The Profile and Content of Polyphenolic Compounds and Antioxidant and Anti-Glycation Properties of Root Extracts of Selected Medicinal Herbs

The objective of our study was to analyse the extracts from six medicinal herb roots (marshmallow, dandelion, liquorice, angelica, burdock, and comfrey) in terms of antioxidant capacity (ABTS, DPPH) and inhibition of advanced glycation end product (AGEs) formation. The quantification of phenolic acids and flavonoids was analysed using the UHPLC-DAD-MS method. Fifteen polyphenolic compounds were detected in the studied herbs. The higher number of polyphenols were found in marshmallows (ten polyphenols), while the lowest was in comfrey (five compounds). Liquorice root revealed the highest individual phenolic concentration (382 µg/g dm) with the higher contribution of kaempferol-3-O-rutinoside. Comfrey root extract was characterised by the most abundant TPC (Total Phenolic Content) value (29.79 mg GAE/ g dm). Burdock and comfrey showed the strongest anti-AGE activity studies with the BDA-GLU model. Burdock root was also characterised by the highest anti-AGE activity in the BSA-MGO model. The highest antioxidant capacity was determined by ABTS (72.12 µmol TE/g dw) and DPPH (143.01 µmol TE/g dw) assays for comfrey extract. The p-coumaric acid content was significantly correlated with anti-AGE activity determined by the BSA-MGO model. This research sheds new light on the bioactivity of root herbs, explaining the role of p-coumaric acid in preventing diabetes. Supplementary Information The online version contains supplementary material available at 10.1007/s11130-024-01180-z.


Extraction procedure
Powdered herb samples (200 mg) were extracted with 1 mL of methanol 80% (v/v), vortexed for 30 s, and placed in the ultrasonic bath for 30 s.The vortex and ultrasound application were repeated three times.After that, the samples were centrifugated at 14,000 rpm (VWR micro star 30R, Radnor, PA, USA) for 10 min at 4 °C.The obtained supernatants were transferred to 5 mL vials and the herb residues were re-suspended in fresh extraction solvent.These steps were repeated 5 times until 5 mL of each herb supernatant had been extracted.Each herb sample was prepared in three replications.
The above procedure was carried out to obtain material for determining the antioxidant capacity (AC), total polyphenol content (TPC), anti-AGEs potential and profile of polyphenols.
However, extracts to analyse the antiglycaemic activity, the solvent was evaporated under nitrogen, and the obtained material was dissolved in a suitable solvent used in the abovementioned test.
The analysis was performed in triplicate.

Total phenolic content (TPC)
The total phenolic content (TPC) was carried out using the Folin-Ciocalteu reagent according to the procedure described by Foss et al. [1].A mixture containing 15 µL of appropriately diluted extract fractions and 240 µL of Folin's phenol reagent were placed into wells of microplates and incubated for 10 min at room temperature (RT).Next, 15 µL of 20% sodium carbonate was added and shaken.Absorbance was measured at 765 nm using a microplates reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany).Obtained result were expressed as mg gallic acid equivalents (GAE) per gram of sample.All measurements were performed in triplicates.

Determination of polyphenols profile by UHPLC-DAD-MS
The analysis of polyphenols was performed according to the methodology described by Sawicki et al. [2].Polyphenols qualitative and quantitative were carried out using a UHPLC system (Nexera XR, Shimadzu, Japan) coupled with a diode area detector (DAD) and mass spectrometer (LCMS-2020, Shimadzu, Japan).Measurement parameters were as follows: eluent 0.01% formic acid in water with 2 mM ammonium formate (A) and 0.01% formic acid in 95% acetonitrile solution with 2 mM ammonium formate (B); flow rate 0.15 mL/min; scanning in negative ionization; column Column C18 BEH (1.7 μm particle size; 100 × 2.1 mm; Waters, Warsaw, Poland); oven temperature was 50 °C; sample injection volume 10 µL.An analysis was conducted in the selected ion monitoring mode (SIM).Analysed compounds were identified based on their qualitative ions, retention times and λmax value with the previously published data [2,3].The quantity of polyphenols was calculated from the UHPLC-DAD-MS peak area against commercially available standards.The UHPLC-DAD-MS data of herb phenolic acids and flavonoids are presented in Table S1.

