Neuroprotective constituent from the seeds of Alpinia katsumadai Hayata

Article abstract of DOI:10.1016/j.phytol.2016.08.024

Two new compounds, named as rhamnocitrin-3-O-β-D-glucopyranosyl-4′-O-β-D-galactosyl-(1?→?3)-O-β-D-glucopyranoside (1), and (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (2), were isolated from the seeds of Alpinia katsumadai Hayata. Their str

Full text of DOI:10.1016/j.phytol.2016.08.024

Phytochemistry Letters 18 (2016) 59–63
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Neuroprotective constituent from the seeds of Alpinia katsumadai
Hayata
a
b
c,
Dong-Yang Chen , Fan Yang , Yu-Qun Lin *
a
Department of Information Engineering, The First Affiliated Hospital of Shantou University Medical College, Shantou 515041, China
Department of Pharmacy, The Second People’s Hospital of Shantou, Shantou 515041, China
The First Affiliated Hospital of Shantou University Medical College, Shantou 515041, China
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
0
Received 13 June 2016
Received in revised form 29 August 2016
Accepted 31 August 2016
Available online xxx
Two new compounds, named as rhamnocitrin-3-O-
b
-D
-glucopyranosyl-4 -O-
b
-D-galactosyl-(1 !3)-O-
b-D-glucopyranoside (1), and (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (2), were
isolated from the seeds of Alpinia katsumadai Hayata. Their structures were elucidated on the basis of
spectroscopic analysis. Additionally, compounds 2 exhibited potent neuroprotective activity on 1-
methyl-4-phenylpyridinium-induced oxidative damage in PC12 cells.
Keywords:
Alpinia katsumadai Hayata
Isocoumarin
ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Flavonol
Neuroprotective activity
PC12 cells
1. Introduction
damage and neuronal cell death (Leuner et al., 2007). Supplemen-
tation of exogenous antioxidants has their effects in curtailing the
Alpinia katsumadai Hayata is mainly distributed in the Southern
and Southeast Asia (Li et al., 2010; Ngo and Brown, 1998). Previous
chemical investigations on this plant have resulted in the isolation
of various constituents such as flavonoids, kavalactones, diary-
lheptanoids, stilbenes, monoterpenes and sesquiterpenes (Nam
and Seo, 2012). Some of these compounds have anti-emetic, anti-
viral, anti-oxidant, anti-tumor and cytoprotective activities
oxidative damage to cellular macromolecules (Wu et al., 2015).
This study was conducted to find new neuroprotective agents
against oxidative stress-induced neurodegeneration.
2. Results and discussion
Compound 1 was obtained as a yellow amorphous powder. Its
(Grienke et al., 2010; Jeong et al., 2007; Li et al., 2011; Xu et al.,
molecular formula C34
42
H O21 was deduced from HR-ESI–MS. The
2
013; Yang et al., 1999). This paper described the isolation and
IR spectrum showed the presence of the carbonyl group(s)
ꢀ1
ꢀ1
characterization of two new compounds, rhamnocitrin-3-O-
glucopyranosyl-4 -O-
b
-
D-
(1675 cm ), hydroxyl group(s) (3365 cm ), and aromatic ring
0
ꢀ1
b-D
-galactosyl-(1 !3)-O-
b
-D
-glucopyrano-
(s) (1553,1462 cm ). The UV spectrum at 262 and 338 nm showed
1
side (1), and (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochro-
man-1-one (2) from the seeds of Alpinia katsumadai Hayata (Fig. 1).
