Comprehensive Qualitative and Quantitative Analysis of Flavonoids in Dandelion (Taraxacum officinale) Flowers and Food Products

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Comprehensive qualitative and quantitative analysis of flavonoids in dandelion (Taraxacum officinale) flowers and food products Journal: Journal of Agricultural and Food Chemistry Manuscript ID Draft Manuscript Type: Article Date Submitted by the Author: n/a Complete List of Authors: Jedrejek, Dariusz; Instytut Uprawy Nawozenia i Gleboznawstwa, Department of Biochemistry and Crop Quality Pawelec, Sylwia; Instytut Uprawy Nawozenia i Gleboznawstwa, ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 1 1Comprehensive qualitative and quantitative analysis 2of flavonoids in dandelion (Taraxacum officinale) 3flowers and food products 4Dariusz Jedrejek,* Sylwia Pawelec 5 6†Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State 7Research Institute, Czartoryskich 8 Str., 24-100 Puławy, Poland 8 9*Corresponding author: E-mail: [email protected]; Phone: 0048 814786886 10 Page 1 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 2 12 ABSTRACT 13 Taraxacum officinale, a cosmopolitan perennial, is widely used as edible, forage, 14 honey and medicinal plant. Unsurprisingly, detailed LC-QTOF-MS profiling of flower 15 extract revealed the presence of several flavonoid signals most likely lacking literature 16 confirmation. Targeted isolation, including DoE planning based extraction, led to the 17 isolation of 11 flavonoids, of which three were new (16, 18 and 33). Their structures 18 were established by the use of NMR technique. Both biflavones (16 and 18) were 19 structured from two luteolin molecules, linked by a C-C bond through IB (C-6′) and IIA 20 (C-6″ or C-8″) rings. Novel flavonolignan (33) composed of tricin and unusual, 21 containing a carboxyl function, lignan moiety. The contents of 28 flavonoids 22 (glycosides, aglycones, biflavones and flavonolignans) was determined, based on LC23 MS/MS method, in raw flowers and dandelion syrups and tincture. Multivariate 24 analyses showed the separation of spring and autumn flowers and a high diversity of 25 food products, and helped identify metabolites correlated with the samples. 26 27 KEYWORDS: dandelion, flavonoids, biflavones, flavonolignans, NMR, UHPLC-MS/MS Page 2 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 3 29 INTRODUCTION 30 The genus Taraxacum, commonly known as dandelion, is one of the better-known and 31 commonly used by humans representatives of Asteraceae plant family. Taraxacum 32 officinale F.H.Wigg. (common dandelion) is a yellow-flowering herbaceous plant native 33 to temperate climates, usually spreading in pastures and wasteland.1 Over the 34 centuries, common dandelion and other local Taraxacum species (e.g. T. mongolicum 35 and T. coreanum) have been used in the European, Chinese and North American folk 36 medicines as a remedy in the treatment of various ailments, such as liver and digestive 37 disorders, diabetes, skin inflammations, arthritic and rheumatic diseases.2 Nowadays, 38 the whole plant (including root, leaves, and flowers) has “generally recognized as safe” 39 status approved by U.S. Food and Drug Administration, and is commonly processed 40 into different edible products – salads, teas, tinctures, syrups, jellies, cakes, wines, 41 honeys and coffee substitutes.3 In addition, T. officinale herb is a Pharmacopeial 42 material that is approved for use as an herbal supplement, in the form of tincture and 43 tea preparations, by European Medicines Agency.4,5 44 Taraxacum