Antioxidant capacity ABTS assay
The measurement was carried out according to the method described by Horszwald and Andlauer [4].Briefly, ABTS•+ solution was diluted with a water:methanol mixture (20:80, v/v) to an absorbance level of 0.70 ± 0.02 at 734 nm.For the spectrophotometric assay, 290 μL of the ABTS•+ solution and 10 μL of the respective extract, Trolox or blank (80% methanol) were mixed, and absorbance was measured at 734 nm directly after 6 min incubation at 30 °C using a microplate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany).The response of the calibration curve was linear from 0.01 to 2.0 mM (R2 = 0.999).The results were expressed as μmol of Trolox equivalents (TE) /g of sample.Analysis was carried out in triplicate.

DPPH assay
Determination of antioxidant properties by the DPPH • method was conducted according to the procedure developed by Horszwald and Andlauer [4].The DPPH • radical solution was prepared by dissolving 10 mg of DPPH• in 250 mL of 80% methanol.To perform the spectrophotometric test, 300 µL of DPPH• solution and 20 µL of appropriately diluted sample or Trolox solution were mixed.The resulting mixture was left for 30 min at room temperature in the dark.Decreasing absorbance of the resulting solution was monitored at 517 nm using a microplate reader FLUOstar Omega (BMG LABTECH, Ortenberg, Germany).The standard curve was plotted based on the length of the lag phase versus Trolox concentrations within the range of 0.01 -2.0 mM (R 2 = 0.998).The results were expressed as μmol of TE/g of sample.
Analysis was carried out in triplicate.

In vitro antiglycation activity BSA-GLU assay
Inhibition of AGEs formation in the BSA-GLU test was assessed following the method of Przygodzka and Zieliński [5].One millilitre of a mixture of D-glucose (1.0 M), BSA (10 mg/mL) and sodium azide (0.1 mg/mL) in phosphate buffer (0.1 M, pH 7.4) was incubated (55 °C for 3 days) with or without 1 mL of the analyzed extract dissolved in phosphate buffer.Three hundred microliters of the obtained material was placed into 96-well plates, and the formation of AGEs was determined based on the measurements of fluorescence at λ = 330 nm (excitation wave) and at λ = 410 nm (emission wave) (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany).The positive control was aminoguanidine.The analysis was carried out in triplicate.
The mathematical formula was used to calculate the percent inhibition of AGEs formation by the tested extracts (Formula 1).% inhibition = (1-( fluorescence of the sample/ fluorescence of the blank)) * 100% (1)

BSA-MGO assay
Inhibition of AGEs formation in the BSA-MGO test was assessed following the method of Przygodzka and Zieliński [5]. 1 mL of a mixture of BSA (1 mg/mL), MGO (5 mM) and sodium azide (0.1 mg/mL) in phosphate buffer (0.1 M, pH 7.4) was incubated (37 °C for 7 days) with or without 1 mL of the tested extracts dissolved in phosphate buffer.Three hundred microliters of the obtained material was placed into 96-well plates, and the formation of AGEs was determined based on the measurements of fluorescence at λ = 340 nm (excitation wave) and at λ = 420 nm (emission wave) (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany).The positive control was aminoguanidine.The analysis was carried out in triplicate.

Statistical analysis
The data are presented as mean values ± standard deviations of triplicate measurement.
The differences between samples were analyzed by a one-way ANOVA with Tukey's test (p < 0.05).Pearson's linear correlation was applied to measure the statistical relationship between anti-AGEs, ABTS, DPPH parameters, individual phenolics, and TPC.Principal component analysis (PCA) was performed based on a correlation matrix to reduce the data's dimensionality and present the samples in a new coordinate system.The statistical analysis was performed using STATISTICA 13.0 (StatSoft Inc., Tulsa, OK, USA).

Table S1 .
The parameter of the analyzed phenolic acids and flavonoids