Flavonoids and fagopyrins in buckwheat leaves and flowers during the growing season and their relationship with weather conditions

2.1 Plant material, sampling, and extraction

The Darja variety (F. esculentum Moench), grown from seeds obtained from the Faculty of Biotechnology (Ljubljana, Slovenia), was used for the cultivation trials. Sowing took place on July 4, 2015, in a field in Gameljne, central Slovenia (46°07′09.2″ N, 14°29′51.5″ E, elevation 310 m above sea level). Plant material was sampled at 14 time points at 4-day intervals between August 3 and September 24, 2015.

Three biological replicates of leaves and flowers were sampled at each time point. Thus, six samples (three leaves and three flowers) were collected at each time point. Each biological replicate consisted of leaves or flowers taken from 30 different individual plants randomly selected throughout the field. The collected plant material was dried and milled to a fine powder using a Waring Commercial 8010 EB blender (Stamford, CT, USA). For extraction, the same protocol was used for each biological replicate: 100 mg of the milled sample was weighed in triplicate into 15 mL plastic flasks and extracted with 10 mL of acetone:water (9:1, v/v) purchased from Panreac (Barcelona, Spain). The flasks were incubated for 2 h at 60 °C. The resulting extracts were filtered through a 0.22 µm syringe filter before all subsequent analyses.

2.2 Spectrophotometric analyses of antioxidant activities and phytochemical content

All spectrophotometric assays were performed in triplicate in 330-µL TPP microplates (Thermo Fisher Scientific, Waltham, MA, USA) using a Macherey-Nagel Nanocolor UV-VIS spectrophotometer (Düren, Germany) and a BioTek Synergy H4 microplate reader (Thermo Fisher Scientific). If the initial absorbance measurement at the wavelength specific to each method was outside the linearity range (0.1–1.0), the sample was diluted to bring the absorbance within the linearity range (ideally around 0.7), and this dilution was used for further absorbance measurements. The average of the three absorbance measurements was used as the result of each spectrophotometric assay.

Antioxidant activity (AA) was determined using three spectrophotometric methods: the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the reducing power (RP) assay. The DPPH assay was performed according to the method of Brand-Williams et al. [9] using the Fluka DPPH reagent (Honeywell, Charlotte, NC, USA). To 9 µL of each sample, 225 µL of DPPH reagent was added. Absorbance was measured at 517 nm after 30 min.

The ABTS assay was performed following the method described by Re et al. [10]. The ABTS reagent was obtained from Sigma-Aldrich (Saint Louis, MO, USA). The ABTS radical cation was generated by reacting a 7 mM ABTS solution with a 140 mM ammonium persulfate (Sigma-Aldrich) solution to prepare the ABTS-persulfate solution. Eight microliters of sample were combined with 312 µL of the ABTS-persulfate solution and incubated for 6 min. Absorbance was then measured at 734 nm.

The reducing power (RP) was determined as described by Oyaizu [11] using K₃Fe(CN)₆ from Kemika (Zagreb, Croatia), trichloroacetic acid (TCA) from Merck (Darmstadt, Germany), and FeCl₃·6H₂O from Sigma-Aldrich. For each sample, 5 µL of the stock solution was combined with 54 µL of 0.1 mol/L phosphate buffer (pH 7.0) and 60 µL of 1 % K₃Fe(CN)₆ solution. The mixture was incubated at 50 °C for 20 min. Then, 30 µL of 10 % TCA and 150 µL of purified water were added. Initial absorbance (A1) was measured at 700 nm. Next, 5 µL of 0.1 % FeCl₃ solution was added, and the absorbance at 700 nm was measured again (A2). AA was calculated using the equation:

The total phenolic content (TPC) was determined using the Folin-Ciocalteu (FC) method following the procedure of Singleton and Rossi [12]. TPC was expressed as mg gallic acid equivalents (GAE) per gram of dry weight (DW) (mg GAE/g DW), calculated using a gallic acid standard. The FC reagent and sodium carbonate were purchased from Merck, and the gallic acid for the control samples was purchased from Sigma-Aldrich. Briefly, 40 µL of extract was mixed with 150 µL of purified water and 10 µL of FC reagent. After 3 min, 20 µL of 20 % sodium carbonate (Na₂CO₃) solution was added, and the mixture was incubated for 60 min. Absorbance was then measured at 750 nm.

