Corresponding author: Paraskev Nedialkov ( pnedialkov@pharmfac.mu-sofia.bg ) Academic editor: Plamen Peikov
© 2021 Zlatina Kokanova-Nedialkova, Denitsa Aluani, Virginia Tzankova, Paraskev Nedialkov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kokanova-Nedialkova Z, Aluani D, Tzankova V, Nedialkov P (2021) Simultaneous quantification of the major flavonoids from wild spinach by UHPLC-HRMS and their neuroprotective effects in a model of H2O2-induced oxidative stress on SH-SY5Y cells. Pharmacia 68(3): 657-664. https://doi.org/10.3897/pharmacia.68.e71030
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A modified UHPLC-HRMS method for simultaneous quantification of eight flavonoids from the aerial parts of the wild spinach (Chenopodium bonus-henricus L.) was re-validated for specificity, the limit of detection and quantitation limit, linearity, accuracy, and precision. The glycosides of spinacetin (Chbhnf-04, Chbhnf-06, and Chbhnf-08) and patuletin (Chbhnf-01) were the predominant compounds. The total amount of assayed flavonoids from the aerial parts of a title plant was estimated to be 1.82% and 1.4% in two different populations from Vitosha Mountain (Bulgaria). The neuroprotective properties of compounds Chbhnf-02, Chbhnf-04, Chbhnf-06, Chbhnf-07, Chbhnf-08 were further assessed using a model of H2O2-induced oxidative stress on human neuroblastoma SH-SY5Y cells. All tested flavonoids demonstrated statistically significant neuroprotective activity close to that of silibinin. Patuletin (Chbhnf-07) and spinacetin (Chbhnf-08) triglycosides showed the most protective effects at the lowest concentration of 50 µM.
Chenopodium bonus-henricus, flavonoids, neuroprotection, oxidative stress, quantification
The genus Chenopodium (Amaranthaceae) numbers a wide range of species (more than 200) and is native to all the continents with exception of Antarctica as well as in some distant archipelagoes (such as Juan Fernandez, New Zealand, and Hawaii) (
As a continuation to our studies on phytochemistry and pharmacology of the aerial parts of C. bonus-henricus L., in the present study, we reported a re-validation of a modified UHPLC-HRMS method for simultaneous quantification of eight major flavonoids from two populations in Vitosha Mountain (Bulgaria). Besides, their neuroprotective effects in a model of H2O2-induced oxidative stress on human neuroblastoma SH-SY5Y cells were established, as well.
UHPLC-HRMS analysis was performed using a Thermo Scientific Dionex Ultimate 3000 RSLC (Germering, Germany) consisting of 6-channel degasser SRD-3600, high-pressure gradient pump HPG-3400RS, autosampler WPS-3000TRS, and column compartment TCC-3000RS coupled to Thermo Scientific Q Exactive Plus (Bremen, Germany) mass spectrometer. All the reagents used were of analytical grade. The main flavonoids (purity 95–96%), patuletin-3-O-[β-apiofuranosyl(1→2)]-β-glucopyranosyl(1→6)-β-glucopyranoside (Chbhnf-01), patuletin-3-O-gentiobioside (Chbhnf-02), 6-methoxykaempferol-3-O-[β-apiofuranosyl(1→2)]-β-glucopyranosyl(1→6)-β-glucopyranoside (Chbhnf-03), spinacetin-3-O-[β-apiofuranosyl (1→2)]-β-glucopyranosyl(1→6)-β-glucopyranoside (Chbhnf-04), spinacetin-3-O-gentiobioside (Chbhnf-06), patuletin-3-O-(5˝´-О-Е-feruloyl)-β-D-apiofuranosyl(1→2)[β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (Chbhnf-07), spinacetin-3-O-(5˝´-О-Е-feruloyl)-β-D-apiofuranosyl (1→2)[β-D-glucopyranosyl(1→6)]-β-D-glucopyranoside (Chbhnf-08), 6-methoxykaempferol-3-O-(5˝´-О-Е-feruloyl)-β-D-apiofuranosyl(1→2)[β-D-glucopyranosyl (1→6)]-β-D-glucopyranoside (Chbhnf-09) were previously isolated from the aerial parts of C. bonus-henricus L. (
The aerial parts of Chenopodium bonus-henricus L. were collected in a flowering stage at altitudes of 1200 m (Zheleznitsa village) and 1734 m (Kumata hut), from Vitosha mountain, Bulgaria in May-June 2020. The plants were identified by P. Nedialkov and the voucher specimens from the plant populations (No. SOM-177438 and No. SOM-177439) were deposited at the National Herbarium, Bulgarian Academy of Sciences, Sofia, Bulgaria.
