5 ml) and sonicate (35 kHz, 250W) at room temperature for 30 min.
      2. Transfer with an Eppendorf pipette 200 μl of the solubilized extracts in an Eppendorf tube (2 ml) and mix it with 200 μl of thiolysis media.
      3. Put the Eppendorf tube in the water bath set at 65 °C for 1 h to carry on the thiolysis. No shaking of the sample is needed. During the thiolysis the flavan-3-ol monomeric units of the condensed tannins are released in their native state (terminal units) as well as their respective thioethers (extension units), as schematically depicted in Figure 2.
        Note: The thiolysis is effective only if condensed tannins are made of catechin and/or gallocatechin units. If tannins are made of fisetinidol, robinetinidol or monomeric units different than flavan-3-ols, the interflavonyl bond will not be cleaved.


        Figure 2.  Scheme of the condensed tannin thiolysis with cysteamine hydrochloride. R1, R2 = H or OH.

      4. The thiolysis is quenched with 1 ml of water and the solution is purified through a PTFE 0.45 μm HPLC filter.
      5. Non-thiolyzed reference samples are prepared by dissolving 200 μl of the solubilized extracts in sequence with 200 μl of methanol and 1.0 ml of water, followed by the same thermal treatment as used during the thiolysis (1 h at 65 °C) and purified through a PTFE 0.45 µm HPLC filter.
      6. Prepare calibration stock solutions of the flavan-3-ols (0.01, 0.02, 0.05, 0.10, 0.25, 0.50 g/L in methanol). The calibration standards solutions for HPLC are then prepared by diluting 200 μl of the calibration stock solutions in sequence with 200 μl of methanol and 1.0 ml of water. The calibration standard solutions are then thermally treated (1 h at 65 °C in a water bath) and purified (PTFE 0.45 µm HPLC-filter) in the same way as the samples.
      7. All samples and calibration standards are sealed in an HPLC vial and stored in a refrigerator between 3 and 8 °C until analysis. The samples remain stable for about 1 week under these conditions.
    2. Measurement
      1. Condition the reversed-phase HPLC-UV system equipped with the Cosmosil column using solution A (see Recipes) at 30 °C.
      2. Inject 25 μl of the sample (e.g., thiolyzed extract, non-thiolyzed extract, calibration standard) in the HPLC column. Before injection the samples should be conditioned at room temperature for at least 1 h. However, it is recommended to limit the exposition of the sample at room temperature to less than 24 h before the HPLC measurement.
      3. The mobile phase gradient described in Table 1 was applied at a flow equal to 1 ml/min. Compositions of solution A and solution B are described in the section Recipes. The temperature of the column is kept at 30 °C all over the measurement.

        Table 1. Mobile phase gradient applied during HPLC measurements


        Notes:
        1. The quasi-isocratic conditions between 5 and 20 min are needed to achieve a sufficient resolution (separation) of the peaks corresponding to epigallocatechin, catechin and their thioethers.
        2. The HPLC measurements should be repeated at least twice on replicated samples, therefore taking into account independent sample preparation, thiolysis, and analysis.
      4. Detect the absorbance at 280 nm. An example of the HPLC chromatograms at 280 nm of bark extracts before and after thiolysis is reported in Figure 3.


        Figure 3. HPLC-UV chromatograms between 10 and 35 min of Silver fir (Abies alba [Mill.]) bark extracts before and after thiolysis. Absorption peaks are associated to various flavanol and flavanol thioethers as follows: 1 = gallocatechin; 2 = gallocatechin thioether; 3 = epigallocatechin; 4 = catechin; 5 = catechin thioether; 6 = epigallocatechin thioether; 7 = epicatechin; 8 = epicatechin thioether.

      5. Integrate the area under the absorption peak(s) that corresponds to the flavan-3-ols and their thioethers.
        Notes:
        1. While analytical standards of native flavan-3-ols are commercially available from chemical substance distributors (e.g., Sigma-Aldrich), no standards are available for flavan-3-ol thioethers. The preparation of flavan-3-ol thioethers standards could be performed only starting from oligomers of known composition (also not easily available) and requires lengthy purification steps (Torres and Bobet, 2001). In the present study the retention times of the flavan-3-ol thioethers were identified by extemporary HPLC-MS measurements performed in an external laboratory using the same column and method and Agilent 1290 Infinity HPLC system equipped with a mass detector (Agilent 6130 quadrupole MS).
        2. Quantify the concentration of the flavan-3-ols in the thiolyzed and non-thiolyzed samples by comparison with the calibration curves developed for each single flavan-3-ol measuring the corresponding calibration standards. As already mentioned, no calibration standard are commercially available for the flavan-3-ol thioethers. Extemporary measurements performed on epicatechin dimers (proanthocyanidin B2) showed that, after complete thiolysis (complete disappearing of the peak corresponding to the epicatechin dimer) the peaks corresponding to epicatechin and epicatechin-thioether had almost an equal absorption area. The concentration of all flavan-3-ol thioethers was then calculated using for the flavanol thioethers the same UV molar absorption (MABS) of the corresponding native flavan-3-ols (e.g., MABS of epicatechin thioether was considered equal to MABS of epicatechin). In Table 2 the MABS for the measured flavan-3-ols are reported.