herb extracts (water and alcoholic) have been shown to exert a wide range 45 of in vitro and in vivo biological actions, such as antioxidant, anti-inflammatory, 46 anticarcinogenic, anti-hyperglycemic, anti-thrombotic, antimicrobial, antiviral, and 47 hepatoprotective, in a number of scientific studies.5–11 Polyphenols, mainly 48 hydroxycinnamic acids and flavonoids, as well as terpenoids and sesquiterpene 49 lactones have been found as major bioactive metabolites of dandelion plant 50 material.12,13 Albeit, despite the generally well-recognized chemical profile of dandelion 51 herb and flowers, new metabolites (or metabolites not yet reported in the taxon) are 52 occasionally documented, as exemplified by the recently characterized flavonoids of 53 T. mongolicum or T. officinale fruits.14,15 In addition, despite the fairly widespread use Page 3 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 4 54 of dandelion herb, the amount of detailed research devoted to the quantitative content 55 of bioactive metabolites (beyond colorimetric measurement of total polyphenols and 56 flavonoids) seems to be negligible and insufficient.15–20 It should be noted that the vast 57 majority of quantitative works on dandelion herb flavonoids include only a few major 58 components (such as quercetin and luteolin and their glycosides) in a single 59 analysis.16–20 60 The work is phytochemically exploratory, with several specific objectives, including 61 detailed HR-QTOF-MS qualitative analysis of Taraxaci Flos, targeted isolation and 62 identification of flavonoid oligomers and flavonolignans, and measurement of the 63 quantitative content of nearly 30 flavonoid compounds in raw plant material (spring and 64 autumn flowers) and food products based on dandelion flowers (syrups and tincture). 65 Page 4 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 5 66 MATERIALS AND METHODS 67 Plant Material. Dandelion flowers were collected from a natural site in southeastern 68 Poland (Kazimierski Landscape Park, 51.378605 N, 22.072647 E) in 2021 (October), 69 2022 (April and October) and 2023 (April). Plant material was preserved by freeze70 drying, then was finely ground with electric mill and sieved (1 mm), and finally stored 71 at 4 °C until use. A voucher specimens are deposited in our laboratory. In addition, 72 three different commercially available dandelion flower-based syrups (syrup I (48% 73 flower decoction), II (60% flower decoction) and III (40% flower decoction), purchased 74 on the Polish market in May 2023) and a handmade ethanol flower tincture (formulation 75 according to the medicinal plant textbooks21–23) were used in the study. Detailed 76 information on syrup compositions and a description of the tincture preparation are 77 given in the Supplementary Materials (Descriptions S1-S2). 78 Instruments and Materials. 1Hand 13C-NMR spectra (including oneand two79 dimensional) were recorded on a Bruker Avance III HD Ascend-500 spectrometer 80 (Bruker BioSpin, Rheinstetten, Germany), equipped with 3 mm broad-band inverse 81 (BBI) probe. HRESIMS spectra, exact masses of compounds, MS/MS fragmentation 82 patterns, and molecular formulas were obtained using a Bruker Impact II HD (Bruker, 83 Billerica, MA, USA) quadrupole time-of-flight (Q-TOF) mass spectrometer coupled with 84 a Thermo Scientific Ultimate 3000 RS chromatographic system. HPLC separations 85 towards extract fractionation and compounds isolation were performed with a Dionex 86 (Sunnyvale, CA, USA) system equipped with a photodiode array detector PDA-100 87 and fraction collector