Neuroprotective Activity of Cerebrosides from Typhonium giganteum by Regulating Caspase-3 and Bax/Bcl-2 Signaling Pathways in PC12 Cells

Article abstract of DOI:10.1021/acs.jnatprod.6b00954

An investigation of the potential neuroprotective natural product constituents of the rhizomes of Typhonium giganteum led to the isolation of two new cerebrosides, typhonosides E (1) and F (2), along with 11 known analogues (3-13). The structures of compounds 1 and 2 were elucidated by spectroscopic data interpretation. The activity of these compounds against glutamate-induced cell apoptosis was investigated in PC12 cells. All compounds exhibited such activity, which was related to the length of the fatty acyl chain. Among them, longan cerebroside II (11), with the longest fatty acyl chain, showed the most potent protective effect in PC12 cells from glutamate injury, with an EC₅₀ value of 2.5 μM. Moreover, at the molecular level, longan cerebroside II (11) downregulated the expression of caspase-9, caspase-3, and Bax, upregulated the expression of Bcl-2, and decreased the level of cytosolic cytochrome c in a concentration-dependent manner.

Full text of DOI:10.1021/acs.jnatprod.6b00954

Article
pubs.acs.org/jnp
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
S Supporting Information
*
ABSTRACT: An investigation of the potential neuroprotective
natural product constituents of the rhizomes of Typhonium
giganteum led to the isolation of two new cerebrosides,
typhonosides B (1) and C (2), along with 11 known analogues
(
3−13). The structures of compounds 1 and 2 were elucidated by
spectroscopic data interpretation. The activity of these compounds
against glutamate-induced cell apoptosis was investigated in PC12
cells. All compounds exhibited such activity, which was related to
the length of the fatty acyl chain. Among them, longan cerebroside
II (11), with the longest fatty acyl chain, showed the most potent
protective effect in PC12 cells from glutamate injury, with an EC50
value of 2.5 μM. Moreover, at the molecular level, longan
cerebroside II (11) downregulated the expression of caspase-9, caspase-3, and Bax, upregulated the expression of Bcl-2, and
decreased the level of cytosolic cytochrome c in a concentration-dependent manner.
troke is a devastating disease with high morbidity and
mortality. Although the current understanding of stroke
cells. First, bioassay-guided fractionation of the ethyl acetate
extract (from the ethanolic extract of rhizomes of T. giganteum)
led to the isolation of individual cerebrosides. Subsequently, the
purified compounds were screened for their putative neuro-
protective activities against glutamate-induced cell apoptosis in
PC12 cells, and structure−activity relationships were also
investigated for these cerebrosides. Finally, the molecular
mechanisms underlying the neuroprotective effects of active
constituents against glutamate-induced PC12 cell apoptosis
were further explored. The results obtained herein showed that
the cerebrosides may protect PC12 cells from being damaged
by glutamate.
1
S
provides a basis for a favorable prognosis, protecting the nerve
cells from being damaged to improve brain function is still a
2
major challenge. The dried rhizomes of Typhonium giganteum
Engl. (Araceae) are widespread in parts of mainland China,
3
India, and Malaysia. The species is recorded in the Chinese
Pharmacopoeia for the treatment of stroke, epilepsia, and
4
tetanus. Previous phytochemical investigations of T. giganteum
have led to the presence of fatty acids, lignans, nucleosides,
5
−9
hydantoins, and cerebrosides. It has been demonstrated that
a mixture of cerebrosides from T. giganteum reduced transient
ischemic brain damage in rats and mice by activating large
1
0
RESULTS AND DISCUSSION
conductance calcium-activated (BK ) channels. Also,
■
Ca
purified cerebrosides (termitomycesphins A and B) from
The air-dried and powdered rhizomes of T. giganteum were
extracted by 75% ethanol (EtOH) to obtain a crude extract,
which was suspended in water and then partitioned with
petroleum ether, EtOAc, and n-BuOH, sequentially. The
EtOAc fractions were purified by column chromatography
and semipreparative HPLC to afford two new (1 and 2) and 11
known (3−13) compounds.
Termitomyces albuminosus were found to activate BK channels
Ca
11
at micromolar concentration. These findings indicated that
cerebrosides, as active components of T. giganteum, may elicit
neuroprotective effects through the activation of BKCa channels.
