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Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis.


Nine monoterpenoid indole alkaloids; naucletine (1), angustidine (2), nauclefine (3), angustine (4), naucline (5), angustoline (6), harmane (7), 3,14-dihydroangustoline (8), strictosamide (9) and one quinoline alkaloid glycoside; pumiloside (10) from Nauclea officinalis were tested for cholinesterase inhibitory activity. All the alkaloids except for pumiloside (10) showed strong to weak BChE inhibitory effect with [IC.sub.50] values ranging between 1.02-168.55 [micro]M. Angustidine (2), nauclefine (3), angustine (4), angustoline (6) and harmane (7) showed higher BChE inhibiting potency compared to galanthamine. Angustidine (2) was the most potent inhibitor towards both AChE and BChE. Molecular docking (MD) studies showed that angustidine (2) docked deep into the bottom gorge of hBChE and formed hydrogen bonding with Ser 198 and His 438. Kinetic study of angustidine (2) on BChE suggested a mixed inhibition mode with an inhibition constant (Ki) of 6.12 [micro]M.


Nauclea officinalis



Molecular docking

Kinetic study


The genus Nauclea consists of 35 species in which about ten species are distributed in tropical Africa, Asia and Australia (Chen and Taylor 2011). N. officinalis (Pierre ex Pitard) Merr. and Chun is distributed in lowland to hill forests and also in swampy areas (Ng and Whitmore 1989). Nauclea species have been reported to produce interesting bioactive phytochemicals, in particular monoterpenoid indole alkaloids such as 10-(anti-proliferative) (Erdelmeier et al. 1992) and subditine (cytotoxic) (Liew et al. 2014).

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal loss which eventually results in low levels of acetylcholine (ACh) neurotransmitters in the affected region of the brain. Natural products, in particular alkaloids, are promising candidates in the development of ChEIs for AD. Galanthamine which is isolated from Galanthus spp. is an example of an alkaloid ChEIs used clinically (Dall'Acqua 2013).

In our continuing search for bioactive phytochemicals, nine indole alkaloids; naucletine (1) (Shafiee and Rashidbaigi 1977), angustidine (2), nauclefine (3), angustine (4) (Abreu and Pereira 1998), naucline (5) (Liew et al. 2012), angustoline (6) (Erdelmeier et al. 1992), harmane (7) (Seki et al. 1993), 3,14-dihydroangustoline (8) (Erdelmeier et al. 1992), strictosamide (9) and one quinoline alkaloid glycoside; pumiloside (10) (Fan et al. 2010) were isolated from bark and leaves of N. officinalis. In this study, the isolation and evaluation of the activity of all ten alkaloids toward AChE and BChE are reported. In addition, the interactions of the active compounds with both AChE (angustidine 2) and BChE (angustidine 2 and naudefine 3) by MD, together with the kinetic studies of the most potent compound (angustidine 2) were also communicated.

Materials and methods

General procedures and plant materials

The general procedures were same as previously described (Liew et al. 2012). The plant materials of N. officinalis were collected at Hutan Simpan Madelc, Keluang, Johor, Malaysia. The voucher specimens (KL 5655) of these plants were deposited at the Herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia.

Extraction and isolation

Compounds from dichloromethane extract of the bark (DEB) (2.0 kg) of N. officinalis were isolated as previously described (Liew et al. 2012). The extraction procedures for the leaves (1.9 kg) were same as the bark but with the additional acid-base extraction to obtain dichloromethane extract of the leaves (DEL). Methanol extract of the leaves (MEL) was subjected to HPLC for purification of polar compounds.

Purification of compounds

DEB gave compounds 1-6. Purification of fractions 6 and 7 by preparative thin layer chromatography (PTLC) yielded 1 (11.8 mg, C[H.sub.2][Cl.sub.2]:MeOH; 99:1) and 4 (15.2 mg, C[H.sub.2][Cl.sub.2]:MeOH; 98:2) respectively. Both compounds 2 (5.5 mg, C[H.sub.2][Cl.sub.2]:MeOH; 98:2) and 3 (8.5 mg, C[H.sub.2][Cl.sub.2]:MeOH; 98:2) were obtained after purification by PTLC from fraction 12 while 5 (12.8 mg, C[H.sub.2][Cl.sub.2]:MeOH; 97:2) and 6 (4.6 mg, C[H.sub.2][Cl.sub.2]:MeOH; 97:3) were obtained from fractions 14 and 15, respectively. DEL yielded 7 (3.2 mg, C[H.sub.2][Cl.sub.2]:MeOH; 96:4) and 8 (2.3 mg, C[H.sub.2][Cl.sub.2]:MeOH; 95:5) by purification of fractions 10 and 12 respectively using PTLC.