        Table 2. Molar absorption factor (MABS) of flavan-3-ols and flavan-3-ol thioethers


  2. MALDI-TOF mass spectrometry
    1. Sample preparation
      1. Dissolve the dried plant extracts (2.5 mg) in 1 ml of aqueous acetone (50%) and sonicate (35 kHz, 250 W) at room temperature for 30 min.
      2. Prepare a matrix solution by dissolving 10 mg of 2,5-dihydroxybenzoic acid in 1 ml of pure acetone.
      3. In Eppendorf tube (1 ml) mix 10 µl of the extract solution with 10 µl of matrix solution and spike it with 1 µl of KCl (10 g/L in water) to enhance the formation of potassiated ions and suppress the formation of sodiated and/or other types of ions.
        Note: Plant tissues might contain different kinds of salts, which may consequently contribute to the ion formation during the MALDI ionization process. For correct spectra interpretation it is therefore essentially important to ensure the formation of only one type of ions (e.g., either sodiated or potassiated, or others). Of particular concern are already mentioned sodium and potassium ions, because these two salts are common constituents in plant tissues. The mass difference between Na+ and K+ is 16 amu, which is also a mass difference between the building units of the condensed tannins (Table 4). Thus, unintended formation of Na+ and K+ ions at the same time may result in errors in spectra interpretation.
      4. Deposit 5 µl of the mixture on the MALDI-TOF stainless steel plate and allow it to dry at room temperature for about 1 h.
      5. The samples should be stored at room temperature and measured at the same day of the preparation.
    2. Measurement
      1. Set the MALDI TOF mass spectrometer in positive linear mode with a monitoring range between 700 and 4,500 m/z.
      2. Calibrate the MALDI TOF mass spectrometer with the calibration standard (e.g., Peptide calibration standard II, Bruker Daltonics, Germany)
      3. Perform the measurement on a MALDI-TOF mass spectrometer collecting about 700 laser shots for each sample.

Data analysis

  1. HPLC-UV after thiolysis
    1. The molar concentration (µmol/L) of each flavan-3-ol monomer released from the condensed tannins (FTANN) is corrected according to the equation (1):
      FTANN = Fth-FNth (1)
      Where,
      Fth = molar concentration (µmol/L) of the flavan-3-ol (or its thioether) in the thiolyzed sample,
      FNth = molar concentration (µmol/L) of the flavan-3-ol (or its thioether) in the non-thiolyzed sample.

      For the flavan-3-ol thioethers no correction is needed, because they are obviously not present as native compounds in the extracts.
      In Table 2 an example of calculation based on the chromatogram of Figure 3 is reported.

      Table 3. Data analysis from the HPLC-UV chromatogram of Figure 3

      *Note: Identification label used in Figure 3.

    2. The measured amounts of the released monomeric units provide the following information on the molecular structure of condensed tannins (Bianchi et al., 2015):
      1. The sum of the molar concentrations of a specific flavanol unit (both as terminal and extension unit) gives an indication about the amount of such monomeric unit in the tannin.
      2. The relative ratios between different monomeric units present in the tannin (also as stereoisomers like catechin and epicatechin) could be estimated by the relative ratios between the associated released flavanols.
      3. Differences between the configuration of terminal units and those of the extension units could be identified.
      4. The mean degree of polymerization (mDP) of the condensed tannins could be estimated with the equation (2):

        Where,
        = molar concentration (µmol/L) of the total released flavan-3-ol extension units (flavan-3-ol thioethers) after thiolysis,
        = molar concentration (µmol/L) of the total released flavan-3-ol terminal units (native flavan-3-ol) after thiolysis.
        For the example in Figure 3 and using the data reported in Table 2, the calculation results as follow:


  2. MALDI-TOF mass spectrometry
    1. Typical MALDI MS spectrum of plant extract containing condensed tannins is shown in Figure 4. The spectrum contains several groups of peak patterns regularly distanced from each other along the m/z axis. Each peak represents a tannin oligomeric chain with a defined composition of tannin monomeric units, which masses are reported in Table 4. The masses of the monomeric units are calculated from the masses of the monomers (Figure 1) by subtracting the mass of 2 hydrogen atoms (e.g., catechin monomeric unit = 290.3 - 2 = 288.3 Da). The distance between the repeating pattern of peaks (Figure 4) permits to identify the type of monomeric unit in the oligomer chain and the corresponding class of tannin (Table 4).
      Note: MALDI-TOF analysis doesn’t permit to distinguish between monomeric units having the same mass (e.g., catechin and robinetinidol).
    2. Within a peak group, peaks distanced by 16 m/z or multiple of 16 m/z (Figure 4) indicate the occurrence of various monomeric (building) units in the same condensed tannin, e.g., gallocatechin (304.3 Da) and catechin (288.3 Da).