FC 204 (Gilson, Middleton, WI, USA) using a Sephadex LH-20 88 (Merck, Warsaw, Poland) column (80 × 2.8 cm i.d.) and reversed-phase (RP) HPLC 89 columns (Atlantis T3, 250 × 19 mm, 10 µm, and 250 × 10 mm, 5 µm, Waters, Milford, 90 MA, USA; and Cosmosil πNAP, 250 × 10 mm, 5 µm, Nacalai Tesque Inc., Kyoto, Page 5 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 6 91 Japan). Quantitative UHPLC analyses were performed using a Waters ACQUITY 92 chromatographic system equipped with PDA and Xevo TQ-S micro Triple Quadrupole 93 (TQD) mass detector and RP column (BEH C18, 100 × 2.1 mm, 1.7 µm, Waters). Plant 94 material, extracts and isolated compounds were dried using Gamma 2–16 LSC freeze 95 dryer (Christ, Osterode am Harz, Germany). 96 Extraction and Isolation of Flavonoids. The powdered plant material (750 g) was 97 defatted with n-hexane (6 L) over 10 h using Soxlet apparatus. The residue (600 g) 98 was twice extracted with 90% methanol (MeOH) (2 × 6 L) at 65 °C and 180 rpm for 55 99 min (Innova 44R shaker, Eppendorf, Hamburg, Germany), based on the optimization 100 results of Design of Experiment (DoE) design (Description S3, Tables S1-S4, Fig. S1101 S2). The raw extract (E1) was filtered and concentrated under reduced pressure, 102 followed by liquid-liquid extraction with ethyl acetate (3 × 1 L). After evaporation of the 103 solvent, the residue was suspended in 50% t-BuOH (v/v) and freeze-dried, yielding 104 10.4 g of flavonoid extract (E2). The E2 was further fractionated on a Sephadex LH-20 105 (Merck) column (100 × 2.8 cm), which was eluted with 95% MeOH (v/v) at a flow rate 106 of 2.4 mL/min. The separation was under the control of HPLC-UV350nm detection and 107 additionally monitored by LC-MS analyses. The seven pooled fractions (A–G) were 108 collected, concentrated, and freeze-dried to give 1.23 g of fraction A, 0.95 g of fraction 109 B, 1.21 g of fraction C, 483 mg of fraction D, 51 mg of fraction E, 49 mg of fraction F, 110 and 31 mg of fraction G. Fractions promising with new metabolites, including A, and 111 D-G, were further separated to individual compounds using reverse-phase HPLC 112 (Dionex HPLC-PDA system). Fraction A was chromatographed in 3 steps using 113 Atlantis T3 (250 × 19 mm, Waters; gradient 20–55% of acetonitrile (MeCN), flow rate 114 7 mL/min, temperature 30 °C) and Cosmosil πNAP (Nacalai Tesque Inc.; gradient 25– 115 30% of MeCN, flow rate 3.5 mL/min, temperature 30 °C; and gradient 60–64% of Page 6 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 7 116 MeOH, flow rate 3 mL/min, temperature 25 °C) columns. Fractions D-G were purified 117 using Atlantis T3 (250 × 10 mm, Waters; gradient 26–29% of MeCN, flow rate 3 118 mL/min, temperature 25 °C) column. Aforementioned chromatographic methods 119 resulted in 11 pure flavonoid metabolites, including compound 16 (17 mg) was 120 recovered from fraction E, compound 18 (14 mg) from fraction G, and compound 33 (9 121 mg) from fraction A. 122 Liquid Chromatography High-Resolution Mass Spectrometric Analysis. HR123 MS/MS data were obtained using a Bruker Impact II HD Q-TOF mass spectrometer 124 coupled with a Ultimate 3000 RS (Thermo Scientific) UHPLC system. The MS operated 125 in electrospray ionization and both polarity modes, with the following settings: mass 126 scan range 50–1400 m/z; capillary voltage 4.0 kV (ESI+) or 3.0 kV (ESI–); nebulizer 127 and drying gas (N2) 2.0 bar and 10 L/min, respectively; dry gas temperature 220 °C. 128 The MS/MS spectra were registered using a collision energy (CE) of 50 eV with 129 stepping between 50–125% of CE. The