The aerial parts of C. bonus-henricus L. were dried in а shade, and the powdered plant materials from Zheleznitsa village (200.24 mg) and Kumata hut (200.22 mg) were extracted with 80 vol. % MeOH (60 mL) by ultrasonic-assisted extraction for 30 min. The MeOH extracts were diluted to 100 mL 80 vol. % MeOH. The resulting solutions were filtered and the first 10 mL were removed. An aliquot (10 mL) of each solution was evaporated to dryness, then dissolved in water, and further purified by solid-phase extraction over RP18. The sorbents were first washed with H2O, then eluted with 80 vol. % MeOH (12 × 500 µL) in 10.0 mL volumetric flasks and diluted to the nominal volume with the same solvent (solution A1 and A2). Subsequently, 2 mL of solutions A1 and A2 were diluted to 25 mL 80 vol. % MeOH (solutions B1 and B2). Further, 1 mL of solutions B1 and B2 were diluted to 5 mL 80 vol. % MeOH (solution C1 and C2). Solutions C1 and C2 were used for LC-MS quantification of flavonoids in the aerial parts of C. bonus-henricus L.
UHPLC-HRMS conditions were published previously (
The quantification of flavonoids was carried out using the external standard method. Each of the flavonoids was dissolved in 25 mL 80 vol. % MeOH (primary solutions). The stock standard solution of eight flavonoids was prepared by combining the aliquots (1 mL) of each primary solution and diluting it to 10 mL with 80 vol. % MeOH. It was stored in the refrigerator at 4 °C. The working standard solutions of appropriate concentration were prepared by diluting the stock standard solution with 80 vol. % MeOH.
External standard calibrations were established on six data points covering the concentration range of 12.875–412 ng/mL for (Chbhnf-01) and (Chbhnf-02), 13.125–420 ng/mL for (Chbhnf-03), (Chbhnf-06) and (Chbhnf-08), 13–416 ng/mL for (Chbhnf-04), 12.750–408 ng/mL for (Chbhnf-07), and 13.250–424 ng/mL for (Chbhnf-09).
The limit of detection (LOD) of an analytical procedure is the lowest analytical concentration at which an analyte(s) could be detected qualitatively. Typically, peak heights are two or three times the noise level. The quantitation limit (LOQ) is also the lowest concentration at that level analyte can be quantitated with acceptable precision, requiring peak heights 10 to 20 times higher than the baseline noise. This signal-to-noise ratio is a good rule of thumb. Limits of detection (LODs) were calculated according to the expression 3.3σ/S, where σ was the standard deviation of the response and S the slope of the calibration curve. Limits of quantification (LOQs) were established from the expression 10σ/S (
Accuracy is the closeness of the analytical results obtained by the analyses to the true values and is usually presented as a percent of nominal (
The precision of an analytical method is the amount of variation in the results obtained from multiple analyses of the homogeneous samples. Intra‐day precision (repeatability), defines the precision obtained using the same operating conditions over a designated short period (typically ≤1 day). Inter‐day precision (intermediate precision), defines the precision obtained using the same operating conditions, typically within the same laboratory, over a designated period (typically ≥1 day) (
Human neuroblastoma cell line SH-SY5Y was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). SH-SY5Y cells were maintained in 75 ml flasks at 37 °C in a humidified atmosphere with 5% CO2. The cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 2 mM L-glutamine. At 95% confluence, they were plated in 96-well plates for the next experiments.