In addition, other natural products (e.g., formononetin,
naringin, and isorhynchophylline) have also shown neuro-
protective effects by regulating the expression of apoptosis-
Typhonoside B (1) was obtained as a white, amorphous
12−14
powder. Its molecular formula was established as C H O N
related proteins, such as Bcl-2, Bax, and caspase-3.
45 85
9
This contribution reports the potential neuroprotective
effects of cerebrosides from T. giganteum by regulating the
caspase cascade and the Bax/Bcl-2 signaling pathways in PC12
Received: October 17, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00954
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showed that their structures were almost identical except for
subtle differences in the chain length of the α-hydroxy acyl
18
moiety and/or nitrogen-containing unit. The signals at 105.4,
7
4.9, 78.3, 71.5, 78.3, and 62.4 in the 13C NMR spectrum and
the anomeric proton signal at 4.91 (d, J = 7.8 Hz) suggested
that the sugar in 1 is a β-glucose moiety. Acidic hydrolysis of 1
gave D-glucose as a sugar residue, which was determined by GC
analysis of its corresponding trimethylsilylated L-cysteine
adduct. The HMBC spectrum of 1 revealed a correlation
from H-1 (δH 4.24 and 4.71) to the anomeric carbon (δC
1
05.7) (Table 1), demonstrating attachment of the glucose
1
1
moiety at C-1. The H− H COSY correlation from H-1 to H-5
(
δ
H
5.92) was used to define the 2-amino-1,3-dioxygenated
1
fragment. The H NMR spectrum showed two olefinic protons
4
at δ 5.99 (H-4) and 5.92 (H-5), attributed to a Δ double
H
4
bond. The Δ double bond was found to be trans, as evidenced
by the vicinal coupling constant (J4 = 15.6 Hz). HMBC
,5
+
correlations from H-5 (δ 5.92) and H-8 (δ 5.47) to C-6 (δ
by its HRESIMS (m/z 806.6116 [M + Na] ), corresponding to
four degrees of unsaturation. The IR spectrum of 1 exhibited
H
H
C
32.9), C-7 (δ
27.5), and C-10 (δ 27.3) were used to locate
C
C
−1
8
absorption bands at 3361 and 1645 cm ascribable to OH/NH
the second double bond at C-8/C-9 in 1 (Figure 1). The Δ
double bond was determined to be cis, as evidenced by the
1
13
and CO groups. The H and C NMR data of 1 (Table 1)
in C D N indicated the presence of a sugar residue, a long-
chemical shifts of δ 27.5 (C-7) and δ 27.3 (C-10). As
C C
5
5
15
chain nitrogen-containing unit, and a nonpolar acyl chain,
comprising a glycosphingolipid. Further comparison of the 1D
NMR spectra of 1 with those of the co-isolated known 9
reported in the previous literature, the signals of carbons next
to a cis alkene bond appear at δ 27−28, while those of a trans
C
double bond appear at δ 32−33. The lengths of the two chains
C
Table 1. NMR Spectroscopic Data for Compounds 1 and 2 in C D N
5
5
1
2
a
b
a
b
position
1
δH
4.71 (dd, 10.8, 6.0)
.24 (dd, 10.8, 4.2)
δC
70.1
δH
δC
69.9
4.71 (dd, 10.2, 6.0)
4.25 (dd, 10.8, 4.2)
4.82 (m)
4
2
3
4
5
6
7
8
9
4.80 (m)
54.6
72.3
54.3
72.1
4.76 (t, 6.0)
5.99 (dd, 15.3, 6.6)
5.92 (dt, 15.3, 6.6)
2.16 (m)
4.76 (t, 6.0)
5.99 (dd, 15.6, 6.0)
5.92 (dt, 15.6, 6.0)
2.17 (m)
132.2
132.1
32.9
131.9
131.8
32.7
2.03 (m)
27.6
2.06 (m)
32.7
5.47 (m)
130.6
129.4
27.3
5.50 (m)
129.7
130.9
32.5
5.46 (m)
5.50 (m)
1
1
1
1
1
1
2
3
4
5
1
2
2
1
2
3
4
5
6
0
2.18 (m)
2.14 (m)
1−15
6
1.24−1.36 (m)
1.27 (m)
29.6−30.0
32.1
1.24−1.36 (m)
1.27 (m)
29.3−29.9
31.9
7
1.27 (m)
22.9
1.27 (m)
22.7
8
0.85 (t, 6.6)
14.3
0.86 (t, 6.6)
14.1
′
′
′
′
175.6
72.5
175.4
72.3
4.56 (m)
4.58 (m)
2.01 (m)
35.6
2.02 (m)
35.4
1.78 (m)
25.9
1.78 (m)
25.7
′−18′
1.24−1.36 (m)
1.27 (m)
29.6−30.0
32.1
1.24−1.36 (m)
1.27 (m)
29.3−29.9
31.9
9′
0′
1′
″
″
″
″
″
″
1.27 (m)
22.9
1.27 (m)
22.7
0.85 (t, 6.6)
4.91 (d, 7.8)
4.02 (m)
14.2
0.86 (t, 6.6)
4.92 (d, 7.8)
4.02 (m)
14.1
105.7
75.1
105.4
74.9
4.22 (m)
78.4
4.21 (m)
78.2
4.21 (m)
71.5
4.21 (m)
71.3
3.89 (m)
78.6
3.90 (m)
78.4
4.50 (m)
62.6
4.51 (m)
62.4
4
.34 (dd, 11.7, 5.4)
4.36 (dd, 11.4, 5.4)
8.36 (d, 9.0)
N−H
8.35 (d, 9.0)
a
b
Data were measured at 600 MHz. Data were measured at 150 MHz.
