A Simple High-Throughput Procedure for Microscale Extraction of Bioactive Compounds from the Flowers of Saint John’s Wort (Hypericum perforatum L.)
Abstract
1. Introduction
2. Materials and Methods
2.1. Plant Material
2.2. Determination of Flower Fresh Weight
2.3. Collection and Extraction of a Bulk Flower Sample
2.4. Collection and Extraction of Single Flower Samples
2.5. LC/DAD/MS Analysis
2.6. LC/UV-Vis Analysis
2.7. Statistical Analysis
3. Results
3.1. Selection of the Stage of Flower Development for Analysis
3.2. Bulk-Flower Extraction and LC-DAD-QTOF Analysis
3.2.1. LC-DAD-QTOF Analysis and Identification of the Compounds
3.2.2. Testing Different Material-to-Solvent Ratios
3.2.3. Testing Different Durations of the Extraction
3.3. Single-Flower Extraction
3.4. Analysis of Plants Obtained Through Apomixis Using the “Single-Flower” Procedure
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
SSR | Simple sequence repeat |
LC-MS | Liquid chromatography–mass spectrometry |
DAD | Diode array detector |
QTOF | Quadrupole time of flight |
ESI | Electrospray ionization |
TIC | Total ion current |
RSD | Relative standard deviation |
UV-Vis | Ultraviolet–visible |
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Flower Stage | Plant 3_29 | Plant 90_44 | Plant 139_28 | Across Genotypes | ||||
---|---|---|---|---|---|---|---|---|
Mean FW | RSD | Mean FW | RSD | Mean FW | RSD | Mean FW | RSD | |
1st | 15.14 | 4.83 | 23.24 | 12.05 | 19.04 | 3.86 | 19.14 ± 4.05 a | 21.16 |
2nd | 21.79 | 4.66 | 41.92 | 14.58 | 34.16 | 7.93 | 32.62 ± 10.15 a | 31.13 |
3rd | 47.36 | 2.36 | 53.63 | 1.24 | 52.58 | 6.02 | 51.19 ± 3.36 b | 6.57 |
4th | 62.27 | 5.96 | 66.91 | 13.90 | 65.23 | 4.30 | 64.80 ± 2.35 b | 3.63 |
5th | 43.48 | 4.32 | 48.92 | 2.43 | 60.08 | 4.31 | 50.83 ± 8.46 b | 16.65 |
Compound | tR min | Identity | Trivial Name | UV λ Max nm | [M−H]− m/z | Δppm | MS2 Fragments | Reference |
---|---|---|---|---|---|---|---|---|
1 * | 5.1 | caffeoylquinic acid | chlorogenic acid | 218, 300, 325 | 353.0885 | 1.96 | 191.06, 179.03, 135.05 | [50,51] |
2 | 7.3 | procyanidin B1 | - | 279 | 577.13583 | 1.17 | 425.09, 407.08, 289.07, 125.02 | [52,53] |
3 * | 8.8 | catechin | - | 278 | 289.0721 | 1.16 | 245.08, 205.05, 125.02 | [54] |
4 * | 10.2 | procyanidin B2 | - | 280 | 577.1357 | 0.94 | 425.09, 407.08, 289.07, 125.02 | [52,53] |
5 * | 11.2 | epicatechin | - | 279 | 289.0720 | 0.82 | 245.08, 205.05, 125.02 | [54] |
6 * | 11.9 | procyanidin C1 | - | 280 | 865.1987 | 0.19 | 695.14, 577.13, 407.08, 287.06, 125.02 | [55] |
7 * | 14.8 | quercetin-3-O-rutinoside | rutin | 257, 358 | 609.1472 | 1.79 | 300.03, 271.02, 255.03, 179.00, 151.00 | [54] |
8 * | 15.2 | quercetin-3-O-galactoside | hyperoside | 255, 355 | 463.0892 | 2.15 | 300.03, 271.02, 255.03, 179.00, 151.00 | [54] |
9 ** | 15.4 | quercetin-3-O-glucoronide | querciturone | 255, 355 | 477.0683 | 1.75 | 301.03, 179.00, 151.00 | [56] |