acquired data were calibrated internally with 130 sodium formate (10 mM solution in 50% 2-propanol) which was injected to the ion 131 source before the sample analysis. Data were processed using DataAnalysis 4.4 132 software (Bruker). Chromatographic separation was carried out on an HSS C18 133 column (100 × 2.1 mm, 1.7 µm, Waters) equipped with a pre-column. A 28 min linear 134 gradient (10→35%) of acetonitrile–water (both acidified with 0.1% formic acid), with a 135 flow rate of 0.4 mL/min, was applied. The column was held at 45 °C. Injection volume 136 was 2 µL. 137 NMR Measurements. 1D and 2D NMR spectra were recorded on Bruker Avance III 138 HD Ascend-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with 139 5 mm broad-band inverse (BBI) probe. The 1H and 13C NMR spectra (at 500 and 125 140 MHz, respectively) were measured in DMSO-d6 at 30 °C. Chemical shifts are given on Page 7 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 8 141 the δ scale. Data processing was performed with the Topspin software (version 3.5pl2, 142 Bruker BioSpin). 143 Quantitative UHPLC Analysis of Flavonoids. For plant material (autumn 2021144 spring 2023), a 250 mg of ground flowers were extracted twice (2 × 4 mL) using 80% 145 MeOH in two steps: I – thermo-shaking (20 min, 60 °C, 800 rpm; Thermomixer, 146 Eppendorf), and II – ultrasonic bath (10 min, 40 °C, 35 kHz). After centrifugation (10 147 min, 5 °C, 12,000 × g), the supernatants were combined and dried in a thermo-block 148 (40 °C) under N2 and re-dissolved in 2 mL of 80% MeOH. Samples of syrups (4 mL) 149 and tincture (15 mL after concentration to 3 mL) were purified using SPE technique 150 (Oasis HLB 500 mg column (Waters)). Polar components were eluted with 2% MeOH, 151 while flavonoids using 95% MeOH (containing 1% ammonia). Purified samples were 152 dried similarly to the extracts and re-dissolved in 1 mL of 80% MeOH. All samples were 153 stored at -20 °C until analysis. Five independent extracts were prepared for ground 154 flowers and four purified samples for syrups and tincture. Prior to analyses, the 155 samples were appropriately diluted with 80% MeOH and filtered (0.22 µm). 156 Quantitative UHPLC-MS/MS (Waters) measurements were based on multiple reaction 157 monitoring (MRM) detection mode and external calibration for the 16 reference 158 flavonoid substances. Detailed information regarding detection: capillary voltage, 159 precursor→fragment ions’ transitions, and collision energy is shown in Table S6; while 160 calibration parameters are placed in Table S7. The general chromatographic 161 separation conditions were as follows: column BEH C18 (100 × 2.1 mm, 1.7 µm, 162 Waters), column temperature 45 °C, a 14 min linear gradient (15→35%) of acetonitrile– 163 water (both acidified with 0.1% formic acid), flow rate of 0.4 mL/min, and injection 164 volume 2 µL. The MS detector was operated in positive ion mode with the following 165 settings: capillary voltage (3.1 kV), source and desolvation gas temperatures (150 °C Page 8 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 15 314 distance and Ward's algorithm) analyses highlighted the great distinctness of spring 315 and autumn flowers, as well as a greater variation in spring material (separation of 316 spring flowers 2022 and 2023 along PC2) compared to autumn samples was observed 317 (Fig. 4A-B). Nonetheless, the separation of autumn flowers from the two harvest years 318 was later evident along PC3 (Fig. S23B). The PCA biplots (Fig. 4C and S23C) and 319 heat map (Fig. 4B) confirmed the seasonal clustering of the flowers with respect to 320 chemical composition, and among the positive correlations recorded were: autumn 321 2021 – compounds 5, 24 and 27; autumn 2022 – compounds 21 and 40-43; spring 322 2022 – compounds 15, 18+19, 22, 25-26, 28, 33 and 37-39; spring 2023 – compounds 323 3, 8, 14, 16 and 20. The current work is the first to attempt to identify seasonal 324 phytochemical variation in dandelion flowers. 