MEDICINAL
Med Chem Res (2012) 21:722 725
CHEMISTRY
DOI 10.1007/s00044-011-9581-9
RESEARCH
ORIGINAL RESEARCH
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 termination of nerve impulse transmission at the choliner-
acetylcholinesterase (AChE) inhibitor, along with nine gic synapses by rapid hydrolysis of ACh. AChE inhibitors
weakly or no active compounds, toddalolactone (2), dictam- that can increase the cholinergic transmission by blocking
nine (3), c-fagarine (4), magnolone (5), (-)-(S)-edulinine (6), the degradation of ACh are therefore considered to be a
zanthodioline (7), edulitine (8), 5,6,7-trimethoxycoumarin promising approach for the treatment of AD. In addition,
(9), and haplopine (10) have been isolated from Zanthoxylum recent researches showed that AChE inhibitors not only
nitidum (Z. nitidum). Skimmianine (1) inhibited 50% of alleviate the cognitive defect of AD patients by elevating
AChE activity at the concentrations of 8.6 Ä… 0.7 lg/ml ACh levels, but also act as disease modifying agents by
when the IC50 value of Physostigmine as a standard was preventing the first step of AD, the assembly of b-amyloid
0.013 Ä… 0.002 lg/ml. Antiacetylcholinesterase activity of peptide into amyloid plaque (Munoz-Torrero and Camps
skimmianine (1) was also supported by TLC bioautographic 2006; Pang et al., 1996). This discovery stimulated a great
assay. The structure activity relationship on the anti-acet- interest in screening natural anti-AChE as lead compounds.
ylcholinesterase activity of the quinoline moiety is also This study was carried out to search for useful leads which
discussed in this article. could become new candidates for the development of
rational drug design against AD.
Keywords Zanthoxylum nitidum Skimmianine Zanthoxylum nitidum (Roxb.) DC (Rutaceae) have been
Acetylcholinesterase inhibitor Quinoline alkaloid used for the treatment of toothache and gastrointestinal
Alzheimer s disease 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,
Introduction such as antiviral and antifungal activities (Yang and Chen
2008), cytotoxic activity (Yang et al., 2008), anticancer
Alzheimer s disease (AD) is the most common neurode- activity (Wang et al., 2007), anti-inflammatory activity (Hu
generative disorder of this century and the most prevalent et al., 2006a), DNA topoisomerase-I inhibitory activity
cause of dementia with aging. Most treatment strategies (Fang et al., 1993), antispasmodic and analgesic activities
have been based on the cholinergic hypothesis which (Zeng et al., 1982). Previous reports have also showed that
postulates that AD is a result of decreased levels of the extracts of Zanthoxylum species have cholinergic activities
neurotransmitter acetylcholine (ACh) in the cortex (Lahiri relevant to the treatment of AD (Carpinella et al., 2010).
et al., 2002). The cholinergic hypothesis claims that ace- However, till now, no previous cholinesterase inhibitory
tylcholinesterase (AChE) plays a significant role in the 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
Z. Yang (&) D. Zhang J. Ren M. Yang
the root of Z. nitidum exhibited significant AChE inhibitory
School of Life Science and Engineering, Lanzhou University
activity with an IC50 value of 10.7 Ä… 0.4 lg/ml when
of Technology, Lanzhou 730050, People s Republic of China
e-mail: [e-mail] physostigmine showed AChE inhibitory activity with an
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Med Chem Res (2012) 21:722 725 723
IC50 value of 0.013 Ä… 0.002 lg/ml. This study further et al., 1961). Briefly, 140 ll of 0.1 M sodium phosphate
investigated the AChE inhibiting constituents of alkaloidal buffer (pH 8.0), 20 ll of 1 mg/ml sample solution, and
extract from Z. nitidum. 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
Materials and methods solution was incubated at 37°C for 20 min. The optical
density was measured at 405 nm. Enzyme activity was
Plant materials calculated by comparing the rate of reaction for the sam-
ples relative to that for the blank. The percentage of
The roots of Z. nitidum (Roxb.) DC (Rutaceae) were pur- inhibitory activity was calculated by subtracting the per-
chased from Huanghe Herb Market in Lanzhou and were centage of enzyme activity from 100. Physostigmine was
identified by Associate Professor Lin Yang who majored in used as a positive control. The experiments were done in
plant classification, School of Life Science and Engineer- triplicate.
ing, Lanzhou University of Technology, Lanzhou, China.
The voucher specimen is deposited at the School of TLC bioautographic assay for AChE inhibitory activity
Life Science and Engineering, Lanzhou University of
Technology. The TLC bioautographic assay for AChE inhibitory
activity was modified by our group (Yang et al., 2009).
