SUPPORTING INFORMATION

Isolation, Characterisation and Quantification of Tricin and Flavonolignans in the Medicinal Rice Njavara (Oryza sativa L.), as Compared to Staple Varieties.

Smitha Mohanlal . Rathnam Parvathy . Vasantha Shalini . Antony Helen. Ananthasankaran Jayalekshmy

S. Mohanlal . R. Parvathy . A. Jayalekshmy (corresponding author)

Chemical Sciences & Technology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Industrial Estate P.O., Thiruvananthapuram-695019, Kerala, India. Tele: +91 471 2515 365. fax: +91 471 2491712. E-mail: ,

V. Shalini . A. Helen

Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram – 695 581.

Isolation of Compounds 1, 2 and 3 and theirQuantification by HPLC-PDA Analysis

About 2 g of diethyl ether soluble residue was chromatographed over silica gel open column using petroleum ether:ethyl acetate gradient to collect A1-A118 fractions. Similar fractions were pooled, based on silica gel GF254 thin layer chromatography, to get B1-B12 fractions and subjected to DPPH activity. Fraction B11 (300 mg), active towards DPPH, was further chromatographed on a Sephadex LH-20 column by elution with MeOH to give C1-C20 fractions. The C18 fraction gave a yellow powder which was purified by re-crystallization with MeOH to give pure compound 1 (24 mg). C5-C6 were subjected to preparative HPLC on a 250 mm x 20 mm i.d., 5μm, YMS-Pack R&D ODS column (YMC Co., Ltd. Japan) with (28:72) CH3CN:H2O at a flow rate of 20 ml/min at 330 nm (λmax) giving impure compounds 2 and 3, which were then recrystallised in HPLC grademethanol to afford 2 (3.1 mg) and 3 (3 mg), respectively. 1H (500MHz), 13C (125 MHz) of the compounds, were recorded in CD3OD and CD3COCD3 using a Bruker AMX 500 spectrometer (Bruker Avance II 500) with TMS as the internal standard. Mass spectrum was recorded under FAB/HRMS at 5000 resolution using JMS 600H (JEOL) mass spectrometer. IR spectra were recorded using Spectrum one FT-IR spectrometer (Perkin Elmer).

A 20-µl aliquot of sample solution, previously filtered through 0.2 µm filter (Millipore Corporation, Billerica, MA), was analysed in Shimadzu HPLC (high performance liquid chromatography) system consisting of an SCL-10Avp system controller, two LC-8A solvent delivery units, an SPD-M20A UV-VIS photo diode array (PDA) detector, and equipped with Multi-PDA LC Solution (software), on a (250 mm x 4.6 mm i.d.) 5µm,YMC-Pack R&D ODS analytical column (YMC Co., Ltd. Japan). The mobile phase consisted of acetonitrile and water (28:72) at a flow rate of 1 ml/min. The separated compounds 1, 2 and 3 were quantified using detector response at 330 nm.

Chemical structures of compounds 1, 2 and 3 isolated from ‘Njavara’.

Spectroscopic data

Tricin (1). Pale yellow crystal. UV: λmax (MeOH) 269, 352. IR: υmax (KBr) 3313, 2928, 2851, 1654 cm-1. HRFAB-MS: m/z 331.26, calcd for CHO [M+H]+, 331.07). 1H NMR (CD3COCD3): δ 13.04 (1H, s, 5-OH), 7.40 (2H, s, H-2’ and 6’), 6.76 (1H, s, H-3), 6.57 (1H, d, J = 2 Hz, H-8), 6.27 (1H, d, J = 2.5 Hz, H-6), 3.99 (6H, s, -OCH3 x 2). 13C NMR (CD3COCD3): δ 183.1 (C-4), 164.8 (C-2), 163.4 (C-9), 158.9 (C-5), 149.1 (C-3’, C-5’), 140.9 (C-4’), 122.3 (C-1’), 105.6 (C-2’, C-6’), 105.2 (C-10), 104.7 (C-3), 99.7 (C-6), 94.9 (C-8), 56.9 (-OCH3 x 2).

Tricin-4’-O-(erythro-β-guaiacylglyceryl)ether (2). Yellow amorphous powder. UV: λmax (MeOH) 272, 288 sh, 305 sh, 335. IR: υmax (KBr) 3367, 2921, 2851, 1646, 1610, 1591 cm-1.