Purification of MEL by HPLC was carried out as follows: A Waters HPLC system equipped with a C18 reversed phase column (10 mm x 150 mm x 5 [micro]M) was employed with a mobile phase flow-rate of 3 ml/min. Gradient elution system: water + formic acid (A) and methanol + formic acid (B) as the mobile phase: 0-2 min, 30-30% B; 2-32 min. 30-100% B; 32-35 min, 100-100% B; 35-37 min, 100-30% B; 37-40 min 30-30% B. Injection volume was 200 [micro]l and the UV spectrum range was 210-400 nm. The respective retention times of 10 and 9 were 17.22 min and 23.64 min.

Chemicals and enzymes

All the chemicals and enzymes used were same as described by Jamila et al. (2014).

In vitro cholinesterase inhibitory assay and MD studies

Cholinesterase inhibitory activity and MD studies were carried out as described by Jamila et al. (2014).

BChE kinetic study

Kinetic studies of BChE inhibition was determined by constructing Lineweaver-Burk (LB) plots; reciprocal plots of 1/V versus 1/[S] at different concentrations of substrate S-butyrylthiocholine chloride (1.75-14.00 mM) in the absence and presence of two different concentrations of inhibitors (33.2 and 66.4 [micro]M). The [K.sub.i] value was estimated from the replots of the slope of the individual LB plots versus the inhibitor concentrations.

Results and discussion

Cholinesterase inhibition studies

DEB and MEL from N. officinalis showed moderate BChE inhibition; [IC.sub.50] values of 97.33 [+ or -] 5.07 [micro]g/ml and 132.00 [+ or -] 12.70 [micro]g/ml, respectively. Therefore, the phytochemicals from both the extracts were isolated and tested for both BChE and AChE. The determination of cholinesterase inhibition of AChE and BChE were evaluated according to colorimetric Ellman's method (Ellman et al. 1961). Compounds 1-6 were obtained from DEB while compounds 7 and 8 were isolated from DEL. Compounds 9 and 10 were obtained from MEL. All of the alkaloids except for pumiloside (10) displayed strong to weak BChE inhibitory effect with the [IC.sub.50] values ranging between 1.02 and 168.55 [micro]W while three compounds (angustidine 2, angustoline 6 and pumiloside 10) showed moderate to weak AChE inhibition with [IC.sub.50] values between 21.71 and 261.89 [micro]M (Table 1). Interestingly, angustidine (2), angustine (4), nauclefine (3), angustoline (6) and harmane (7) were more potent BChE inhibitors compared to galanthamine. Angustidine (2) (Fig. 1) was found to be the most potent inhibitor of both AChE and BChE with a higher selectivity towards BChE (selectivity index = 21.09).

Despite being 23 times less potent than galanthamine against AChE, upon a molar basis comparison, angustidine (2) was 28 times more potent as an inhibitor of BChE compared to galanthamine. Both enzymes complement one another for their role in cholinergic neurotransmission. Based on recent studies, it was postulated that progressive decline in activity of AChE in certain brain regions during the progression of AD, is replaced by progressive increase in BChE activity, which may act as a compensatory mechanism for ACh hydrolysis (Cokugras 2003). Owing to the growing importance of BChE, an agent that inhibits BChE would be beneficial especially during the later stages of AD.

The alkaloids isolated in the present study showed comparable inhibitory activity with previously reported alkaloids (Ahmed et al. 2013), however these isolated alkaloids were more BChE selective. To the knowledge of the authors, this is the first report on the BChE inhibition potential of naucletine (1), nauclefine (3), naucline (5), angustoline (6), 3,14-dihydroangustoline (8) and angustidine (2), with the last being the most potent.

MD of angustidine (2) and nauclefine (3)

In order to understand the binding mechanisms of these compounds with cholinesterase enzymes, MD studies were performed on the most potent inhibitor; angustidine (2) and nauclefine (3). MD of angustine (4) was not performed as it was reported before (Passos et al. 2013). The findings indicated that angustidine (2) docked deep into the bottom gorge of hBChE, forming hydrogen bonds with Ser 198 and His 438 (Table 2). The hydrogen bonds formed with the amino acid residues at the catalytic site could be responsible for the potency of angustidine (2) as a BChE inhibitor. On the other hand, angustidine (2) was well accommodated, forming [pi]-[pi] stacking interaction with Trp 84 at the choline binding site, which anchored this compound to the TcAChE active site gorge (Fig. 2). Meanwhile, nauclefine (3) formed hydrogen bond with Gly 116 at the oxyanionic hole and [pi]-[pi] interactions were observed between His 438 of liBChE and the aromatic ring of nauclefine (3). Since the active site of BChE has many of its channel-lining aromatic residues replaced by residues with aliphatic side chains, it is able to accommodate bulkier compounds compared to AChE. Due to space availability, both angustidine (2) and nauclefine (3) were able to accommodate and docked completely into the base of the active site and was held in place by the hydrogen bond. In contrast, Passos et al. (2013) reported that angustine (4) was mainly stabilized by hydrophobic interactions involving its aromatic moieties with Trp 82, Trp 231, Leu 286 and Phe 329 residues of BChE.