The SH-SY5Y cells were seeded in 96-well microplates at a density of 2 × 104 cells/well and allowed to attach to the well surface for 24 h at 37 °C in a humidified atmosphere with 5% CO2 (24 h). Five different concentrations of flavonoids (50, 100, 200, 400, and 800 µM) were added to cells and incubated for 24 h. For each concentration, a set of at least 8 wells were used. The cell viability was estimated by MTT-dye reduction assay (
The model of oxidative stress damage on neuroblastoma SH-SY5Y cell line was achieved by H2O2 treatment of cells. SH-SY5Y cells were seeded at a density of 3.5 × 104 cells/well in 96-well plates and allowed to attach at the bottom of the wells for 24 h. Furthermore, the cell medium was aspirated, and the cells were treated with solutions of flavonoids (50, 100, 200, 400, and 800 µM) in RPMI for 60 min before H2O2 exposure. Afterward, the SH-SY5Y cells were washed with phosphate-buffered saline (PBS) to remove the extracellular amount of the tested compounds. Subsequently, the treatment with a solution of hydrogen peroxide (H2O2, 1 mM) in PBS for 15 min accomplished the SH-SY5Y damage. Silibinin was used as a reference compound because of its protective activity (
Statistical analysis was performed by one-way analysis of variance with Dunnett’s post hoc test. Differences were accepted to be significant when P < 0.05. All statistical analysis was carried out on Graph Pad 6 software (GraphPad Software, Inc., La Jolla, CA, USA).
A modified ultra-high performance liquid chromatography – high-resolution mass spectrometry (UHPLC-HRMS) was used to establish the quantity of the main flavonoids in the aerial parts of C. bonus-henricus L. from two populations in Vitosha mountain (Bulgaria) in this work.
The efficiency of the extraction procedure and chromatographic conditions were given in a previous report (
Quantitative determination of the main flavonoids in the aerial parts of C. bonus-henricus L. was performed by the method of the external standard. Eight previously isolated flavonoids from a title plant were used as external standards (
The calibration curves were linear over the concentration range of 12.875–412 ng/mL for (Chbhnf-01) and (Chbhnf-02), 13.125–420 ng/mL for (Chbhnf-03), (Chbhnf-06) and (Chbhnf-08), 13–416 ng/mL for (Chbhnf-04), 12.750–408 ng/mL for (Chbhnf-07), and 13.250–424 ng/mL for (Chbhnf-09). All calibration curves showed very good linear regressions and the correlation coefficients were R2 > 0.998 (Table
Marker compound | Linear range (ng/mL) | Regression equations | R2 | LOD (ng/mL) | LOQ (ng/mL) |
---|---|---|---|---|---|
Chbhnf-01 | 12.875 ÷ 412 | y = 53052x − 440933 | 0.9987 | 1.59 | 4.81 |
Chbhnf-02 | 12.875 ÷ 412 | y = 81917x − 686762 | 0.9987 | 1.14 | 3.44 |
Chbhnf-03 | 13.125 ÷ 420 | y = 53868x − 467219 | 0.9994 | 1.99 | 6.05 |
Chbhnf-04 | 13.000 ÷ 416 | y = 56272x − 384872 | 0.9995 | 1.31 | 3.96 |
Chbhnf-06 | 13.125 ÷ 420 | y = 112020x − 847350 | 0.9994 | 1.16 | 3.52 |
Chbhnf-07 | 12.750 ÷ 408 | y = 30118x − 248618 | 0.9990 | 1.56 | 4.71 |
Chbhnf-08 | 13.125 ÷ 420 | y = 40092x − 281735 | 0.9998 | 1.97 | 5.98 |
Chbhnf-09 | 13.250 ÷ 424 | y = 31702x − 259428 | 0.9997 | 0.97 | 2.95 |
The method showed that LODs and LOQs were 1.59 ng/mL and 4.81 ng/mL (Chbhnf-01), 1.14 ng/mL and 3.44 ng/mL (Chbhnf-02), 1.99 ng/mL and 6.05 ng/mL (Chbhnf-03), 1.31 ng/mL and 3.96 ng/mL (Chbhnf-04), 1.16 ng/mL and 3.52 ng/mL (Chbhnf-06), 1.56 ng/mL and 4.71 ng/mL (Chbhnf-07), 1.97 ng/mL and 5.98 ng/mL (Chbhnf-08) and 0.97 ng/mL and 2.95 ng/mL (Chbhnf-09), respectively (Table
The accuracy of the analytes was checked by addition of a standard solution mixture at three concentrations (32.96, 65.92, and 98.88 ng/mL for Chbhnf-01 and Chbhnf-02; 33.60, 67.20, and 100.80 ng/mL for Chbhnf-03, Chbhnf-06, and Chbhnf-08; 33.28, 66.56 and 99.84 ng/mL for Chbhnf-04; 32.64, 65.28 and 97.92 ng/mL for Chbhnf-07; 33.92, 67.84 and 101.76 ng/mL for Chbhnf-09) close to that expected in the real plant samples.
