O R I G I N A L R E S E A R C H

Skimmianine, a furoquinoline alkaloid from Zanthoxylum nitidum
as a potential acetylcholinesterase inhibitor

Zhong-duo Yang

•

Dong-bo Zhang

•

Jin Ren

•

Ming-jun Yang

Received: 15 July 2010 / Accepted: 19 January 2011 / Published online: 4 February 2011
Ó Springer Science+Business Media, LLC 2011

Abstract

Skimmianine (1), a newly discovered strong

acetylcholinesterase (AChE) inhibitor, along with nine
weakly or no active compounds, toddalolactone (2), dictam-
nine (3), c-fagarine (4), magnolone (5), (-)-(S)-edulinine (6),
zanthodioline (7), edulitine (8), 5,6,7-trimethoxycoumarin
(9), and haplopine (10) have been isolated from Zanthoxylum
nitidum (Z. nitidum). Skimmianine (1) inhibited 50% of
AChE activity at the concentrations of 8.6 ± 0.7 lg/ml
when the IC

50

value of Physostigmine as a standard was

0.013 ± 0.002 lg/ml. Antiacetylcholinesterase activity of
skimmianine (1) was also supported by TLC bioautographic
assay. The structure activity relationship on the anti-acet-
ylcholinesterase activity of the quinoline moiety is also
discussed in this article.

Keywords

Zanthoxylum nitidum

 Skimmianine 

Acetylcholinesterase inhibitor

 Quinoline alkaloid 

Alzheimer’s disease

Introduction

Alzheimer’s disease (AD) is the most common neurode-
generative disorder of this century and the most prevalent
cause of dementia with aging. Most treatment strategies
have been based on the cholinergic hypothesis which
postulates that AD is a result of decreased levels of the
neurotransmitter acetylcholine (ACh) in the cortex (Lahiri
et al.,

2002

). The cholinergic hypothesis claims that ace-

tylcholinesterase (AChE) plays a significant role in the

termination of nerve impulse transmission at the choliner-
gic synapses by rapid hydrolysis of ACh. AChE inhibitors
that can increase the cholinergic transmission by blocking
the degradation of ACh are therefore considered to be a
promising approach for the treatment of AD. In addition,
recent researches showed that AChE inhibitors not only
alleviate the cognitive defect of AD patients by elevating
ACh levels, but also act as disease modifying agents by
preventing the first step of AD, the assembly of b-amyloid
peptide into amyloid plaque (Munoz-Torrero and Camps

2006

; Pang et al.,

1996

). This discovery stimulated a great

interest in screening natural anti-AChE as lead compounds.
This study was carried out to search for useful leads which
could become new candidates for the development of
rational drug design against AD.

Zanthoxylum nitidum (Roxb.) DC (Rutaceae) have been

used for the treatment of toothache and gastrointestinal
diseases in traditional medicine (Zheng and Xing,

2009

;

Wan et al.,

2005

). In recent years, extracts of the herb have

been reported to show various pharmacological activities,
such as antiviral and antifungal activities (Yang and Chen

2008

), cytotoxic activity (Yang et al.,

2008

), anticancer

activity (Wang et al.,

2007

), anti-inflammatory activity (Hu

et al.,

2006a

), DNA topoisomerase-I inhibitory activity

(Fang et al.,

1993

), antispasmodic and analgesic activities

(Zeng et al.,

1982

). Previous reports have also showed that

extracts of Zanthoxylum species have cholinergic activities
relevant to the treatment of AD (Carpinella et al.,

2010

).

However, till now, no previous cholinesterase inhibitory
activity of this plant has been reported. As part of our
ongoing search for AChE inhibitors from Chinese medic-
inal plants, it was found that the total alkaloidal extract of
the root of Z. nitidum exhibited significant AChE inhibitory
activity with an IC

50

value of 10.7 ± 0.4 lg/ml when

physostigmine showed AChE inhibitory activity with an

Z. Yang (

&)  D. Zhang  J. Ren  M. Yang

School of Life Science and Engineering, Lanzhou University
of Technology, Lanzhou 730050, People’s Republic of China
e-mail: [e-mail]

123

Med Chem Res (2012) 21:722–725

DOI 10.1007/s00044-011-9581-9

MEDICINAL

CHEMISTRY

RESEARCH

IC

50

value of 0.013 ± 0.002 lg/ml. This study further

investigated the AChE inhibiting constituents of alkaloidal
extract from Z. nitidum.

Materials and methods

Plant materials

The roots of Z. nitidum (Roxb.) DC (Rutaceae) were pur-
chased from Huanghe Herb Market in Lanzhou and were
identified by Associate Professor Lin Yang who majored in
plant classification, School of Life Science and Engineer-
ing, Lanzhou University of Technology, Lanzhou, China.
The voucher specimen is deposited at the School of
Life Science and Engineering, Lanzhou University of
Technology.