B
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a
Table 2. Effects of Glutamate on PC12 Cell Viability
glutamate concentration (mM)
0
cell viability (%)
100 ± 1.82
1
1
1
1
2
0
68.7 ± 3.34**
47.2 ± 1.46**
38.0 ± 2.58**
28.4 ± 3.18**
18.6 ± 1.94**
2.5
5
7.5
0
a
Each value represents the mean ± SD, n = 6, ** p < 0.01 vs control
group.
It was found that PC12 cell viability increased up to 70%
Figure 1. Key COSY and HMBC correlations of compounds 1 and 2.
after being treated with 10 μg/mL of the ethyl acetate extract of
T. giganteum (Figure S19, Supporting Information). Sub-
sequently, compounds 1−13 were tested for their cytotoxicity
against PC12 cells using the MTT method. The results showed
that all the compounds had no discernible cytotoxicity for
PC12 cells at a concentration of 20 μM (Figure S20,
Supporting Information). The compounds 1−13 were further
screened for neuroprotective effects against glutamate-induced
neurotoxicity in PC12 cells, and their EC50 values are
summarized in Table 3. Compounds 11 and 12 were the
of 1 were identified by ESIMS/MS analysis (Figure S9,
Supporting Information). ESIMS fragments at m/z 279 and
3
41 were the two main ions of compound 1 by McLafferty
rearrangement (Figure S9, Supporting Information). Thus, the
number of carbons in nitrogen-containing and acyl units was
determined to be 18 and 21, respectively. The chemical shifts at
δC 70.1 (C-1), 54.6 (C-2, chiral carbon), 72.3 (C-3, chiral
carbon), 175.7 (C-1′), and 72.5 (C-2′, chiral carbon) in 1 were
virtually identical to those reported for (2S,3R,2′R)-phytos-
8
Table 3. Effects of Compounds 1−13 on Glutamate-Induced
Injury in PC12 Cells
phingosine moieties, suggesting that the configurations at C-2,
C-3, and C-2′ of 1 are also 2S,3R,2′R. Accordingly, the
structure of typhonoside B (1) was assigned as 1-O-β-D-
glucopyranosyl-(2S,3R,4E,8Z)-2-[2′(R)-hydroxyheneicosanoyl-
amino]-4,8-octadecadiene-1,3-diol.
Typhonoside C (2) was observed to be an isomer of 1 due to
their closely comparable NMR data but different signals for C-7
and C-10 (Table 1). Comparison of the NMR data of 2 with
those of 1 showed that the 8,9 alkene bond of 2 is trans, which
a
compound
EC50 (μM)
1
2
3
4
5
6
7
8
9
8.9 ± 0.20
8.8 ± 0.09
27.1 ± 0.33
29.7 ± 0.40
20.1 ± 0.25
22.2 ± 0.19
12.0 ± 0.08
12.4 ± 0.12
6.5 ± 0.18
6.9 ± 0.15
2.5 ± 0.28
2.7 ± 0.41
6.6 ± 0.07
9.2 ± 0.17
was confirmed by the signals at δ C-7 (δ 32.7) and C-10 (δ
C
C
C
3
2.5). Thus, the structure of typhonoside C (2) was defined as
-O-β-D -glucopyranosyl-(2S,3R,4E,8E)-2-[2′(R)-
1
hydroxyheneicosanoylamino]-4,8-octadecadiene-1,3-diol.
