Neurotrophic compounds from Indonesian Zingiberceae plant

Neurotrophic compounds from Indonesian Zingiberceae plant, BANGLE
Miwa Kubo1, Megumi Nakai2, Kenichi Harada1, Nobuaki Matsui1, Masaaki Akagi1, Midori Suenaga1, Yoichi Matsunaga1, Yoshiyasu Fukuyama1
1Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, 770-8514, Japan;
2Department of Pharmacy, Kochi Medical Graduate School, Nankoku, Kochi 783-8505, Japan; As part of our efforts to discover natural products with neurotrophic properties, we investigated the MeOH extract of the root of BANGLE (Zingiber purpurenum) that
exhibited neuritogenesis activity in PC12 cells at 25 μg/mL, resulting in the isolation of neurotrophic phenylbutenoid dimers 1 and 2, and 3 – 5 as new compounds.
Compounds 3 and 4 are the first example of curcumin coupled with phenylbutenoid. Compounds 1 and 2 found not only to significantly induce neurite sprouting of PC12 cells,
but also to increase the neurite length and number of neurites in primary cultured rat cortical neurons, and also showed protective activity against cell death caused by
deprivation of serum. Furthermore, chronic treatment of these compounds enhanced hippocampal neurogenesis in dementia model OBX mice. Compounds 3 and 4 had
s i g n i fi c a n t N G F - p o t e n t i a t i n g e f f e c t s o n P C 1 2 c e l l s . A d d i t i o n a l l y, c o m p o u n d 3 a c c e l e r a t e d t h e p r e v e n t i o n o f A β 4 2 a g g r e g a t i o n .
N. Matui, Y. Kido, H. Okada, M. Kubo, M. Nakai, N. Fukuishi, Y. Fukuyama, M. Akagi, Neuroscience Letters, 513 (1), 72-77 (2012).
Effects of Neurotrophins Differentiation
Extract
Neuronal
Maturation
Ageing
Death
Astrocyte
Neurons
Oligodendrocyte
NMR spectrum (600 MHz, C6D6) of 3
1H
+
-
+
++
-
O
3
1
H
7
11
4''
8''
6''
OMe
OH
OH
2
4
OMe
OMe
Cassumunarin A (4) 1
23.0
A
69.7
6
43.8
H
[α]D = + 0 (c 0.1, MeOH)
IR ν max : 3389 (OH), 1658 (C=O), 1579 (arom.) cm-1
UV λ max (log ε) : 359 (2.65), 287 (3.87), 259 (3.92) nm
FAB-MS : m/z 609[M+K]+
HR-FAB-MS : found 609.1819
calcd 609.1819 for C34H34O8K
B
OCH3-8''
OCH3-8'
H-2' (d, J = 15.4)
H-2'' (d, J = 15.5)
H-12
H-8 H-9'
H-11
H-9''
9
149.3
11
OCH3-9
OCH3-10
149.3
H-6
H-3
15
HO
HO
CHO
16
MeO
OH
O
MeO
OH
HO
MeO
18: R=OH
19: R=OMe
17
20 : R=OH
21 : R=OMe
22
23
24
OMe
O
OH
OH
OMe
OH
MeO
OH
O
OMe
MeO
O
O
S-CoA HO
O
S-CoA
O
HO
CoASH
110.6
111.1
8''
127.2
3''
146.9
O
Screening
R
S-CoA
O
O
R
MeO
R
CO2
R
2
Column : 10 × 250mm COSMOSIL 5C18-AR-II"
Mobile phase : MeOH : H2O = 83 : 17
Flow rate : 2.0 mL/min, Detector : UV 254nm
Injection volume : 3µL (4 mg/mL)
OMe
R OMe
1
OMe
OH
H
H-H COSY
HMBC
R=
OMe
O
5
OH
10
15 20 25
30 35 40
time (min)
OMe
OH
OMe
OH
OMe
OMe
OMe
O
OMe
12%!