Chemicals Firstly, 0.1 ll of 10 mg/ml compound (1) was spotted on a
silica gel TLC plate and migrated by 35:1 chloro-
Acetylcholinesterase (EC3.1.1.7, Sigma product no C2888), form:methanol solution. The plate was dried absolutely
Fast Blue B salt, acetylthiocholine iodide (ATCI), 5,50- with a hair dryer. Then AChE (1 U/ml in buffer pH 7.8)
dithiobis [2-nitrobenzoic acid] (DTNB), physostigmine and and 1-naphthyl acetate (1.5 mg/ml in 40% ethanol solu-
Huperzine A were purchased from Sigma (St. Louis, MO, tion) were sprayed onto TLC plate subsequently. After
USA). 1-Naphthyl acetate and 1-naphthol were obtained each solution was sprayed, TLC plate was blown quickly
from Sinopharm Chemical Reagent CO., Ltd (Shanghai, with cold wind from a hair dryer until no free liquid was
China). MCI-GEL CHP 20P (75 150 lm) were from found on it. Secondly, the plate was incubated at 37°C for
Mitsubishi Chemical Holdings Corp. Silica gel GF254 20 min in a humid atmosphere. At last, Fast Blue B salt
plates and silica gel (200 300 mesh) were purchased from (0.5 mg/ml in distilled water) was sprayed onto the plate.
Qingdao Haiyang Chemical Co., Ltd (Qingdao, China). Huperzine A was used as a positive control. White spots on
a purple background showed AChE inhibitory activity.
Extraction and isolation False-positive results due to inhibition of 1-naphthol
reaction with Fast Blue B salt were eliminated by the
The roots of Z. nitidum (10 kg) were powdered and method of Yang et al., (2009). Another TLC plate identical
extracted with 95% ethanol (50 l 9 2) under reflux for 2 h to the one in the TLC assay was prepared. The developed
to obtain alcohol extract, which was evaporated to dryness TLC plate was sprayed with tris HCl (pH 7.8), 1-naphthol
and then suspended in distilled water. The suspension was (1.5 mg/ml in 40% ethanol solution) and Fast Blue B salt
adjusted to pH 2 by the addition of HCl (4 M). The acid (0.5 mg/ml in distilled water) in sequence. Appearance of
aqueous solution was filtered after one night. The filtrate white spots on a purple background indicated false-positive
solution was basified to pH 11 with NaOH (1 M), and then results.
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 Results and discussion
gel column chromatography combined with microplate
assay for AChE activity to obtain ten compounds: 1 In the course of searching for AChE inhibitors from Chi-
(380.6 mg), 2 (2.6 mg), 3 (16.2 mg), 4 (18.5 mg), 5 nese herbal medicines, it was found that total alkaloidal
(5.6 mg), 6 (49.8 mg), 7 (18.4 mg), 8 (24.8 mg), 9 extract from the root of Z. nitidum strongly inhibited the
(5.6 mg), and 10 (27.9 mg). AChE activity with an IC50 value of 10.7 Ä… 0.4 lg/ml
when physostigmine showed AChE inhibitory activity with
Microplate assay for AChE inhibitory activity an IC50 value of 0.013 Ä… 0.002 lg/ml. Further bioactivity-
guided chromatographic fractionation led to obtain a newly
The ten compounds were tested for AChE inhibiting discovered strong AChE inhibitor along with nine weakly
activities by a slightly modified Ellman s method (Ellman or no active compounds, which were identified as
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724 Med Chem Res (2012) 21:722 725
OCH3
Table 1 The inhibitory activities on AChE of compounds 1 10 and
5
4
4a 3 total alkaloid from Z. nitidum
6
1'
7
2'
Sample Inhibition rate at IC50 value
2
R1 8 8a N O OCH3
0.1 mg/ml Ä… SD (%) (lg/ml) Ä… SD
1 OH
R2 1 R1=R2=OCH3
(final concentration)
3 R1=R2=H
OH
4 R1=H, R2=OCH3
H3CO O O Total alkaloid 89.7 Ä… 2.7 10.7 Ä… 0.4
10 R1=OH, R2=OCH3
2
Skimmianine (1) 91.3 Ä… 2.0 8.6 Ä… 0.7
Toddalolactone (2) 33.9 Ä… 1.2 Null
Dictamnine (3) 12.6 Ä… 1.6 Null
O
H OCH3
CH2OH
H3CO OH c-Fagarine (4) 24.3 Ä… 1.5 Null
H
Magnolone (5) 9.9 Ä… 1.8 Null
O
H3CO O OH
(-)-(S)-Edulinine (6) 19.4 Ä… 1.0 Null
N O
O
Zanthodioline (7) 34.4 Ä… 2.3 Null
5 6
Edulitine (8) 45.1 Ä… 2.8 [100
5,6,7-Trimethoxycoumarin 49.7 Ä… 1.8 [100
(9)
OH
OCH3 Haplopine (10) 51.9 Ä… 2.5 [100
O
H
H
Physostigmine 0.013 Ä… 0.002
OCH3
OH
H3CO
Values are expressed as mean Ä… SD (n = 3)
N O
N O
H
OCH3
OCH3
H3CO O O
7 8 9
Fig. 1 Chemical structures of compounds 1 10
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.
