SUPPLEMENTARY MATERIAL Isolation and structural identification of the main anthocyanin monomer in Vitis amurensis Rupr

Many types of anthocyanins are present in Vitis amurensis Rupr of ‘Beibinghong’, which are grown in Northeast China and have high antioxidant activity. However, the anthocyanin with the highest content in V. amurensis Rupr has not yet been identified. In this study, pulsed electric field extraction and semi-preparative liquid phase separation were used to isolate the anthocyanin monomer from ‘Beibinghong’. UV-Vis spectroscopy, FTIR spectroscopy, mass spectra (MS) and nuclear magnetic resonance (NMR) were used to identify the anthocyanin monomer. The antioxidant activities of the anthocyanin monomer were also analyzed. Malvidin-3,5-O-diglucoside was identified as the main anthocyanin in V. amurensis Rupr, which could be used as a raw material for its extraction. Furthermore, malvidin-3,5-O-diglucoside can be potentially used as a functional food, and a novel therapeutic and preventive agent for oxidative stress-related diseases. This study provides technical information for the future purification and structural identification of anthocyanins.


UV-Vis spectroscopy
The anthocyanin was dissolved in 0.1% hydrochloric acid methanol (A), and scanned at 200-700 nm with UV-Vis spectroscopy. Three drops of 5% AlCl 3 in methanol was added to the solution (B), and the mixture was left undisturbed for 10 min. The scanning was repeated to observe any shift in the maximum absorption wavelength.
Each sample was injected in duplicate. Identity assignment was based on the retention times and MS/MS spectral analysis with a QTrap 4500 Detector AB Applied Biosystems MDS Sciex (Framingham, MA, USA). Positive ionization mode was performed under the following parameters: mass range 200-900 m/z, capillary temperature 550 °C, 5500 V of ion spray voltage, curtain gas 30 psi, ion source gas I 60 psi and ion source gas II 70 psi.

NMR spectrometry analysis
The sample was dissolved in CF 3 COOD-CD 3 OD (5:95; v/v) solvent at a concentration of 10 mg/mL, and measured at 600 MHz 1 H and 150 MHz 13 C, respectively, using a Bruker AVANCE III 600 MHz spectrometer (Bruker Biospin Co., Karlsruhe, Germany) with CPQCI probe. The chemical shift is displayed on the δ (ppm) scale, with TMS or the solvent signal as the internal standard. Sample temperatures were stabilized at 298 K (Lee et al. 2009;Srivastava & Vankar 2010;Cruz et al. 2015;Matera et al. 2015).

DPPH assay
A volume of 1.5 mL of 0.004% ethanolic DPPH solution was mixed with 0.5 mL sample and kept for 30 min at room temperature in the dark. The absorbance value at 517 nm was measured as A 1 , with three replicates for each sample. The absorbance of sample combined with ethanol was recorded as A 2 , while ethanol combined with DPPH was A 0 . Anhydrous ethanol was used as blank control.

ABTS assay
ABTS was obtained by mixing an equal proportion of 7 mmol/L ABTS and 2.45 mmol/L K 2 S 2 O 8 , and kept in the dark room at room temperature for 12-16 h. The ABTS solution was diluted with ethanol until the absorbance reached 0.7 ± 0.02 at 734 nm.
The anthocyanin solution (200 μL) was added to 4 mL ABTS solution, incubated at 37 ºC in a water bath for 10 min, and the absorbance value at 734 nm was measured as A 1 , with three replicates for each sample. The absorbance of the sample combined with ethanol was recorded as A 2 , while ethanol combined with ABTS as A 0 . Anhydrous ethanol was used as blank control.

Statistical analysis
Each sample were analyzed by UV-Vis spectroscopy, FTIR spectroscopy, UPLC-MS and NMR for twice. Data of UV-Vis, FTIR and MS/MS spectra were processed using Origin 9.0 software. NMR spectra data were analyzed by MestReNova software. All experiments of antioxidant activity analysis were carried out three times. Data were expressed as the mean ± the standard deviation (SD).

Acknowledgements
We thank Professor Yongguang Yin of Jilin University for his technical support in the purification process, and Chunyu Wang for his assistance in NMR testing. We also thank the Jilin Agricultural University Agricultural Products Processing and Storage

Laboratory and the National Engineering Laboratory for Deep Processing of Wheat and
Corn for their support. Figure S1. Semi-prep HPLC chromatogram. Figure S2. UV-Vis spectra of the anthocyanin monomer. Figure S3. FTIR spectrum of the anthocyanin monomer. Figure S4. MS/MS spectrum of the anthocyanin monomer.    Figure S8. DPPH scavenging rate. Figure S9. ABTS scavenging rate. Table S1. 1 H and 13 C spectral data (ppm) of malvidin-3,5-O-diglucoside. Figure S1. Semi-prep HPLC chromatogram.
Peak 3-the main anthocyanin monomer, was collected and studied. Figure S2. UV-Vis spectra of the anthocyanin monomer.