Accelerative effects of carbazole-type alkaloids from Murraya koenigii on neurite outgrowth and their derivative’s in vivo study for spatial memory

Article abstract of DOI:10.1007/s11418-020-01388-8

Murraya koenigii is a medicinal plant that contains several carbazole-type alkaloids as its characteristic constituents. Blood–brain barrier permeable constituents of M. koenigii accelerated neurite outgrowth in PC-12 cells. Nine compounds were isolated f

Full text of DOI:10.1007/s11418-020-01388-8

Journal of Natural Medicines
https://doi.org/10.1007/s11418-020-01388-8
ORIGINAL PAPER
Accelerative efects of carbazole‑type alkaloids from Murraya koenigii
on neurite outgrowth and their derivative’s in vivo study for spatial
memory
Mamiko Yano1 · Souichi Nakashima1 · Shiori Kasa1 · Seikou Nakamura1 · Kaneyasu Nishimura2 · Yoshimi Oda1,3
Kazuyuki Takata2 · Hisashi Matsuda1
·
Received: 17 December 2019 / Accepted: 7 January 2020
© The Japanese Society of Pharmacognosy 2020
Abstract
Murraya koenigii is a medicinal plant that contains several carbazole-type alkaloids as its characteristic constituents. Blood–
brain barrier permeable constituents of M. koenigii accelerated neurite outgrowth in PC-12 cells. Nine compounds were iso-
lated from M. koenigii and their efects on neurite outgrowth were examined. Murrayamine-E (8) at 10 μM showed signifcant
efect. Focusing on the carbazole skeleton, we synthesized derivatives to attenuate cytotoxicity. 9-Benzyl-9H-carbazol-4-ol
(15) exhibited strong neurite outgrowth accelerative efect. In addition, the novel object recognition test and the Morris
water maze test were performed to evaluate memory improvement of 15 in APdE9 mice. Compound 15 tended to improve
spatial memory in the Morris water maze test. These results suggest that carbazole derivative 15 would be a seed compound
for Alzheimer’s disease drug.
Keywords Carbazole · Alkaloid · Murraya koenigii · PC-12 · Neurite outgrowth · Alzheimer’s disease
Introduction
treat symptoms. To discover drugs for fundamental treat-
ment, we focused on neurogenesis, which is diferentiation
of neural stem cell into neuron. This occurs in not only fetal
and infant brains but also in adult brain [1, 2]. In AD brains,
neuronal death has resulted in characteristic symptoms. If
neurogenesis is accelerated, lost brain function would be
complemented and AD would be treatable [3].
AD is a neurodegenerative disease characterized by cogni-
tive impairment. The rapid increase in the number of AD
patients as a result of the aging population has become a
critical problem. Recently, donepezil, an acetylcholinester-
ase inhibitor, and memantine, an N-methyl-d-aspartic acid
receptor antagonist, have been used to treat AD patients.
However, these drugs only retard AD progression. AD
patients need curable therapies and drugs, not drugs that
The PC-12 cell line is derived from a pheochromocy-
toma of rat adrenal medulla. Stimulation of nerve growth
factor (NGF) on PC-12 cells leads neuron-like morphologi-
cal change and getting sensitivity for dopamine. With these
characteristics, PC-12 cells have been used as the model
of neurite cells. In this study, PC-12 cells were used as the
model of neural stem cells in the frst screening.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-020-01388-8) contains
supplementary material, which is available to authorized users.
On the other hand, alkaloids account for about 70% of
the drugs in the world, and 89.6% of central nervous system
(CNS) agents in Japan are alkaloids. Under such circum-
stances, in this study, we focused on alkaloids as seed for
CNS agents. We have been studying the neurite outgrowth
accelerative efects of plants containing alkaloids. In the
past, we investigated the neurite outgrowth accelerative
efect of the fower of Nelumbo nucifera, which contains
aporphine-type alkaloids [5].