Oxidative stress has been considered as a main factor in the
pathogenesis of neural diseases, which can be caused by cytotoxic
agents (Lee et al., 2005; Luo et al., 2012). Exposure of neurons to
such cytotoxic agents will result in the activation of intracellular
toxic events to increase the mitochondrial membrane permeabili-
ty, release cytochrome-c from the mitochondrial to nucleus,
activate caspase-related apoptotic proteins and facilitate the
formation of apoptosome complex, finally resulting in DNA
the characteristic signals of a flavonoid. The H NMR data of 1
(Table 1) showed the signal of hydroxyl group at
d
12.59 (1H, s, H-
8.17 (2H, d,
5). The spectrum showed four aromatic protons at
d
0
0
0
0
0
J = 9.0 Hz, H-2 , H-6 ) and 7.11 (2H, d, J = 9.0 Hz, H-3 , H-5 ),
0
1
suggesting an AA BB system. Additionally, the H NMR spectrum
also displayed two meta-coupled alkene protons at 6.71 (1H, d,
d
J = 2.0 Hz, H-8) and 6.42 (1H, d, J = 2.0 Hz, H-6). The acid hydrolysis
with HPLC analysis confirmed the presence of D-galactose and D-
glucose in the ratio of 1:2 (Tanaka et al., 2007). The proton signals
0
000
0
000
at
4
d 5.51 (1H, d, J = 7.5 Hz, H-1 ), 5.07 (1H, d, J = 7.5 Hz, H-1 ), and
0
.25 (1H, d, J = 8.0 Hz, H-1 ) were attributed to three sugar
1
moieties in the H NMR spectrum, which further confirmed that
these three sugar residues were -pyranosyl configurations. One
-glucosyl moiety was linked to the aglycone at C-3, indicated by
b
*
Corresponding author.
b-D
E-mail address: yuqunlinyq@163.com (Y.-Q. Lin).
http://dx.doi.org/10.1016/j.phytol.2016.08.024
874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1

6
0
D.-Y. Chen et al. / Phytochemistry Letters 18 (2016) 59–63
Fig. 1. Structure and key HMBC correlations of compounds 1 and 2.
correlation from a methoxyl proton signal
d 3.89 (3H, s) to d 165.4
Table 1
(
C-7) revealed that a methoxyl unit was linked to aglycone at C-7.
1
H NMR (500 MHz) and 13C NMR (125 MHz) spectral data of compounds 1 and 2 in
Therefore, the structure of compound 1 was elucidated as
6
DMSO-d (d in ppm, J in Hz).
0
rhamnocitrin-3-O-
1 !3)-O- -glucopyranoside.
Compound 2 was obtained as a white amorphous powder. Its
b
-D
-glucopyranosyl-4 -O-
b
-D-galactosyl-
Position
1
Position
2
(
b-D
d
H
d
C
H C
d d
HR-ESI–MS and NMR data gave the molecular formula C13
which indicated 6 of unsaturation. Compound 2 appearing purple
in the 10% ethanol sulfate solution and showing positive reaction
H
16
O
5
,
2
3
4
–
–
–
157.6
134.1
178.9
159.5
98.9
165.4
93.8
156.3
105.2
124.8 8a
131.3
116.8 10
161.1 11
116.8 12
1
3
4
ꢀ164.3
ꢁ
ꢀ84.1
2.71 (1H, d, J = 13.5) 28.6
2.76 (1H, d, J = 13.5)
ꢀ113.6
5
6
7
8
9
-OH
12.59 (1H, s)
6.42 (1H, d, J = 2.0)
with FeCl
3
3
-K
3
[Fe(CN)
283 cm , indicated the presence of phenolic hydroxyl group. The
UV spectrum at 233 and 281 nm and the IR spectrum (1711, 1633,
6
], together with the IR spectrum at 3361,
4a
5
6
7
8
ꢀ
1
–
ꢀ143.1
6.71 (1H, d, J = 2.0)
–
ꢀ146.3
ꢀ1
ꢀ138.8
