Phytochemical analysis, UPLC-ESI-Orbitrap-MS analysis, biological activity, and toxicity of extracts from Tripleurospermum limosum (Maxim.) Pobed

2.2.1 Preparation of TL extracts for quantitative phytochemical analysis and ultra-performance liquid chromatography (UPLC)-mass spectroscopy (MS) analysis

Collected samples of TL were dried in a place that was dry, airy, and out of direct sunlight, and pulverised to powder. Twenty grams of this powder was added to a single-neck round-bottomed flask (glass, 500 mL), followed by addition of 200 mL of various solvents (water, methanol, ethanol, acetone, ethyl acetate, ethyl ether, dichloromethane or hexane) and refluxing using a hotplate magnetic stirrer employing methyl silicone oil as the heating medium for 6 h at the respective boiling points of the solvents. Extracts were filtered through a Whatman No.1 filter paper and evaporated under reduced pressure at < 50 °C until dry using a rotary evaporator. All dried extracts were weighed and stored at − 20 °C until use. Yield was calculated as % yield = (weight of dry extract/initial weight of dry sample) × 100.

2.2.2 Preparation of preliminary experimental solutions for qualitative phytochemical analysis

2.3.1 Tests for proteins

2.3.2 Tests for carbohydrates

2.3.3 Tests for phenols

2.3.4 Tests for organic acids

2.3.5 Tests for tannins

2.3.6 Tests for flavonoids

2.3.7 Tests for saponins

2.3.8 Tests for steroids and triterpenoids

2.3.9 Tests for terpenoids

2.3.10 Tests for alkaloids

2.3.11 Tests for anthraquinones

2.3.12 Tests for coumarins and lactones

2.3.13 Tests for volatile oils and fats

2.3.14 Tests for cardiac glycosides

2.3.15 Tests for cyanogenic glycosides

2.5.1 Determination of total carbohydrate content (TCC)

The TCC was measured according to our previous method (Zhang et al. 2020). Briefly, 250 μL of TL extract in distilled water, 125 μL of phenol solution (5%), and 625 μL of H₂SO₄ were mixed in an Eppendorf tube and incubated for 30 min. Subsequently, 200 μL of the sample was pipetted from each Eppendorf tube onto a microplate. A calibration curve was produced based on glucose (0–200 mg/L) as a standard. The absorbance of the sample was recorded at 490 nm against a blank sample consisting of TL extract with distilled water. The mean of three readings was used and TCC was expressed in milligrams of glucose equivalents (GE)/g of TL extract.

2.5.2 Determination of total protein content (TP_roC)

The TP_roC was measured according to our previous method (Guo et al. 2022). Briefly, 200 μL of bicinchoninic acid (BCA) working solution and 20 μL of TL extract in distilled water were mixed in a microplate and incubated at 37 °C for 30 min. A calibration curve was produced based on bovine serum albumin (BSA) (0–500 mg/L) as a standard. The absorbance of the sample was recorded at 562 nm against a blank sample consisting of TL extract with distilled water. The mean of three readings was used and TP_roC was expressed in milligrams of BSA equivalents (BSAE)/g of TL extract.

2.5.3 Determination of total triterpenoid content (TT_riC)

The TT_riC was measured according to the method with slight modifications (Fiallos-Jurado et al. 2016). Briefly, 180 μL of TL extract in acetic anhydride and 20 μL of H₂SO₄ were mixed in a microplate and incubated at room temperature for 10 min. A calibration curve was produced based on ginsenoside Re (0–400 mg/L) as a standard. The absorbance of the sample was recorded at 350 nm against a blank sample consisting of TL extract with acetic anhydride. The mean of three readings was used and TT_riC was expressed in milligrams of ginsenoside Re equivalents (GRE)/g of TL extract.