* Hisashi Matsuda
matsuda@mb.kyoto-phu.ac.jp
1
Department of Pharmacognosy, Kyoto Pharmaceutical
University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
2
Division of Integrated Pharmaceutical Sciences, Kyoto
Pharmaceutical University, Misasagi, Yamashina-ku,
Kyoto 607-8414, Japan
3
N.T.H Co., Ltd., 1-8-11 4F Sky-ebisu Bldg., Ebisu,
Shibuya-ku, Tokyo 150-0013, Japan
Vol.:(0123456789)
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Journal of Natural Medicines
Carbazole-type alkaloids have many biological activi-
ties, including anti-infammatory, antitumor [5], antibiotic,
anti-oxidant, neuronal cell protective, and anti-prion activi-
ties [6]. In particular, the anti-oxidant and neuronal cell-
protective efects are valuable for treating neurodegenerative
disorders, Alzheimer’s disease (AD), etc.[7] and carbazole
derivatives were reported to be promising agents for AD
[8, 9]. A dibromo-aminopropyl carbazole was reported to
show proneurogenic and neuroprotective efect [10]. How-
ever, there are few reports of the efects of carbazoles with-
out bromo groups on neurogenesis [9, 11]. Therefore, in
this study, we focused on carbazole as its basic chemical
structure.
Reagents for bioassay
RPMI-1640, EMEM, and Ham’s F12 were purchased from
Sigma-Aldrich (St. Louis, MO, USA); FBS (fetal bovine
serum), HS (horse serum), penicillin, and streptomycin
were purchased from Thermo Fisher Scientifc (Waltham,
MA, USA); donepezil hydrochloride was purchased from
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); and
other chemicals were purchased from FUJIFILM Wako Pure
Chemical Co., Ltd. (Osaka, Japan).
Cell culture
Murraya koenigii is a medicinal plant belonging to fam-
ily Rutaceae and is distributed in tropical and subtropical
regions including India, Nepal, Malaysia, and Sri Lanka.
The leaves are called “curry leaf” and have been used as a
spice around the world. The plant has been used tradition-
ally as stomachic and analgesic, and for the treatment of
diarrhea and anxiety disorder. Previous studies reported that
it has anti-oxidant [12] and anti-bacterial efects [13]. The
plant contains carbazole-type alkaloids as characteristic con-
stituents [14] as well as sesquiterpenes [15]. In our previous
study, we found that some alkaloids from the leaves exert an
inhibitory efect on melanogenesis [16].
PC-12 cells (JCRB0733) were obtained from JCRB Cell
Bank (Japanese Collection of Research Bioresources Cell
Bank; Osaka, Japan). The cells were cultured in RPMI-1640
supplemented with 10% FBS, 5% HS, 4 mM l-glutamine,
penicillin (100 units/mL), and streptomycin (100 µg/mL)
at 37 °C in 5% CO2/air. The cells were used for subsequent
bioassays as required.
Synthesis of 9‑benzyl‑9H‑carbazole (13)
and 9‑benzyl‑9H‑carbazol‑4‑ol (15)
In this study, to search for seed compounds for AD defni-
tive therapy from the perspective of neurogenesis, we eval-
uated the accelerative efects of carbazole-type alkaloids
including compounds from M. koenigii and synthesized
derivatives on neurite outgrowth. In addition, in vivo study
using AD model mice for spatial memory was performed for
promising compound.
A solution of a 4-hydroxy-9H-carbazole (11, 200 mg,
1.09 mmol) in DMF (5.0 mL) was treated with TBSCl
(TBDMSCl, 414 mg, 2.75 mmol) in the presence of imi-
dazole (187 mg, 2.75 mmol) and the solution was stirred
under N2 at room temperature for 12 h. The reaction mix-
ture was quenched with water and extracted three times with
EtOAc. The EtOAc layer was washed with brine, then dried
over Na2SO4 powder. The mixture was fltered and the sol-
vent was removed under reduced pressure. The residue was
purifed by silica gel column chromatoraphy (CC) [n-hex-
ane–EtOAc (5:1, v/v)] to give 4-[(tert-butyldimethylsilyl)
oxy]-9H-carbazole (12, 325 mg, quant.). Next, a solution of
the obtained compound 12 (100 mg, 0.34 mmol)] in THF
(4.0 mL) was treated with sodium hydride (60%, disper-
sion in parafn liquid, 20 mg, 0.51 mmol), and the mixture
was stirred for 30 min at 0 °C. Benzyl bromide (0.06 mL,
0.51 mmol) was added to the solution and the mixture was
stirred under N2 at room temperature for 2 h. The reaction
mixture was quenched with ice-water and extracted four
times with EtOAc. The EtOAc layer was washed with brine,
then dried over Na2SO4 powder. The mixture was fltered
and the solvent was removed under reduced pressure. The
residue was purifed by silica gel CC [n-hexane–EtOAc
(30:1, v/v)] to give 9-benzyl-4-[(tert-butyldimethylsilyl)
oxy]-9H-carbazole (14, 111 mg, 85%). Next, a solution of
the obtained compound 14 (30 mg, 0.077 mmol) in THF
(1.0 mL) was treated with tetrabutylammonium fuoride
Materials and methods
Plant material
The methanolic extracts of the leaves of M. koenigii and
its constituents (1–9) were obtained as described previously
[16].
