Flavonoids

Using a FFQ, participants reported the intake of foods consumed during the previous month. The FFQ was designed for the Dutch population and based on the VetExpress, a 104-item FFQ, valid for estimating the intake of energy, total fat, saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA), and cholesterol in adults [5 (link)]. The VetExpress was updated and extended with vegetables, fruit, and foods for estimating the intake of specific PUFA’s, vitamins, minerals, and flavonoids. To identify relevant foods and food groups for this questionnaire, food consumption data of the Dutch National Food Survey of 1998 were used. Foods that contributed >0.1% to the intake of one of the nutrients of interest of adults were added in this survey. Thus, the FFQ is expected to include foods that cover the daily intake of each nutrient of food of interest for at least 90%. In a final step, foods were clustered to food items and extended with new foods on the market and foods to guarantee face validity. The FFQ was sent to each study participant, and after completing it, the participants returned the FFQ in an envelope free of postal charge. A dietician went through each FFQ to check for completeness. If necessary, she contacted the participants by telephone and obtained information on unclear or missing items. The FFQ also included questions on adherence to a special diet as well as questions about the use of dietary supplements.
Some of the offspring and their partners who completed the general questionnaire of the LLS were invited to the clinic for additional measurements at the Leiden University Medical Center. These measurements lasted a half day and couples were invited for the morning program or the afternoon program, which were slightly different due to practical reasons. The first 24-hour recall was performed in those participants who came to the clinic for the measurement in the morning program [N=128 (Noffspring=62, Ncontrol=66)]. A dietician asked the participants about their dietary intake of the previous day covering all foods and beverages consumed from waking up until the next morning. The dieticians received standardized training, using a formal protocol, to reduce the impact of the interview on the reporting process. For the two remaining recalls, the dietician contacted the participants by telephone within the next seven days. The 24-hour recalls were performed throughout the year and the days were chosen non-consecutively. They include a randomly assigned combination of days of the week with all days of the week represented (80% weekdays and 20% weekend days), for each individual.
The food data from both dietary assessment methods were converted into energy and nutrient intake by using the NEVO food composition database of 2006 [6 ]. Furthermore, foods were categorized into 24 major food groups. Age was calculated from date of birth and completion date of the FFQ. For subjects with missing information on the date of completing the FFQ, we used the median date of the other subjects.

The aluminum chloride colorimetric method was used for the determination of the total flavonoid content of the sample [21 –24 ]. For total flavonoid determination, quercetin was used to make the standard calibration curve. Stock quercetin solution was prepared by dissolving 5.0 mg quercetin in 1.0 mL methanol, then the standard solutions of quercetin were prepared by serial dilutions using methanol (5–200 μg/mL). An amount of 0.6 mL diluted standard quercetin solutions or extracts was separately mixed with 0.6 mL of 2% aluminum chloride. After mixing, the solution was incubated for 60 min at room temperature. The absorbance of the reaction mixtures was measured against blank at 420 nm wavelength with a Varian UV-Vis spectrophotometer (Cary 50 Bio UV-Vis Spectrophotometer, Varian). The concentration of total flavonoid content in the test samples was calculated from the calibration plot (Y = 0.0162x + 0.0044, R² = 0.999) and expressed as mg quercetin equivalent (QE)/g of dried plant material. All the determinations were carried out in triplicate.

Total flavonoid content was determined following a method by Park et al (2008) [28 (link)]. In a 10 ml test tube, 0.3 ml of extracts, 3.4 ml of 30% methanol, 0.15 ml of NaNO₂ (0.5 M) and 0.15 ml of AlCl₃.6H₂O (0.3 M) were mixed. After 5 min, 1 ml of NaOH (1 M) was added. The solution was mixed well and the absorbance was measured against the reagent blank at 506 nm. The standard curve for total flavonoids was made using rutin standard solution (0 to 100 mg/l) under the same procedure as earlier described. The total flavonoids were expressed as milligrams of rutin equivalents per g of dried fraction.

Pearson’s correlation analysis was conducted by calculating the correlation coefficient between anthocyanin content and the expression of DEGs enriched in the flavonoid–anthocyanin biosynthesis pathway (ko00941 and ko00942). Furthermore, R2R3-MYB and bHLH TFs with differential expression levels were used to perform a correlation analysis with differentially accumulated anthocyanins. Interaction networks between DEGs and differentially accumulated anthocyanins were visualized using Cytoscape 2.8.2 (Cho et al., 2016 (link)).

Flavonoids were extracted from the seeds of litchi for LC-MS/MS analysis. The details of the extraction of the flavonoids from litchi seeds and the procedure of LC-MS/MS analysis for the identification and structural confirmation of the flavonoids can be found in the Supplementary Material.

An Autoflex Speed MALDI time-of-fight (TOF)/TOF mass spectrometer (Bruker Daltonics) with a MALDI source equipped with a 2,000-Hz solid-state Smartbeam Nd : YAG UV laser (355 nm, Azura Laser AG, Berlin, Germany) was used for profiling and imaging (Figure 1D).
To acquire in situ (+) MS profiling data of flavonoids from the tissue sections, all mass spectra were obtained over the m/z range of 100 to 700, each mass spectrum included an accumulation of 50 laser scans, and each scan was amassed from 500 laser shots. Three biological replicates of the sample and three technical replicates of each biological replicate were performed for MALDI-MS data acquisition (n = 3 × 3). To acquire the images of flavonoids, a 250-μm laser raster step-size was utilized for flavonoid in situ detection in tissues, and each pixel (scan spot) included 300 laser shots. With the use of FlexImaging 4.1 (Bruker Daltonics), the three “teaching points” for the correct positioning of the solid-state UV laser (Smartbeam Nd : YAG) for spectral acquisition were marked around a tissue section using a white ink correction pen. The m/z values of the compound ions that can be used for external mass calibration were listed as follows: His ([M+H]⁺, m/z 156.0768), Gly-Gly-Leu (tripeptide, [M+H]⁺, m/z 246.1448), Ala-His-Lys (tripeptide, [M+H]⁺, m/z 355.2088), Leu-Leu-Tyr (tripeptide, [M+H]⁺, m/z 408.2493), and Arg-Gly-Asp-dTyr-Lys (pentapeptide, [M+H]⁺, m/z 620.3151). Gly-Gly-Leu (tripeptide, [M+H]⁺, m/z 246.1448) and Arg-Gly-Asp-dTyr-Lys (pentapeptide, [M+H]⁺, m/z 620.3151) ions were selected in combination with the matrix ion of 2-MBT([M+H]⁺, m/z 167.9942) for internal mass calibration in the cubic enhanced mode. For the MALDI-TOF-MS analysis, MS/MS spectra were acquired in collision-induced dissociation (CID) mode, and argon was used as the collision gas. The flavonoid fragment ions were acquired under the following condition: ion source 1, 19.0 kV; ion source 2, 17.4 kV; lens, 8.8 kV; reflector 1, 21.0 kV; reflector 2, 9.8 kV; and accelerating voltage, 20.0 kV. The UV laser power ranged from 65% to 90%. MS/MS spectra were recorded based on no less than 5,000 laser shots over the m/z range of 0 to 100 with a sampling rate of 2.00 G/s, a detector gain of 9.5×, and an electronic gain of 100 mV.