2.5.4 Determination of total phenolic content (TP_heC)

The TP_heC was measured according to our previous method (Zhang et al. 2020). Briefly, 100 μL of Folin & Ciocalteu’s phenol reagent (FC reagent) (1 M) and 200 μL of TL extract in distilled water were mixed in an Eppendorf tube and incubated for 5 min. Subsequently, 500 μL of Na₂CO₃ solution (20%) was added and allowed to stand at room temperature for 40 min in the dark (with mixing every 10 min). Subsequently, 200 μL of the sample was pipetted from each Eppendorf tube onto a microplate. A calibration curve was produced based on gallic acid (0–100 mg/L) as a standard. The absorbance of the sample was recorded at 750 nm against a blank sample consisting of TL extract with distilled water and Na₂CO₃. The mean of three readings was used and TP_heC was expressed in milligrams of gallic acid equivalents (GAE)/g of TL extract.

2.5.5 Determination of total flavonoid content (TFC)

The TFC was measured according to our previous method (Zhang et al. 2020). Briefly, 100 μL of AlCl₃ (2%) in methanol and 100 μL of TL extract in methanol were mixed in a microplate and incubated at room temperature for 10 min. A calibration curve was produced based on quercetin (0–100 mg/L) as a standard. The absorbance of the sample was recorded at 415 nm against a blank sample consisting of TL extract with methanol. The mean of three readings was used and TFC was expressed in milligrams of quercetin equivalents (QE)/g of TL extract.

2.5.6 Determination of total phenolic acid content (TPAC)

The TPAC was measured according to the method with slight modifications (Mihailović et al. 2015). Briefly, 20 μL of TL extract in distilled water, 20 µL of Arnow reagent, 20 µL of HCl solution (0.1 M), 120 µL of distilled water and 20 µL of NaOH solution (1 M) were mixed in a microplate and recorded immediately at 490 nm against a blank sample (Arnow reagent was replaced with distilled water). A calibration curve was produced based on caffeic acid (0–100 mg/L) as a standard. The mean of three readings was used and TPAC was expressed in milligrams of caffeic acid equivalents (CAE)/g of TL extract.

2.5.7 Determination of total tannin content (TT_anC)

The TT_anC was measured according to our previous method (Guo et al. 2022). Briefly, 200 μL of FC reagent (1 M) and 200 μL of TL extract in distilled water were mixed in an Eppendorf tube and incubated for 5 min. Subsequently, 100 μL of Na₂CO₃ solution (20%) and 1500 μL of distilled water were added and allowed to stand at room temperature for 30 min in the dark (with mixing every 10 min). Subsequently, 200 μL of the sample was pipetted from each Eppendorf tube onto a microplate. A calibration curve was produced based on tannic acid (0–200 mg/L) as a standard. The absorbance of the sample was recorded at 725 nm against a blank sample consisting of TL extract with distilled water and Na₂CO₃. The mean of three readings was used and TT_anC was expressed in milligrams of tannic acid equivalents (TAE)/g of TL extract.

2.5.8 Determination of condensed tannin content (CTC)

The CTC was measured according to the method with slight modifications (Mihailović et al. 2015). Briefly, 4 mg of phloroglucinol was added to 2 mL of TL extract in distilled water. Subsequently, 1 mL of HCl solution and 1 mL of formaldehyde solution were added and mixed in an Eppendorf tube and incubated at room temperature overnight. The precipitate was separated by filtration, the unprecipitated phenols were measured in the filtrate according to Section 2.5.4.

2.5.9 Determination of gallotannin content (GC)

The GC was measured according to the method with slight modifications (Mihailović et al. 2015). Briefly, 875 µL of TL extract in methanol and 375 µL of saturated KIO₃ solution were mixed in an Eppendorf tube and incubated at 15 °C for 120 min. A calibration curve was produced based on gallic acid (0–400 mg/L) as a standard. The absorbance of the sample was recorded at 550 nm against a blank sample (KIO₃ was replaced with distilled water). The mean of three readings was used and GC was expressed in milligrams of gallic acid equivalents (GAE)/g of TL extract.