1451, 1342, 859 cm ) showed the presence of the aromatic ring
and conjugated ketone. The H NMR spectrum (Table 1) of 2
1
0
–
6.96 (1H, s) 107.3
ꢀ115.8
1
0
0
0
0
0
0
1
2
3
4
5
6
7
–
exhibited one aryl proton signal at
methylene signals at 2.71 (1H, d, J = 13.5 Hz, H-4
d, J = 13.5 Hz, H-4 ), one methine signal at 1.92 (1H, d, H-9), three
methyls at 0.93 (3H, d, J = 6.5 Hz, H-10), 0.82 (3H, d, J = 6.5 Hz, H-
1), 1.18 (3H, s, H-12), and three hydroxyl groups at 9.52 (1H, brs,
H-5), 8.81 (1H, brs, H-7). C NMR spectrum
(Table 1) showed 13 signals, associated with one carbonyl ( 164.3,
C-1); six olefinic carbons including one methine ( 107.3, C-8), two
quaternary carbons ( 113.6, C-4a; 115.8, C-8a) and three
oxygenated quaternary olefinic carbons ( 143.1, C-5; 146.3, C-6;
138.8, C-7); one quaternary carbon connecting to oxygen ( 84.1, C-
3); one methine ( 35.8, C-9); one methylene ( 28.6, C-4) and three
methyls ( 15.6, C-10; 17.1, C-11; 21.8, C-12). The correlation
d
6.96 (1H, s, H-8), two
8.17 (1H, d, J = 9.0)
7.11 (1H, d, J = 9.0)
–
9
1.92 (1H, s) 35.8
0.93 (3H, d, J = 6.5) 15.6
0.82 (3H, d, J = 6.5) 17.1
1.18 (3H, s) 21.8
d
a
) and 2.76 (1H,
d
b
d
7.11 (1H, d, J = 9.0)
8.17 (1H, d, J = 9.0)
3.89 (3H, s)
d
d
131.3 5,6,7-OH 9.52, 9.37, 8.81 (brs)
56.0
1
d
d
-OMe
13
d
9.37 (1H, brs, H-6), d
3
1
2
3
4
5
6
4
1
2
3
4
5
6
-Glc
d
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.51 (1H, d, J = 7.5)
3.35 (1H, m)
3.24 (1H, m)
3.19 (1H, m)
3.49 (1Hm)
101.7
74.9
76.6
70.9
77.2
d
d
d
d
3.68 (1H, br d, J = 11.0) 60.3
3.52 (1H, m)
d
d
-Glc
d
00
5.07 (1H, d, J = 7.5)
2.99 (1H, m)
3.31 (1H, m)
3.02 (1H, m)
3.34 (1H, m)
3.61 (1H, m)
100.8
73.3
80.0
70.1
77.8
61.9
1
1
between H-9 and H-10/H-11 in H- H COSY spectrum, and the
correlation between H-10, H-11 and C-9, H-10 and C-11 in HMBC
spectrum suggested the presence of an isopropyl group. In the
HMBC spectrum (Fig. 1), the correlation between H-8 and C-8a/C-
00
00
00
00
00
7
/C-1, indicated the benzene ring connecting an ester. The
3
.46 (1H, m)
correlation between H-4 and C-4a/C-8a/C-5, indicated the methy-
lene at C-4a. The correlation between H-12 and C-3, H-9, H-10, H-
11 and C-3, indicated both the methyl and isopropyl group at C-3.
0
0
0
0
0
0
0
00
-Gal
3
1
2
3
4
5
6
000
000
000
000
000
000
4.25 (1H, d, J = 8.0)
3.36 (1H, m)
3.23 (1H, m)
3.15 (1H, m)
3.45 (1H, m)
3.75 (1H, m)
103.2
75.8
73.3
77.6
77.1
13
Based on the C NMR and HMBC, C-3 was confirmed as quaternary
carbon connecting to oxygen. Combining the unsaturation degree,
the lactone ring was deduced in compound 2. The correlation
between H-12 and C4/C-9, H-4 and C-1/C-9 further established the
structure of compound 2. The absolute configuration at C-3 was
61.9
3
.39 (1H, m)
assigned by comparing the optical rotation of compound 2 ([
D = ꢀ55.7) and (3R)-3,4-dihydro-3-methyl-5,6,8-trihydroxy-1H-2-
benzopyran-1-one ([
these data, compound 2 was elucidated as (3R)-5,6,7-trihydroxy-3-
isopropyl-3-methylisochroman-1-one.