1
1
1
1
0
1
2
3
16
The known compounds soya-cerebroside II (3), soya-
1
6
cerebroside I (4), 1-O-β-D-glucopyranosyl-(2S,3R,4E,8Z)-2-
[
(
2′(R)-hydroxyoctadecanoylamino]-4,8-octadecadiene-1,3-diol
17
5),
1-O-β-D-glucopyranosyl-(2S,3R,4E,8E)-2-[2′(R)-
b
edaravone
17
hydroxyoctadecanoylamino]-4,8-octadecadiene-1,3-diol (6),
a
The EC50 value is defined as the concentration giving half-maximal
1
-O-β-D-glucopyranosyl-(2S,3R,4E,8E)-2-[2′(R)-hydroxy-
protection against glutamate-induced cell death and is expressed as the
17
leicosanoylamino]-4,8-octadecadiene-1,3-diol (7), 1-O-β-D-
glucopyranosyl-(2S,3R,4E,8Z)-2-[2′(R)-hydroxyeicosanoyl-
b
mean ± SD of triplicate determinations. Positive control.
17
amino]-4,8-octadecadiene-1,3-diol (8), 1-O-β-D-glucopyrano-
syl-(2S,3R,4E,8Z)-2-[2′(R)-hydroxydocosanoylamino]-4,8-oc-
most potent cerebrosides, with EC50 values of 2.5 and 2.7 μM,
respectively, which were more potent than edaravone, the
positive control used for this assay. Additionally, compounds 1,
2, 9, 10, and 13 showed EC50 values of 6.5−8.9 μM.
1
8
9
tadecadiene-1,3-diol (9), typhonoside A (10), longan
1
9
7
cerebroside II (11), longan cerebroside I (12), and
typhonoside (13) were identified by spectroscopic data
8
analysis as well as by comparison with the literature data.
Effects of Compounds 1−13 on PC12 Cell Viability.
Glutamate-mediated toxicity is an important mechanism of
neuronal death in various pathological conditions including
ischemia, trauma, epileptic seizures, and neurodegenerative
Preliminary Structure−Activity Relationships of the
Compounds. To analyze the preliminary structure−activity
relationships of the compounds, the neuroprotective activity
between the cis- and trans-isomers 1 and 2 was compared. As
shown in Figure 2A, there were no significant differences of the
cell viability between compound 1 (cis-) and compound 2
(trans-) groups (p > 0.05). For the other five pairs of
compounds (3 vs 4, 5 vs 6, 7 vs 8, 9 vs 10, and 11 vs 12),
similar results were also observed. Compounds 9 and 13 differ
in the presence/absence of a double bond at C-4/C-5 on the
nitrogen-containing unit. As shown in Figure 2B, the cell
viability of the compound 9 group (containing a double bond at
C-4/C-5) was almost the same as that of compound 13 (not
2
0−24
disorders.
PC12 cells, a sympathetic nerve cell line derived
from the rat pheochromocytoma, have been used widely for
25
neurological studies. Using a MTT assay, the cytotoxicity of
glutamate was evaluated in PC12 cells. As shown in Table 2,
the cell viability decreased along with the increase of glutamate
in the range 0−20 mM, and the IC value of glutamate was
5
0
about 12.5 mM. Thus, 12.5 mM glutamate was chosen for
26
subsequent experiments.
C
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Figure 2. (A) Effects of the double-bond configuration of compounds 1−12 on glutamate-induced PC12 cell death. (B) Effects of the occurrence of
4,5
a Δ double bond of compounds 9 and 13 on glutamate-induced PC12 cell death. (C) Effects of the fatty acyl chain length of compounds 1, 3, 5, 7,
9
, and 11 on glutamate-induced PC12 cell death. (D) Effects of longan cerebroside II (11) on glutamate-induced PC12 cell viability. (A−C) PC12
cells were treated with 12.5 mM glutamate with each compound for 24 h. (D) PC12 cells were treated with 12.5 mM glutamate with different
concentrations (0.1, 1, 5, 10, 15, and 20 μM) of longan cerebroside II (11) for 24 h. Cell viability was measured by the MTT assay. Each value
#
#
represents the mean ± SD, n = 6, p < 0.01 vs control group, *p < 0.05, **p < 0.01 vs model group; NS, not significant.