2.6.1 DPPH assay

The DPPH scavenging activity was assayed according to our previous method (Zhang et al. 2020). Briefly, 100 µL of TL extract in methanol and 100 µL of DPPH in methanol (50 µM) were mixed in a microplate and allowed to stand at room temperature for 20 min in the dark. The absorbance of the sample was recorded at 515 nm. Butylated hydroxytoluene (BHT), L-ascorbic acid and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were used as positive references. The Half-maximal inhibitory concentration (IC₅₀) values were calculated and expressed as the mean ± standard deviation (SD) in μg/mL.

2.6.2 ABTS assay

The ABTS scavenging activity was assayed according to our previous method (Zhang et al. 2020). Briefly, 190 μL of diluted ABTS solution and 10 μL of TL extract in DMSO were mixed in a microplate and incubated for 20 min in the dark. The absorbance of the sample was recorded at 734 nm. BHT, L-ascorbic acid and trolox were used as positive references.The IC₅₀ values were calculated and expressed as the mean ± SD in μg/mL.

2.6.3 Hydroxyl radical assay

The hydroxyl radical scavenging activity was assayed according to our previous method (Zhang et al. 2020). Briefly, 50 µL of TL extract in DMSO, 50 µL of FeSO₄ solution (3 mM) and 50 µL of H₂O₂ solution (3 mM) were mixed in a microplate and incubated for 10 min. After then 50 µL of salicylic acid solution (6 mM) was added and incubated at room temperature for 30 min in the dark. The absorbance of the sample was recorded at 492 nm. BHT, L-ascorbic acid and trolox were used as positive references. The IC₅₀ values were calculated and expressed as the mean ± SD in μg/mL.

2.6.4 Superoxide radical assay

The superoxide radical scavenging activity was assayed according to our previous method (Guo et al. 2022). Briefly, 45 µL of TL extract in DMSO (10 mg/mL), 15 µL of p-nitroblue tetrazolium chloride (NBT) in DMSO (1 mg/mL) and 150 µL of NaOH in DMSO (50 μM) were mixed in a microplate and the absorbance of the sample was recorded immediately at 560 nm against a blank sample (NBT was replaced with DMSO). Curcumin was used as a positive reference. The scavenging activity was expressed as % scavenging rate and was calculated as follows: $$\%scavenging=\left(1-\frac{\mathrm{\Delta }{A}_{\mathit{sample}}}{\mathrm{\Delta }{A}_{\mathit{control}}}\right)\times 100\%$$

2.6.5 FRAP assay

The FRAP assay was assayed according to our previous method (Zhang et al. 2020). Briefly, 20 µL of TL extract in DMSO and 180 µL of FRAP reagent were mixed in a microplate and incubated at 37 °C for 30 min in the dark. A calibration curve was produced based on FeSO₄ (0–600 mg/L) as a standard. The absorbance of the sample was recorded at 595 nm. BHT, L-ascorbic acid and trolox were used as positive references. The FRAP was expressed as the Trolox Equivalent Antioxidant Capacity (TEAC_FRAP).

2.6.6 CUPRAC assay

The CUPRAC assay was assayed according to our previous method (Zhang et al. 2020). Briefly, 20 µL of CuCl₂ solution (100 mM), 50 µL of neocuproine in 96% ethanol (7.5 mM), 50 µL of NH₄Ac solution, 20 µL of TL extract in DMSO, and 30 µL of distilled water were mixed in a microplate and incubated at 50 °C for 20 min. This mixture was allowed to stand at room temperature for 10 min. The absorbance of the sample was recorded at 450 nm. BHT, L-ascorbic acid and trolox were used as positive references. The CUPRAC was expressed as the Trolox Equivalent Antioxidant Capacity (TEAC_CUPRAC).