Chemicals

Acetylcholinesterase (EC3.1.1.7, Sigma product no C2888),
Fast Blue B salt, acetylthiocholine iodide (ATCI), 5,5

0

-

dithiobis [2-nitrobenzoic acid] (DTNB), physostigmine and
Huperzine A were purchased from Sigma (St. Louis, MO,
USA). 1-Naphthyl acetate and 1-naphthol were obtained
from Sinopharm Chemical Reagent CO., Ltd (Shanghai,
China). MCI-GEL CHP 20P (75–150 lm) were from
Mitsubishi Chemical Holdings Corp. Silica gel GF254
plates and silica gel (200–300 mesh) were purchased from
Qingdao Haiyang Chemical Co., Ltd (Qingdao, China).

Extraction and isolation

The roots of Z. nitidum (10 kg) were powdered and
extracted with 95% ethanol (50 l 9 2) under reflux for 2 h
to obtain alcohol extract, which was evaporated to dryness
and then suspended in distilled water. The suspension was
adjusted to pH 2 by the addition of HCl (4 M). The acid
aqueous solution was filtered after one night. The filtrate
solution was basified to pH 11 with NaOH (1 M), and then
extracted with chloroform (15 l 9 2) to obtain the total
alkaloid extract (9.3 g). The alkaloidal extract was
repeatedly isolated and purified by the MCI-GEL and silica
gel column chromatography combined with microplate
assay for AChE activity to obtain ten compounds: 1
(380.6 mg), 2 (2.6 mg), 3 (16.2 mg), 4 (18.5 mg), 5
(5.6 mg), 6 (49.8 mg), 7 (18.4 mg), 8 (24.8 mg), 9
(5.6 mg), and 10 (27.9 mg).

Microplate assay for AChE inhibitory activity

The ten compounds were tested for AChE inhibiting
activities by a slightly modified Ellman’s method (Ellman

et al.,

1961

). Briefly, 140 ll of 0.1 M sodium phosphate

buffer (pH 8.0), 20 ll of 1 mg/ml sample solution, and
15 ll of 0.28 U/ml AChE were mixed and pre-incubated at
4

°C for 20 min. The reaction was started by adding 10 ll

of 0.01 M DTNB and 10 ll of 0.075 M ATCI, and the total
solution was incubated at 37

°C for 20 min. The optical

density was measured at 405 nm. Enzyme activity was
calculated by comparing the rate of reaction for the sam-
ples relative to that for the blank. The percentage of
inhibitory activity was calculated by subtracting the per-
centage of enzyme activity from 100. Physostigmine was
used as a positive control. The experiments were done in
triplicate.

TLC bioautographic assay for AChE inhibitory activity

The TLC bioautographic assay for AChE inhibitory
activity was modified by our group (Yang et al.,

2009

).

Firstly, 0.1 ll of 10 mg/ml compound (1) was spotted on a
silica gel TLC plate and migrated by 35:1 chloro-
form:methanol solution. The plate was dried absolutely
with a hair dryer. Then AChE (1 U/ml in buffer pH 7.8)
and 1-naphthyl acetate (1.5 mg/ml in 40% ethanol solu-
tion) were sprayed onto TLC plate subsequently. After
each solution was sprayed, TLC plate was blown quickly
with cold wind from a hair dryer until no free liquid was
found on it. Secondly, the plate was incubated at 37

°C for

20 min in a humid atmosphere. At last, Fast Blue B salt
(0.5 mg/ml in distilled water) was sprayed onto the plate.
Huperzine A was used as a positive control. White spots on
a purple background showed AChE inhibitory activity.

False-positive results due to inhibition of 1-naphthol

reaction with Fast Blue B salt were eliminated by the
method of Yang et al., (

2009

). Another TLC plate identical

to the one in the TLC assay was prepared. The developed
TLC plate was sprayed with tris–HCl (pH 7.8), 1-naphthol
(1.5 mg/ml in 40% ethanol solution) and Fast Blue B salt
(0.5 mg/ml in distilled water) in sequence. Appearance of
white spots on a purple background indicated false-positive
results.

Results and discussion

In the course of searching for AChE inhibitors from Chi-
nese herbal medicines, it was found that total alkaloidal
extract from the root of Z. nitidum strongly inhibited the
AChE activity with an IC

50

value of 10.7 ± 0.4 lg/ml

when physostigmine showed AChE inhibitory activity with
an IC

50

value of 0.013 ± 0.002 lg/ml. Further bioactivity-

guided chromatographic fractionation led to obtain a newly
discovered strong AChE inhibitor along with nine weakly
or no active compounds, which were identified as

Med Chem Res (2012) 21:722–725

723

123

skimmianine (1) (Chakravarty et al.,

1999

), toddalolactone;

(2) (Ye et al.,

1989

), dictamnine; (3) (Snider and Wu

2006

), c-fagarine; (4) (Li et al.,

2008

), magnolone; (5) (Yu

et al.,

1998

), (-)-(S)-edulinine; (6) (Boyd et al.,

2000

),

zanthodioline; (7) (Chen et al.,

1997

), edulitine; (8) (Imai

et al.,

1989

), 5,6,7-trimethoxycoumarin; (9) (Hu et al.,

2006b

) and haplopine (10) (Tang et al.,

1995

) (Fig.