General experimental procedures
For EIMS and HREIMS measurements, a JEOL JMS-
GCMATE mass spectrometer was used; for measurements of
1H-and 13C-NMR spectra, JEOL JNM-ECA600 (600 MHz)
and JNM-ECS400 (400 MHz) spectrometers were employed
with tetramethylsilane as an internal standard. The follow-
ing conditions were used in chromatography: normal-phase
silica gel column chromatography (CC), Silica gel BW-200
(Fuji Silysia Chemical, Ltd., 150–350 mesh); TLC, pre-
coated TLC plates with Silica gel 60F254 (Merck, 0.25 mm).
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Journal of Natural Medicines
(TBAF, ca. 1 mol/L in THF, 0.31 mL, 0.31 mmol) and the
solution was stirred under N2 at 0 °C for 30 min. The reaction
mixture was poured into water and the whole was extracted
three times with EtOAc. The EtOAc layer was washed with
brine, then dried over Na2SO4 powder. The mixture was fl-
tered and the solvent was removed under reduced pressure.
The residue was purifed by silica gel CC [n-hexane–EtOAc
(10:1, v/v)] to give 9-benzyl-9H-carbazol-4-ol (15, 20 mg,
94%). 9-benzyl-9H-carbazole (13, 277 mg, 90%) was syn-
thesized from carbazole (10, 200 mg, 1.20 mmol) accord-
ing to the same procedure for the synthesis of compound
14. The obtained compounds 14 and 15 were identifed by
comparison of their physical data (1H-NMR, 13C-NMR and
MS) with reference data [17, 18].
Starting at 6–7 months of age, groups of female APdE9
mice were intraperitoneally administered 9-benzyl-9H-car-
bazol-4-ol (15) at the dose of 10 mg/mL/kg (suspended in
25 mg/mL CMC sodium in saline, n=5 per group) or saline
(10 mL/kg, n = 5) once daily for 30 days. Groups of age-
matched nontransgenic littermates received saline (10 mL/
kg, n=5).
Mice were housed in a 12-h light–dark cycle in a temper-
ature-controlled (22 1 °C) environment with free access
to food and water. The decrease in body weight was<10%
compared with the frst administration.
Novel object recognition (NOR) test
The novel object recognition test was performed after
administering test compound for 1 month, as described
previously [20]. One mouse was placed in an open-feld
(40×40×30 cm [length×width×height]) under 20 lx light
for 10 min. After 30 min of frst break, two indistinguishable
objects (A and B) were placed in the arena and the mouse
was allowed to explore for 10 min.
Neurite outgrowth in PC‑12
The screening assay for PC-12 neurite outgrowth was per-
formed as described previously [5, 19] with slight modif-
cations. In brief, PC-12 cells (1.0×105 cells/400 μL/well)
were seeded into 24-well type I collagen-coated multiplates
(AGC Techno Glass Co., Ltd., Tokyo, Japan). After culture
for 24 h, a test compound and nerve growth factor (NGF,
2 ng/mL at fnal concentration) were added and further incu-
bation was carried out for 48 h. Neurite outgrowth was quan-
tifed by microscopy and expressed as the proportion of cells
bearing neurite processes > 1 cell diameter in length. The
test compound was dissolved in DMSO, and fnal DMSO
concentration in the medium was 0.1%. Neurite outgrowth
positive ratio of PC-12 cells was calculated using the fol-
lowing formula:
After 30 min of the second break, one of the objects were
replaced by a novel object (C) and the mouse was allowed to
explore for 10 min. The behavior of the mouse was recorded
by the video-tracking system Etho Vision XT 11.5 (Noldus
Information Technology). The positions of the two objects
remained consistent throughout the paradigm. The object
preference index, which was used as a measure of nonspatial
working memory function, was calculated as follows:
Object preference index (%) = B or C∕(A + B or C) × 100.