      Table 4. Typical monomeric units for different classes of condensed tannins



      Figure 4. MALDI-TOF mass spectrum between 800 and 2,000 m/z of Silver fir (Abies alba [Mill.]) bark extract showing mass peaks associated to condensed tannins containing catechin (or robinetinidol), gallocatechin and gallate esters

    3.  The identification of the monomeric unit composition in correspondence of a peak of the MALDI-TOF spectrum, can be performed using the equation (3):
      M + Cat+ = x∙272.3 + y∙288.3 + z∙304.3 + MCat + 2.0 (3)
      Where,
      x, y and z are the number of fisetinidol, catechin (or robinetinidol) and gallocatechin units in the oligomeric chain, respectively,
      M is m/z value of the oligomeric chain observed in the mass spectrum,
      MCat is the mass of the cationization ion (e.g., Na+, K+),
      2.0 is the weight of the two hydrogen atoms at the ends of the oligomeric chain.
      In the presence of building units different than flavan-3-ols (e.g., stilbenes) or moieties (e.g., glycosides, gallic acid ester), additional repeating peak groups will be detectable in the mass spectrum, shifted from the non-esterified series by a gap corresponding to the mass of the non-flavanol unit or of the moiety (Bianchi et al., 2014). E.g., the presence of an oligomer series containing a gallate ester moiety (152.1 Da) results in peak groups distanced from the non-esterified series by 152 m/z, as reported in Figure 4. In this case the general Eq. 3 should be modified including the detected moiety, as shown in Eq. 4. In this case, for simplicity, the 272.3 unit representing fisetinidol was deleted (as it was not detected in the spectrum of Figure 4) and the mass of the cationization ion K+ (39.1) is explicitly reported. The variables y and z have the same meaning as in Eq. 3 and w is the number of gallate ester moieties in the tannin oligomer.
      M + K+ = y∙288.3 + z∙304.3 + w∙152.1 + 39.1 + 2.0 (4)
      The application of such equation is shown in Table 5.

      Table 5. Interpretation of the MALDI-TOF spectrum reported in Figure 4


      The degree of polymerization (DP) of each tannin oligomer is defined by equation (5):
      DP = x + y + z (5)
      Where,
      x, y and z have the same meaning as in equation (3).
      In presence of glycosil or ester moieties, their number should not be accounted in the calculation of the DP, because they are considered as an integrant part of the monomeric unit, e.g., flavanol glucosides, flavanol gallates (Bianchi et al., 2014; Bianchi et al., 2015).

  3. Concluding remarks
    1. Combination of MALDI-TOF mass spectrometry with HPLC-UV after acid thiolysis of hot water extracts from plant tissues permits to obtain various indicators on the condensed tannin structure present in the extracts.
    2. Both methods provide indication on the predominant monomeric unit, and therefore the class of the condensed tannins present in the extracts.
    3. HPLC-UV combined with thiolysis gives information on the mean degree of polymerization and could also distinguish between monomeric units with different stereochemistry. Its main limitation is the reduced thiolysis efficacy in case of side chain (branching) and the non-cleavability of interflavonyl bonds between monomeric units different than catechin, gallocatechin or their epimers.
    4. MALDI-TOF mass spectrometry provides information on the degree of polymerization, where obviously peaks with the highest detected m/z ratio indicate the maximal (observed) polymerization degree of the condensed tannin in the measured sample. Furthermore, the presence of monomeric units different than simple flavan-3-ols could also be elucidated by the MALDI-TOF mass spectra.

Recipes

  1. Thiolysis media
    500 mg cysteamine hydrochloride HPLC grade
    0.2 ml hydrochloric acid 37% HPLC grade
    9.3 ml methanol HPLC grade
    Preparation of thiolysis media:
    1. Weigh 500 mg of cysteamine hydrochloride on an analytical balance in a 20 ml glass flask.
    2. Add 9.3 ml of methanol with a calibrated Eppendorf pipette and shake the mixture until complete dissolving of any solid residual.
    3. Add 0.2 ml of concentrated HCl (37%) with a calibrated Eppendorf pipette and sonicate the mixture (35 kHz, 250 W) at room temperature for 5 min.
    4. Store the prepared thiolysis media in refrigerator (3-8 °C) protecting from light and air until use.
  2. HPLC mobile phases
    1. Solution A: 0.10% v/v trifluoracetic acid in water (about 55 ml/sample)
    2. Solution B: 0.08% v/v TFA in acetonitrile water mixture (ACN:Water = 4:1, v/v) (about 25 ml/sample)

Acknowledgments

We thank the Swiss National Research Program 'Resource Wood' (NRP66) for the financial support of this work. This protocol was developed starting from the procedures originally described in Matthews et al. (1997), Torres and Lozano (2001), Pasch et al. (2001) and Monagas et al. (2010).