Blank samples from the same unspiked plant material were analyzed at the same time as the spiked samples and the measured values were subtracted. Besides, the related compounds showed overall recoveries ranging from 95.33% to 99.77% with RSD ranging from 0.65% to 2.99%. A good agreement between the spiked and determined concentrations indicated acceptable accuracy (Table
Flavonoids | Added (ng/mL) | Founda (ng/mL) | Recoverya (%) | RSD (%) |
---|---|---|---|---|
Chbhnf-01 | 32.96 | 32.29 ± 0.87 | 97.98 ± 2.64 | 2.69 |
65.92 | 64.79 ± 0.69 | 98.28 ± 1.05 | 1.07 | |
98.88 | 97.95 ± 0.95 | 99.06 ± 0.96 | 0.97 | |
Chbhnf-02 | 32.96 | 31.94 ± 0.39 | 96.89 ± 1.18 | 1.22 |
65.92 | 64.72 ± 1.08 | 98.17 ± 1.64 | 1.67 | |
98.88 | 97.58 ± 0.92 | 98.68 ± 0.93 | 0.94 | |
Chbhnf-03 | 33.60 | 32.35 ± 0.48 | 96.29 ± 1.44 | 1.49 |
67.20 | 65.76 ± 1.03 | 97.85 ± 1.53 | 1.56 | |
100.80 | 98.60 ± 2.11 | 97.82 ± 2.09 | 2.14 | |
Chbhnf-04 | 33.28 | 32.21 ± 0.96 | 96.78 ± 2.89 | 2.99 |
66.56 | 64.17 ± 1.73 | 96.41 ± 2.60 | 2.69 | |
99.84 | 99.61 ± 2.22 | 99.77 ± 2.22 | 2.23 | |
Chbhnf-6 | 33.60 | 32.42 ± 0.70 | 96.50 ± 2.08 | 2.16 |
67.20 | 64.20 ± 1.17 | 95.54 ± 1.75 | 1.83 | |
100.80 | 98.03 ± 2.03 | 97.26 ± 2.01 | 2.07 | |
Chbhnf-07 | 32.64 | 31.12 ± 0.20 | 95.33 ± 0.62 | 0.65 |
65.28 | 62.90 ± 1.11 | 96.36 ± 1.69 | 1.76 | |
97.92 | 95.56 ± 1.79 | 97.59 ± 1.82 | 1.87 | |
Chbhnf-08 | 33.60 | 32.11 ± 0.68 | 95.56 ± 2.02 | 2.11 |
67.20 | 64.25 ± 0.85 | 95.61 ± 1.26 | 1.32 | |
100.80 | 99.08 ± 2.43 | 98.29 ± 2.41 | 2.45 | |
Chbhnf-09 | 33.92 | 32.57 ± 0.26 | 96.01 ± 0.76 | 0.79 |
67.84 | 65.88 ± 1.04 | 97.11 ± 1.53 | 1.58 | |
101.76 | 98.67 ± 0.69 | 96.97 ± 0.67 | 0.69 |
The precision of the retention times was determined by analyzing the calibration samples during a single day and on three different days, respectively. The RSDs of retention times of the analytes were ≤ 0.15 for intra‐day and ≤ 0.14 for inter‐day precision assays, respectively. Also, the related compounds showed overall recoveries ranging from 96.83% to 102.52% (for intra‐day and inter‐day precision assays) with RSDs from 0.35% to 2.34%. (Tables
Evaluation of intra‐day precision (repeatability) of the UHPLC-HRMS method.
