Effect of Harvest Age on Total Phenolic, Total Anthocyanin Content, Bioactive

1 Supplementary Material

2 Article 3 Effect of harvest age on total phenolic, total 4 anthocyanin content, bioactive antioxidant capacity 5 and antiproliferation of black and white glutinous 6 rice sprouts

7 Visessakseth So 1, Piman Pocasap 2,3, Khaetthareeya Sutthanut 2,3, Benjabhorn Sethabouppha 4, 8 Wipawee Thukhammee 3,5, Jintanaporn Wattanathorn 3,5, and Natthida Weerapreeyakul 2,3,*

9 1 Graduate School (in the program of Pharmaceutical Sciences), Faculty of Pharmaceutical Sciences, Khon 10 Kaen University, Khon Kaen 40002, Thailand; [email protected] (V.S.) 11 2 Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon 12 Kaen 40002, Thailand; [email protected] (P.P.); [email protected] (N.W.); [email protected] 13 (K.S.) 14 3 Human High Performance and Health Promotion Research Institute, Khon Kaen University, Khon Kaen 15 40002, Thailand; [email protected] (N.W.); [email protected] (J.W.); [email protected] (W.T.); 16 [email protected] (K.S.); [email protected] (P.P.) 17 4 Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand; 18 [email protected] (B.S.) 19 5 Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand; 20 [email protected] (W.T.); [email protected] (J.W.) 21 * Correspondence: [email protected]; Tel.: +66-4320-2378

22 Received: 7 September 2020; Accepted: 4 October 2020; Published: date

Appl. Sci. 2020, 10, 7051; doi:10.3390/app10207051 www.mdpi.com/journal/applsci

BGR Test TPC TAC DPPH FRAP Cyto-24h Cyto-48h DCFH-DA GSH Apoptosis Caspase 0.698* 0.747* -0.690** 0.314 0.949** 0.562 -0943** 0.922** 0.824* 0.782** (p<0.05) (p<0.05) (p<0.005) (p=0.9254) (p<0.0001) (p=0.057) (p<0.00001) (p<0.001) (p<0.05) (p<0.005) TPC 0.698* 1.00 0.312 -0.549 0.891** 0.465 0.769* -0.762** 0.868** 0.883** 0.976** (p<0.05) (p=0.413) (p=0.064) (p<0.001) (p=0.127) (p<0.05) (p<0.005) (p<0.005) (p<0.001) (p<0.000001) TAC 0.747* 0.312 1.00 -0.340 -0.089 0.810* 0.184 -0.704* 0.571 0.502 0.421 (p<0.05) (p=0.413) (p=0.371) (p=0.819) (p<0.05) (p=0.636) (p<0.05) (p=0.109) (p=0.168) (p=0.259) DPPH -0.690** -0.549 -0.340 1.00 -0.419 -0.593* -0.311 0.587* -0.663 -0.761** -0.608* (p<0.005) (p=0.064) (p=0.371) (p=0.120) (p<0.05) (p=0.260) (p<0.05) (p=0.052) (p<0.005) (p<0.05) FRAP 0.314 0.891** -0.089 -0.419 1.00 0.027 0.663* -0.446 0.590 0.686* 0.834** (p=0.9254) (p<0.001) (p=0.819) (p=0.120) (p=0.924) (p<0.05) (p=0.146) (p=0.095) (p<0.05) (p<0.001) BGR Cyto-24h 0.949** 0.465 0.810* -0.593* 0.027 1.00 0.400 -0.840** 0.778* 0.656* 0.545* (p<0.0001) (p=0.127) (p<0.05) (p<0.05) (p=0.924) (p=0.140) (p<0.005) (p<0.05) (p<0.05) (p<0.05) Cyto-48h 0.562 0.769* 0.184 -0.311 0.663* 0.400 1.00 -0.649* 0.579 0.589* 0.545* (p=0.057) (p<0.05) (p=0.636) (p=0.260) (p<0.05) (p=0.14) (p<0.05) (p=0.102) (p<0.05) (p<0.05) DCFH-DA -0943** -0.762** -0.704* 0.587* -0.446 -0.840** -0.649* 1.00 -0.891* -0.786** -0.849** (p<0.00001) (p<0.005) (p<0.05) (p<0.05) (p=0.146) (p<0.005) (p<0.05) (p<0.05) (p<0.005) (p<0.001) GSH 0.922** 0.868* 0.571 -0.663 0.590 0.778* 0.579 -0.891* 1.00 0.914* -0.928** (p<0.001) (p<0.05) (p=0.109) (p=0.052) (p=0.095) (p<0.05) (p=0.102) (p<0.05) (p<0.05) (p<0.001) Apoptosis 0.824* 0.883** 0.502 -0.761** 0.686* 0.656* 0.589* -0.786** 0.914* 1.00 0.897** (p<0.05) (p<0.001) (p=0168) (p<0.005) (p<0.05) (p<0.05) (p<0.05) (p<0.005) (p<0.05) (p<0.0001) Caspase 0.782** 0.976** 0.421 -0.608* 0.834** 0.545* 0.545* -0.849** -0.928** 0.897** 1.00 (p<0.005) (p<0.000001) (p=0.259) (p<0.05) (p<0.001) (p<0.05) (p<0.05) (p<0.001) (p<0.001) (p<0.0001)