325 The quantitative flavonoid analysis of dandelion food products revealed their high 326 variability (6.5-30.9 µg/mL of total flavonoids, Table 3), and they were generally 327 arranged in the following order: syrup I ≥ tincture > syrup II > syrup III. The proportion 328 of the specified flavonoid groups in the samples was also heterogeneous: glycosides 329 (70% of total flavonoids) and aglycones (27%) predominated in syrup I, while in syrups 330 II-III the proportion was reversed (aglycones – about 70% and glycosides – about 331 25%), similarly, aglycones (83%) definitely predominated in the tincture, but 332 flavonolignans (11%) were second. In each product, total biflavones were a minor 333 fraction (less than 2% of total flavonoids), but in the tincture (0.35 µg/mL) their level 334 was several times (3-15) higher than those in syrups (0.02–0.1 µg/mL) (Table 3). 335 Surprisingly, the three commercial syrups differed significantly in their quantitative 336 flavonoid profile, which, in addition, was not simply related to the manufacturer’s 337 declared dandelion decoction content (syrups I-III – 48%, 60% and 40%, respectively). 338 According to Table 3, the distinguishing feature for syrup III was the lowest recorded Page 15 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 16 339 concentrations of all flavonoids, for syrup I the highest levels of the three luteolin 340 glycosides, and for the tincture the highest concentrations of most other compounds 341 (aglycones, biflavones and flavonolignans). It can be noted that the number of 342 publications devoted to quantitative chemical analysis of dandelion food products, like 343 flowers, is negligible. Overall, the flavonoid levels in commercial syrups, especially I-II, 344 are in fairly close agreement with the result obtained for the handmade syrup (22 µg 345 luteolin eq/mL) in the paper by Majewski et al. 2022.11 On the other hand, our alcoholic 346 flower tincture (85 g/1000 mL) had a significantly higher flavonoid content (269 µg/g 347 dw) compared to herb tincture (50 g/500 mL, 90 µg/g dw) developed by Epure et al. 348 2023.20 349 Subsequently, multivariate statistical methods were used to graphically illustrate the 350 differences/similarities between dandelion food products. The results of PCA (95% 351 variability covered by the first two components, Fig. S24) and HCA (Ward's algorithm 352 and Euclidean distance) analyses confirmed the high variability of the products 353 (separate grouping of samples, Fig. 4D-E). Similarly, the PCA-biplots (Fig. 4F and S24) 354 and heat map (Fig. 4E) confirmed the sample clustering with respect to chemical 355 composition, which was previously highlighted at the level of Table 3 (e.g., the positive 356 correlation of syrup I with glycosides, as well as tincture with flavone aglycones). 357 Dandelion is a well-known useful plant with many documented biological properties. 