Total flavonoid content (TFC) was determined using the aluminum chloride (AlCl₃) method described by Zhishen et al. [13] and expressed as mg rutin equivalents (RE) per gram of dry weight (mg RE/g DW), calculated using a rutin standard. The AlCl₃ solution was prepared with AlCl₃·6H₂O purchased from Sigma-Aldrich and methanol from Carlo Erba Reagents (Val de Reuil, France). To 200 µL of extract, 20 µL of 5 % AlCl₃ solution in methanol was added. After a 30-min incubation, absorbance was measured at 425 nm.

2.3 HPLC analysis

Analyses were performed on a Shimadzu Prominence high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) using a modified method based on Tavčar et al. [4]. For separation, a Phenomenex Kinetex XB-C18 column (100 × 4.6 mm, 2.7 µm) from Phenomenex (Torrance, CA, USA) was used. Acetonitrile and water were purchased from Panreac, and TFA was obtained from Roth. Separation was carried out at 40 °C with an injection volume of 5 µL. The mobile phase consisted of (A) water with 5 % acetonitrile and 0.1 % trifluoroacetic acid (TFA), and (B) acetonitrile with 5 % water and 0.1 % TFA, applied with a gradient program at 2 mL/min. The gradient program was as follows: 0 % B (0–0.5 min), increasing to 49 % B (0.5–6.0 min), then to 100 % B (6.0–30.0 min), held at 100 % B (30.0–36.0 min), and returning to 0 % B (36.0–40.0 min). Rutin and quercitrin were detected using an SPD-M20A photodiode array detector (PDA) at 254 nm, while fagopyrins were detected using a Shimadzu RF-10A XL fluorescence detector (excitation wavelength 330 nm, emission wavelength 590 nm).

The linearity and limits of quantification (LOQ) of the method were validated by measuring the areas under the curve (AUC) of seven dilutions of respective reference standards. The rutin standard was purchased from Sigma-Aldrich, the quercitrin dihydrate standard from Roth (Karlsruhe, Germany), and the hypericin standard (used as the external standard for quantification of fagopyrins) from Sigma-Aldrich. Standards of quercitrin and hypericin were first dissolved to prepare 0.1 mg/mL stock solutions (i.e., the least diluted solution for the calibration curve), while the rutin stock solution had a concentration of 2.0 mg/mL. Further dilutions for the standard calibration curves were prepared from each of these stock solutions. The most diluted solutions reached the lower LOQs (0.003 mg/mL for rutin, 1.2 × 10⁻⁵ mg/mL for quercitrin, 1.23 × 10⁻⁴ mg/mL for hypericin). AUCs of analyzed samples were converted to concentrations using equations derived from the standard calibration curves with the coefficients of determination (R ²) of 0.997 for rutin, 1.000 for quercitrin, and 0.999 for hypericin. The concentrations of quercitrin were corrected for the dihydrate form of the reference standard using the equation:

where conc is concentration, q is quercetin, and M is molar mass.

2.4 Meteorological data

Meteorological data (high and low temperature, precipitation, and sunshine duration) for 2015 were obtained from the database of the Slovenian Environment Agency [14]. The data were recorded at the Ljubljana-Bežigrad weather station, located 8 km south of the buckwheat field. The raw data were summarized into 4-day intervals corresponding to the sampling periods. Four-day averages were calculated for both maximum and minimum temperatures, while 4-day totals were used for precipitation and sunshine duration. For these totals, only the precipitation and sunshine duration in the four days preceding each sampling were considered, not the cumulative sums from the first sampling onward. The intervals were classified as “wet” (W, total precipitation > 1 mm) or “dry” (D, total precipitation ≤ 1 mm; Table 1). The 1 mm threshold was used because this amount of precipitation is equivalent to one wet day according to the definition of the World Meteorological Organization [15].