A means the number of nose touches during exploration
time (10 min) to familiar object (A), B means to familiar
object (B), and C means to novel object (C).
Neurite outgrowth positive ratio = B∕A.
Where A and B indicate the proportion of cells bearing
neurite processes in vehicle- and test-compound-treated
groups, respectively.
Morris water maze (MWM) test
The MWM test was performed after the NOR test, as
described previously [21]. A circular pool (diameter
100 cm, height 30 cm, painted white) was flled with water
(23 1 °C) and a transparent platform (diameter 10 cm)
was submerged 1 cm beneath the water surface. The water
was made white by adding nontoxic water-based pigment.
As distal spatial visual cues, pictures of diferently shaped
objects were placed along the pool wall. A video camera was
installed on the ceiling at the center of the pool and attached
to Etho Vision to monitor swim paths and latencies. Acqui-
sition trials (four trials per block per day for 5 days) were
initiated by placing the mouse in the pool facing the wall
at randomly chosen start positions and the time required to
fnd the submerged platform was recorded. Each trial lasted
a maximum of 60 s until the mouse found the platform. If the
Blood–brain barrier (BBB) permeability
We examined the permeability of the constituents with a
BBB Kit™ (RBT-24H, PharmaCo-Cell Co., Ltd., Nagasaki,
Japan), an in vitro model of BBB. The examination was car-
ried out as described previously [5].
Animal experiments
All mice were provided by the Bioscience Research Center,
Kyoto Pharmaceutical University. All animal experiments
were approved by the Committee for Animal Research
at Kyoto Pharmaceutical University (Protocol No.:
PCOG-18–036).
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Journal of Natural Medicines
mouse failed to fnd the platform within 60 s, it was guided
to the platform and placed on it for 15 s. After the comple-
tion of each block of four trials, the mouse was dried and
returned to its home cage. In the trial phase, escape latency,
swimming speed*** and path length were recorded. On day
6, the platform was removed from the pool and a 60-s probe
test was performed. In the probe test, the mouse was placed
in the pool facing the wall at the position opposite to the
removed platform and the frequency of crossing the place
where the platform was formerly set recorded.
of the nose touch frequency between wild-type and APdE9
mice injected with saline and 15. In the MWM test, data
are presented as means S.E.M. Statistical signifcance was
determined using one-sided t test, with genotype and test
day of acquisition trials or treatment group and test day of
acquisition trials as sources of variation. In probe tests, the
frequency of crossing the platform were compared between
wild-type and APdE9 mice injected with saline and 15 with
one-sided t test.
Statistical analysis
Results
In the PC-12 assay, data are expressed as the total number
of cells. Fisher’s exact test was used to determine the sta-
tistical signifcance of the diferences between the control
group and the test compound treated groups. p<0.05 was
considered statistically signifcant. In the NOR test, one-
sided t test was used to determine statistical signifcance
First, we examined the efect of the methanolic (MeOH)
extract of M. koenigii on neurite outgrowth in PC-12 cells.
The results are shown in Table 1. The MeOH extract tended
to have a signifcant efect at 1 μg/mL (p=0.092) and was
thus inferred to contain compounds that accelerate neurite
outgrowth.
One of the important considerations for CNS drugs is
BBB permeability. In this study, BBB permeability was
examined in an in vitro BBB model composed of brain
microvascular endothelial cells, pericytes and astrocytes. As
a result, BBB-permeable constituents in the MeOH extract
tended to accelerate neurite outgrowth in PC-12 cells at the
concentration of 100 μg/mL on the blood side (4.8% posi-
tive, p=0.11).