Figure 3. Effects of longan cerebroside II (11) on the apoptosis rate of PC12 cells treated with or without glutamate (detected using an annexin V-
FITC/PI assay). PC12 cells were treated with or without 12.5 mM glutamate along with low, medium, or high concentrations (0.1, 1, and 10 μM) of
longan cerebroside II (11) for 24 h. The control group did not contain glutamate. The apoptotic cells were detected in the right lower and right
upper quadrants. (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), (G) percentages of apoptotic cells in
#
#
different experimental conditions. The data are represented as means ± SD, n = 3, p < 0.01 vs control group, **p < 0.01 vs model group.
D
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Figure 4. Effects of longan cerebroside II (11) on the expression levels of caspase-9, caspase-3, Bcl-2, and Bax and the content of cytosolic
cytochrome c in PC12 cells treated with or without glutamate. (A) Western blotting analysis for caspase-9 in PC12 cells. (B) Western blotting
analysis for caspase-3 in PC12 cells. (C) Western blotting analysis for Bcl-2 and Bax in PC12 cells. (D) Western blotting analysis for the content of
cytosolic cytochrome c in PC12 cells. The PC12 cells were treated with or without 12.5 mM glutamate along with low, medium, or high
concentrations (0.1, 1, and 10 μM) of longan cerebroside II (11) and the 10 μM longan cerebroside II (11) group for 24 h, respectively. The data
#
#
are represented as means ± SD, n = 3, p < 0.01 vs control group, *p < 0.05, **p < 0.01 vs model group.
containing a double bond at C-4/C-5). In turn, to investigate
whether the activity of these compounds is correlated with the
length of the nonpolar acyl chain, the cell viability of
compounds 1, 3, 5, 7, 9, and 11, which differ in the length of
the nonpolar acyl chain, was assayed. It was found that there
were significant differences in cell viability after treatment with
compounds 1, 3, 5, 7, 9, and 11 at the same concentration. As
shown in Figure 2C, the cell survival rate gradually increased
along with the increase of the nonpolar acyl chain length of
those compounds (3, 5, 7, 1, 9, and 11). These results indicated
that the longer the nonpolar acyl chain, the higher the
neuroprotective activity. In summary, the structure−activity
relationship results suggested that (i) the configuration of the
double bond at C-8/C-9 on the nitrogen-containing unit is not
a prerequisite for the activity; (ii) the presence/absence of a
double bond at C-4/C-5 on the nitrogen-containing unit does
not affect the activity; and (iii) the length of the nonpolar acyl
chain affects the activity, continuously increasing the activity
along with an increase in its length.
To further confirm the effects of the nonpolar acyl chain of
cerebroside on its neuroprotective activity, the dose−effect
relationship of longan cerebroside II (11) was evaluated by the
MTT assay. As shown in Figure 2D, the cell survival rate of the
longan cerebroside II (11) group was significantly increased in
a concentration-dependent manner compared with that of the
glutamate group. Longan cerebroside II (11) showed a
cytoprotection as low as 0.1 μM, and it almost completely
prevented PC12 cells from being damaged by glutamate at 10
μM.
Longan Cerebroside II (11) Inhibits Apoptosis in
Glutamate-Treated PC12 Cells. As cell death, apoptosis
plays an important role in both acute and chronic neurological
27−29
diseases.
To examine whether longan cerebroside II (11)
might inhibit the apoptosis mediated by glutamate, an
annexinV-FITC/PI double staining method was used to detect
E
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the apoptotic rate of PC12 cells by flow cytometry. As shown in
Figure 3A and B, compared with the untreated control group,
the cells treated with glutamate showed an increase in the
number of total apoptotic cells (2.10% vs 46.6%). However, the
apoptosis of PC12 cells induced by glutamate was markedly
reduced after longan cerebroside II (11) treatment (Figure
evaluated, longan cerebroside II (11), having a long lipid acyl
chain, showed the most potent cytoprotection against
glutamate injury. Flow cytometry results indicated that longan
cerebroside II (11) protected PC12 cells from glutamate injury
due to its concentration-dependent antiapoptotic effects.
Furthermore, longan cerebroside II (11) suppressed gluta-
mate-induced apoptosis by regulating the caspase cascade and
the Bax/Bcl-2 pathway in a concentration-dependent manner.