10 ** | 15.5 | quercetin-3-O-glucoside | isoquercitrin | 255, 355 | 463.0889 | 1.5 | 300.03, 271.03, 255.03 | [57,58] |
11 * | 16.9 | quercetin-3-O-rhamnoside | quercitrin | 255, 345 | 447.0941 | 1.81 | 300.03, 271.02, 255.03, 179.00, 151.00 | [58] |
12 * | 24.9 | I3, II8-biapigenin | amentoflavone | 268, 330 | 537.0834 | 1.26 | 443.04, 385.07, 151.00 | [53] |
13 * | 43.0 | protopseudohypericin | - | - | 521.0875 | −0.59 | 477.10, 423.09 | [59] |
14 * | 47.1 | pseudohypericin | - | 592 | 519.0722 | 0.08 | 487.05, 475.08, 503.04, 449.07, 421.07 | [59] |
15 * | 52.1 | protohypericin | - | - | 505.0939 | 1.99 | 461.10, 407.09 | [60] |
16 | 54.0 | furohyperforin | - | - | 551.3733 | −1.63 | 411.25, 482.30, 329.18 | [61] |
17 * | 54.7 | hyperforin | - | 223, 272 | 535.3789 | −0.72 | 466.11, 398.24, 383.22, 315.16 | [61] |
18 * | 55.54 | hypericin | - | 592 | 503.0773 | 0.12 | 459.09, 487.04, 443.07, 405.08 | [61] |
3_29 | 90_44 | 139_28 | Mean RSD (%) per Compound | ||||
---|---|---|---|---|---|---|---|
Compound | Mean (%) | RSD | Mean (%) | RSD | Mean (%) | RSD | |
1 | 3.18 ± 0.40 b | 12.70 | 1.82 ± 0.2 a | 11.03 | 2.08 ± 0.08 a | 3.71 | 9.15 ± 4.78 |
3 | 4.15 ± 0.28 a | 6.83 | 1.45 ± 0.08 b | 5.53 | 2.81 ± 0.19 c | 6.86 | 6.41 ± 0.76 |
4 | 1.16 ± 0.0.14 a | 12.10 | 1.42 ± 0.04 b | 3.15 | 1.17 ± 0.06 a | 4.95 | 6.73 ± 4.73 |
5 | 3.88 ± 0.35 a | 3.09 | 5.34 ± 0.3 b | 5.69 | 4.51 ± 0.23 c | 5.01 | 4.60 ± 1.35 |
6 | 1.04 ± 0.03 a | 3.13 | 0.82 ± 0.01 b | 0.90 | 0.93 ± 0.05 c | 5.75 | 3.26 ± 2.43 |
7 | 13.37 ± 0.90 a | 6.73 | 0.08 ± 0.07 b | 7.83 | 4.78 ± 0.38 c | 7.99 | 7.52 ± 0.69 |
8 | 13.24 ± 0.75 a | 5.67 | 21.14 ± 1.73 b | 8.25 | 14.54 ± 0.85 a | 5.82 | 6.58 ± 1.45 |
9+10 | 8.26 ± 0.91 a | 11.03 | 14.80 ± 0.68 b | 4.62 | 10.52 ± 0.85 c | 8.05 | 7.90 ± 3.21 |
11 | 5.41 ± 0.58 a | 10.69 | 5.90 ± 0.44 a | 7.62 | 5.55 ± 0.65 a | 11.72 | 10.01 ± 2.13 |
12 | 11.26 ± 1.74 b | 15.45 | 7.78± 0.67 a | 8.62 | 9.68 ± 1.48 ab | 15.27 | 13.11 ± 3.89 |
13 | 0.97 ± 0.10 a | 10.37 | 0.56 ± 0.13 a | 22.97 | 0.86 ± 0.3 a | 34.92 | 22.75 ± 12.28 |
14 | 6.60 ± 0.71 a | 10.81 | 8.17 ± 0.89 a | 10.98 | 7.38 ± 0.66 a | 8.94 | 10.24 ± 1.13 |
15 | 0.41 ± 0.05 ab | 11.34 | 0.55 ±0.15 b | 27.06 | 0.29 ± 0.1 a | 36.51 | 24.97 ± 12.71 |
17 | 23.38 ± 3.04 a | 12.99 | 21.94 ± 2.94 a | 13.53 | 30.93 ± 2.42 b | 7.83 | 11.45 ± 3.15 |
18 | 3.68 ± 0.50 a | 13.65 | 8.21 ± 0.94 b | 11.53 | 3.98 ± 0.25 a | 6.17 | 10.45 ± 3.86 |
Mean RSD per plant | 9.77 ± 3.79 | 9.95 ± 7.01 | 11.30 ± 10.33 | 10.34 ± 6.08 | |||
13+14 * | 7.58 ± 0.81 a | 10.74 | 8.74 ± 0.86 a | 9.80 | 7.38 ± 0.66 a | 8.94 | 9.66 |
15+18 * | 4.10 ± 0.55 a | 13.35 | 8.76 ± 0.92 b | 10.52 | 8.24 ± 0.53 b | 6.38 | 9.33 |
Compound | Apomictic Line Hp93 | Three Independent Genotypes | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
MEAN | RSD | MIN | MAX | MIN/MEAN | MAX/MEAN | MEAN | RSD | MIN | MAX | MIN/MEAN | MAX/MEAN | |
1 | 2.06 | 8.01 | 1.79 | 2.34 | 86.67 | 113.55 | 2.36 | 30.49 | 1.82 | 3.18 | 77.22 | 134.64 |