The meteorological variables were defined as follows:

1. Average 4-day maximum temperature: sum of maximum temperatures in the four days preceding the sampling divided by the number of days (e.g., for the August 19 sampling, the sum of the maximum temperatures on August 15, 16, 17, and 18 was used, divided by 4).

2. Average 4-day minimum temperature: sum of minimum temperatures in the four days preceding the sampling divided by the number of days.

3. 4-day sum of precipitation: sum of precipitation in the four days preceding the sampling.

4. 4-day sum of sunshine duration: sum of sunshine duration in the four days preceding the sampling.

2.5 Statistical analysis

The analysis used the following 12 variables (4 meteorological and 8 non-meteorological):

Average 4-day maximum temperature; average 4-day minimum temperature; 4-day sum of precipitation; 4-day sum of sunshine duration; antioxidant activity (AA) – DPPH method; AA – ABTS method; AA – reducing power method; concentration of total phenolic compounds – FC method; concentration of total flavonoids – AlCl₃ method; concentration of rutin – HPLC; concentration of quercitrin – HPLC; concentration of fagopyrins – HPLC.

Pairwise Pearson correlation matrices were calculated in Excel (Microsoft, Redmond, WA, USA), resulting in 11 correlations tested for each variable. Separate pairwise correlation matrices for leaves and flowers were created for all, wet, and dry intervals. p-Values were calculated from the Pearson correlation coefficients using SPSS version 26 software (IBM, Armonk, NY, USA), and the Benjamini-Hochberg method [16] was used for false discovery rate (FDR) correction. Corrected p-values less than 0.05 were considered significant. The pairwise correlation matrices with all Pearson coefficients and corresponding p-values are available in an Excel worksheet in the Research data (Digital Commons Data dataset). The dataset also includes data values for all variables and time points.

3.1 Weather conditions

Between July 30 and September 23, 2015, there were seven dry 4-day periods (D1–D7) with total precipitation per period of less than 1 mm, while the other seven 4-day periods were classified as wet (W1–W7) (Table 1).

The wet periods included the first sampling period (July 30 – August 2), during which only leaf samples were collected because the plants were not yet flowering. Therefore, the statistical analysis for leaves across all periods was based on 14 time points (n = 14); for flowers across all periods, n = 13; for both leaves and flowers in dry periods, n = 7; for leaves in wet periods, n = 7; and for flowers in wet periods, n = 6. Meteorological data (temperature, precipitation, sunshine) for the 14 sampling intervals are presented in Figure 1.

Figure 1:

Weather conditions during the 2015 growing season. Notes: bars representing sunshine hours during wet periods are marked with a blue border. Labels: T, temperature; h, hours; D1–D7, dry sampling periods; W1–W7, wet sampling periods.

3.2 Concentration of secondary metabolites

Analysis of the phytochemical content revealed that buckwheat flowers contained more secondary metabolites than the leaves during the main flowering period (approximately August 7 to August 30), but the concentrations of phenolic compounds in the flowers decreased rapidly after this period (Figure 2).

Figure 2:

Concentrations of secondary metabolites in buckwheat leaves and flowers. Notes: concentrations are expressed in mg substance/g dried plant material (dry weight). Concentrations of rutin, quercitrin, and fagopyrins were measured with high-performance liquid chromatography (HPLC). Error bars represent standard error. Labels: phenols, concentration of total phenolic compounds – FC method; flavonoids, concentration of total flavonoids – AlCl₃ method; D1–D7, dry sampling periods; W1–W7, wet sampling periods.