a]20
0
0
the HMBC correlation from
d
5.51 (H-1 ) to
d
134.1 (C-3). The HMBC
a
]20 D = ꢀ61.7) (Krohn et al., 1997). Based on
0
00
0
correlations from 5.07 (H-1 ) to
d
d
161.1 (C-4 ), from
3.31 (H-3 ) to
-glucosyl unit was attached to C-
-galactosyl moiety was linked to C-3 . The HMBC
d
4.25 (H-
0
000
000
000
0000
1
) to
d
80.0 (C-3 ), and from
d
d
103.2 (C-1
)
exhibited that the remaining
b-D
Overproduction of ROS can cause lipid peroxidation, protein
denaturation, nucleic acid damage, and mitochondrial dysfunction,
0
000
4
, and the
b
-D

D.-Y. Chen et al. / Phytochemistry Letters 18 (2016) 59–63
61
thereby impairing cellular function and integrity (Adibhatla and
Hatcher, 2010; Wang and Michaelis, 2010). This type of damage has
been considered as a major cause of neuronal damages in some
neurodegenerative disorders (Birben et al., 2012; Surendran and
Rajasankar, 2010). Therefore, the imbalance between the intracel-
lular oxidative and anti-oxidative defense systems, requires the
supplement of external antioxidants to eliminate ROS as the
potential neuroprotective therapeutics (Xin et al., 2014).
In this study, we demonstrated that compound 2 increased the
+
viability of PC12 cells, and inhibited cellular apoptosis in MPP -
induced oxidative stress model (Figs. 2–5 ). Compound 2 effectively
+
protected PC12 cells against MPP -induced injury, most possibly
through mitochondria pathway, closely related to the presence of
hydroxyl groups with powerful antioxidant action and effective
free radical-scavenging ability. These results warrant further study
as the potential agent for the treatment of neurodegenerative
disease.
Fig. 3. Effect of compound 2 on mitochondrial membrane potential in MPP+-
damaged PC12 cells assayed by JC-1. Values were expressed as mean ꢃ SD (n = 5).
#
#
P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs MPP+ group.
3. Conclusions
We isolated and identified two new compounds, rhamnocitrin-
0
3-O-
b-D
-glucopyranosyl-4 -O-
b
-
D
-galactosyl-(1 !3)-O-
b-D-glu-
copyranoside (1), and (3R)-5,6,7-trihydroxy-3-isopropyl-3-meth-
ylisochroman-1-one (2), from the seeds of Alpinia katsumadai
Hayata for the first time. Compound 2 exhibited potent neuro-
+
protective activity against MPP -induced damage in PC12 cells
through preventing apoptosis via mitochondria pathway.
4. Experimental
4.1. General experimental procedure
Optical rotations were measured on P-2000 polarimeter
(
JASCO). IR spectra were recorded on a PerkinElmer Spectrum
One FT-IR spectrometer with KBr pellets. The UV spectra were
recorded using a Shimadzu UVmini-1240 spectrophotometer with
PhotoMultiplier Tube. The NMR spectra were performed on a
Bruker AV-500 spectrometer, using TMS as an internal standard.
HR-ESI–MS spectra were measured on a Waters API QSTAR Pular-1
mass spectrometer. HPLC was performed by Shimadzu apparatus
+
Fig. 4. Effect of compound 2 on DNA fragmentation of MPP -damaged PC12 cells.
#
#
Values were expressed as mean ꢃ SD (n = 5). P < 0.01 vs control group; *P < 0.05,
*
*P < 0.01 vs MPP+ group.
(
LC-6AD pump, SPD-20A UV detector, YMC ODS-A (20 mm ꢂ 250
mm, 10 m), detection at UV 265 nm). Column chromatography
was performed with D101 macroporous resin and reversed-phase
18 silica gel (Merck, Germany), and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden).
m
4
.2. Plant materials
C
Alpinia katsumadai Hayata was purchased from Anguo City,
Hebei province, China, in June 22nd 2015, and authenticated by
Prof. Jinkai Liu, Kunming Institute of Botany, Chinese Academy of
Sciences. Avoucher specimen (No. 20150622) was deposited at The
First Affiliated Hospital of Shantou University Medical College.
4
.3. Extraction and isolation
Air-dried seeds of Alpinia katsumadai Hayata (20.0 kg) were
pulverized, and then extracted with 90% ethanol (50 L ꢂ 3 ꢂ 4 h)
under reflux. The plant residue was extracted with distilled water
and the solvent was evaporated to afford a residue (A, 1066 g). The
9
7
0% ethanol collected was evaporated to yield a crude extract (B,
70 g). The residue A was chromatographed on a D101 macro-
porous resin (HPD-110, 20 L) column (20 cm ꢂ 200 cm) with
gradient elution of H O/ethanol (100:0–0:100, 50 L) to yield 6
fractions (Fr. 1–6). Fr. 3 (353 g) was chromatographed on a RP C18
silica gel eluted with gradient H O/methanol (100:0-0:100) to
afford 5 subfractions (Fr. 3.1–3.5). Fr. 3.3 (14.6 g) was purified by
preparative HPLC using MeOH/H O (40:60, /v) as the mobile
phase (1.5 mL/min) to afford compound 1 (16.3 mg, t = 24.5 min).
