SUPPLEMENTARY MATERIAL

Hepatoprotective and neuroprotective tocopherol analogues isolated from the peels

of Citrus unshiu Marcovich

Kyeong-Hwa Seoa, Dae-Young Leeb, Dong-Sung Leec, Hee-Cheol Kangd, Hak-Soo

Kime, Youn-Chul Kimcand Nam-In Baeka*

aGraduate School of Biotechnology and Department of Oriental Medicine

Biotechnology, Kyung Hee University, Yongin 446-701, Republic of KoreabDepartment of Herbal Crop Research, National Institute of Horticultural and Herbal

Science, RDA, Eumseong 369-873, Republic of KoreacCollege of Pharmacy, Wonkwang University, Iksan 570-749, Republic of KoreadR&D Center, GFC Co., Ltd., Yongin 446-908, Republic of KoreaeSEOULCOSMETICS, Co., Ltd, R&D center, Incheon 405-821, Republic of Korea

Three tocopherol analogues methoxytocopherol (1), α-tocopherol (2)

and γ- tocopherol (3),were isolated from the peels of Citrus unshiu

Marcovich. The protective effects of the isolated compounds against

tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in human

liver-derived HepG2 cells and glutamate-induced oxidative stress in

HT22-immortalized hippocampal cells were evaluated. Compounds 1-3

were significantly protective in HepG2, cells with EC50 values of 21.22

± 2.01, 25.21 ± 2.11, and 25.25 ± 1.21 μM, respectively, and in HT22

cells, compounds 1-3 had EC50 values of 20.62 ± 1.36, 6.44 ± 1.65, and

9.52 ± 1.54 μM, respectively.

Keywords:Citrus unshiu, hepatoprotective, HepG2, HT22,

methoxytocopherol, neuroprotective, tocopherol

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Structures and Protective effects of Compounds 1-3

Table S1. Protective effects of compounds 1-3, isolated from the peels of Citrus unshiu

Marcovich, on glutamate-induced oxidative injury in HT22 cells and t-BHP-

induced hepatotoxicity in HepG2 cells

Figure S1. Structures of compounds 1-3 from the peels of Citrus unshiu.

Figure S2.Effects of compounds 1-3 on t-BHP-induced oxidative injury in HepG2 cells.

Cells were pretreated with compounds 1-3 for 12 h and then incubated for 6

h with t-BHP (50 μM). Curcumin (20 μM) was used as a positive control.

Data are presented as the means ± S.D. of three independent experiments;

*P < 0.05 vs. control.; #P < 0.05 vs. t-BHP.

Figure S3. Protective effects of compounds 1-3 on glutamate-induced oxidative

neurotoxicity in HT22 cells. Cells were treated with compounds 1-3 for 4 h

and then incubated for 12 h with glutamate (5 mM). Trolox (100 μM) was

used as a positive control. Data are presented as the means ± S.D. of three

independent experiments; *P < 0.05 vs. control.; #P < 0.05 vs. glutamate.

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4647484950515253545556575859606162636465666768697071

Table S1. Protective effects of compounds 1-3, isolated from the peels of Citrus unshiu

Marcovich, on glutamate-induced oxidative injury in HT22 cells and t-BHP-induced

hepatotoxicity in HepG2 cells

Compound HT22 (EC50, μM) HepG2 (EC50, μM)

Positive control Trolox (22.42 ± 3.23) Curcumin (8.78 ± 2.41)

1 20.62 ± 1.36 21.22 ± 2.01

2 6.44 ± 1.65 25.21 ± 2.11

3 9.52 ± 1.54 25.25 ± 1.21

The HT22 and HepG2 cells were treated with 1-3 for 4 h and then incubated for 12 h

with glutamate (5 mM). Next, the cells were incubated for 6 h with t-BHP (50 μM). The

resulting half-maximal effective concentrations (EC50) are expressed as the percentage

of viability compared to the control. Trolox and curcumin were used as positive

controls. Data represent the mean values ± SD of three experiments.

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8283848586

Figure S1. Structures of compounds 1-3 from the peels of Citrus unshiu.

Figure S2. Effects of compounds 1-3 on t-BHP-induced oxidative injury in HepG2

cells. Cells were pretreated with compounds 1-3 for 12 h and then incubated

for 6 h with t-BHP (50 μM). Curcumin (20 μM) was used as a positive

control. Data are presented as the means ± S.D. of three independent

experiments; *P < 0.05 vs. control.; #P < 0.05 vs. t-BHP.

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Figure S3. Protective effects of compounds 1-3 on glutamate-induced oxidative

neurotoxicity in HT22 cells. Cells were treated with compounds 1-3 for 4 h

and then incubated for 12 h with glutamate (5 mM). Trolox (100 μM) was

used as a positive control. Data are presented as the means ± S.D. of three

independent experiments; *P < 0.05 vs. control.; #P < 0.05 vs. glutamate.

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