The concentration of the most abundant flavonoid, rutin, in the flowers decreased 3.7-fold from 66 mg/g dry weight (DW) at the end of August to 18 mg/g DW in the last sampling period (September 20–23). In contrast, the concentration of fagopyrins in the flowers remained constant across all sampling periods and did not decrease sharply in September. The concentration of the second most abundant flavonoid, quercitrin, in the flowers reached about a quarter of the rutin concentration during the main flowering period, while in the leaves, the quercitrin concentration was approximately 100-fold lower than the rutin concentration throughout August and September.

3.3 Antioxidant activity

The ABTS and RP assays showed a peak in antioxidant potential in the flowers during intense flowering, followed by a sharp decline, while AA in the leaves generally remained constant (Figure 3).

Figure 3:

Antioxidant activity in buckwheat leaves and flowers. Notes: error bars represent standard error. Antioxidant activity coefficients for different methods are not linearly related and therefore are not directly comparable. Labels: RP, reducing power method; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method; DPPH, 2,2-Diphenyl-1-picrylhydrazyl method; D1–D7, dry sampling periods; W1–W7, wet sampling periods.

These results in the flowers reflect the trends observed for phenolic compounds. According to both the ABTS and RP methods, AA in the flowers decreased by approximately 4- to 5-fold between August 30 and September 23.

3.4 Correlations between the meteorological and non-meteorological variables

Correlations between the meteorological and non-meteorological variables, such as concentrations of secondary metabolites and antioxidant activities, are shown in Table 2 as Pearson correlation coefficients (r). p-Values (both unadjusted and FDR-adjusted) are included when unadjusted p-values were below 0.05. FDR-corrected p-values less than 0.05 were considered significant.

In buckwheat leaves, there was a strong and significant positive correlation during dry periods between fagopyrin concentration and average minimum temperature (r = 0.95, FDR-adjusted p = 0.011, n = 7). Under higher minimum nighttime temperatures, which are common during summer heatwaves, fagopyrin concentration in the leaves increased. In buckwheat flowers, antioxidant activity (RP method) showed a significant positive correlation with both average maximum temperature (r = 0.95, FDR-adjusted p = 0.0091, n = 7) and average minimum temperature (r = 0.90, FDR-adjusted p = 0.029, n = 7) during dry periods, but the positive correlation with sunshine did not reach significance (r = 0.73, FDR-adjusted p = 0.15). Thus, during summer heatwaves, the antioxidant activity of the flowers increased. Other correlations in leaves and flowers, regardless of the period, were not significant when the FDR correction was applied. Figure 4 shows scatter plots for the observed significant correlations between the meteorological and non-meteorological variables.

Figure 4:

Observed significant correlations (FDR-adjusted p < 0.05) between the meteorological and non-meteorological variables (antioxidant activities and concentrations of secondary metabolites). Labels: minT, minimum temperature; maxT, maximum temperature; RP, antioxidant activity – reducing power method; fagopyrins (L), concentration of fagopyrins in buckwheat leaves – HPLC method; R ², coefficient of determination.

Four-day sunshine duration showed no correlation with rutin (r = 0.10) or quercitrin (r = 0.10) in buckwheat flowers across all periods. However, when dry and wet periods were analyzed separately, strong positive correlations were observed during dry periods (rutin: r = 0.83; quercitrin: r = 0.87), and strong negative correlations during wet periods (rutin: r = −0.88; quercitrin: r = −0.92) (Table 2). These correlations do not imply causation, and they were no longer significant at the 0.05 level after FDR correction (e.g., FDR-adjusted p = 0.059 for the correlations of both rutin and quercitrin with sunshine duration in buckwheat flowers in dry periods), although the unadjusted p-values were below the 0.05 threshold (e.g., unadjusted p-values for the correlation with sunshine duration in flowers in dry periods were 0.021 for rutin and 0.011 for quercitrin).