HR FAB-MS: m/z 527.42 calcd for CHO [M+H]+, 527.15. 1H NMR (CD3COCD3): δ12.78 (1H, s, 5-OH), 7.29 ( 2H, s, H-2’, 6’), 6.94 (1H, d, J = 2 Hz, H-3), 6.74 (1H, dd,J= 8, 2 Hz, H-19), 6.70 (1H, s, H-15), 6.66 (1H, d, J = 8 Hz, H-18), 6.45 (1H, d, J = 2 Hz, H-8), 6.15 (1H, d, J = 2 Hz, H-6), 4.90 (1H, d, J = 5 Hz, H-13), 4.25-4.22 (1H, m, H-12), 3.88 (6H, s, 3’, 5’-OCH3), 3.78 (1H, dd, J = 12, 5.5 Hz, 11-CH2), 3.71 (3H, s, H-16 -OCH3), 3.41(1H, dd, J = 12, 3.5 Hz, 11-CH2). 13C NMR (CD3COCD3): δ183.1 (C-4), 165.4 (C-7), 164.6 (C-2), 163.3 (C-5), 158.9 (C-9), 154.6 (C-3’, 5’), 148.0 (C-16), 146.6 (C-17), 140.1 (C-4’), 133.7(C-14), 127.6 (C-1’), 120.1 (C-19), 115.3 (C-18), 111.0 (C-15), 106.0 (C-3), 105.3 (C-10), 105.1 (C-2’, 6’), 99.9 (C-6), 95.0 (C-8), 88.1 (C-12), 73.6 (C-11), 61.2 (C-13), 57.0 (C-3’, 5’–OCH3), 56.3 (C-16 –OCH3).

Tricin-4’-O-(threo-β-guaiacylglyceryl) ether (3). Yellow amorphous powder. UV: λmax (MeOH) 272, 287 sh, 300 sh, 334. IR: υmax (KBr) 3390, 2920, 2851, 1651, 1613, 1590 cm-1. HRFAB-MS: m/z 527.19, calcd. for CHO [M+H]+,527.15. 1H NMR (CD3COCD3): δ12.78 (1H, s, 5-OH), 7.29 ( 2H, s, H-2’, 6’), 6.95 (1H, d, J = 1.5 Hz, H-3), 6.80 (1H, dd, J = 8.3, 1.8 Hz, H-19), 6.68 (1H, s, H-15), 6.65 (1H, d, J = 8.5 Hz, H-18), 6.46 (1H, br s, H-8), 6.15 (1H, d, J = 1 Hz, H-6), 4.92 (1H, d, J = 7 Hz, H-13), 4.06-4.03 (1H, m, H-12), 3.90 (6H, s, 3’, 5’-OCH3), 3.70 (3H, s, H-16-OCH3), 3.62(1H, dd, J = 12.3, 3.3 Hz, 11-CH2), 3.27(1H, J = 12, 2.3 Hz, 11-CH2). 13C NMR (CD3COCD3): δ 183.2 (C-4),165.3 (C-7),164.2 (C-2), 163.3 (C-5), 158.8 (C-9), 154.3 (C-3’, 5’), 148.0 (C-16), 146.8 (C-17), 140.5 (C-4’), 133.6 (C-14), 127.7 (C-1’), 120.6 (C-19), 115.2 (C-18), 111.4 (C-15), 106.0 (C-3), 105.1 (C-10), 105.0 (C-2’, 6’), 99.9 (C-6), 95.1 (C-8), 89.8 (C-12), 73.9 (C-11), 61.6 (C-13), 57.0 (C-3’, 5’–OCH3), 56.2 (16 –OCH3).

Spectra (1H NMR, 13C NMR, HRMS, UV), HPLC chromatogram and corresponding PDA of compound 1, 2 and 3

Compound 1

1H NMR spectra

13C spectra

Mass spectra

IR spectra

UV spectra

HPLC spectra and corresponding PDA

Compound 2

1H NMR spectra

13C spectra

Mass Spectra

IR spectra

UV spectra

HPLC chromatogram and corresponding PDA

Compound 3

1H NMR spectra

13C spectra

Mass spectra

IR spectra

UV spectra

HPLC chromatogram and corresponding PDA

NB diethyl ether extract

HPLC chromatogram and corresponding PDA

SJ diethyl ether extract

HPLC chromatogram and corresponding PDA

PM diethyl ether extract

HPLC chromatogram and corresponding PDA

Animals

Female rats (Sprague Dawley strain) (120-200 g) bred in the Biochemistry department animal house were used for this study. They were kept in an environment with controlled temperature (24–26 0C), humidity (55-60%) and photoperiod (12:12 h) light–dark cycle. A commercially balanced diet (Amrut laboratory Animal feeds, Maharashtra, India) and tap water were provided ad libitum. The animals received human care, in compliance with the present institutional guidelines. All experiments were conducted as per the guidelines of the Animal Ethics Committee (IA EC-KU-12/08-09-BC-AH (13)) according to Government of India accepted principles for laboratory animals’ use and care.