SUPPLEMENTARY MATERIAL

Sassarandainol: A new neolignan and anti-inflammatory constituents

from the stem of Sassafras randaiense

Ya-Ling Houa, Hsun-Shuo Changa,b, Hui-Chun Wang a, Sheng-Yang Wangc, Tze-Ying

Chend, Chu-Hung Linb & Ih-Sheng Chena,b,*

a Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical

University, Kaohsiung 807, Taiwan.; b School of Pharmacy, College of Pharmacy,

Kaohsiung Medical University, Kaohsiung 807, Taiwan.; c Department of Forestry, National Chung Hsing University, Taichung 402, Taiwan.; d

Department of Forestry and Natural Resources, National Ilan University,

Ilan 260, Taiwan.

Abstract

A new neolignan (R)-(–)-sassarandainol (1), together with 10 known compounds (2–11)

were isolated from the stem of Sassafras randaiense. The structures were determined by

spectroscopic techniques. Among these isolates, compounds: γ-tocopherol (5),

subamolide B (7), and β-sitosterone (9) exhibited moderate iNOS inhibitory activity on

nitrite production induced (%) by 30.51, 28.68, and 16.96, repectively.

Keywords: Sassafras randaiense; Lauraceae; stem; neolignan; (R)-(–)-sassarandainol;

anti-inflammatory activity.

*Corresponding author, E-mail: m635013@kmu.edu.tw

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SUPPLEMENTARY MATERIAL

Figure S1. Sassafras randaiense

Figure S2. Branch of Sassafras randaiense

Figure S3. NOESY correlations of (R)-(–)-sassarandainol (1)

Figure S4. HMBC correlations of (R)-(–)-sassarandainol (1)

Figure S5. COSY correlations of (R)-(–)-sassarandainol (1)

Figure S6. 1H NMR spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S7. 13C NMR spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 100 MHz

Figure S8. HSQC spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S9. COSY spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S10. NOESY spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S11. HMBC spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Table S1. Data of 1H NMR (400 MHz, CDCl3), and 13C NMR (100 MHz, CDCl3) of

compound 1

Table S2. Effects of isolates from the stem of S. randaiense on LPS-induced nitrite production and cell viability in RAW 264.7 CellsTable S3. Effects of extracts from the stem of S. randaiense on LPS-induced nitrite

production and cell viability in RAW 264.7 Cells

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Figure S1. Sassafras randaiense

Figure S2. Branch of Sassafras randaiense

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Figure S3. NOESY correlations of (R)-(–)-sassarandainol (1)

Figure S4. HMBC correlations of (R)-(–)-sassarandainol (1)

Figure S5. COSY correlations of (R)-(–)-sassarandainol (1)

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Figure S6. 1H NMR spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S7. 13C NMR spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 100 MHz

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Figure S8. HSQC spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S9. COSY spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

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Figure S10. NOESY spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

Figure S11. HMBC spectrum of (R)-(–)-sassarandainol (1) in CDCl3 at 400 MHz

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Table S1. Data of 1H NMR (400 MHz, CDCl3), and 13C NMR (100 MHz, CDCl3) of

compound 1

Positions1

1H (J in Hz) 13C

1 130.4

2 7.10 (d, 8.6) 130.6

3 6.75 (d, 8.6) 115.1

4 154.0

5 6.75 (d, 8.6) 115.1

6 7.10 (d, 8.6) 130.6

7 3.00 (dd, 13.8, 5.9) 41.7

2.73 (dd, 13.8, 6.5)

8 4.47 (m) 75.0

9 1.27 (d, 6.4) 19.3

1′ 132.2

2′ 7.08 (d, 9.0) 129.5

3′ 6.81 (d, 9.0) 116.0

4′ 156.2

5′ 6.81 (d, 9.0) 116.0

6′ 7.08 (d, 9.0) 129.5

7′ 3.32 (d, 6.6) 39.3

8′ 5.95 (ddt, 16.9, 9.8, 6.6) 137.8

9′ 5.06 (dd, 16.9, 1.8) 115.4

5.04 (dd, 9.8, 1.8)

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Table S2. Effects of isolates from the stem of S. randaiense on LPS-induced nitrite production and cell viability in RAW 264.7 CellsAll experiments were repeated at least three times. Results are shown as the mean ±

standard error of means (SE) from three independent experiments. aEmax indicates mean

maximum inhibitory effect of nitrite production, at a concentration of 20 μM, expressed

as a percentage inhibition induced by LPS (200 ng/mL) in the presence of vehicle. The

Emax of iNOS inhibitors aminoguanidine (AG) was evaluated at a concentration of 100

μM. b positive control (a selective iNOS inhibitor)

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compoundsNitrite

Production (%)Emax (%)a Cell Viability (%)

magnolol (2) 82.29 ± 0.01 17.71 ± 0.01 71.43 ± 146erythro-7'-hydroxy strebluslignanol (3)

83.37 ± 0.04 16.63 ± 0.04 111.70 ± 0.78

threo-7'-hydroxy strebluslignanol (4)

88.94 ± 0.09 11.06 ± 0.09 115.91 ± 0.59

γ-tocopherol (5) 30.51 ± 0.04 69.49 ± 0.04 111.51 ±1.432β-methoxyclovan-9α-ol (6) 86.78 ± 0.03 13.22 ± 0.03 90.51 ± 3.00subamolide B (7) 28.68 ± 0.04 71.32 ± 0.04 111.42 ± 0.39

palmitic acid (8) 85.98 ± 0.08 14.02 ± 0.08 94.49 ± 3.1

β-sitosterone (9) 16.96 ± 0.07 83.04 ± 0.07 84.13 ± 0.42mixture of β-sitosterol (10) and stigmasterol (11)

74.81 ± 0.07 25.19 ± 0.07 95.26 ± 1.95

Aminoguanidineb 21.27 ± 0.64 78.74 ± 0.64 88.87 ± 2.98

Table S3. Effects of extracts from the stem of S. randaiense on LPS-induced nitrite production and cell viability in RAW 264.7 Cells

SamplesNitrite

Production (%)Emax (%)a Cell Viability (%)

MeOH ext. 39.68 ± 4.28 60.30 ± 4.28 32.77 ± 6.55

EtOAc layer 41.78 ± 4.74 58.22 ± 4.74 12.06 ± 0.23

H2O layer 78.76 ± 3.62 21.24 ± 3.62 99.24 ± 0.72

Aminoguanidineb (100 µM) 33.62 ± 1.08 – –

All experiments were repeated at least three times. Results are shown as the mean ± standard error of means (SE) from three independent experiments. aEmax indicates mean maximum inhibitory effect of nitrite production, at a concentration of 100 μg/mL, expressed as a percentage inhibition induced by LPS (200 ng/mL) in the presence of vehicle. The Emax of iNOS inhibitors aminoguanidine (AG) was evaluated at a concentration of 100 μM. b positive control (a selective iNOS inhibitor)

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