3 | 1.38 | 8.08 | 1.19 | 1.64 | 86.81 | 119.04 | 2.80 | 48.07 | 1.45 | 4.15 | 51.77 | 147.91 |
4 | 1.77 | 8.80 | 1.48 | 2.20 | 83.71 | 124.71 | 1.25 | 11.77 | 1.16 | 1.42 | 92.63 | 113.57 |
5 | 5.00 | 9.13 | 4.55 | 6.17 | 90.95 | 123.49 | 4.58 | 16.02 | 3.88 | 5.34 | 84.83 | 116.75 |
6 | 1.53 | 11.83 | 1.27 | 1.98 | 82.78 | 129.55 | 0.93 | 12.19 | 0.82 | 1.04 | 87.82 | 112.20 |
7 | 0.12 | 15.33 | 0.09 | 0.16 | 80.06 | 136.35 | 6.08 | 110.85 | 0.08 | 13.37 | 1.33 | 219.95 |
8 | 15.48 | 8.60 | 13.80 | 18.33 | 89.15 | 118.39 | 16.31 | 26.00 | 13.24 | 21.14 | 81.18 | 129.66 |
9+10 | 16.04 | 7.84 | 13.89 | 17.98 | 86.61 | 112.10 | 11.20 | 29.67 | 8.26 | 14.80 | 73.82 | 132.23 |
11 | 6.34 | 12.63 | 5.34 | 8.29 | 84.25 | 130.71 | 5.62 | 4.51 | 5.41 | 5.90 | 96.23 | 105.00 |
12 | 11.40 | 27.52 | 6.59 | 16.32 | 57.82 | 143.13 | 9.57 | 18.19 | 7.78 | 11.26 | 81.29 | 117.62 |
13 | 0.38 | 37.29 | 0.21 | 0.63 | 55.22 | 165.87 | 0.80 | 26.82 | 0.56 | 0.97 | 70.10 | 126.06 |
14 | 4.54 | 10.72 | 3.76 | 5.98 | 82.88 | 131.54 | 7.39 | 10.63 | 6.60 | 8.17 | 89.40 | 109.85 |
15 | 0.50 | 45.01 | 0.26 | 0.86 | 51.68 | 172.70 | 0.42 | 31.60 | 0.29 | 0.55 | 68.59 | 131.42 |
17 | 27.39 | 7.68 | 21.04 | 29.76 | 76.81 | 108.65 | 25.42 | 18.99 | 21.94 | 30.93 | 86.32 | 121.68 |
18 | 6.15 | 13.11 | 5.22 | 8.22 | 84.82 | 133.59 | 5.29 | 47.94 | 3.68 | 8.21 | 69.59 | 155.60 |
13+14 * | 4.92 | 11.74 | 4.08 | 6.55 | 82.79 | 133.09 | 8.19 | 7.09 | 7.58 | 8.74 | 92.58 | 106.71 |
15+18 * | 6.65 | 14.11 | 5.49 | 8.98 | 82.61 | 135.13 | 5.71 | 46.40 | 4.10 | 8.76 | 71.78 | 153.55 |
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Rusanova, M.; Rusanov, K.; Alekova, M.; Georgieva, L.; Georgieva, P.; Zagorcheva, T.; Atanassov, I. A Simple High-Throughput Procedure for Microscale Extraction of Bioactive Compounds from the Flowers of Saint John’s Wort (Hypericum perforatum L.). Appl. Sci. 2025, 15, 7334. https://doi.org/10.3390/app15137334
Rusanova M, Rusanov K, Alekova M, Georgieva L, Georgieva P, Zagorcheva T, Atanassov I. A Simple High-Throughput Procedure for Microscale Extraction of Bioactive Compounds from the Flowers of Saint John’s Wort (Hypericum perforatum L.). Applied Sciences. 2025; 15(13):7334. https://doi.org/10.3390/app15137334
Chicago/Turabian StyleRusanova, Mila, Krasimir Rusanov, Marina Alekova, Liliya Georgieva, Pavlina Georgieva, Tzvetelina Zagorcheva, and Ivan Atanassov. 2025. "A Simple High-Throughput Procedure for Microscale Extraction of Bioactive Compounds from the Flowers of Saint John’s Wort (Hypericum perforatum L.)" Applied Sciences 15, no. 13: 7334. https://doi.org/10.3390/app15137334
APA StyleRusanova, M., Rusanov, K., Alekova, M., Georgieva, L., Georgieva, P., Zagorcheva, T., & Atanassov, I. (2025). A Simple High-Throughput Procedure for Microscale Extraction of Bioactive Compounds from the Flowers of Saint John’s Wort (Hypericum perforatum L.). Applied Sciences, 15(13), 7334. https://doi.org/10.3390/app15137334