OH
OH
O
OH
OMe
Comparison of HPLC Chromatograms between MeOH
Extract and Compound 1
OMe
O
c
OMe
OMe
1
R
Curcumin
OMe
OMe
OH
H2O
OCH3
OCH3
NGF-like compounds from BANGLE
MeO
O
R
O
b
OH
7''
OCH3
OMe
28
27
O
OMe
122.8
5''
26
OMe
R
8'
O
a
CoASH
O
R
146.9
R
CO2
malonyl-CoA
OH
7'
OMe
25 µg/mL
Plausible biosynthesis of compound 3#
123.8
143.4
OMe
25
O
OMe
OMe
OMe
OMe
Ctrl#
( 0.5% EtOH )#
OCH3
H-3' (d, J = 15.4)
RO
OH
MeO
OMe
C
121.1
D
H
MeO
MeO
HO
13 : R = Me
14 : R = H
OMe
144.7
3'
127.1
195.2
O
MeO
MeO
MeO
MeO
CHO
RO
O
H
115.2
122.9
10
MeO
OMe
5'
O
7 132.5
OMe
OH
119.1
1''
O
HO
11 : R = Me
12 : R = H
O
OH
*
*
196.1
1
MeO
RO
MeO
6
5
MeO
OMe
+
H
O
O
O
OMe
OMe
3: R =H
4 : R = OMe
MeO
MeO
OMe
OMe
R
9
MeO
MeO
OH
OMe
2 : R = OMe
8 : R = OH
MeO
MeO
OMe
O
OMe
R
OMe
feruloyl-CoA
OH
OMe
OMe
1 : R = OMe
7 : R = OH
Absence of NGF
R
OMe
O
R H
MeO
OMe
OMe
Cassumunarin A#
δC = 43.6 ppm#
OH
OMe
OMe
3
H-5 H-4
MeO
OMe
OMe
H-3'' (d, J = 15.5)
OMe
NGF-like Activity of BANGLE #
in PC12 Cells #
HMBC correlation of 3
OH
9'
3''
O
9
7'
5'
3'
1'
1''
OH
OH
O
O
5
Compounds isolated from BANGLE
This plant is used as a spice
and also used for traditional
Indonesian medicine ʻjamuʼ.
Purpose : Fever, Headache,
Stomach pain, Rheumatism,
Obesity
The ingredients of herbal
medicine in women after
childbirth. etc…#
result
CABE JAWA
LEMPUYANG
DELINGO
BANGLE
MAHONI
TEMU HITAM
KUNIT MANGGA
GAMBIR
JAMBU BOL
JEDAM
SAHAMBUNTIT
PUPUK POLAJADI
CEPLE SARI
Mangosteen
Delima
precursor cells
Development
BANGLE (Zingiber purpureum)
Screening of Indonesian
Food Plant Extract by
PC12 Cells#
Neurotrophins
(NGF, BDNF, NT-3, NT-4/5 )
Neuronal stem cells
Cassumunarin A
OMe
3
1
MeO
(10 mM)
OMe
H-2
OMe
OMe
19
1
1) Tom J. Mabry, Tetrahedron Lett., 35, 981-984 (1994).!
5
A
B
70
**
60
RFU!
Compound 4!
60!
500!
0!
0!
1!
5!
2000!
10!
1500!
50!
100!
1!
5!
10!
50!
500!
0!
475!
100!
495!
515!
535!
555!
575!
0!
475!
595!
495!
515!
535!
555!
575!
595!
Concentration (uM)!
Reduction in β-sheet formation by compounds 1 and 3. (A): Percentage change of β-sheet. (B) or (C): Relative
fluorescence units (RFU) of thioflavin T. Aβ42 (12 μM) in PBS were incubated with compounds 1 and 3 at the indicated
concentrations (0, 1, 5, 10, 50 and 100 μM) at 36°C for 1 h. The samples were diluted 10-fold and incubated with 5 μM ThT for 30 min at 4°C. Fluorescence intensity was measured with excitation at 440 nm, and emission at 475- 650 nm using a
spectrophotometric plate. β-sheet formation was detected as a peak at 490 nm. 20
10
3
(3 µM)
2500!
1000!
20!
**
NGF
1
(2ng/mL) (30 µM)
3000!
2000!
1000!
0!
30
2500!
1500!
40!
40
3 (3 µM)
3
1
3500!
Compound 1!
80!
**
50
4000!
3000!
!"#$
% of beta-sheet!
80
0
time (min)
Compound 41
4500!
Compound 13
100!
**
90
1 (30 µM)
30 35 40
C
3500!
100
NGF 2ng/mL
15 20 25
4000!
120!