358 HR-QTOF-MS/MS analysis of the flower extract of this plant revealed the presence of 359 numerous flavonoids, among which were well-known and bioactive metabolites such 360 as luteolin and its glycosides, chrysoeriol, tricin, apigenin. In addition, more than 20 361 other flavonoid compounds were detected, including several oligomers and more than 362 a dozen flavonolignans (lignan derivatives of tricin). Targeted isolation of metabolites 363 using extraction optimized by DoE planning and various chromatographic methods led Page 16 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 17 364 to the isolation of 11 metabolites whose chemical structures were determined by 365 spectroscopic data. Among them were four biflavones, including philonotisflavone 366 (previously reported in the taxon) and dicranolomin (reported for the first time in the 367 taxon), as well as two new compounds, 6',8''-biluteolin (16) and 6',6''-biluteolin (18). 368 On the other hand, among the seven flavonolignans isolated, there was one new 369 compound, taraxalignan A (33), as well as six already described metabolites, except 370 that two methoxycalquiquelignan derivatives (40-41) have not yet been reported in the 371 Taraxacum. The quantitative content of more than 30 flavonoids was determined, 372 based on UHPLC-ESI-MS/MS analysis, in raw flowers and dandelion food products. 373 Comparative and chemometric analysis (including PCA, HCA and PLS-DA) using 374 quantitative flavonoid data showed seasonal clustering of flower samples (spring vs. 375 autumn), with compounds 27, 24, 34, 29, 35, 16, 5, 32 and 25 among the traits with 376 the greatest impact. A similar approach for food products (three commercial syrups 377 and a handmade tincture) showed their high variation in phytochemical composition. 378 Our work is the first such comprehensive study of flavonoids in dandelion flowers, and 379 fills many phytochemical knowledge gaps for this plant and the food products made 380 from it. 381 Page 17 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 18 382 ACKNOWLEDGEMENTS 383 We thank Lukasz Pecio for performing NMR experiments, Agata Soluch for technical 384 assistance and Anna Rybicka for hosting the UHPLC-MS instrument. 385 386 FOUNDING SOURCES 387 The work was partially supported from the Polish National Science Centre project 388 PRELUDIUM 2022/45/N/NZ9/03440. 389 390 SUPPORTING INFORMATION 391 Supplementary Materials include Descriptions S1-S3, Tables S1-S7 and Figures S1392 S24. The material is available free of charge via Internet at http:// pubs.acs.org. 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No RT (min) UV λmax (nm) Observed [M+H]+, m/z Formula Error (ppm) MS/MS fragments Identity 1 1.69 323, 295sh 163.0393/181.0498 C9H9O4 -1.2 89.0384, 117.0336, 136.0516 caffeic acid* 2 2.73 308 147.0445/165.0547 C9H7O2 -0.6 - coumaric acid* 3 3.78 254, 336 449.1084 C21H21O11 -1.1 287.0564 luteolin-7-O-glucoside* 4 4.83 326, 295sh 499.1241/517.1351 C25H25O12 -1.4 163.0397 3,5-di-caffeoylquinic acid* 5 5.55 267, 337 449.1086 C21H21O11 -1.8 287.0555 luteolin-4'-O-glucoside* 6 6.36 326, 295sh 499.1241/517.1348 C25H25O12 -1.5 163.0395 di-caffeoylquinic acid 7 6.59 250, 269, 345 463.1240 C22H23O11 -1.1 301.0712 chrysoeriol-O-hexoside 8 6.84 267, 340 449.1083 C21H21O11 -0.9 287.0558 luteolin-3'-O-glucoside* 9 7.01 - 442.2071 [M+NH4]+ C21H32NO9 0.0 171.1172, 217.1227 taraxinic acid-1-O-glucoside* 10 8.49 254, 341 855.1186 C45H27O18 0.7 285.0400, 419.0771, 703.1097 flavonoid (tri-luteolin) 11 8.98 254, 344 855.1187 C45H27O18 0.6 285.0401, 153.0188, 703.1081 flavonoid (tri-luteolin) 12 9.31 256, 344 855.1183 C45H27O18 1.0 285.0403, 153.0186, 703.1076 flavonoid (tri-luteolin) 13 9.80 327, 295sh 513.1395/531.1500 C26H27O12 -0.5 163.0395 caffeoyl-feruloylquinic acid 14 10.16 252, 346 287.0557 C15H11O6 -2.3 153.0188, 135.0446 