Compds. | Intra‐day precision (repeatability) | |||
---|---|---|---|---|
RT ± SD (min) | RSD | Recovery ± SD (%) | RSD (%) | |
Chbhnf-01 | 7.01 ± 0.010 | 0.15 | 101.00 ± 2.16 | 2.14 |
Chbhnf-02 | 8.40 ± 0.007 | 0.08 | 100.76 ± 1.98 | 1.96 |
Chbhnf-03 | 9.33 ± 0.010 | 0.11 | 101.68 ± 1.25 | 1.23 |
Chbhnf-04 | 10.20 ± 0.013 | 0.13 | 100.23 ± 1.89 | 1.88 |
Chbhnf-06 | 12.90 ± 0.009 | 0.07 | 98.18 ± 1.48 | 1.50 |
Chbhnf-07 | 15.66 ± 0.006 | 0.04 | 99.30 ± 2.05 | 2.06 |
Chbhnf-08 | 16.18 ± 0.006 | 0.04 | 97.53 ± 1.82 | 1.87 |
Chbhnf-09 | 16.33 ± 0.005 | 0.03 | 97.03 ± 0.75 | 0.77 |
Compds. | Inter‐day precision (intermediate precision) | |||
---|---|---|---|---|
RT±SD (min) | RSD | Recovery ± SD (%) | RSD (%) | |
Chbhnf-01 | 7.01 ± 0.010 | 0.14 | 100.13 ± 2.17 | 2.16 |
Chbhnf-02 | 8.40 ± 0.010 | 0.12 | 100.43 ± 2.35 | 2.34 |
Chbhnf-03 | 9.33 ± 0.009 | 0.09 | 102.52 ± 0.73 | 0.72 |
Chbhnf-04 | 10.21 ± 0.009 | 0.09 | 98.79 ± 0.88 | 0.89 |
Chbhnf-06 | 12.91 ± 0.006 | 0.05 | 99.66 ± 0.64 | 0.64 |
Chbhnf-07 | 15.66 ± 0.007 | 0.04 | 97.53 ± 0.34 | 0.35 |
Chbhnf-08 | 16.18 ± 0.006 | 0.04 | 97.83 ± 0.93 | 0.95 |
Chbhnf-09 | 16.33 ± 0.005 | 0.03 | 96.83 ± 0.77 | 0.80 |
The modified UHPLC-HRMS method was applied for quantification of eight major flavonoids in the aerial parts of C. bonus-henricus L. from two different populations (Zheleznitsa and Kumata hut) in Vitosha mountain (Bulgaria). The results show that the flavonoids of spinacetin (Chbhnf-04, Chbhnf-06 and Chbhnf-08) and patuletin (Chbhnf-01) were the predominant compounds and reached 0.53%, 0.25%, 0.29%, 0.26% in the aerial parts of C. bonus-henricus from Kumata hut (KH) and 0.32%, 0.19%, 0.21%, 0.20% in the aerial parts collected from Zheleznitsa (ZH), respectively (Table
Content of flavonoids in the aerial parts of C. bonus-henricus L. from Kumata hut (KH) and Zheleznitsa (ZH).
Compds. | Flavonoids | Content of flavonoids (%) | |
---|---|---|---|
KH | ZH | ||
Chbhnf-01 | patuletin-3-O-[β-apiofuranosyl(1→2)]- | 0.26 ± 0.0036 | 0.20 ± 0.0025 |
β-glucopyranosyl(1→6)-β-glucopyranoside | |||
Chbhnf-02 | patuletin-3-O-gentiobioside | 0.10 ± 0.0012 | 0.09 ± 0.0004 |
Chbhnf-03 | 6-methoxykaempferol-3-O-[β-apiofuranosyl(1→2)]- | 0.17 ± 0.0035 | 0.15 ± 0.0015 |
β-glucopyranosyl(1→6)-β-glucopyranoside | |||
Chbhnf-04 | spinacetin-3-O-[β-apiofuranosyl(1→2)]- | 0.53 ± 0.0137 | 0.32 ± 0.0024 |
β-glucopyranosyl(1→6)-β-glucopyranoside | |||
Chbhnf-06 | spinacetin-3-O-gentiobioside | 0.25 ± 0.0031 | 0.19 ± 0.0011 |
Chbhnf-07 | patuletin-3-O-(5˝´-О-Е-feruloyl)-β-D-apiofuranosyl | 0.14 ± 0.0012 | 0.15 ± 0.0012 |
(1→2)[β-D-glucopyranosyl (1→6)]- | |||
β-D-glucopyranoside | |||
Chbhnf-08 | spinacetin-3-O-(5˝´-О-Е-feruloyl)-β-D-apiofuranosyl (1→2)[β-D- | 0.29 ± 0.0058 | 0.21 ± 0.0020 |
glucopyranosyl(1→6)]- | |||
β-D-glucopyranoside | |||
Chbhnf-09 | 6-methoxykaempferol-3-O-(5˝´-О-Е-feruloyl)-β-D-apiofuranosyl(1→2)[β-D- | 0.08 ± 0.0019 | 0.09 ± 0.0009 |
glucopyranosyl (1→6)]- | |||
β-D-glucopyranoside | |||
The total content of flavonoids (%) | 1.82 | 1.4 |
The neuroprotective properties of compounds Chbhnf-02, Chbhnf-04, Chbhnf-06, Chbhnf-07, and Chbhnf-08 were further assessed using a model of H2O2-induced oxidative stress on SH-SY5Y cells. SH-SY5Y cells possess morphological and biochemical characteristics of human neurons and represent a suitable in vitro model for studying the mechanisms of damage and neuroprotection in different neurodegenerative diseases as Parkinson’s, Alzheimer’s diseases, etc. (