1

) by

comparing the NMR and Mass spectral data with previ-
ously published literature. Compound (5) was isolated from
Zanthoxylum genus for the first time.

The AChE inhibitory activities of these compounds

were tested by Ellman’s method in 96-well microplates and
the results are shown in Table

1

. Among these ten com-

pounds, only skimmianine (1) showed strong AChE
inhibitory activity with an IC

50

value of 8.6 ± 0.7 lg/ml

when physostigmine showed AChE inhibitory activity with
an IC

50

value of 0.013 ± 0.002 lg/ml. Compounds 2–7

were scarcely able to inhibit AChE at 0.1 mg/ml, while
8

–10 showed low activities.

Antiacetylcholinesterase activity of skimmianine (1)

was also supported by the results of modified TLC bioau-
tographic assay (Fig.

2

). It was found that white spots on a

purple background were seen only in the TLC assay, and
not in the false-positive assay, skimmianine (1) was
therefore considered to be true activity. The results of the
TLC assay were consistent with those of the microplates
assay.

The alkaloid is the most important type of AChE

inhibitor and there are several types of alkaloids which
have been obtained as AChE inhibitors (Houghton et al.,

2006

). As early as in 2006, Rahman et al., (

2006

) have

reported two quinoline alkaloids from Skimmia laureola
(Rutaceae), methyl isoplatydesmine and ribalinine which
have similar skeletal structures with skimmianine (1), and
showed moderate AChE inhibitory activity. However, of
these quinoline alkaloids from Z. nitidum, only compound
(1) has remarkable AChE inhibitory activity. By comparing
the structure activity relationship of compounds 1, 3, 4, and

N

O

R

2

R

1

OCH

3

1

2

3

4

5

6

7

8

4a

8a

1'

2'

1 R

1

=R

2

=OCH

3

3 R

1

=R

2

=H

4 R

1

=H, R

2

=OCH

3

10 R

1

=OH, R

2

=OCH

3

OH

H

3

CO

O

OH

O

OCH

3

2

O

O

O

H

3

CO

H

3

CO

O

H

CH

2

OH

H

5

N

OCH

3

OH

O

OH

6

N

O

OCH

3

O

OH

H

OH

H

7

N

H

O

OCH

3

OCH

3

8

O

H

3

CO

H

3

CO

OCH

3

O

9

Fig. 1

Chemical structures of compounds 1–10

Table 1

The inhibitory activities on AChE of compounds 1–10 and

total alkaloid from Z. nitidum

Sample

Inhibition rate at
0.1 mg/ml ± SD (%)
(final concentration)

IC

50

value

(lg/ml) ± SD

Total alkaloid

89.7 ± 2.7

10.7 ± 0.4

Skimmianine (1)

91.3 ± 2.0

8.6 ± 0.7

Toddalolactone (2)

33.9 ± 1.2

Null

Dictamnine (3)

12.6 ± 1.6

Null

c-Fagarine (4)

24.3 ± 1.5

Null

Magnolone (5)

9.9 ± 1.8

Null

(-)-(S)-Edulinine (6)

19.4 ± 1.0

Null

Zanthodioline (7)

34.4 ± 2.3

Null

Edulitine (8)

45.1 ± 2.8

[100

5,6,7-Trimethoxycoumarin

(9)

49.7 ± 1.8

[100

Haplopine (10)

51.9 ± 2.5

[100

Physostigmine

0.013 ± 0.002

Values are expressed as mean ± SD (n = 3)

Fig. 2 a

Huperzine A (1 9 10

-4

lg) and 1 lg compound (1) were

applied to a silica gel G plate and the TLC bioautographic assay was
carried out. b Huperzine A (1 9 10

-4

lg) and 1 lg compound (1)

were applied to a silica gel G plate and the false-positive test was
carried out

724

Med Chem Res (2012) 21:722–725

123

10

, an inhibitory effect of substituents on the quinoline

moiety against AChE was observed. It was found that the
presence of a methoxy group at C-7 significantly enhanced
the inhibitory activity. This finding indicates that a meth-
oxy group at C-7 is important for AChE inhibition.

It has been reported that skimmianine (1), a furoquino-

line alkaloid which is abundant in the Rutaceae, has vari-
ous pharmacological activities such as antiviral and
antifungal activities (Yang and Chen

2008

), trypanocidal

activity (Ambrozin et al.,

2005

), antiplatelet aggregation

activity (Chen et al.,

2000

), spontaneous motor activity

(Cheng,

1986

), cytotoxic activity (Chaturvedula et al.,

2003

). In this study, we first demonstrated that the total

alkaloidal extract from the root of Z. nitidum possessed
potent AChE inhibitory activity and skimmianine (1) was
responsible compound for this activity. These results
indicated that anti-acetylcholinesterase activity of skim-
mianine (1) may be valuable in the treatment of AD.

Acknowledgments

This study is supported by the National Natural

Science Foundation of China (no. 20802031) and the Excellent
Young Teachers Program of Lanzhou University of Technology
(no. Q200904).

Conflict of interest

None.

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