Table 1 Neurite outgrowth accelerative efects of MeOH extract of
M. koenigii leaves in PC-12 cells
Sample
Conc. (μg/mL) and ratio of cell with neurite outgrowth
(%)
0
0.1
1
10
MeOH ext 2.8 (20/728) 3.1 (29/938) 4.2 (26/616) 2.8 (25/898)
We examined the neurite outgrowth accelerative efects
of the alkaloids (Fig. 1) from M. koenigii and derivatives
Values mean “ratio of cells with neurite outgrowth (number of cells
with neurite outgrowth/total cells)” (n=4)
Fig. 1 Chemical structures of compounds from M. koenigii leaves
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Journal of Natural Medicines
Fig. 2 Synthesis scheme of
carbazole derivatives
Table 2 Neurite outgrowth
accelerative efects of
Sample
Conc. (μM) and ratio of cell with neurite outgrowth (%)
compounds from M. koenigii
leaves in PC-12 cells
0
0.1
1
10
Mahanimbicine (1)
Mahanimbine (2)
3.9 (18/464)
5.0 (20/410)
4.7 (28/601)
4.1 (22/542)
5.2 (24/466)
3.1 (17/556)
3.7 (18/485)
4.1 (22/538)
3.7 (23/615)
4.8 (29/604)
2.2 (14/617)
5.8 (25/428)
5.5 (38/695)
4.4 (21/482)
5.6 (37/666)
3.5 (19/537)
4.3 (21/494)
4.2 (24/573)
3.7 (20/546)
6.6 (34/513)
3.8 (21/560)
6.9 (28/407)
5.2 (26/496)
4.1 (23/564)
5.0 (25/495)
4.2 (22/530)
6.0 (32/534)
5.2 (27/519)
4.8 (26/546)
7.1 (42/588)
4.9 (25/514)
7.6 (32/422)
6.6 (38/576)
4.8 (25/525)
5.0 (27/536)
4.0 (26/650)
3.2 (19/593)
7.2 (35/487)*
5.5 (31/565)
7.5 (55/736)*
Murrayamine-B (3)
O-Methylmahanine (4)
Pyrayafoline D (5)
Eustifoline-C (6)
Euchrestine-B (7)
Murrayamine-E (8)
Koenimbine (9)
Donepezil (positive control)
Values mean “ratio of cells with neurite outgrowth (number of cells with neurite outgrowth/total cells)”
(n=4)
Signifcantly diferent from control group, *p<0.05
Table 3 Neurite outgrowth accelerative efects of carbazole and its
benzyl group linked to the nitrogen atom of the hetero-
cycle. It was reported that the benzyl group attenuated
cytotoxicity induced by oxidative stress [22]. As shown
in Table 3, 15 at 1 μM accelerated neurite outgrowth
in PC-12 cells (p = 0.079). In addition, because 13 did
not accelerate neurite outgrowth, a hydroxy group at the
four position might be needed to accelerate the neurite
outgrowth.
derivatives in PC-12 cells
Sample
Conc. (μM) and ratio of cell with neurite
outgrowth (%)
0
0.1
1
Carbazole (10)
2.6 (14/532) 1.5 (9/602) 2.5 (14/568)
3.3 (19/582) 3.1 (17/549) 2.4 (13/534)
3.9 (22/565) 3.0 (14/460) 3.2 (16/502)
4-Hydroxycarbazole (11)
9-Benzyl-9H-carbazole
Compound 15 tends to accelerate neurite outgrowth and
the yield was sufcient. To determine whether 15 improves
object cognitive ability, the NOR test was carried out. The
frequency of nose touches of the APdE9 group slightly
decreased the scores compared to that of WT. APdE9 trans-
genic mice administered 15 showed a subtle increase in fre-
quency (p=0.08, Fig. 3).
(13)
9-Benzyl-9H-carbazol-4-ol 3.1 (18/584) 3.5 (17/483) 4.9 (25/506)
(15)
Values mean “ratio of cells with neurite outgrowth (number of cells
with neurite outgrowth/total cells)” (n=4)
The MWM test was employed to evaluate the efect of 15
on spatial learning and memory. In the training phase, the
escape latency decreased with time and no diferences were
observed among the three groups (Fig. 4a). In the probe
test, the group of APdE9 transgenic mice administered 15
showed increased platform crossing frequency compared to
the group of APdE9 transgenic mice administered saline
(p=0.08, Fig. 4b). Thus, compound 15 tended to improve
spatial memory in APdE9 transgenic mice.
(Fig. 2). From the results in Table 2, murrayamine-E (8)
potentiated neurite outgrowth signifcantly at 10 μM. It
was considered that the functional group at the nitrogen
atom might be important for the neurite outgrowth accel-
erative efect as the prenyl group of 8 was related to the
efect. Carbazole (10) and 4-hydroxycarbazole (11) found
that they have no neurite outgrowth accelerative efects
(Table 3). Synthesized two derivatives, 9-benzyl-9H-car-
bazole (13) and 9-benzyl-9H-carbazol-4-ol (15) have a
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Journal of Natural Medicines
diseases. In this study, we focused on neurogenesis as the
target AD therapeutic agent.