2.6.7 Iron chelating assay

The chelating activity for ferrous ions was assayed according to our previous method (Zhang et al. 2020). Briefly, 50 µL of TL extract in methanol, 110 µL of ultra-pure water, and 20 µL of FeCl₂ solution (0.5 mM) were mixed in a microplate and incubated for 5 min. Subsequently, 20 µL of ferrozine solution (2.5 mM) was added and incubated for 10 min. The absorbance was recorded at 562 nm against a blank sample (ferrozine solution was replaced with water). Ethylenediaminetetraacetic acid disodium salt (EDTANa₂) was used as a positive reference. The IC₅₀ values were calculated and expressed as the mean ± SD in μg/mL.

2.6.8 Copper chelating assay

The chelating activity for copper ions was assayed according to our previous method (Guo et al. 2022). Briefly, 40 µL of TL extract in ultra-pure water, 140 µL of acetic acid-sodium acetate buffer solution (pH 6.0, 50 mM), and 10 µL of CuSO₄ solution (5 mM) were mixed in a microplate and incubated for 30 min. Subsequently, 10 µL of pyrocatechol violet solution (4 mM) was added and incubated for 30 min. The absorbance was recorded at 632 nm against a blank sample (pyrocatechol violet was replaced with water). EDTANa₂ was used as a positive reference. The IC₅₀ values were calculated and expressed as the mean ± SD in μg/mL.

2.6.9 H₂O₂ assay

The H₂O₂ scavenging activity was assayed according to our previous method (Guo et al. 2022). Briefly, 70 µL of phenol solution (pH 7.0, 12 mM, in 84 mM phosphate buffer (PBS)), 20 µL of 4-aminoantipyrine solution (pH 7.0, 0.5 mM, in 84 mM PBS), 32 μL of H₂O₂ solution (pH 7.0, 0.7 mM, in 84 mM PBS), 8 µL of horseradish peroxidise (EC 1.11.1.7) solution (pH 7.0, 1 U/mL, in 84 mM PBS) and 70 µL of TL extract (pH 7.0, in 84 mM PBS) were mixed in a microplate and the absorbance of the sample was recorded immediately at 504 nm against a blank sample (phenol solution was replaced with PBS). Ferulic acid was used as a positive reference. The IC₅₀ values were calculated and expressed as the mean ± SD in μg/mL.

2.6.10 β-Carotene bleaching assay

The β-carotene method was carried out according to our previous method (Zhang et al. 2020). Briefly, β-carotene solution was prepared by dissolving β-carotene (2 mg) in CHCl₃ (10 mL). Then, 2 mL of the solution was pipetted into a flask and vortex-mixed with linoleic acid (40 mg) and Tween 40 (400 mg). After the removal of CHCl₃, 100 mL of oxygenated ultrapure water was added, and the emulsion shaken vigorously. Aliquots (2.4 mL) of the emulsion were pipetted into different test tubes containing 0.1 mL of TL extract in methanol (5 mg/mL). BHT and butyl hydroxyanisole (BHA) were used as positive controls. In the control group, TL extract was replaced with water. When the sample was added to the emulsion, it was recorded as t = 0 min. The tubes were capped and placed in a water bath at 60 °C. The absorbance was recorded at 470 nm every 15 min until 120 min. Antioxidant activity coefficient (AAC) was calculated according to the following equation: $$\mathit{AAC}=\frac{A_{A(120)}-A_{C(120)}}{A_{C(0)}-A_{C(120)}}\times 1000$$ where A_A(120) is the absorbance of the antioxidant at 120 min, A_C(120) is the absorbance of the control at 120 min, and A_C(0) is the absorbance of the control at 0 min.