The extract B was subjected to column chromatography eluted
2
2
+
Fig. 2. Effect of compound 2 on the cell viability of MPP -damaged PC12 cells by
MTT assay. Pre-incubation with compound 2 or NAC (positive control) for 24 h
2
v
protected PC12 cells from MPP+ toxicity (0.5 mM, 24 h). NAC: N-acetyl-
Values were expressed as mean ꢃ SD (n = 5). P < 0.01 vs control group; *P < 0.05,
L
-cysteine.
##
R
+
*
*P < 0.01 vs MPP group.

6
2
D.-Y. Chen et al. / Phytochemistry Letters 18 (2016) 59–63
Fig. 5. Effect of compound 2 on caspase activity of MPP -damaged PC12 cells. Values were expressed as mean ꢃ SD (n = 5). ##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs
+
+
MPP group.
ꢀ
with petroleum ether, ethyl acetate, acetone and methanol. The
ethyl acetate portion (77.3 g) was chromatographed over a silica gel
with gradient elution of petroleum ether/ethyl acetate (100:0–
Table 1. HR-ESI–MS: m/z found 251.0922 [M-H] (calcd. for
C H O , 251.0927).
13 15 5
0
:100) to obtain 6 fractions (Fr. 1–Fr. 6). Fr. 4 (11.2 g) was subjected
4.7. Cell culture and treatment
to Sephadex LH-20 column chromatography eluted with methanol
to give 6 subfractions (Fr. 4.1–Fr. 4.6). Fr. 4.3 (1.8 g) was further
Differentiated PC12 cells were maintained in DMEM medium
purified on HPLC (MeOH/H
2
O, 55:45, v/v, 1.5 mL/min) to give
supplemented with 10% FBS, 100 U/mL penicillin G and 100
m
g/mL
ꢁ
compound 2 (20.2 mg, t = 22 min).
R
streptomycin at 37 C in humidified 95% air/5% CO
2
. PC12 cells were
treated as: cells were incubated for 24 h with compound 2 at 1, 3,
10 M or N-acetyl- -cysteine (NAC, 10 M, positive control),
4.4. Analytical acid hydrolysis
m
L
m
respectively; cells treated by DMSO as the negative control.
Consequently, PC12 cells were treated by 0.5 mM MPP for 24 h.
ꢁ
+
Compound 1 (1 mg) was hydrolyzed with 2 M HCl at 85 C for
1
h and then extracted with ethyl acetate. The aqueous layer was
evaporated and dissolved in pyridine (1 mL). After adding 1 mg
L-
4.8. Cell viability measurement
cysteine methyl ester hydrochloride, the mixture was heated at
ꢁ
60 C for 1 h. 20
m
L O-tolyl isothiocyanate was then added and the
Cell viability was determined by MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay. After treatment
ꢁ
mixture was heated at 60 C for another 1 h. An aliquot (2 mL) of
the supernatant was subjected to HPLC analysis. The mobile phase
consisted of acetonitrile and 0.1% formic acid. The sugar residues of
compounds 1 were identified by comparing the retention times of
with compound 2 at 1, 3, 10 mM or NAC at 10 mM for 24 h and then
+
0.5 mM MPP for 24 h, 0.5 mg/mL MTT solution was added in each
ꢁ
well and incubated for another 4 h at 37 C. Culture medium was
the corresponding derivative with the standard D-glucose (t
5.65 min) and -galactose (t = 31.86 min).
R
=
removed and the formazan crystals were solubilized with DMSO.
The absorbance was measured at 570 nm by a microplate reader.
Cell viability was expressed as the percentage of the negative
control, which was set to 100%.