Fig. 2 a Huperzine A (1 9 10-4 lg) and 1 lg compound (1) were
The AChE inhibitory activities of these compounds
applied to a silica gel G plate and the TLC bioautographic assay was
were tested by Ellman s method in 96-well microplates and
carried out. b Huperzine A (1 9 10-4 lg) and 1 lg compound (1)
the results are shown in Table 1. Among these ten com- were applied to a silica gel G plate and the false-positive test was
carried out
pounds, only skimmianine (1) showed strong AChE
inhibitory activity with an IC50 value of 8.6 Ä… 0.7 lg/ml
when physostigmine showed AChE inhibitory activity with
an IC50 value of 0.013 Ä… 0.002 lg/ml. Compounds 2 7 The alkaloid is the most important type of AChE
were scarcely able to inhibit AChE at 0.1 mg/ml, while inhibitor and there are several types of alkaloids which
8 10 showed low activities. have been obtained as AChE inhibitors (Houghton et al.,
Antiacetylcholinesterase activity of skimmianine (1) 2006). As early as in 2006, Rahman et al., (2006) have
was also supported by the results of modified TLC bioau- reported two quinoline alkaloids from Skimmia laureola
tographic assay (Fig. 2). It was found that white spots on a (Rutaceae), methyl isoplatydesmine and ribalinine which
purple background were seen only in the TLC assay, and have similar skeletal structures with skimmianine (1), and
not in the false-positive assay, skimmianine (1) was showed moderate AChE inhibitory activity. However, of
therefore considered to be true activity. The results of the these quinoline alkaloids from Z. nitidum, only compound
TLC assay were consistent with those of the microplates (1) has remarkable AChE inhibitory activity. By comparing
assay. the structure activity relationship of compounds 1, 3, 4, and
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Med Chem Res (2012) 21:722 725 725
Fang SD, Wang LK, Hecht SM (1993) Inhibitors of DNA topoiso-
10, an inhibitory effect of substituents on the quinoline
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Chem 58:5025 5027
presence of a methoxy group at C-7 significantly enhanced
Houghton PJ, Ren YH, Howes MJ (2006) Acetylcholinesterase
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Hu J, Xu XK, Liu RH, Zhang C, Li HL, Liang MJ, Zhang WD
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(2006a) Study on lignans constituents of Zanthoxylum nitidum.
It has been reported that skimmianine (1), a furoquino-
Pharmaceut Care Res 6:51 53
line alkaloid which is abundant in the Rutaceae, has vari-
Hu J, Zhang WD, Liu RH, Zhang C, Shen YH, Li HL, Liang MJ, Xu
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XK (2006b) Benzophenanthridine alkaloids from Zanthoxylum
nitidum (Roxb.) DC, and their analgesic and anti-inflammatory
antifungal activities (Yang and Chen 2008), trypanocidal
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activity (Chen et al., 2000), spontaneous motor activity
Constituents of the root bark of Murraya-paniculata collected in
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drug targets for alzheimer s disease treatment. Drug Dev Res 56:
alkaloidal extract from the root of Z. nitidum possessed
267 281
potent AChE inhibitory activity and skimmianine (1) was
Li X, Tang HZ, Gou XJ, Tu J (2008) Study on chemical constituents
responsible compound for this activity. These results
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Munoz-Torrero D, Camps P (2006) Dimeric and hybrid anti-
mianine (1) may be valuable in the treatment of AD.
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Pang YP, Quiram P, Jelacic T, Hong F, Brimijoin S (1996) Highly
Acknowledgments This study is supported by the National Natural
potent, selective, and low cost bis-tetrahydroaminacrine inhib-
Science Foundation of China (no. 20802031) and the Excellent
itors of acetylcholinesterase steps toward novel drugs for
Young Teachers Program of Lanzhou University of Technology
treating alzheimer s disease. J Biol Chem 271:23646 23649
(no. Q200904).
Rahman AU, Khalid A, Sultana N, Ghayur MN, Mesaik MA, Khan
MR, Gilani AH, Choudhary I (2006) New natural cholinesterase
Conflict of interest None.
inhibiting and calcium channel blocking quinoline alkaloids.
J Enzyme Inhib Med Chem 21:703 710
Snider BB, Wu XX (2006) Synthesis of (?)-myrtopsine, (?)-7,8-
dimethoxymyrtopsine, and related 2,3-dihydro-3-hydroxy-2-
(1-hydroxy-1-methylethyl) benzofuran natural products. Heterocy-
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