JFDA Supporting Information 20200101.Pdf

1 The Chemical Aspects of Raman Spectroscopy: Statistical Structure-Spectrum

2 Relationship in The Analyses of Bioflavonoids 3 Chih-Hsien Wanga, Chia-Chi Huang*a, Wenlung Chen*a, and Yen-Shi Laib 4 aDepartment of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 60083, 5 Taiwan 6 bColossus BioPharma Consultants Company, Ltd., NO 15, LN 98, Jhengde Road, Kaohsiung 80449, 7 Taiwan 8

25 26 *Corresponding authors: 27 E-mail address: [email protected] (C.-C. H.); [email protected] (WL. C.) 28

1 1. Materials 2 Flavonol, Flavone, Apigenin, Genistein, (+)-Catechin hydrate, (−)-Gallocatechin, (−)-Gallocatechin 3 gallate, (-)-Epicatechin, and (-)-Epigallocatechin were purchased from SIGMA; Kaempferol, Myricetin, 4 Luteolin, Daidzein, (−)-Catechin hydrate, and gallic acid from Fluka; and Quercetin and Flavanone from 5 Aldrich. (-)-Epicatechin 3-gallate and (-)-Epigallocatechin 3-gallate were obtained from Sigma- 6 Aldrich. Cyanidin, Dephinidin, Malvidin, Pelargonidin, Peonidin, Petuidin, Callistephin, Malvin, 7 Oenin, and Pelargonin were purchased from Extrasynthese (Genay Cedex, France). Acetonitrile and 8 Trifluoroacetic acid (HPLC grade) were obtained from Merck. All these chemicals were used as 9 received. Fresh yam tuber of Dioscorea alata L., D. alata L. var. purpurea was purchased from a local 10 agricultural cultivation station (Ming-Chien, Taiwan). 11 2. Purifications of anthocyanins by High Performance Liquid Chromatography (HPLC) 12 Extraction of anthocyanins was reported earlier [1]. The HPLC (HITACHI, Ltd, Tokyo, Japan) system 13 included pressurizing pump (Model L-7100), UV-Vis detector (Model L-7420), injector (Rheodyne 14 Model 7725) and analysis software (D-7000 HSM). The operation conditions were: C18 column 15 (Thermo Hypersil-keystone BDS HYPERSIL C18, 250 mm × 10 mm i.d. with a particle size of 5 μm);

-1 16 flow rate, 1 mL min ; Solvent A: 0.5% acetic acid in H2O; Solvent B: 0.2% acetic acid in methanol; 17 Mobile phase gradient: Solvent B: 0% at 0 min, 100% at 20 min,100% at 25 min; Injection volume: 18 100 μL; Detection wavelength: 530 nm. 19 Ten fractions of anthocyanin were collected and evaporated to remove volatile solvents under 20 reduced pressure. Powder samples were obtained by subsequent lyophilizing cycles. 21

1 Figure and Table Legends 2 3 Fig. S1 HPLC chromatogram of the principal anthocyanins fraction of Dioscorea purpurea. 4 Fig. S2 FT-Raman spectrum of peak No. 5 from Fig. S1. 5 Table S1 Mass spectrometry and database-predicted molecular ion structures of the HPLC fractions 6 of Dioscorea purpurea. 7 Table S2 Recoded scale values for the structure indices by SPSS. 8 9 Supplemental file: FT-Raman spectra and structures of 28 flavonoid standards. 10

Fig. S1

Intensity

Retention Time/min

Fig. S2

Table S1

Peak Rt Molecular MS/MS (m/z) compound (min) ion [M+] (m/z) 1 2 3 4 5 710 692, 656 732 673 979 817, 449, 287 cyanidin + 3 glucose + sinapic acid vitisin B-pyranopeonidin + 3 glucose + sinapic acid 1017 855, 487, 325 or 5-methylpyranocyanidin + 3 glucose + sinapic acid 1155, 979, 949, 817, 655, 625, cyanidin + 4 glucose + ferulic acid + sinapic 1317 287 acid 1199, 933, 831, 669, 625, 463, 1361 peonidin + 4 glucose + 2 sinapic acid 301 1347 1185, 979, 817, 655, 449, 287 cyanidin + 4 glucose + 2 sinapic acid 1347, 1141, 979, 817, 655, 1509 cyanidin + 5 glucose + 2 sinapic acid 449, 287 6 vitisin B-pyranopeonidin + 2 glucose 649 487, 325 or 5-methylpyranocyanidin + 2 glucose 787 625, 449, 287 cyanidin + 2 glucose + ferlic acid 817 655, 449, 287 cyanidin + 2 glucose + sinapic acid vitisin B-pyranopeonidin + 2 glucose + H2O 873 711, 549, 531, 325 or 5-methylpyranocyanidin + 2 glucose + H2O 993 831, 463, 301 peonidin + 3 glucose + sinapic acid 993, 817, 787, 655, 625, 449, cyanidin + 3 glucose + ferulic acid + sinapic 1155 287 acid 1185 817, 655, 287 cyanidin + 3 glucose + 2 sinapic acid 1199 831, 669, 301 peonidin + 3 glucose + 2 sinapic acid 1347 1185, 979, 817, 655, 449, 287 cyanidin + 4 glucose + 2 sinapic acid 1347, 1141, 979, 817, 449, 1509 cyanidin + 5 glucose + 2 sinapic acid 287 7 451 433, 395 Vitisin A-pyranopelargonidin + 2 glucose 663 339 or 5-mehtylpyranopeonidin + 2 glucose peonidin + 2 glucose + sinapic acid 801 301, 271 and pelargonidin + 2 glucose + sinapic acid 831 625, 463, 301 peonidin + 2 glucose + sinapic acid vitisin B-type pyranopeonidin + 2 glucose + sinapic acid 853? 855 325 or 5-methylpyranocyanidin + 2 glucose + sinapic acid 817 449, 287 cyanidin + 2 glucose + sinapic acid