2.6.11 NO assay

The NO scavenging activity was assayed according to our previous method (Guo et al. 2022). Briefly, 3 mL of TL extract in methanol (1 mg/mL) and 3 mL of sodium nitroprusside solution (pH 7.4, 5 mM, in 0.1 M PBS) were mixed in an Eppendorf tube and incubated at 25 °C for 150 min. At intervals, 100 μL of the sample was pipetted from each Eppendorf tube onto a microplate containing 100 µL of Griess reagent. In the control group, TL extract was replaced with methanol. The absorbance was recorded at 546 nm against a blank sample (Griess reagent was replaced with distilled water). Curcumin (0.1 mg/mL) was used as a positive reference.

2.7.1 α-Glucosidase inhibition assay

The α-glucosidase inhibitory activity was assayed according to our previous method (Zhang et al., 2020). Briefly, 20 μL of TL extract in DMSO (10 mg/mL) and 100 µL of yeast α-glucosidase (EC 3.2.1.20) solution (pH 6.9, 0.1 U/mL, in 0.1 M PBS) were mixed in a microplate and incubated at 25 °C for 10 min. Subsequently, 50 μL of p-nitrophenyl-α- D-glucopyranoside solution (pH 6.9, 5 mM, in 0.1 M PBS) was added and incubated at 25 °C for 5 min. Before and after incubation, the absorbance was recorded at 405 nm. In the control group, TL extract was replaced with DMSO. Acarbose was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{\mathrm{\Delta }{A}_{\mathit{sample}}}{\mathrm{\Delta }{A}_{\mathit{control}}}\right)\times 100\%$$

2.7.2 α-Amylase inhibition assay

The α-amylase inhibitory activity was assayed according to our previous method (Zhang et al. 2020). Briefly, 20 μL of TL extract in DMSO, 80 µL of distilled water, and 100 µL of porcine pancreatic α-amylase (EC 3.2.1.1) solution (pH 6.9, 4 U/mL, in 20 mM PBS) were mixed in an Eppendorf tube and incubated at 25 °C for 5 min. Subsequently, 200 µL of 0.5% starch solution (pH 6.9, in 20 mM PBS) was added and incubated at 25 °C for 3 min. Then, 200 µL of the mixture was removed from the Eppendorf tube and added to a separate Eppendorf tube containing 100 µL of 3,5-dinitrosalicylic acid reagent solution and placed in a 85 °C water bath. After 15 min, the mixture was removed from the water bath and diluted with 900 µL of distilled water. The absorbance was recorded at 540 nm. In the control group, TL extract was replaced with DMSO. In the blank group, enzyme solution was replaced with PBS. Acarbose was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{A_{\mathit{sample}}-A_{\mathit{blank}}}{A_{\mathit{control}}}\right)\times 100\%$$

2.7.3 AChE inhibition assay

The AChE inhibitory activity was assayed according to our previous method (Zhang et al. 2020). Briefly, 20 μL of TL extract in 10% DMSO (1 mg/mL), 120 µL of PBS (pH 8.0, 0.1 M) and 20 µL of AChE (EC 3.1.1.7) solution (pH 8.0, 0.8 U/mL, in 0.1 M PBS) were mixed in a microplate and incubated at 25 °C for 15 min. Subsequently, 20 μL of acetylthiocholine iodide (ATCI) solution (pH 8.0, 1.78 mM, in 0.1 M PBS) and 20 μL 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) solution (pH 8.0, 1.25 mM, in 0.1 M PBS) were added and incubated at 25 °C for 5 min. Before and after incubation, the absorbance was recorded at 405 nm. In the control group, TL extract was replaced with 10% DMSO. Donepezil was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{\mathrm{\Delta }{A}_{\mathit{sample}}}{\mathrm{\Delta }{A}_{\mathit{control}}}\right)\times 100\%$$