luteolin* 15 11.85 253, 341 571.0882 C30H19O12 -1.9 419.0775, 153.0192, 285.0389 philonotisflavone# 16 12.53 254, 344 571.0881 C30H19O12 -1.8 419.0778, 153.0195, 445.0560 taraxabiluteolin A# 17 13.23 254, 341 855.1200 C45H27O18 -0.9 153.0186, 551.0983, 703.1094 flavonoid (tri-luteolin) 18 13.66 254, 342 571.0885 C30H19O12 -2.5 419.0778, 445.0562 taraxabiluteolin B# 19 13.68 254, 340 571.0887 C30H19O12 -2.6 419.0778, 445.0563 dicranolomin# 20 14.09 265, 338 271.0609 C15H11O5 -3.1 153.0183 apigenin* 21 15.52 250, 269, 346 301.0710 C16H13O6 -1.0 258.0529, 286.0478 chrysoeriol* 22 15.67 250, 269, 351 331.0816 C17H15O7 -1.1 315.0501, 203.0338 tricin* 23 16.25 255, 342 853.1036 C45H25O18 -0.1 153.0188, 269.0439, 497.0499 flavonoid oligomer 24 17.34 268, 339 497.1441 C26H25O10 0.2 331.0815, 315.0503 calquiquelignan D# 25 18.00 269, 339 527.1550 C27H27O11 -0.5 331.0824, 315.0506 salcolin B# 26 18.12 268, 330 331.0816 C17H15O7 -1.0 315.0507, 270.0528 apometzgerin* 27 18.74 268, 337 497.1447 C26H25O10 -0.9 331.0818, 315.0504 calquiquelignan E# 28 19.58 269, 337 527.1549 C27H27O11 -0.2 331.0822, 315.0507 salcolin A# 29 20.25 269, 342 675.2076 C36H35O13 -0.5 331.0820, 315.0510, 270.0533 flavonolignan (tricin derivative) 30 20.69 270, 337 495.1286 C26H23O10 -2.0 284.0687, 331.0821, 255.0661 flavonolignan (tricin derivative) 31 20.86 297, 310 787.3688 C45H55O12 -1.9 147.0444, 204.1030, 641.3354 N-coumaroyl-spermine derivative 32 21.16 269, 341 705.2183 C37H37O14 -0.7 331.0830, 315.0512 flavonolignan (tricin derivative) 33 21.46 270, 321 525.1399 C27H25O11 -1.5 331.0826, 284.0693, 255.0664 taraxalignan A# 34 21.51 269, 340 675.2080 C36H35O13 -1.2 331.0822, 315.0511 flavonolignan (tricin derivative) 35 22.03 269, 340 675.2079 C36H35O13 -1.0 331.0823, 315.0512 flavonolignan (tricin derivative) Page 21 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 22 36 22.35 268, 339 479.0979 C25H19O10 -1.4 257.0456, 435.1101, 287.0557 flavonoid 37 22.56 269, 341 705.2185 C37H37O14 -1.1 331.0830, 315.0512 flavonolignan (tricin derivative) 38 23.14 269, 341 705.2186 C37H37O14 -1.1 331.0832, 315.0513 flavonolignan (tricin derivative) 39 23.89 269, 341 495.1299 C26H23O10 -2.8 287.0560, 315.0509 flavonolignan (tricin derivative) 40 24.03 268, 340 511.1614 C27H27O10 -3.0 331.0828, 315.0513 7''-methoxycalquiquelignan D# 41 24.58 268, 339 511.1610 C27H27O10 -2.2 331.0826, 315.0515 7''-methoxycalquiquelignan E# 42 25.10 269, 340 541.1715 C28H29O11 -1.9 331.0821, 315.0509 flavonolignan (methoxysalcolin B) 43 25.13 269, 339 541.1713 C28H29O11 -1.7 331.0822, 315.0507 flavonolignan (methoxysalcolin A) * the identity of the compound was confirmed by comparison with reference substance; # compound was isolated and identified in this study Page 22 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 23 Table 2. 1H and 13C NMR data for new flavonoid compounds 16, 18 and 33 (in DMSO-d6). 16 (taraxabiluteolin A) 18 (taraxabiluteolin B) 33 (taraxalignan A) No δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) 2 165.9 - 166.1 - 162.6 - 3 106.2 5.97, s 105.9 5.99, s 104.8 6.99, s 4 181.1 - 181.3 - 181.6 - 5 161.2 - 161.3 - 161.3 - 6 98.7 6.08, d (2.1) 98.8 6.11, d (2.0) 99.3 6.18, br s 7 164.1 - 164.2 - 165.6 - 8 93.2 5.74, d (2.0) 93.4 5.97, d (2.0) 94.4 6.51, br s 9 157.2 - 157.4 - 157.5 - 10 103.2 - 103.4 - 103.3 - 1' 123.0 - 122.8 - 125.5 - 2' 115.9 7.29, s 115.6 7.22, s 104.2 7.30, s 3' 145.1 - 144.9 - 152.2 - 4' 148.3 - 148.1 - 139.3 - 5' 119.9 6.82, s 119.9 6.74, s 152.2 - 6' 122.9 - 123.8 - 104.2 7.30, s 3',5'-OMe - - - - 56.3 3.77, s 1'' - - - - 139.3 - 2'' 