References

  1. Bianchi, S., Gloss, A., Kroslakova, I., Mayer, I., Pichelin, F. (2014). Analysis of the structure of condensed tannins in water extracts from bark tissues of Norway spruce (Picea abies [Karst.]) and Silver fir (Abies alba [Mill.]) using MALDI-TOF mass spectrometry. Ind. Crop Prod 61: 430-437.
  2. Bianchi, S., Kroslakova, I., Janzon, R., Mayer, I., Saake, B. and Pichelin, F. (2015). Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry 120: 53-61.
  3. Cheynier, V., Labarbe, B., Moutounet, M. (2001). Estimation of procyandin chain lenght. Method in Enzymology 335, 82-94.
  4. Garnier, S., Pizzi, A., Vorster, O.C., Halasz, L. (2001). Comparative rheological characteristics of industrial polyflavonoid tannin extracts. J Appl Poly Sci 81: 1634-1642.
  5. Hümmer, W. and Schreier, P. (2008). Analysis of proanthocyanidins. Mol Nutr Food Res 52(12): 1381-1398.
  6. Jerez, M., Selga, A., Sineiro, J., Torres, J.L. and Núñez, M.J. (2007). A comparison between bark extracts from Pinus pinaster and Pinus radiata: Antioxidant activity and procyanidins composition. Food Chem 100(2): 439-444.
  7. Matthews, S., Mila, I., Scalbert, A., Pollet, B., Lapierre, C., Hervé du Penthoat, C.L.M., Rolando, C., Donnelly, D.M.X. (1997). Method for estimation of proanthocyanidins based on their acid depolymerization in the presence of nucleophiles. J Agric Food Chem 45(4): 1195-1201.
  8. Monagas, M., Quintanilla-Lopez, J.E., Gomez-Cordoves, C., Bartolomé, B., Lebron-Aguilar, R. (2010). MALDI-TOF MS analysis of plant proanthocyanidins. J Pharm Biomed Anal 51(2): 358-372.
  9. Pasch, H., Pizzi, A., Rode, K. (2001). MALDI-TOF mass spectrometry of polyflavonoid tannins. Polymer 42(18): 7531-7539.
  10. Pizzi, A., Stephanou, A. (1994). Fast vs. slow-reacting non-modified tannin extracts for exterior particleboard adhesives. Holz als Roh- und Werkstoff 52(4): 218-222.
  11. Torres, J. L. and Bobet, R. (2001). New flavanol derivatives from grape (Vitis vinifera) byproducts. Antioxidant aminoethylthio-flavan-3-ol conjugates from a polymeric waste fraction used as a source of flavanols. J Agric Food Chem 49(10): 4627-4634.
  12. Torres, J. L., Lozano, C. (2001). Chromatographic characterization of proanthocyandins after thiolysis with cysteamine. Chromatographia 54(7): 523-526.
  13. Yoneda, S., Nakatsubo, F. (1998). Effects of the hydroxylation patterns and degrees of polymerization of condensed tannins on their metal-chelating capacity. J Wood Chem Technol 18(2): 193-205.

简介

从植物组织提取的缩合单宁是酚醛树脂的合适替代物。它们的可能影响它们的化学反应性的分子结构可以通过在酸硫解和MALDI-TOF质谱之后使用HPLC-UV来评估。用半胱胺盐酸盐在酸性甲醇中溶解植物提取物导致缩合的单宁寡聚体的单体单元的释放,其可以通过与分析标准比较通过反相HPLC-UV进一步定量。使用2,5-二羟基苯甲酸作为基质和K sup +作为阳离子化试剂的MALDI-TOF质谱分析突出了单宁的分子结构特征(例如单体单元序列)低聚物。该方法允许估计平均和最大(可观察)聚合度,单体单元的类型和单宁单体的糖基化和/或酯化的存在。

[背景] 缩合单宁是由可从几种植物组织(例如软木树皮)中提取的黄烷-3-醇单体单元组成的多酚低聚物。它们已被认为是树脂配方(例如木材粘合剂和泡沫材料)中合成酚醛树脂的合适替代品。在缩合鞣酸中检测到的最常见的黄烷-3-醇单体,其羟基化模式和立体化学不同,如图1所示。