vitisin B-type pyranopeonidin + 3 glucose + sinapic acid 1017 855, 487, 325 or 5-methylpyranocyanidin + 3 glucose + sinapic acid cyanidin + 3 glucose + sinapic acid + ferulic 1155 817, 787, 655, 625, 449, 287 acid 1185 817, 655, 449, 287 cyanidin + 3 glucose + 2 sinapic acid 1347 1185, 979, 817, 655, 449, 287 cyanidin + 4 glucose + 2 sinapic acid vitisin B-type pyranopeonidin + 5 glucose + 2 sinapic acid 1547 1179, 855, 487, 325 or 5-methylpyranocyanidin + 5 glucose + 2 sinapic acid 1553, 1347, 1185, 1023, 817, 1715 cyanidin + 5 glucose + 3 sinapic acid 449, 287 8 451 433, 395, 377, 331 malvidin + ? 466 448, 309 5-methylpyranopelargonidin + ??? 637 581, 525 652 523, 505, 487 801 463, 301 peonidin + 2 glucose + ferulic acid 831 669, 463, 301 peonidin + 2 glucose + sinapic acid vitisin B-type pyranopeonidin + 3 glucose + sinapic acid 1017 487, 325 or 5-methylpyranocyanidin + 3 glucose + sinapic acid Vitisin A-pyranopelargonidin + 3 glucose + sinapic acid 1031 869, 339 5-methylpyranopeonidin + 3 glucose + sinapic acid 1347 1185, 979, 817, 655, 449, 287 cyanidin + 4 glucose + 2 sinapic acid 1347, 1141, 817, 611, 449, 1509 cyanidin + 5 glucose + 2 sinapic acid 287 1523, 1317, 993, 817, 655, cyanidin + 5 glucose + 2 sinapic acid + 1685 449, 287 ferulic acid 9 Vitisin A-pyranopeonidin+ ?? 483 386, 369 5-methylpyranomalvidin 511 414, 397 595 466, 448, 309 5-methylpyranopelargonidin 679 vitisin B-type pyranopeonidin + 2 glucose + sinapic acid 855 487, 325 or 5-methylpyranocyanidin + 2 glucose + sinapic acid vitisin B-type pyranopeonidin + 3 glucose + sinapic acid 1017 855, 487, 325 or 5-methylpyranocyanidin + 3 glucose + sinapic acid caftaric acid-petunidin + ? 1039 925, 715, 627 or fertaric acid-delphinidin + ?

Vitisin A-pyranopelargonidin + 4 glucose + 2 sinapic acid 1399 1031, 707, 339 5-methylpyranopeonidin + 4 glucose + 2 sinapic acid 10 5-methylpyranomalvidin 483 386, 369, 330 or Vitisin A-type pyranopeonidin 511 397, 358, 340 ?? 637 581, 525 ?? 680 423, 233 vitisin B-type pyranopeonidin + 2 glucose + ferulic acid 825 663, 487, 325 or 5-methylpyranocyanidin + 2 glucose + ferulic acid vitisin B-type pyranopeonidin + 2 glucose + sinapic acid 855 693, 487, 325 or 5-methylpyranocyanidin + 2 glucose + sinapic acid Vitisin A-pyranopelargonidin + 2 glucose + sinapic acid 869 339 5-mehtylpyranopeonidin + 2 glucose + sinapic acid Vitisin A-pyranopelargonidin + 2 glucose + ferulic acid 841 339 5-mehtylpyranopeonidin + 2 glucose + ferulic acid 1185 817, 655, 449, 287 cyanidin + 3 glucose + 2 sinapic acid

Table S2

Structure indices Assigned values αH 1 αOGal,βH 2 αOH,βH 3 βH 4 βOGal,αH 5 βOH,αH 6 H 7 H,H 8 null 9 OGlu 10 OH 11 OMe 12 OO 13

Supplemental file – FT-Raman spectra of 28 bioflavonoid standards.