2.7.4 BChE inhibition assay

The BChE inhibitory activity was assayed according to our previous method (Zhang et al. 2020). Briefly, 20 μL of TL extract in 10% DMSO (1 mg/mL), 120 µL of PBS (pH 8.0, 0.1 M), and 20 µL of BChE (EC 3.1.1.8) solution (pH 8.0, 0.8 U/mL, in 0.1 M PBS) were mixed in a microplate and incubated at 25 °C for 15 min. Subsequently, 20 μL of S-butyrylthiocholine chloride solution (pH 8.0, 0.4 mM, in 0.1 M PBS) and 20 μL of DTNB solution (pH 8.0, 1.25 mM, in 0.1 M PBS) were added and incubated at 25 °C for 5 min. Before and after incubation, the absorbance was recorded at 405 nm. In the control group, TL extract was replaced with 10% DMSO. Donepezil was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{\mathrm{\Delta }{A}_{\mathit{sample}}}{\mathrm{\Delta }{A}_{\mathit{control}}}\right)\times 100\%$$

2.7.5 Tyrosinase inhibition assay

The tyrosinase inhibitory activity was assayed according to the method with slight modifications (Liang et al. 2012). Briefly, 100 μL of L-tyrosine solution (pH 6.8, 5 mM, in 0.1 M PBS), 20 µL of PBS (pH 6.8, 0.1 M) and 40 µL of TL extract in 10% DMSO (5 mg/mL) were mixed in a microplate. Subsequently, 40 μL of mushroom tyrosinase (EC 1.14.18.1) solution (pH 6.8, 200 U/mL, in 0.1 M PBS) was added and incubated at 37 °C for 20 min. The absorbance was recorded at 450 nm. In the control group, TL extract was replaced with 10% DMSO. In the blank group, enzyme solution was replaced with PBS. Arbutin was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{A_{\mathit{sample}}-A_{\mathit{blank}}}{A_{\mathit{control}}}\right)\times 100\%$$

2.7.6 Urease inhibition assay

The urease inhibitory activity was assayed according to the method with slight modifications (Aslam et al. 2011 & Sedaghati et al., 2021). Briefly, 60 μL of urea solution (pH 7.4, 100 mM, in 0.01 M PBS), 15 µL of jack bean urease (EC 3.5.1.5) solution (pH 7.4, 5 U/mL, in 0.01 M PBS) and 15 µL of TL extract in PBS (pH 7.4, 0.01 M) were mixed in a microplate and incubated at 37 °C for 30 min. Subsequently, 60 μL of phenol reagents and 60 μL of alkali reagent were added and incubated at 37 °C for 30 min. The absorbance was recorded at 625 nm. In the control group, TL extract was replaced with PBS. In the blank group, enzyme solution was replaced with PBS. Thiourea was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{A_{\mathit{sample}}-A_{\mathit{blank}}}{A_{\mathit{control}}}\right)\times 100\%$$

2.7.7 XO inhibition assay

The XO inhibitory activity was assayed according to the method with slight modifications (Nguyen et al. 2004). Briefly, 50 μL of TL extract in PBS (pH 7.5, 5 mg/mL, 0.07 M), 35 µL of PBS (pH 7.5, 0.07 M) and 30 µL of XO (EC 1.17.3.2) solution (pH 7.5, 0.01 U/mL, in 0.07 M PBS) were mixed in a quartz microplate and incubated at 25 °C for 15 min. Subsequently, 60 μL of xanthine solution (pH 7.5, 150 μM, in 0.07 M PBS) was added and the solution was incubated at 25 °C for 30 min. The reaction was stopped by adding 25 µL of 1 M HCl. The absorbance was recorded at 290 nm. In the control group, TL extract was replaced with PBS. The enzyme solution was added to the microplate after adding HCl. In the blank group, enzyme solution was replaced with PBS. Allopurinol was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{A_{\mathit{sample}}-A_{\mathit{blank}}}{A_{\mathit{control}}}\right)\times 100\%$$