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图1.在缩合单宁结构中确定的最常见的单体

  低聚物中单体单元的具体结构和聚合度强烈影响单宁的化学反应性和物理性质,例如与醛的缩合反应速率,重金属螯合能力和水溶液的粘度溶液(Pizzi和Stephanou,1994; Yoneda和Nakatsubo,1998; Garnier等人,2001)。因此,鉴定单宁的分子结构对于更好地确定其可能的利用是重要的。
  缩合单宁单体的结构的分析已经用不同的方法进行,例如尺寸排阻色谱法(SEC),正相和反相高效液相色谱法(HPLC),基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF MS)和核磁共振(NMR)(Hümmer和Schreier,2008)。
 在酸性甲醇中和在甲苯-α-硫醇或半胱胺盐酸盐(硫解)的存在下,然后通过反相HPLC-UV的单宁的解聚被认为是用于估计缩合单宁的平均聚合度的合适方法,它们的构建单元的构型(Matthews等人,1997; Cheynier等人,2001; Jerez等人, ,2007; Bianchi等人,2015)。同样,已经显示MALDI-TOF MS在确定缩合单宁的结构方面是成功的(Pasch等人,2001; Monagas等人,2010 ; Bianchi 等。,2014)。
   用于分析从植物组织提取的缩合单宁的所提出的方法代表了先前公布的酸性硫解和MALDI-TOF MS之后的HPLC-UV程序的优化。进行HPLC分析中流动相梯度的仔细调节,以便在释放的黄烷醇和它们相应的硫醚之间达到充分的分离。在制备用于MALDI-TOF MS的样品中的阳离子化试剂的选择也被仔细地测量,特别是考虑到含有相对高量的无机化合物如树皮提取物的样品。
 该方法成功地用于表征从软木树种,例如银杉,欧洲落叶松,挪威云杉,道格拉斯杉木和苏格兰松树提取的缩合单宁(Bianchi等人。,2015)。

关键字:缩合单宁, 硫解, 高效液相色谱, MALDI-TOF, 植物提取物

材料和试剂

  1. 1 ml  Eppendorf管
  2. 2 ml  Eppendorf管 >
  3. HPLC过滤器17mm,PTFE,0.45μm(infochroma,目录号:8817-P-4)
  4. Cosmosil Protein-R 4.6×250mm HPLC柱(NACALAI TESQUE,目录号:06527-11)
  5. 植物组织的干提取物(例如,软木树皮)
    干燥的植物提取物可以通过不同的方法获得。对于单宁的提取,建议在溶剂如甲醇,丙酮或水中浸渍细研磨组织(尺寸小于1mm)。萃取温度应在30至90℃之间,萃取时间为10至60分钟。提取物的干燥必须避免提取物尽可能多的后改性(例如氧化)。因此,建议使用真空干燥和/或冷冻干燥。干燥后,提取物应储存在冰箱(3-8°C)中,避光和空气
  6. 去离子水(18.2MΩ-cm)
  7. 2,5-二羟基苯甲酸(MALDI MS的基质物质,HPLC级)(Sigma-Aldrich,目录号:85707)
  8. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  9. 半胱胺盐酸盐(HPLC级)(Sigma-Aldrich,目录号:49705)
  10. 盐酸(HCl)(37%)(Sigma-Aldrich,目录号:320331)
  11. 甲醇(用于HPLC)(Sigma-Aldrich,目录号:34860)
  12. 丙酮(用于HPLC)(Sigma-Aldrich,目录号:270725)
  13. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:302031)
  14. 乙腈(ACN)(Sigma-Aldrich,目录号:34998)
  15. Flavan-3-ol分析标准:
    (+) - 儿茶素(Sigma-Aldrich,目录号:43412) ( - ) - 表儿茶素(Sigma-Aldrich,目录号:68097) ( - ) - 高粱酸酯(Sigma-Aldrich,目录号:01338) ( - ) - 表没食子儿茶素(Sigma-Aldrich,目录号:08108)
  16. 质谱校准标准覆盖约750至4000m/z(例如,Peptide calibration standard II,Bruker Daltonics,Germany)的范围。
  17. 硫解介质(参见配方)
  18. HPLC流动相(参见配方)

设备

  1. Eppendorf移液器1-10μl
  2. Eppendorf移液器100-1,000μl
  3. HPLC系统,配有UV-光电二极管阵列检测器。在我们的研究中,使用Agilent 1100 LC系统(Agilent,Waldbronn)
  4. 水浴(Polyscience,UK)
  5. 超声波浴(BANDELIN electronic,型号:Sonorex RK510)
  6. 装配有在UV波长中发射的激光的MALDI-TOF质谱仪。在我们的研究中,使用MALDI-TOF质谱仪Reflex III(Bruker Daltonics,Germany)。
  7. 分析天平
  8. 冰箱
  9. HPLC小瓶(1ml)