3
C−E). With the increased longan cerebroside II (11)
concentration (0.1, 1, and 10 μM), the apoptotic rate of
PC12 cells decreased proportionally (32.42%, 23.58%, and
EXPERIMENTAL SECTION
3.13%, respectively). The influence of longan cerebroside II
■
(11) on the cell cycle treated with or without 12.5 mM
General Experimental Procedures. Optical rotations were
measured on a Gyromat-Hp digital automatic polarimeter. UV spectra
were recorded on a Shimadzu UV-2501 spectrophotometer. IR spectra
were determined on a Bruker Tensor 27 infrared spectrophotometer
with KBr disks. NMR spectra were acquired on a Bruker DRX 600
NMR spectrometer. ESIMS were measured on an Agilent 6410B
Triple Quad LC-ESIMS/MS spectrometer. HRESIMS were recorded
on an Agilent 6520 Accurate-Mass Q-TOF LC/MS. HPLC was
performed on a Shimadzu LC-20AT equipped with an SPD-M20A
PDA detector, using a Phenomenex C18 column (250 × 10 mm, 5 μm)
and a Phenomenex C18 column (250 × 10 mm, 2.6 μm). Gas
chromatographic (GC) analysis was performed on an Agilent HP5890
gas chromatograph equipped with a 30QC2/AC-5 quartz capillary
column (30 × 0.32 mm, 0.25 μm) and flame ionization detection.
Silica gel (200−300 mesh) was obtained from Qingdao Marine
Chemical Factory. Sephadex LH-20 was purchased from Pharmacia
Biotech. All other chemicals were of analytical grade. TLC analyses
were carried out on silica gel 60 F254 plates.
Plant Material. The rhizomes of T. giganteum were collected from
Bozhou, Anhui Province, People’s Republic of China, in October 2011
and were identified by Prof. Li-Na Chen of Nanjing Medical
University. A voucher specimen (TG-2011-01) has been deposited
in the herbarium of Nanjing Medical University.
Extraction and Isolation. The air-dried and powdered rhizomes
of T. giganteum (30 kg) were extracted with 75% EtOH (90 L × 3) at
0 °C, filtered, and concentrated to give an EtOH extract (1.1 kg),
glutamate was also investigated by flow cytometry measure-
ment. However, the results showed that longan cerebroside II
(11) had no effect on the cell cycle (Figure S21, Supporting
Information). These results indicate that the cytoprotective
activity of longan cerebroside II (11) may be due to its
antiapoptosis effect.
Longan Cerebroside II (11) Suppresses Glutamate-
Induced Apoptosis by Regulating the Caspase-9,
Caspase-3, and Bax/Bcl-2 Signaling Pathways. The
caspase and Bcl-2 families are important regulators of apoptotic
cell death in experimental models of excitotoxic, ischemic, and
traumatic brain injury. As the key members of the caspase
family, caspase-9 and caspase-3 play a dominant role in the
30,31
activation of the caspase cascade.
mechanism of the antiapoptosis effect of longan cerebroside II
11), the expression of these two caspases was analyzed by
To explore the molecular
(
Western blotting. As shown in Figure 4, the expression levels of
caspase-9 (Figure 4A) and caspase-3 (Figure 4B) increased
greatly when the PC12 cells were exposed to 12.5 mM
glutamate. In contrast, the levels of caspase-9 and caspase-3 in
PC12 cells significantly decreased in a concentration-dependent
manner after being treated with 0.1, 1, and 10 μM longan
cerebroside II (11) (Figure 4A and B). These results suggest
that longan cerebroside II (11) can protect PC12 cells from
glutamate-induced apoptosis by inhibiting the expression levels
of caspase-9 and caspase-3.
7
which was then suspended in water (3 L) and partitioned successively
with petroleum ether (60−90 °C), ethyl acetate (EtOAc), and n-
butanol (n-BuOH) (3 L × 3) to give three fractions (Fr.A to Fr.C).
The EtOAc portion (Fr.B, 50 g) was separated by silica gel column
chromatography (CC) and eluted with a CH
system (100:0−0:100) to afford eight fractions (Fr.B1 to Fr.B8). Fr.B3
7.5 g) was subjected to silica gel CC eluted with a gradient of
The Bcl-2 family proteins represent the central regulator of
cytochrome c release and caspase activation. Activation of the
Bcl-2 family member Bax results in the release of cytochrome c
from the intermembrane space of the mitochondria into the
2 2
Cl −MeOH gradient
(
CH Cl −MeOH (20:1−5:1) to give three subfractions (Fr.B3-1 to
2
2
3
2−36
Fr.B3-3). Subfraction Fr.B3-2 (1.5 g) was chromatographed on silica
cytosol, while Bcl-2 blocks cytochrome c release.