In the frst screening, the neurite outgrowth accelera-
tive efect of M. koenigii was examined. Murrayamine-E
(8) showed the neurite outgrowth accelerative efect and the
functional group linked to the nitrogen atom of the hetero-
cycle might be related to the efect. Aminopropyl carba-
zole P7C3, which has been reported to induce neurogenesis
[10], also has a functional group at nitrogen atom. Next,
we synthesized carbazole derivatives (13 and 15). Com-
pound 15 showed the neurite outgrowth accelerative efect
and the hydroxy group at the 4-position might be needed to
generate the efect. Unfortunately, in SH-SY5Y cells, often
used as in vitro models in the research for neuronal func-
tion, 8 and 15 had no efect on neurite outgrowth (Table S1).
Kinds of the cell derivation and mechanisms on outgrowth
might be parts of the reasons why 8 and 15 showed difer-
ent efects. In addition, in human-induced pluripotent stem
cells (hiPSCs)-derived cortical neuroprogenitors, 15 showed
tendency of accelerative efect on increase of the expression
of doublecortin (DCX) and class III β-tubulin (TUBB3) in
mRNA level (Fig. S1). Our results suggested that 15 tended
to promote the neuronal maturation from neuroprogenitors
to immature neuron stage. Further studies are required to
assess the efects in dentate gyrus and on other types of
neurons because neurogenesis was reported to happen in
the region [1].
Fig. 3 Novel object recognition test. Nose touch frequency was used
as preference index in the novel object recognition test. Data are pre-
sented as means S.E.M. (n=4–5). Compound 15 was administered
at the dose of 10 mg/kg, i.p. a: p=0.08
Discussion
Alzheimer’s disease (AD) is one of the most urgent problems
in the world, but its cause has been well known. AD drugs
are limited, and new drug development is going through dif-
fculties. Although most of the drug development has been
based on the amyloid β hypothesis, it was reported that the
structure of amyloid β in AD brain was difcult to reproduce
in laboratories [23] and this might be the reason for the dif-
fculties in AD drug development. It was reported that neu-
rogenesis occurs in adult human brain [2] and neurogenesis
could be one of the strategies for treating neurodegenerative
The derivatives of indoline, which is a component of car-
bazole, have been reported to show efects on neurosphere
formation and BBB permeability [24]. This partly agrees
with the results of this study, and it was considered that the
indoline structure could be needed for BBB permeability.
In addition, WT mice and APPSwe/PS1dE9 (APdE9)
transgenic mice were used. APdE9 transgenic mice, which
carry both human APP with the Swedish mutation and
the presenilin-1 A246E mutation and have been shown to
Fig. 4 Morris water maze test.
a Escape latency was evalu-
ated in the training phase in the
MWM test. Data are expressed
as means S.E.M. (n=4–5).
Compound 15 was administered
at the dose of 10 mg/kg, i.p. b
Platform crossing frequency
was evaluated in the probe test
in the MWM test. Data are
expressed as means S.E.M.
(n=4–5). a: p=0.08 Compound
15 was administered at the dose
of 10 mg/kg, i.p
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Journal of Natural Medicines
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overproduction, have been widely used. In these transgenic
mice, the levels of p-Tau and amyloid β and its deposits
(senile plaques) in hippocampus and cortical cortex increase
with age, similar to AD patients [25]. Therefore, the APdE9
mouse model is a good model for evaluating seed com-
pounds for AD drugs. For comparison, donepezil (1 mg/
kg, i.p.) was reported to ameliorate cognitive impairment,
by in vivo study [26]. In this study, the NOR test and the
MWM test were performed, which are widely used to inves-
tigate mouse behavior [27, 28]. Memory and learning ability
are evaluated in the NOR test and spatial learning ability is
evaluated in the MWM test.
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memory but had no efect on short-term memory. In con-
clusion, carbazole-type alkaloids from M. koenigii and their
derivatives accelerated neurite outgrowth in PC-12 cells.
Carbazole derivative 15 tended to improve spatial memory
and thus could be a seed compound for AD therapeutic
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