程序

  1. 硫解后的HPLC-UV
    1. 样品和标准品制备
      1. 将干燥的植物提取物(5mg)溶解在甲醇(5ml)中并在室温下超声处理(35kHz,250W)30分钟。
      2. 用Eppendorf移液管转移200μl溶解的提取物在Eppendorf管(2ml)中,并与200μl硫解介质混合。
      3. 将Eppendorf管置于65℃的水浴中1小时进行硫解。不需要摇动样品。在硫解过程中,缩合鞣酸的黄烷-3-醇单体单元以其天然状态(末端单元)以及它们各自的硫醚(延伸单元)释放,如图2中示意性所示。 注意:只有当缩合单宁由儿茶素和/或没食子儿茶素单元组成时,硫解才有效。如果单宁是由非瑟酮醇,罗替尼诺或不同于黄烷-3-醇的单体单元组成,则交联基不会被裂解。


        图2.使用半胱胺盐酸盐的缩合单宁硫解方案。 R1,R2 = H或OH。

      4. 硫化用1ml水淬灭,溶液通过PTFE0.45μmHPLC过滤器纯化
      5. 通过将200μl溶解的提取物与200μl甲醇和1.0ml水顺序溶解,接着进行与硫解期间所用相同的热处理(65℃1小时),并通过PTFE0.45μmHPLC过滤器
      6. 制备黄烷-3-醇(0.01,0.02,0.05,0.10,0.25,0.50g/L,在甲醇中)的校准储备溶液。然后通过用200μl甲醇和1.0ml水依次稀释200μl校准储备溶液来制备HPLC的校准标准溶液。然后以与样品相同的方式对校准标准溶液进行热处理(在水浴中在65℃下1小时)和纯化(PTFE0.45μmHPLC-过滤器)。
      7. 将所有样品和校准标准品密封在HPLC小瓶中并储存在3-8℃的冰箱中直到分析。样品在这些条件下保持稳定约1周
    2. 测量
      1. 使用溶液A(参见配方)在30℃条件下安装有Cosmosil柱的反相HPLC-UV系统。
      2. 在HPLC柱中注入25μl样品(例如,硫解提取物,非硫化提取物,校准标准品)。在注射前,样品应在室温下调节至少1小时。然而,建议将样品在室温下的暴露限制在HPLC测量之前小于24小时
      3. 在等于1ml/min的流量下施加表1中所述的流动相梯度。溶液A和溶液B的组成在食谱部分中描述。在整个测量过程中柱的温度保持在30℃
        表1. HPLC测量期间应用的流动相梯度


        注意:
        1. 需要5至20分钟之间的准等度条件以实现对应于表没食子儿茶素,儿茶素和它们的硫醚的峰的充分分辨率(分离)。
        2. 对于重复的样品,HPLC测量应重复至少两次,因此考虑到独立的样品制备,硫解和分析。
      4. 检测280nm处的吸光度。图3报告了在硫解之前和之后的树皮提取物在280nm的HPLC色谱图的实例

        图3.在硫解之前和之后银枞提取物在10分钟和35分钟之间的HPLC-UV色谱图。吸收峰与各种黄烷醇和黄烷醇硫醚如下:1 =没食子儿茶素; 2 =没食子儿茶素硫醚; 3 =表没食子儿茶素; 4 =儿茶素; 5 =儿茶素硫醚; 6 =表没食子儿茶素硫醚; 7 =表儿茶素; 8 =表儿茶素硫醚
      5. 积分对应于黄烷-3-醇及其硫醚的吸收峰下的面积。
        注意:
        1. 虽然天然黄烷-3-醇的分析标准可从化学物质分配器(例如,Sigma-Aldrich)商购获得,但是没有可用于黄烷-3-醇硫醚的标准。黄酮-3-醇硫醚标准品的制备只能从已知组成的寡聚物(也不容易获得)开始进行,并且需要长时间的纯化步骤(Torres和Bobet,2001)。在本研究中,黄烷-3-醇硫醚的保留时间通过在外部实验室中使用相同的柱和方法以及装备有质量检测器的Agilent 1290 Infinity HPLC系统(Agilent 6130四极杆MS )。
        2. 通过与测量相应校准标准的每??种单一黄烷-3-醇开发的校准曲线比较,定量硫解和非硫解样品中黄烷-3-醇的浓度。如已经提到的,没有市售的用于黄烷-3-醇硫醚的校准标准。对表儿茶素二聚体(原花色素B2)进行的临时测量表明,在完全硫解(对应于表儿茶素二聚体的峰的完全消失)之后,对应于表儿茶素和表儿茶素 - 硫醚的峰具有几乎相同的吸收面积。然后使用对于黄烷醇硫醚,计算相应的天然黄烷-3-醇的相同的UV摩尔吸光度(M sup/ABS),计算所有黄烷-3-醇硫醚的浓度(例如, ABS 视为等于表儿茶素的M ABS )。在表2中,报道了测量的黄烷-3-醇的M ABS