To
gel using CH Cl −MeOH (9:1) as eluent to obtain three subfractions
2
2
address the effect of longan cerebroside II (11) on the signaling
cascade of caspase activation during glutamate-induced
apoptosis in PC12 cells, the involvement of the Bcl-2 family
and the release of cytochrome c in the cytosol were measured
by Western blotting. In the present study, the Bcl-2 protein
levels markedly decreased, whereas the Bax protein levels and
the cytochrome c levels in the cytosol significantly increased in
glutamate-treated PC12 cells when compared with the
untreated control group (Figure 4C and D). However, the
Bcl-2 protein levels gradually increased with exposure to
increasing concentrations of longan cerebroside II (11), and
the Bax protein and the cytochrome c levels in the cytosol
continuously decreased (Figure 4C and D). These results
indicate that longan cerebroside II (11) effectively suppresses
glutamate-induced apoptosis by upregulating the expression of
the Bcl-2 protein, while downregulating the expression of the
Bax protein and decreasing the content of cytosolic cytochrome
c in a concentration-dependent manner.
(
Fr.B3-2-1 to Fr.B3-2-3). Subfraction Fr.B3-2-1 (0.4 g) was separated
using a Sephadex LH-20 column (CHCl −MeOH, 1:1) and then
3
purified by semipreparative HPLC eluted with MeOH−H O gradient
2
mixtures (90:10−100:0) to furnish seven subfractions (Fr.B3-2-1-1 to
Fr.B3-2-1-7). Compounds 3 (15 mg) and 4 (13 mg) were purified
from Fr.B3-2-1-1 by HPLC (MeOH−H O, 88:12). Similarly,
compounds 5 (12 mg) and 6 (10 mg) were isolated from Fr.B3-2-1-
2
2
by HPLC (MeOH−H O, 90:10); compounds 7 (13 mg) and 8 (11
2
mg) were separated from Fr.B3-2-1-3 by HPLC (MeOH−H O, 92:8);
2
compounds 1 (14 mg) and 2 (13 mg) were isolated from Fr.B3-2-1-4
by HPLC (MeOH−H O, 94:6); compounds 9 (11 mg) and 10 (10
2
mg) were purified from Fr.B3-2-1-6 by HPLC (MeOH−H O, 97:3);
2
compounds 11 (9 mg) and 12 (7 mg) were separated from Fr.B3-2-1-
7 by HPLC (MeOH−H O, 98:2), and compound 13 (9 mg) was
2
separated from Fr.B3-2-1-5 by HPLC (MeOH−H O, 95:5).
2
2
3
Typhonoside B (1): white, amorphous powder; [α] +4.0 (c 0.20,
D
CHCl −MeOH, 1:1); IR (KBr) ν 3361, 1650, 1540, 1090, 720
3
max
−
1
1
13
cm ; UV (MeOH) λmax (log ε) 205 (4.05) nm; H and C NMR
+
data, see Table 1; HRESIMS m/z 806.6116 [M + Na] (calcd for
C H NO Na, 806.6138).
45
85
9
In conclusion, 13 cerebrosides were isolated from the ethyl
acetate extract of T. giganteum rhizomes, and two of these (1
and 2) are reported for the first time. Of the compounds
23
Typhonoside C (2): white, amorphous powder; [α]D +3.6 (c 0.20,
CHCl −MeOH, 1:1); IR (KBr) ν 3361, 1650, 1540, 1090, 720
3
max
−
1
1
13
cm ; UV (MeOH) λmax (log ε) 205 (3.96) nm; H and C NMR
F
DOI: 10.1021/acs.jnatprod.6b00954
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
+
data, see Table 1; HRESIMS m/z 806.6125 [M + Na] (calcd for
enhanced chemiluminescence (ECL) reagent. The intensities of bands
were quantified using the ImageJ software (NIH).
C H NO Na, 806.6123).
4
5
85
9
Acidic Hydrolysis of Compound 1. As reported in previous
Statistical Analysis. Data were expressed as means ± standard
deviations (SD). Statistical analyses were done by using the one-way
ANOVA test and Fisher’s least significant difference t-test to compare
the different groups. A probability (P value) of less than 0.05 was
considered to be statistically significant.