2.7.8 ACE inhibition assay

The ACE inhibitory activity was assayed according to the method with slight modifications (Liu et al. 2020). Briefly, 50 μL of ACE (EC 3.4.15.1) solution (pH 8.2, 0.025 U/mL, in 0.08 M 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer with 0.3 M NaCl), 100 µL of TL extract in HEPES (pH 8.2, 1 mg/mL, 0.08 M HEPES buffer with 0.3 M NaCl) and 50 µL of N-[3-(2-Furyl)acryloyl]-Phe-Gly-Gly solution (pH 8.2, 1 mM, in 0.08 M HEPES buffer with 0.3 M NaCl) were mixed in a microplate and incubated at 37 °C for 30 min in the dark. Before and after incubation, the absorbance was recorded at 340 nm. Captopril was used as a positive reference. In the control group, TL extract was replaced with HEPES. In the blank group, enzyme solution was replaced with HEPES. The inhibitory activity was expressed as % inhibition and was calculated as follows: $$\%inhibition=\left(1-\frac{A_{S(0)}-A_{S(30)}}{A_{C(0)}-A_{C(30)}}\right)\times 100\%$$ where A_S(0) is the absorbance of the sample at 0 min, A_S(30) is the absorbance of the sample at 30 min, A_C(0) is the absorbance of the control at 0 min, and A_C(30) is the absorbance of the control at 30 min.

2.8.1 pH stability

pH and thermal stabilities of methanol extract were evaluated according to the method with slight modifications (Mihailović et al. 2015). The stability in acidic and basic environments was investigated using a methanol extract dissolved in deionised water with the pH adjusted to 1, 3, 5, 7, 9, or 11 using 1 M HCl or 1 M NaOH. The final concentration of methanol extract was 50 mg/mL. After incubation at room temperature for 1 h, the pH of the mixture was adjusted to 7 and the total peak area for thirteen peaks was calculated. Peaks 1, 8, 9, 12, and 18 were excluded from this calculation because the compounds either could not be identified (peaks 8, 9, and 12) or had no antioxidant activity (peaks 1 and 18). The pH stability was calculated for the total peak area of the thirteen peaks at pH 7. The TP_heC and ABTS scavenging abilities were examined.

2.8.2 Thermal stability

To evaluate the thermal stability, methanol extract dissolved in deionised water (50 mg/mL, pH 7) was placed in test tubes with screw caps. The test tubes were placed in a boiling water bath (100 °C). Samples were removed after 0, 15, 30, 60, 120, 180, and 240 min and cooled in an ice-water bath. The thermal stability was calculated for the total peak area of the thirteen peaks. The TP_heC and ABTS scavenging abilities were examined.

2.8.3 Modeling of the stability in the gastrointestinal tract

The digestion process of methanol extract was performed according to the method with slight modifications (Mihailović et al. 2015). 100 mL of methanol extract in distilled water (5 mg/mL) were mixed with 10 mL of PBS (pH 6.8, 10 mM) and incubated at 37 °C for 2 min (oral condition). Then 0.5 mL of 1 M HCl-KCl buffer (pH 1.5) and 5 mL of pepsin solution (pH 1.5, 32 U/mL in 1 M HCl-KCl buffer) were added to samples. The mixtures incubated at 37 °C for 60 min (stomach condition). Thereafter, in the mixture was added 1 mL of 1 M NaHCO₃ together with 1 mL of mixture of bile and pancreatic juice (pH 8.2, 10 mg/mL of pancreatin, 14,600 U/mL of trypsin, 13.5 mg/mL of bile extract in 10 mM PBS) and the pH was adjusted to 6.8. The mixtures incubated at 37 °C for 3 h (duodenal condition). Samples for calculation of the total peak area of thirteen peaks were taken at 0, 0.5, 1, 2, 3, and 4 h. The results were used for determination of TP_heC and ABTS scavenging abilities of methanol extract during simulated gastrointestinal (GI) digestion. The thermal and GI stabilities were calculated using the total peak area of the thirteen peaks at 0 h.