Appl. Sci. 2020, 10, 7051; doi:10.3390/app10207051 www.mdpi.com/journal/applsci

Appl. Sci. 2020, 10, 7051

25 Abbreviation: 26 BGR = black glutinous rice, TPC = total phenolic content, TAC = total anthocyanin content, DPPH = DPPH radical scavenging activities, FRAP = ferric reducing 27 antioxidant power, Cyto-24h = cytotoxicity at 24 h in Jurkat cells, Cyto-48h = cytotoxicity at 48 h in Jurkat cells, DCFH-DA = intracellular hydrogen peroxide level in 28 Jurkat cells, GSH = endogenous glutathione level in Jurkat cells, Apoptosis = apoptosis induction effect of BGR in Jurkat cells, Caspase: Caspase 3/7 activity of BGR in 29 Jurkat cells. 30

31 Figure S1. Correlation coefficient (r) of BGR group at different harvest ages with antioxidant capacity (TPC, TAC, DPPH, FRAP, DCHF-DA and GSH), and anticancer 32 effect (cytotoxicity, apoptosis, and caspase activity). If the r value is close to +1 or -1, it will indicate a strong positively relationship or inverse relationship, respectively. 33 If r is near to 0, it will indicate weak or no relationship. *Correlation is significant at p < 0.05.

34 35

RD6 Test TPC TAC DPPH FRAP 1.00 0.447 0.073 0.606* 0.348 (p=0.228) (p=0.851) (p<0.05) (p=0.204) TPC 0.447 1.00 0.440 -0.479 0.986* (p=0.228) (p=0.236) (p=0.136) (p<0.05) RD6 TAC 0.073 0.440 1.00 -0.612 0.539 (p=0.851) (p=0.236) (p=0.106) (p=0.168) DPPH 0.606* -0.479 -0.612 1.00 -0.229 (p<0.05) (p=0.136) (p=0.106) (p=0.4111) FRAP 0.348 0.986* 0.539 -0.229 1.00 (p=0.204) (p<0.05) (p=0.168) (p=0.4111)

37 Abbreviation: 38 TPC = total phenolic content, TAC = total anthocyanin content, DPPH = DPPH radical scavenging 39 activities, FRAP = ferric reducing antioxidant power

40 Figure S2. Correlation coefficient (r) of antioxidant capacity (TPC, TAC, DPPH, and FRAP) of RD6 41 at different harvest ages. If the value is close to +1 or -1, it will indicate a strong positively 42 relationship or inverse relationship, respectively. If r is near to 0, it will indicate weak or no 43 relationship. *Correlation is significant at p < 0.05.

46 47

Appl. Sci. 2020, 10, 7051; doi:10.3390/app10207051 www.mdpi.com/journal/applsci

Appl. Sci. 2020, 10, 7051

(A)

24 h BGR

Cell Jurkat HCT116

1.00 0.952** -0.951**

(p<0.0001) (p<0.0001)

BGR Jurkat 0.952** 1.00 -0.817**

(p<0.0001) (p<0.001)

HCT116 -0.951** -0.817** 1.00

(p<0.0001) (p<0.001)

(B)

48 h BGR

Cell Jurkat HCT116

1.00 0.589* -0.609*

(p<0.05) (p<0.05)

BGR Jurkat 0.589* 1.00 -0.407*

(p<0.05) (p=0.132)

HCT116 -0.609* -0.407* 1.00

(p<0.05) (p=0.132)

50 Figure S3. Correlation coefficient of cytotoxicity between Jurkat and HCT116 after BGR treatment 51 (A) at 24 h and (B) at 48 h. If the value is close to +1 or -1, it will indicate a strong positively 52 relationship or inverse relationship, respectively. If r is near to 0, it will indicate weak or no 53 relationship. *Correlation is significant at p < 0.05.