37,38
literature,
compound 1 (5 mg) was hydrolyzed with 2 M HCl−
dioxane (1:1, 4 mL) under reflux at 80 °C for 6 h. The reaction
mixture was extracted with CHCl (2 mL × 3). The aqueous layer was
3
neutralized with 2 M NaOH and then dried to obtain one or more
monosaccharides. Then the dry powder was dissolved in pyridine (2
mL), L-cysteine methyl ester hydrochloride (1.5 mg) was added, and
the mixture was heated at 60 °C for 1 h. Next, trimethylsilylimidazole
ASSOCIATED CONTENT
■
*
S
Supporting Information
(
1.5 mL) was added to the reaction mixture in ice water and kept at 60
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00954.
°
C for 30 min. The mixture was directly subjected to GC analysis
under the following conditions: column temperature, 185−285 °C;
programmed increase, 3 °C/min; carrier gas, N (1 mL/min); injector
and detector temperature, 250 °C; injection volume, 4 μL; and split
ratio, 1:50. The configuration of D-glucose for compound 1 was
determined by comparing the retentions time with the derivative of an
authentic sample (D-glucose: 18.06 min).
2
UV, IR, and NMR spectra, ESIMS/MS, and HRESIMS
of typhonosides B (1) and C (2) and information on
antibodies used in the Western blotting experiments
(PDF)
Biological Assay Materials. Glutamate, DMSO, and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin,
Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine
serum (FBS) were obtained from Gibco-BRL (Grand Island, NY,
USA). Annexin V-FITC/PI was purchased from BD Biosciences (San
Diego, CA, USA). Compounds 1−13 were dissolved in DMSO at a
AUTHOR INFORMATION
Corresponding Authors
*E-mail (S.-H. Du): shuhudu@njmu.edu.cn. Tel/Fax: +86-25-
6868476.
■
8
1
000-fold final concentration and kept at −20 °C without light. A
*E-mail (A.-X. Zhang): aixia.zhang@njmu.edu.cn. Tel/Fax:
+86-25-86868480.
stock solution of the glutamate was prepared as a culture medium with
a 100-fold final concentration.
ORCID
Cell Culture and Treatment. PC12 cells were purchased from the
Cell Bank of Shanghai Institute of Biochemistry and Cell Biology
Shu-Hu Du: 0000-0002-4819-0822
(
Chinese Academy of Sciences, Shanghai, People’s Republic of China).
Author Contributions
The cells were maintained in DMEM supplemented with 10% (v/v)
⊥
Y. Jin and J.-T. Fan contributed equally to this work.
FBS and incubated at 37 °C under 5% CO . To determine the proper
2
concentrations to be used in this glutamate-induced PC12 cell injury
model, cells were seeded in 96-well plates (10 000 cells/well). After
culturing for 24 h, PC12 cells were treated with various concentrations
of glutamate (0, 10, 12.5, 15, 17.5, and 20 mM) for 24 h. To study the
protective effects of compounds 1−13 on glutamate-induced death in
PC12 cells, cells were seeded in 96-well plates (10 000 cells/well).
After being cultured for 24 h, PC12 cells were treated with medium
containing different concentrations of compounds (2.5, 5, 10, and 20
μM) and 12.5 mM glutamate for 24 h. The control group was not
treated with compounds 1−13 and glutamate.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by the National Natural Science
Foundation of China (Nos. 21275075, 31500278, and
61605084) and the Priority Academic Program Development
of Jiangsu Higher Education Institutions (No. 13KJB360002).
MTT Assay. After 24 h of incubation, 20 μL of MTT (5 mg/mL)
was added to each well by additional incubation for 4 h at 37 °C, and
the formazan crystals were dissolved in DMSO (150 μL). Absorbance
of the formazan solution was measured with a microplate reader at 570
nm. Cell survival was expressed as a relative percentage of the
untreated control.
Flow Cytometry Analysis. Briefly, the treated cells were digested
and collected by trypsin without EDTA. The collected cells were
washed with cold phosphate buffer solution (PBS, pH 7.4) twice and
suspended in 100 μL of 1× binding buffer. Next, 5 μL of FITC-
annexin V and 5 μL of propidium iodide were added. The suspension
was mixed and incubated for 15 min at room temperature in the dark,
and then an additional 400 μL of 1× binding buffer was added to each
suspension. Finally, analysis of apoptosis was performed using a flow
cytometer (BD FACSCalibur, San Jose, CA, USA).
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