        表2.黄烷-3-醇和黄烷-3-醇硫醚的摩尔吸收系数(M ABS


  2. MALDI-TOF质谱法
    1. 样品制备
      1. 将干燥的植物提取物(2.5mg)溶解在1ml丙酮水溶液(50%)中并在室温下超声处理(35kHz,250W)30分钟。
      2. 通过将10mg 2,5-二羟基苯甲酸溶解在1ml纯丙酮中制备基质溶液
      3. 在Eppendorf管(1毫升)混合10微升的提取物溶液与10微升的基质溶液,并与1微升的KCl(10克/升在水中)加强,以加强形成的钾化离子和抑制形成钠化和/或其他类型的离子 注意:植物组织可能含有不同种类的盐,这可能因此有助于在MALDI电离过程中的离子形成。因此,对于正确的光谱解释,确保仅形成一种类型的离子(例如,钠化的或钾化的或其它离子)是非常重要的。特别关注的是钠和钾离子,因为这两种盐是植物组织中的常见成分。 Na +和K + sup/+之间的质量差为16amu,这也是缩合单宁的结构单元之间的质量差异(表4)。因此,Na + 和K + 离子的意外形成同时可能导致光谱解释的错误。
      4. 将5μl混合物沉积在MALDI-TOF不锈钢板上,并使其在室温下干燥约1小时。
      5. 样品应在室温下储存,并在制备的同一天进行测量
    2. 测量
      1. 将MALDI TOF质谱仪设置为正线性模式,监测范围为700至4,500 m/z
      2. 用校准标准品(例如,Peptide calibration standard II,Bruker Daltonics,Germany)校准MALDI TOF质谱仪。
      3. 在MALDI-TOF质谱仪上进行测量,每个样品收集约700个激光照射

数据分析

  1. 硫解后的HPLC-UV
    1. 根据等式(1)校正从缩合单宁释放的每个黄烷-3-醇单体的摩尔浓度(μmol/L)(
      sub> Nth (1)
      在哪里,
      F th th =在硫解样品中黄烷-3-醇(或其硫醚)的摩尔浓度(μmol/L),
      F th N th =未硫解样品中黄烷-3-醇(或其硫醚)的摩尔浓度(μmol/L)。

      对于黄烷-3-醇硫醚,不需要校正,因为它们显然不作为提取物中的天然化合物存在。
      在表2中,报告了基于图3的色谱图的计算实例
      表3.来自图3的HPLC-UV色谱图的数据分析
      *注意:图3中使用的识别标签。

    2. 释放的单体单元的测量量提供了缩合单宁的分子结构的以下信息(Bianchi等人,2015):
      1. 特定黄烷醇单元(作为末端和延伸单元)的摩尔浓度的总和给出关于单宁中这种单体单元的量的指示。
      2. 存在于单宁中的不同单体单元(也作为立体异构体,如儿茶素和表儿茶素)之间的相对比例可以通过相关释放的黄烷醇之间的相对比例来估计。
      3. 可以识别终端单元的配置与扩展单元的配置之间的差异。
      4. 缩合单宁的平均聚合度(mDP)可以用公式(2)估计:

        在哪里,
        =总释放的黄烷-3-醇延伸的摩尔浓度(μmol/L)单元(黄烷-3-醇硫醚)硫化后,
        =总摩尔浓度(μmol/L)释放的黄烷-3-醇末端单元(天然黄烷-3-醇)硫化后 对于图3中的示例,使用表2中报告的数据,计算结果如下:


  2. MALDI-TOF质谱法
    1. 包含缩合单宁的植物提取物的典型MALDI MS光谱显示在图4中。该光谱包含沿着m/z轴彼此规则地间隔开的几组峰图案。每个峰表示具有单宁单体单元的确定组成的单宁寡聚链,该单体单元的质量报告在表4中。单体单元的质量通过从单体的质量计算(图1),减去2个氢原子的质量(例如儿茶素单体单元= 290.3-2 = 288.3Da)。峰的重复图案(图4)之间的距离允许识别低聚物链中的单体单元的类型和相应类别的单宁(表4)。
      注意:MALDI-TOF分析不允许区分具有相同质量的单体单元(例如儿茶素和顺式丁胺醇)。
    2. 在峰组内,距离16m/z或16m/z的倍数的峰(图4)表明在相同的缩合单宁中存在各种单体(建筑)单元,例如,没食子儿茶素(304.3Da)和儿茶素(288.3Da)。