The study started with the determination of the potential cytotoxic effects of five tested flavonoids on SH-SY5Y cells after 24 h incubation. The cell viability was determined by MTT assay as a marker for mitochondrial function. The cytotoxicity was classified according to ISO 10993-5 standard: cell viability above 80% was considered as non-cytotoxic; within 80%–60% as weak; 60%–40% as moderate and below 40% as strong cytotoxicity (
Furthermore, the neuroprotective effects of flavonoids (Chbhnf-02, Chbhnf-04, Chbhnf-06, Chbhnf-07, and Chbhnf-08) were explored in a model of H2O2-induced oxidative stress in SH-SY5Y cells. H2O2 is commonly used as an inducer of oxidative stress in vitro models. The mechanism of H2O2-induced cell damages includes the production of reactive hydroxyl radicals and by the products of Fenton’s reaction that further interact directly with cellular components to damage proteins, lipids and DNA.
Patuletin diglycoside (Chbhnf-02) showed protection against H2O2-oxidative stress damage in the high concentration range from 200 to 800 µM. Triglycosides of patuletin (Chbhnf-07) and spinacetin (Chbhnf-08) showed a high protective effect at concentrations up to 400 µM. Spinacetin glycosides Chbhnf-04 and Chbhnf-06 possessed protective effects in the entire studied concentration range (50–800 µM). The most prominent neuroprotection in vitro was observed with triglycosides of patuletin and spinacetin, respectively compounds Chbhnf-07 and Chbhnf-08. Their protective cell viability effects were seen at the lowest concentration of 50 µM (Figure
Effect of flavonoids and silibinin on the viability of SH-SY5Y cells in a model of H2O2-induced toxicity. Data are presented as means from three independent experiments ± SD (n = 8). ***P < 0.001, vs. untreated control; +++P < 0.001, vs. H2O2 group. (one-way analysis of variance with Dunnet’s post hoc test).
The results from the present study showed that in the model of H2O2-induced oxidative stress in SH-SY5Y cells, the flavonoids Chbhnf-07 and Chbhnf-08 possessed the comparable effect as those of silibinin, while the effects of the other flavonoids were close to silibinin to less extent. Additionally, we suggested that the neuroprotective effects of tested patuletin, 6-methoxykaempferol, and spinacetin glycosides are attributed to their antioxidant activity and ability to scavenge ROS.
The results of this study were in good agreement with previously published research on neuroprotective activities of these flavonoids on isolated rat brain synaptosomes using a 6-hydroxydopamine in vitro model (
A modified UHPLC-HRMS method for simultaneous quantification of eight flavonoids from wild spinach (Chenopodium bonus-henricus L.) was re-validated for specificity, the limit of detection, and quantitation limit, linearity, accuracy, and precision. The flavonoids of spinacetin and patuletin (Chbhnf-01) were the predominant compounds. The total amount of assayed flavonoids was estimated to be 1.82% in the aerial parts from Kumata hut and 1.4% in Zheleznitsa. All tested flavonoids demonstrated statistically significant neuroprotective activity in a model of H2O2-induced oxidative stress in vitro, which resembles those of natural antioxidant silibinin. Patuletin (Chbhnf-07) and spinacetin (Chbhnf-08) triglycosides possessed the most protective effects at the lowest concentration of 50 µM.