2
D
R
0
4.5. Rhamnocitrin-3-O-
b-D-glucopyranosyl-4 -O-b-D-galactosyl-
(
1 !3)-O- -glucopyranoside (1)
b-D
4
.9. Measurement of mitochondrial membrane potential
UV (MeOH)
lmax (log e) 338(3.12), 262(3.22) (nm). IR (KBr) n
max
3
365, 2878, 1675, 1553, 1462, 1232, 825 and 665 cm . H and 13C
ꢀ
1 1
The mitochondrial membrane potential (MMP) was measured
NMR spectroscopic data see Table 1. HR-ESI–MS: m/z found
by the fluorescent probe JC-1. JC-1 is sensitive to MMP, and the
changes in the ratio between aggregate (red) and monomer (green)
fluorescence can indicate the condition of MMP. After treatment
+
+
809.2116 [M+Na] (cacld. for C34
42
H O21Na , 809.2111).
4
.6. (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one
with compound 2 at 1, 3, 10 mM or NAC at 10 mM for 24 h and then
+
(
2)
0.5 mM MPP for 24 h, PC12 cells were incubated with JC-1 for
ꢁ
1
5 min at 37 C in the dark. After two more rinses, the fluorescence
[
a
]20 D = ꢀ55.7 (c 0.08, CHCl
3
). UV (MeOH)
l
max (log
e
): 233
intensity was determined on a fluorescence microplate reader at
an excitation of 490 nm and emission of 530 nm (green fluorescent
monomers) and 590 nm (red fluorescent aggregates) respectively.
(
1
2.29), 281 (2.26). IR (KBr): max 3361, 3283, 2992, 1711, 1633, 1451,
342, 1015, 859 cm . H and C NMR spectroscopic data see
n
ꢀ1
1
13

D.-Y. Chen et al. / Phytochemistry Letters 18 (2016) 59–63
63
The change of MMP was expressed as a percentage of the negative
control, which was set to 100% (Wu et al., 2015).
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4.10. Measurement of DNA fragmentation
Quantification of DNA fragmentation was measured by Cell
plus
Death Detection ELISA
, 3,10
kit. After treatment with compound 2 at
+
1
m
M or NAC at 10
m
M for 24 h and then 0.5 mM MPP for 24 h,
PC12 cells were washed and lyzed for 30 min. After a centrifugation
ꢁ
at 1000 rpm for 10 min at 4 C, 20
m
L supernatant was transferred
to a streptavidin-coated microplate and incubated with a mixture
of anti-histone-biotin and anti-DNA-peroxidase. The peroxidase
0
amount in the immunocomplex was quantified by adding 2,2 -
azinobis (3-ethylbenzthiazoline-6-sulfonic acid) as the substrate.
The absorbance of the reaction mixture was measured by a
microplate reader at 405 nm. The extent of DNA fragmentation was
expressed as a percentage of the negative control, which was set to
+
Lee, C.S., Park, S.Y., Ko, H.H., Song, J.H., Shin, Y.K., Han, E.S., 2005. Inhibition of MPP -
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The caspase activity was measured by the assay kit. After
treatment with compound 2 at 1, 3, 10
4 h and then 0.5 mM MPP for 24 h, PC12 cells were washed twice
m
M or NAC at 10
mM for
+
2
and lyzed for 30 min on ice. After a centrifugation at 20,000 rpm
ꢁ
for 10 min at 4 C, aliquots of supernatants containing 25 mg
protein were added to a reaction buffer supplemented with 0.1%
CHAPS, 100 mM PMSF and 5 mM DTT. The reactions were initiated
after addition of the following fluorescent substrates (50 mM at
the final concentration): Ac-DEVD-Amc for caspase-3 activity, Ac-
ꢁ
LEDH-Afc for caspase-9 activity. After incubation at 37 C for 2 h,
the cleavage of the substrates was measured (Amc: 390/475 nm;
Afc: 400/505 nm) by a microplate reader. The activity of caspase
was expressed as a percentage of the negative control (Wu et al.,
2
015).
4
.12. Statistical analysis
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Values were statistically analyzed by one-way analysis of
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0
0
variance (ANOVA) using the Sigma Stat statistical software (SPSS
Inc., Chicago, IL). Differences were considered as significant at
P < 0.05.
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