Qualitative and quantitative analyses of unbound phenolic compounds from pollen grains of pine (Pinus uncinata)

Abstract

The research in this master project is performed within an interdisciplinary RCN (The Research Council of Norway) research project (QUEST-UV: Quantitative estimates of past UV-B irradiance from fossil pollen) and includes characterization of phenolic compounds, as well as quantification of p-coumaric acid (p-CA), extracted from Pinus uncinata pollen. The pine pollen is protected by an outer exine/wall, mainly consisting of sporopollenin – a very chemical stable complex biopolymer. Empty pollen grains can be found maintained in fossil biomasses while the pollen inside will be degraded. Sporopollenin contains p-CA, so called wall bound p-CA, and the amounts of this p-CA is used as a proxy for UV-B exposure – for both modern and fossil sporopollenin – in Palaeo chemistry research. Nevertheless, modern pollen grains still have the pollen inside – and the pollen, or cytoplasmic content, also contains p-CA, so called free p-CA. The wall-bound p-CA are normally extracted from sporopollenin with relative harsh conditions as pyrolysis followed by derivatization before GC-MS quantification. However, modern pollen grains will potentially contain free p-CA as well - in addition to the wall-bound p-CA. Thus, these free amounts need to be extracted and quantified to see whether the amounts could affect the analyses of the modern wall-bound p-CA or not, challenging the traditional used methods for determining UV-B exposure on modern pollen grains. An Accelerated Solvent Extractor (ASE) was used in this project to facilitate extraction of the free p-CA and other phenolic compounds from modern pollen grains. An ASE-method was developed after initial optimization using elevated temperature (up to 90 °C) and pressure (1500 psi) for shorter periods in combination with a solvent series starting with hexane, followed by acetone and methanol. The ASE extracts were evaporated and redissolved in MeOH, and analyzed with DAD-HPLC, LR-LC MS and high field NMR spectroscopy. Free p-CA was identified with NMR (1D and 2D), DAD-HPLC and LR-LC-MS analyses, and a standard was used for comparison/verification (Merck). Quantification of the free p-CA in the pollen extracts was performed by using DAD-HPLC and a prepared p-CA standard curve. An estimate of the p-CA amounts suggests that free p-CA occupies 5.36 ppm of the pollen extract and 1.78 ppm of the total pollen sample. However structural elucidation of compounds in the extracted material revealed additional p-CA derivatives. When including the confirmed p-CA derivatives into the quantification this increased the total p-CA quantification up to 107.20 ppm of the extract and 35.60 ppm of the total pollen grain sample, a 20-fold increase. Adding the tentatively identified p-CA derivatives as well, the free p-CA constitute 117.92 ppm of the extract and 39.16 ppm of the total grain pollen sample, a 22-fold increase. Fourteen (1-14) compounds obtained in the pollen extracts were examined, whereas eleven of them (including p-CA (3)) were identified as phenolics with DAD-HPLC, LR-LC-MS and NMR analyses. Five of these with full structural elucidation (three p-CA derivatives (11, 13 and 14), naringenin (7) and chalcone naringenin (8)). Naringenin is a flavonoid often found as both a free and bound compound in pine pollen (Cheng et al., 2023; Strohl & Seikel, 1965). Chalcone naringenin is not as common in pine pollen, nevertheless it is the precursor of naringenin in the flavonoid synthesis (Figure 5), whereas the reaction between the two is in equilibrium. p-CA has been found both as a free and unbound compound in pine pollen (Cheng et al., 2023; Strohl & Seikel, 1965). The three p-CA derivatives on the other hand (11, 13 and 14), have not, to our knowledge, been identified as part of the free content of pine pollen before. In spite of that, similar compounds have been identified as a part of the complex biopolymer sporopollenin (Li et al., 2019), which is part of the pollen grain wall. In addition, one p-CA derivative was tentatively identified as a O-methyl coumaric acid (6), while two (9 and 10) were tentatively identified as p-CA derivatives similar to 11, 13 and 14. Taxifolin (4) and hydroxy benzoic acid (1) were tentatively identified as well, both found in pine pollen before (Cheng et al., 2023; Strohl & Seikel, 1965). Benzoic acid (2) was tentatively identified; however, this is not a biosynthetically derived phenolic compound.