      表4.不同类别的缩合单宁的典型单体单元



      图4.在800-2000m/z的银枞("Abies alba"[Mill。])树皮提取物的MALDI-TOF质谱,显示与含有儿茶素的缩合单宁相关的质量峰(或罗替尼多),没食子儿茶素和没食子酸酯

    3. 可以使用公式(3)进行与MALDI-TOF光谱的峰对应的单体单元组成的识别:
      + + em>?288.3 + z ?304.3 + M Cat + 2.0 (3)
      在哪里,
      x , y 和 z 分别是寡聚链中的fisetinidol,儿茶素(或robinetinidol) > M 是质谱中观察到的低聚链的m/z值,
      是 ,K + ),
      2.0是在低聚链端的两个氢原子的重量 在不同于黄烷-3-醇(例如,二苯乙烯)或部分(例如,,糖苷,没食子酸酯)的结构单元存在时,另外的重复峰基团在质谱中是可检测的,其从非酯化系列移动对应于非黄烷醇单元或部分的质量的间隙(Bianchi等人,2014)。如图4所示,含有没食子酸酯部分(152.1Da)的低聚物系列的存在导致与非酯化系列间隔152m/z的峰基团。在该实施例中,情况。 3应当被修饰,包括检测的部分,如Eq。在这种情况下,为了简单起见,代表fisetinidol的272.3单位被删除(因为它在图4的光谱中未检测到),并且阳离子化离子K + (39.1)报告。变量 y 和 z 具有与等式3和 w 是单宁低聚物中没食子酸酯部分的数量 M + K + = y ?288.3 + z ?304.3 + > w ?152.1 + 39.1 + 2.0 (4)
      这种方程的应用如表5所示
      表5.图4中报道的MALDI-TOF光谱的解释


      每种单宁低聚物的聚合度(DP)由等式(5)定义:
      DP = x + y + z (5)
      在哪里,
      x , y 和 z 具有与等式(3) 在糖基或酯部分存在下,它们的数目不应在DP的计算中考虑,因为它们被认为是单体单元的整合部分,例如,黄烷醇葡糖苷,黄烷醇没食子酸酯Bianchi等人,2014; Bianchi等人,2015年)。

  3. 结束语
    1. 在植物组织的热水提取物酸解后,MALDI-TOF质谱法与HPLC-UV的组合允许获得提取物中存在的缩合单宁结构上的各种指标。
    2. 两种方法都提供主要单体单元的指示,因此提取物中存在的缩合单宁类别
    3. HPLC-UV与硫解组合得到关于平均聚合度的信息,并且还可以区分具有不同立体化学的单体单元。其主要限制是在侧链(支化)的情况下降低硫解功效和在不同于儿茶素,没食子儿茶素或其差向异构体的单体单元之间的异丙基磺酸键的不可裂解性。
    4. MALDI-TOF质谱法提供聚合度的信息,其中具有最高检测m/z比的峰显示所测量样品中缩合单宁的最大(观察)聚合度。此外,不同于单纯黄烷-3-醇的单体单元的存在也可以通过MALDI-TOF质谱来阐明。

食谱

  1. 溶血介质
    500mg半胱胺盐酸盐HPLC级
    0.2ml盐酸37%HPLC级
    9.3ml甲醇HPLC级
    硫解介质的制备:
    1. 在20ml玻璃烧瓶中的分析天平上称量500mg半胱胺盐酸盐
    2. 用校准的Eppendorf移液管加入9.3ml甲醇,摇动混合物,直到任何固体残留物完全溶解
    3. 用校准的Eppendorf移液管加入0.2ml浓HCl(37%),并在室温下将混合物(35kHz,250W)超声处理5分钟。
    4. 将制备的硫解介质储存在冰箱(3-8°C)中,避光和空气直至使用
  2. HPLC流动相
    1. 溶液A:0.10%v/v三氟乙酸水溶液(约55ml /样品)
    2. 溶液B:0.08%v/v TFA的乙腈水混合物(ACN:水= 4:1,v/v)(约25ml /样品)

致谢

我们感谢瑞士国家研究计划"资源木材"(NRP66)为这项工作提供财政支持。该协议是从最初在Matthews等人中描述的程序开始开发的。 (1997),Torres和Lozano(2001),Pasch等人。 (2001)和Monagas等人。 (2010)。

参考文献

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引用:Bianchi, S., Kroslakova, I. and Mayer, I. (2016). Determination of Molecular Structures of Condensed Tannins from Plant Tissues Using HPLC-UV Combined with Thiolysis and MALDI-TOF Mass Spectrometry. Bio-protocol 6(20): e1975. DOI: 10.21769/BioProtoc.1975.
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