Catechin

Free and bound phenolics were extracted as described in Verardo et al. (2011) (link). The analysis of BSG free and bound polyphenols was carried out with the use of an ACQUITY Ultra Performance LC system equipped with photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, United States) series with a mass detector Q/TOF micro mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source operating in negative mode at the following conditions: capillary voltage, 2300 kV; source temperature, 100°C; cone gas flow, 40 L/Hr; desolvatation temperature, 500°C; desolvatation gas flow, 11,000 L/h; and scan range, m/z 50–1500. Separations of individual polyphenols were carried out using an ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, United States) at 40°C. The elution gradient was carried out using water containing 1% acetic acid (A) and acetonitrile (B), and applied as follows: 0 min, 1% B; 2.3 min, 1% B; 4.4 min, 7% B; 8.1 min, 14% B; 12.2 min, 24% B; 16 min, 40% B; 18.3 min, 100% B, 21 min, 100% B; 22.4 min, 1% B; 25 min, 1% B. The sample volume injected was 2 μL and the flow rate used was 0.6 mL/min. The compounds were monitored at 280 nm. Integration and data elaboration were performed using MassLynx 4.1 software (Waters Corporation, United States). For the quantification of phenolic compounds, solutions of ferulic acid, chlorogenic acid, catechin, and quercetin in methanol were prepared and used as standard.

Phenolics in crude extracts and each fraction of lingonberries were identified and quantified by HPLC-PDA (Waters e2695 Alliance system, Waters, Milford, MA, USA) according to Raudone et al. with some modifications [27 (link)]. Chromatographic separation was carried out on an ACE C18 reversed-phase column (250 mm × 4.6 mm, particle size 3 µm; ACT, UK) with a gradient elution consisting of 0.1% trifluoroacetic acid in water (eluent A) and acetonitrile (eluent B) at a flow rate of 0.5 mL/min, injection volume of 10 µL, and column temperature maintained at 35 °C. The gradient pattern was 0 min, 10% B; 0–40 min, 30% B; 40–60 min, 70% B; 60–64 min, 90% B; 64–70 min, 10% B. Prior to HPLC-PDA analysis, all samples were dissolved in 70% ethanol until complete dissolution, obtaining a concentration of 1 mg/mL, and filtered through a pore size 0.2 µm PVDF syringe filters (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
The described modified method was validated following international guidelines [28 ]. The selectivity of peaks was evaluated and phenolic compounds were identified by comparing the retention times, UV spectra of the analytes with those of the reference compounds, and on the basis of previous reports on lingonberry phenolics. The PDA detector was set at a wavelength of 280 nm for proanthocyanidins and catechins, 360 nm for flavonols, 520 nm for anthocyanins, 330 for hydroxycinnamic acids, and 260 nm for hydroxybenzoic acids. All phenolics were quantified according to 5–7 points linear calibration curves of external standards, except well-known predominant compounds of lingonberry leaves—quercetin-3-O-(4”-(3-hydroxy-3-methylglutaryl)-rhamnoside (quercetin-HMG-rhamnoside), and 2-O-caffeoylarbutin, because of commercially unavailable standards. They were tentatively quantified using calibration curves of standard substances with similar chemical structures. Limits of detection (LOD) and of quantification (LOQ) were determined via the signal-to-noise ratio method. The trueness of the method was expressed as percent recoveries of phenolics at low, medium, and high concentrations of range, each analyzed in triplicate. To assess the repeatability and intermediate precision of the method, relative standard deviation percentages (% RSD) of peak areas of each quantified phenolic compound were calculated within (six times per day) and between days (three consecutive days), respectively, resulting in total repeatability of 18 replicates.