163.8 - 163.8 - 111.7 7.51, d (2.0) 3'' 102.4 6.59, s 102.9 6.69, s 147.5 - 4'' 181.8 - 181.7 - 152.2 - 5'' 160.2 - 158.3 - 114.9 6.88, d (8.3) 6'' 98.5 6.31, s 111.8 - 123.6 7.58, dd (8.3, 2.0) 7'' 161.4 - 161.5 - 83.3 5.52, t (5.6) 8'' 107.1 - 93.4 6.53, s 62.5 3.78, o 3.84, o 9'' 153.8 - 156.0 - 194.0 - 10'' 103.4 - 103.4 - - - 3''-OMe - - - - 55.6 3.81, s 1''' 121.3 - 121.4 - - - 2''' 113.6 7.04, d (2.3) 113.3 7.42, d (2.3) - - 3''' 145.7 - 145.8 - - - 4''' 149.8 - 149.9 - - - 5''' 115.6 6.76, d (8.3) 116.1 6.90, d (8.2) - - 6''' 118.6 7.02, dd (2.2, 8.3) 119.0 7.43, dd (2.3, 8.2) - - Page 23 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 24 Table 3. Flavonoid content of dandelion flowers and food products based on them (syrups and tincture) (mean ± SD). Compound numbering is consistent with Table 1. Flowers Food products No 2021-Autumn 2022-Spring 2022-Autumn 2023-Spring Syrup I (48% decoction) Syrup II (60% decoction) Syrup III (40% decoction) Tincture (85 g/1000 mL) µg/g (flowers) or ng/mL food products 5 213.41 ± 44.9a 10.15 ± 0.50b 8.74 ± 0.99b 11.45 ± 0.19b 211.84 ± 8.05a 59.52 ± 0.85c 68.76 ± 0.79b 76.96 ± 2.03b 15 35.79 ± 1.54c 64.08 ± 2.89a 37.23 ± 5.84bc 45.23 ± 7.85b 38.79 ± 1.88c 91.34 ± 5.23b 24.06 ± 2.39d 317.65 ± 3.88a 16 2.53 ± 0.18b 3.08 ± 0.11b 1.89 ± 0.15c 4.30 ± 0.57a tracec 3.36 ± 0.11b trace 11.96 ± 0.09a 18+19a 1.65 ± 0.08b 2.84 ± 0.14b 1.67 ± 0.24b 1.95 ± 0.34a trace 2.85 ± 0.35b trace 15.68 ± 0.61a 20 10.48 ± 0.86a 8.88 ± 0.87b 7.06 ± 0.35c 10.75 ± 0.43a 24.25 ± 0.69c 53.71 ± 0.34b 17.25 ± 0.17d 158.70 ± 3.01a 26 8.85 ± 0.35b 12.85 ± 0.89a 6.32 ± 0.6c 9.66 ± 0.34b 294.98 ± 6.74c 312.59 ± 7.87b 60.59 ± 0.52d 577.92 ± 7.86a 29b 1.17 ± 0.03a 0.98 ± 0.10b 1.08 ± 0.07ab 0.72 ± 0.02c 6.12 ± 0.19c 7.57 ± 0.30b 1.38 ± 0.02d 12.16 ± 0.07a 30b trace trace trace trace trace trace trace trace 32b 0.46 ± 0.02c 0.75 ± 0.06a 0.48 ± 0.02c 0.58 ± 0.03b 6.54 ± 0.20a 3.31 ± 0.06c 1.12 ± 0.01d 5.66 ± 0.05b 33 21.19 ± 0.64b 30.65 ± 6.26b 18.76 ± 1.17b 23.71 ± 1.94a 45.16 ± 0.82c 65.47 ± 2.14b 10.45 ± 0.57d 231.39 ± 1.12a 34b 0.78 ± 0.02a 0.65 ± 0.05b 0.72 ± 0.05a 0.49 ± 0.02c 2.29 ± 0.09c 3.41 ± 0.12b trace 8.01 ± 0.18a 35b 0.86 ± 0.02a 0.73 ± 0.07b 0.81 ± 0.05ab 0.54 ± 0.02c 1.97 ± 0.07c 3.04 ± 0.08b trace 9.76 ± 0.11a 36b trace trace trace trace trace trace trace trace 37b 0.31 ± 0.01c 0.51 ± 0.04a 0.32 ± 0.02c 0.37 ± 0.02b 2.42 ± 0.10b 1.59 ± 0.06c trace 3.42 ± 0.06a 38b 0.40 ± 0.01c 0.70 ± 0.06a 0.42 ± 0.03c 0.49 ± 0.03b 2.31 ± 0.09b 1.67 ± 0.03c trace 5.48 ± 0.13a 39b 4.91 ± 0.28c 7.64 ± 0.76a 6.23 ± 0.96b 5.24 ± 0.69bc 9.20 ± 0.21c 10.80 ± 0.42b 2.52 ± 0.03d 64.25 ± 0.28a 40b 0.43 ± 0.02b 0.35 ± 0.06b 0.57 ± 0.09a 0.41 ± 0.03b 0.49 ± 0.01c 0.94 ± 0.04a trace 0.72 ± 0.06b 41b 0.55 ± 0.03ab 0.45 ± 0.09b 0.59 ± 0.09a 0.51 ± 0.04ab 0.69 ± 0.01c 1.35 ± 0.10a trace 1.07 ± 0.08b 42+43b 5.01 ± 0.25bc 4.76 ± 0.34c 6.78 ± 1.59a 6.35 ± 0.47ab 1.59 ± 0.03c 2.61 ± 0.22b 0.72 ± 0.12d 6.15 ± 0.16a mg/g (flowers) or µg/mL food products 3 0.87 ± 0.05a 0.87 ± 0.05a 0.69 ± 0.04b 0.92 ± 0.12a 10.72 ± 0.38a 1.96 ± 0.05b 0.84 ± 0.01c 0.81 ± 0.03c 8 0.87 ± 0.05a 0.87 ± 0.05a 0.69 ± 0.04b 0.92 ± 0.12a 10.75 ± 0.39a 1.71 ± 0.04b 0.79 ± 0.01c 0.75 ± 0.02c 14 2.22 ± 0.08a 2.27 ± 0.20a 1.76 ± 0.06b 2.43 ± 0.12a 6.60 ± 0.07c 9.61 ± 0.16b 4.04 ± 0.03d 21.08 ± 0.33a 21 0.16 ± 0.00a 0.15 ± 0.02a 0.17 ± 0.02a 0.16 ± 0.00a 0.59 ± 0.01c 0.93 ± 0.01b 0.33 ± 0.00d 2.14 ± 0.03a 22 0.06 ± 0.00c 0.10 ± 0.01a 0.05 ± 0.00c 0.07 ± 0.01b 0.71 ± 0.01a 0.44 ± 0.00c 0.14 ± 0.00d 0.64 ± 0.01b Page 24 of 30 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry