Supporting Information

Neuroprotective Activity of Cerebrosides from Typhonium giganteum by Regulating

Caspase-3 and Bax/Bcl-2 Signaling Pathways in PC12 Cells

Yang Jin,†,‡,⊥ Jun-Ting Fan,

†,⊥ Xiao-Ling Gu,† Li-Ying Zhang,

† Jing Han,

§ Shu-Hu Du,

*,†, ‡

and Ai-Xia Zhang*,†

†School of Pharmacy and

‡Key Laboratory of Cardiovascular and Cerebrovascular Drug

Research of Jiangsu Province, Nanjing Medical University, Nanjing 211166, People’s

Republic of China

§School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s

Republic of China

CONTENTS

Figure S1. UV spectrum of compound 1

Figure S2. IR spectrum of compound 1

Figure S3. 1H-NMR spectrum of compound 1 (600 MHz, C5D5N)

Figure S4. 13

C-NMR spectrum of compound 1 (150 MHz, C5D5N)

Figure S5. 1H-

1H COSY spectrum of compound 1

Figure S6. HSQC spectrum of compound 1

Figure S7. HMBC spectrum of compound 1

Figure S8. HR-ESI-MS of compound 1

Figure S9. ESI-MS/MS of compound 1

Figure S10. UV spectrum of compound 2

Figure S11. IR spectrum of compound 2

Figure S12. 1H-NMR spectrum of compound 2 (600 MHz, C5D5N)

Figure S13. 13

C-NMR spectrum of compound 2 (150 MHz, C5D5N)

Figure S14. 1H-

1H COSY spectrum of compound 2

Figure S15. HSQC spectrum of compound 2

Figure S16. HMBC spectrum of compound 2

Figure S17. HR-ESI-MS of compound 2

Figure S18. ESI-MS/MS of compound 2

Figure S19. Effects of ethyl acetate extract from T. giganteum on glutamate-induced PC12

cell death

Figure S20. Effects of compounds 1−13 on PC12 cell viability

Figure S21. Effects of longan cerebroside II (11) on cell cycle of PC12 cells treated

with/without glutamate

Table S1. The information on antibodies used in the western blotting experiments

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 nm

0

5

10

15

20

25

30

35

mAU 23.03/ 1.00

198

282

203

Figure S1. UV spectrum of compound 1

E:\天然药化\金阳\TGD-18-1.0 Au GSH APS--00 29/10/2015

3363.2

7

2921.3

52851.6

4

1724.5

0

1644.6

9

1537.7

6

1465.3

1

1380.6

01348.7

91319.5

31260.9

4

1082.5

11046.8

5

962.2

7

900.3

7

800.6

7

721.3

4

653.7

3610.2

9

5001000150020002500300035004000

Wavenumber cm-1

020

40

60

80

100

120

140

Tra

nsm

itta

nce

[%

]

Figure S2. IR spectrum of compound 1

Figure S3.

1H-NMR spectrum of compound 1 (600 MHz, C5D5N)

Figure S4. 13

C-NMR spectrum of compound 1 (150 MHz, C5D5N)

Figure S5. 1H-

1H COSY spectrum of compound 1

Figure S6. HSQC spectrum of compound 1

Figure S7. HMBC spectrum of compound 1

Figure S8. HR-ESI-MS of compound 1

620.40000

Figure S9. ESI-MS/MS of compound 1

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 nm

0

5

10

15

20

25

30

35

mAU 23.03/ 1.00

198

282

203

Figure S10. UV spectrum of compound 2

E:\天然药化\金阳\TGD-18-2.0 Au GSH APS--00 29/10/2015

3360.5

7

2921.0

02851.5

7

2031.2

2

1725.3

1

1644.8

11623.2

5

1537.5

8

1465.6

0

1380.7

11348.7

91319.9

41261.0

5

1156.1

9

1082.8

01046.1

1

961.9

2

900.4

0

800.6

5

721.3

4

653.2

3610.5

0

430.6

8

5001000150020002500300035004000

Wavenumber cm-1

020

40

60

80

100

120

140

Tra

nsm

itta

nce

[%

]

Figure S11. IR spectrum of compound 2

Figure S12. 1H-NMR spectrum of compound 2 (600 MHz, C5D5N)

Figure S13. 13

C-NMR spectrum of compound 2 (150MHz, C5D5N)

Figure S14. 1H-

1H COSY spectrum of compound 2

Figure S15. HSQC spectrum of compound 2

Figure S16. HMBC spectrum of compound 2

Figure S17. HR-ESI-MS of compound 2

620.40000

602.60000

341.00000

306.00000

279.20000

Figure S18. ESI-MS/MS of compound 2

0

20

40

60

80

100

120

C Glu 1 5 10 20 40

Glu (12.5 mM)

Su

rviv

al

rate

(%

)

##

**

**

**

EA

( µg/mL)

**

Figure S19. Effects of the ethyl acetate (EA) extract on glutamate-induced PC12 cell death.

PC12 cells were treated with 12.5 mM glutamate with EA (1, 5, 10, 20, 40 μg/mL) for 24 h.

Cell viability was measured by MTT assay. Each value represents the mean SD. n = 6, ##

p <

0.01 vs control group, **p < 0.01 vs model group.

0

20

40

60

80

100

120

C 1 2 3 4 5 6 7 8 9 10 11 12 13

20 μM

Su

rviv

al

rate

(%

) *

Figure S20. Effects of compounds 1−13 on PC12 cell viability. Cells were treated with

compounds 1−13 at 20 μM for 24 h. Cell viability was measured by MTT assay.

(A) (B) (C)

(D) (E) (F)

Figure S21. Effects of longan cerebroside II (11) on cell cycle of PC12 cells treated

with/without glutamate. PC12 cells were treated with/without 12.5 mM glutamate along with

low, medium, or high concentrations (0.1, 1, 10 μM) of longan cerebroside II (11) for 24 h.

The control group did not contain glutamate. (A) Control group, (B) glutamate model group,

(C) 0.1 µM longan cerebroside II (11) + glutamate, (D) 1 µM longan cerebroside II (11) +

glutamate, (E) 10 µM longan cerebroside II (11) + glutamate, (F) 10 µM longan cerebroside II

(11).

Table S1. Summary of Antibodies and Working Conditions Used in the Experiments

Antibody Species Source Dilution

Primary antibodies

Anti-caspase-9 Rabbit, polyclonal Cell Signaling Technology Inc., MA, USA 1:1000

Anti-caspase-3 Rabbit, polyclonal Bioworld Technology Inc., MN, USA 1:1000

Anti-cytochrome c Rabbit, monoclone Cell Signaling Technology Inc., MA, USA 1:1000

Anti-Bcl-2 Rabbit, polyclonal Bioworld Technology Inc., MN, USA 1:1000

Anti-Bax Rabbit, polyclonal Bioworld Technology Inc., MN, USA 1:1000

Tubulin Mouse, polyclonal Bioworld Technology Inc., MN, USA 1:5000

Secondary antibodies

Anti-rabbit IgG (HRP) Goat Bioworld Technology Inc., MN, USA 1:5000

Anti-mouse (HRP) Rabbit Bioworld Technology Inc., MN, USA 1:5000