Catechins and caffeine were extracted from the samples according to the method described by Shan et al. [46 ] with minor modifications. Briefly, 0.1 g of freeze-dried tea leaf tissue was ground in liquid nitrogen with a mortar and pestle and extracted with 3 mL 80 % methanol in an ultrasonic sonicator for 10 min at 4 °C. After centrifugation at 6,000 rpm for 10 min, the residues were re-extracted twice as described above. The supernatants were combined and diluted with 80 % methanol to a volume of 10 mL. The obtained supernatants were filtered through a 0.22 μm organic membrane before HPLC analysis.
The catechin and caffeine contents in the extracts were measured using a Waters 2695 HPLC system equipped with a 2489 ultraviolet (UV)-visible detector. A reverse-phase C18 column (Phenomenex 250 mm × 4.6 mm, 5 micron) was used at a flow rate of 1.0 mL/min. The detection wavelength was set to 278 nm, and the column temperature was 25 °C. The mobile phase consisted of 0.17 % (v/v) acetic acid (A) in water, 100 % acetonitrile (B), and the gradient elution was as follows: B 6 % from 0 to 4 min, to 14 % at 16 min, to 15 % at 22 min, to 18 % at 32 min, to 29 % at 37 min, to 45 % at 45 min, to 45 % at 50 min, to 6 % at 51 min and to 6 % at 60 min. Then, 10 μL of the filtrate was injected into the HPLC system for analysis. The filtered sample (10 μL) was injected into the HPLC system for analysis. Samples from each stage of leaf development were analyzed in triplicate.
Amino acids were extracted with hot water [47 , 48 (link)]. Specifically, 0.15 g of freeze-dried tea leaves was ground in liquid nitrogen with a mortar pestle and extracted with 5 mL deionized water for 20 min in a water bath at 100 °C. After centrifugation at 6,000 rpm for 10 min, the residues were re-extracted once as described above. The supernatants were combined and diluted with water to a volume of 10 mL. The supernatants were also filtered through a 0.22 μm membrane before HPLC analysis. Theanine in tea was detected using a Waters 600E series HPLC system equipped with a quaternary pump and a 2489 ultraviolet (UV)-visible detector. A reverse-phase C18 column (Phenomenex 250 mm × 4.6 mm, 5 micron) was used at a flow rate of 1.0 mL/min. The column oven temperature was set to 25 °C. The detection wavelength was set to 199 nm for analysis [49 ]. The mobile phase consisted of 0.05 % (v/v) trichloroacetic acid (A) in water, 50 % acetonitrile (B), and the gradient elution was as follows: B 0 % (v/v) to 100 % at 40 min, to 100 % at 45 min and to 0 % at 60 min [31 ]. Then, 5 μL of the filtrate was injected into the HPLC system for analysis.
Amino acids in tea were detected using a Waters 600E series HPLC system equipped with a quaternary pump, a 2475 fluorescence detector and a 2489 ultraviolet (UV)-visible detector. The Waters AccQ•Tag method [50 (link)] with a Waters AccQ•Tag column (Nova-Pak C18, 4 μm, 150 mm × 3.9 mm) was employed to detect various amino acids according to the protocol of the AccQ•Fluor Reagent Kit [51 , 52 (link)]. To determine the linearity of the chromatographic techniques, calibration plots of standards were constructed based on peak areas (y) using solutions of various concentrations (x). All plots were linear in the examined ranges; the linear ranges for different concentrations of standard compounds are shown in the plots (μg mL⁻¹). The R² value refers to the correlation coefficient of the equation for calculating the content of a compound. The standard compounds C, EC, EGC, ECG, EGCG, GC, theanine and caffeine were purchased from Shanghai Winherb Medical Technology, Ltd., China.
Anthocyanin was extracted as follows: 0.1 g freeze-dry tea leaf tissue was ground in liquid nitrogen and extracted with 5 mL extraction solution (80 % methanol: 1 % hydrochloric acid [HCl]) using an ultrasonic sonicator for 10 min at room temperature. After centrifugation at 6,000 rpm for 10 min, the residues were re-extracted twice as described above. The supernatants were combined and diluted with extraction solution to 10 mL, followed by extraction with trichloromethane. The anthocyanin content was determined by colorimetry at 525 nm [53 (link)].

Dry lotus (Nelumbo) leaves was provided by Hunan Zhenxing Traditional Chinese Medicine Co., Ltd. (Changsha, Hunan, China). Cellulase (CAS 9012-54-8, S10041) was bought from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Total dietary fiber assay kit (TDF-200A) was bought from Megazyme International Ireland Ltd (Bray, Ireland). Rutin, hyperoside, isoquercitrin, astragalin and quercetin were bought from Chengdu Aifa Biotechnology Co., Ltd (Chengdu, Sichuan, China). Catechin, myricetin and kaempferol were bought from Hefei Bome Biotechnology Co. Ltd (Hefei, Anhui, China). DPPH and ABTS were brought from Shanghai Maclean Biochemical Technology Co. Ltd (Shanghai, China). The rest of reagents were brought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and were analytical pure.

The phenolic profile of PME was determined in the dried extract re-dissolved in a methanol:water (80:20, v/v) mixture by liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry (LC-DAD-ESI/MSⁿ) (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA), as previously described by Bessada et al.^{20 (link)}. For the double online detection, 280 and 370 nm were used as preferred wavelengths for diode array detection (DAD) and in a mass spectrometer (MS) connected to HPLC system via the DAD cell outlet. The MS detection was performed in negative mode, using a Linear Ion Trap LTQ XL mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. The identification of the phenolic compounds was performed using standard compounds, when available, by comparing their retention times, UV–Vis and mass spectra; and also, comparing the obtained information with available data reported in the literature giving a tentative identification. For quantitative analysis, a 7-level calibration curve for each available phenolic standard was constructed based on the UV signal [catechin (y = 84.950x − 23.200, R² = 0.999, LOD (Limit of detection) = 0.17 μg/mL; LOQ (Limit of quantification) = 0.68 μg/mL, peaks 1, 2, 3, 4, and 5), myricetin (y = 23287x − 581,708, R² = 0.9988, LOD = 61.21 µg/mL and LOQ = 185.49 µg/mL, peaks 6 and 7) and quercetin-3-O-glucoside (y = 34843x − 160,173, R² = 0.9998; LOD = 0.21 μg/mL; LOQ = 0.71 μg/mL, peak 8)]. For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of the most similar available standard. The results were expressed as mg/g of extract DW.