control
10
Reduction in β-sheet formation by compounds 1 and 3. Neurite Outgrowth of PC12 Cells Promoted by 3#
4
(3 µM)
4
(10 µM)
4 (10 µM)
4 (3 µM)
Neurotrophic Effects of 1 and 2 in Cultured Cells
Neurodifferentiation of PC12 Cells by 1 and 2#
Control (0.5 % EtOH)
Neurite Outgrowth of Cultured Rat Cortical Neurons Promoted by 1 and 2
1 (30µM)
NGF (10ng/mL)
Control (0.5 % EtOH)
2 (30µM)
1 (3 µM)
bFGF (10ng/mL)
Neurite length
PC12 cells were cultured in 24-well plates in DMEM + 10% HS and 5% FBS for 24 h at a density of 2x103 cells/cm2, and then
medium was changed to DMEM + 2% HS and 1% FBS containing 1 and 2. After 4 days cells with neurites were counted.#
2 (3 µM)
Neurite number
Viability
MeO
OMe
300
##
60
40
##
40
##
20
0
C
20
NGF
10
30
10ng/mL Comp.1 (µM)
0
C
50.0
50
54.4
53.1
40
33.1
30
20
7.0
194.9
**
172.2
170.3
157.9
150
100
50
0
0.4
ctrl
0.5%
EtOH
0.4
NGF
10 ng/mL
3
1.2
10
0.5
20
30
40
(+)-19
(+)-11
( (µM)
µM )
3
2.5
10
1.3
20
0
30
40
(-)-19
(-)-1
( (µM)
µM )
ctrl bFGF
0.5% 10 ng/
EtOH mL
0.03 0.3
3
19
1
((µM)
µM )
0.03 0.3
20
2
((µM)
µM )
3
0.03 0.3
3
0.03 0.3
(+)-19
(+)-1
((µM)
µM )
(-)-19
(-)-1
((µM)
µM )
3
5.0
6.6
**
6.2
**
6.0
161.6
8.3
10
NGF
10
30
10ng/mL Comp.2 (µM)
199.9
**
182.6 185.4
*
*
195.9
**
200
8.0
6.8
**
5.7
**
5.2
5.9
**
4.9
4.7
4.0
350
194
**
200
150
110
113
109
112
97
100
250
188
178
185
200
129
150
3.0
146
125 150
159
MeO
100
2.0
OMe
100
50
OMe
50
1.0
0.0
ctrl
0.5%
EtOH
bFGF
10 ng/mL
0.03
0.3
3
0.03
19
1
( (µM)
µM )
0.3
20
2
( (µM)
µM )
3
OMe
250
132
*
100
OMe
1
342
**
300
188
**
158
**
Viability ( % ）
##
60
229.5
**
Viability ( % ）
##
60
68.4
70
231.5
**
223.0
**
Average of neurite number
80
250
230.1
**
250
Average of neurite length ( µm ）
80
80
Percentage of cells with neurites ( % )
% cells with neurites
Neuritogenesis
254.4
**
0
ctrl bFGF 0.03 0.3 3 30
0.5% 10 ng/
EtOH mL
19
1
( (µM)
µM )
0.03 0.3 3
20
2
((µM)
µM )
30
0
2
ctrl bFGF 0.03 0.3 3 30
0.5% 10 ng/
EtOH mL
(+)-19
(+)-1
((µM)
µM )
0.03 0.3 3
OMe
30
(-)-19
(-)-1
( µM )
(µM)
Neurogenesis Effects of 1 and 2 in OBX Mice
Neurogenesis of Phenylbutenoid Dimers in OBX Mice Protocols
memory impairment
deppression
Olfactory Bulbectomized (OBX )Mice
Brain
immunohistochemistry
neurodegenerative model mice olfactory bulb MeO
OBX
Animals
ddY (8W)
MeO
OMe
OMe
OMe
OMe
1
H
N
Schedule
0
7
14
21
28 day
OMe
OMe
2
H
Double-labeled for NeuN and BrdU
BrdU(green)
Merged (yellow)
O
CF3
Fluoxetine
SSRI
BrdU (50mg/kg I.p.)
Brain
Comp.1
Comp.2
400
300
200
100
0
ng / tissue
Plasma
500
sham
OBX
OBX!
Veh. (i.p.)
IHC : Immunohistochemistry
NeuN: Neuron specific nuclear protein
BrdU: 5-Bromo-2 -deoxyuridine
The concentration of 1 and 2 in plasma and brains of
mice after oral administration
600
non
IHC (BrdU / NeuN)
Comp.1 (50mg/kg/day p.o.)
Comp.2 (50mg/kg/day p.o.)
fluoxetine (10mg/kg/day i.p.)
ng / mL
NeuN(red)
Quantitative analysis of the number of
BrdU and NeuN coexpressing cells.
Confocal laserscanning
microscope
SSRI : Selective Serotonin Reuptake Inhibitors
OBX
Confocal microscopy images of double staining for BrdU and NeuN in DG regions
of the hippocampus.
hippocampal
600
Comp.1
500
Comp.2
400
BrdU NeuN Merged
OBX!
FLU. (i.p.)
300
OBX!
Veh. (p.o.)
OBX!
1 (p.o.)
OBX!
2 (p.o.)
200
100
0.5
1
2
time (hr)
24
0
0.5
1
2
time (hr)
24
Compounds 1 and 2 were administrated orally to the mice. The mice were sacrificed at various
time points, and blood and brain tissue were collected. The concentrations of 1 and 2 in plasma
and brain tissue were analyzed by HPLC/MS.
Acknowledgement#
We would like to thank Shinya Hosoda and Eishin Kato (Hosoda SHC Co., Ltd) for their assistance.
"

Download Report