BChE kinetic study of angustidine (2)

Enzyme kinetic study was performed for the most potent inhibitor; angustidine (2) on BChE. Graphical analysis of the Lineweaver-Burk (LB) plot constructed for BChE suggested a mixed type inhibition (Fig. 3) with [K.sub.i] value of 6.12 [micro]M.

Conflict of interest

No conflict to disclose.


BChE                   butyrylcholinesterase
AChE                   acetylcholinesterase
MD                     molecular docking
N. officinalis         Nauclea officinalis
AD                     Alzheimer's disease
ACh                    acetylcholine
ChEIs                  cholinesterase inhibitors
C[H.sub.2][Cl.sub.2]   dichloromethane
MeOH                   methanol
DEB                    dichloromethane extract of bark
DEL                    dichloromethane extract of leaves
MEL                    methanol extract of leaves
HPLC                   high performance liquid chromatography
PTLC                   preparative thin layer chromatography
TcAChE                 Torpedo californica acetylcholinesterase
hBChE                  human butyrylcholinesterase
LB                     Lineweaver-Burk
[K.sub.i]              inhibition constant


Article history:

Received 23 June 2014

Revised 12 September 2014

Accepted 12 November 2014


This work was supported by University of Malaya research grants (UM.C/625/1/HIR/MOHE/SC/37 and PV050/2012A) and Centre National de la Recherche Scientifique (CNRS) grant (57-02-03-1007). The authors thank D.M. Nor and R. Syamsir for collection of plant material and T. L. Eng for botanical identification. The plant collection was carried out within the framework of an official agreement between the CNRS and the University of Malaya (Malaysia).


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Sook Yee Liew (a), (1), Kooi Yeong Khaw (b), (1), Vikneswaran Murugaiyah (b), Chung Yeng Looi (c), Yi Li Wong (c), Mohd Rais Mustafa (c), Marc Litaudon (d), Khalijah Awang (a), *

(a) Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

(b) Discipline of Pharmacology, School of Pharmaceutical Sciences, Universiti Sams Malaysia, 11800 Penang, Malaysia

(c) Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

(d) Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, Cedex, France

* Corresponding author. Tel.: +60 3 79674064; fax: +60 3 79674193. E-mail address: (K. Awang).

(1) These authors contributed equally to this work.

Table 1
Cholinesterase inhibitory activities of alkaloids from N. officinalis.

Compounds                  % inhibition at 100 [micro]g/ml

                           AChE                   BChE

Naucletine(l)              34.19 [+ or -] 7.06    62.02 [+ or -] 1.37
Angustidine (2)            82.62 [+ or -] 2.35    95.88 [+ or -] 0.50
Nauclefine (3)             34.61 [+ or -] 4.84    75.31 [+ or -] 16.61
Angustine (4)              40.19 [+ or -] 0.65    83.97 [+ or -] 1.35
Naudine (5)                35.27 [+ or -] 4.74    82.47 [+ or -] 1.10
Angustoline (6)            77.53 [+ or -] 4.40    82.50 [+ or -] 0.67
Harmane (7)                58.42 [+ or -] 4.98    96.72 [+ or -] 1.53
3,                         38.55 [+ or -] 5.94    72.82 [+ or -] 1.60
Strictosamide (9)          34.76 [+ or -] 3.27    56.01 [+ or -] 0.93
Pumiloside (10)            54.96 [+ or -] 12.15   17.38 [+ or -] 5.47
Calanthamine (standard)    --                     --

Compounds                  [IC.sub.50]


                           [micro]g/ml           [micro]M

Naucletine(l)              ND                    ND
Angustidine (2)             6.54 [+ or -] 0.37    21.72
Nauclefine (3)             ND                    ND
Angustine (4)              ND                    ND
Naudine (5)                ND                    ND
Angustoline (6)            86.72 [+ or -] 5.41   261.89
Harmane (7)                54.75 [+ or -] 0.88   300.68
3,                         ND                    ND
Strictosamide (9)          ND                    ND
Pumiloside (10)            60.62 [+ or -] 4.98   118.36
Calanthamine (standard)     0.27 [+ or -] 0.07     0.94

Compounds                  [IC.sub.50]


                           [micro]g/ml           [micro]M

Naucletine(l)              20.78 [+ or -] 3.06    63.14
Angustidine (2)             0.31 [+ or -] 0.07     1.03
Nauclefine (3)              2.21 [+ or -] 0.03     7.70
Angustine (4)               1.56 [+ or -] 0.05     4.98
Naudine (5)                12.17 [+ or -] 2.23    38.25
Angustoline (6)             8.31 [+ or -] 1.25    25.10
Harmane (7)                 2.40 [+ or -] 0.13    13.18
3,                         16.58 [+ or -] 1.35    49.77
Strictosamide (9)          83.97 [+ or -] 0.61   168.54
Pumiloside (10)            ND                    ND
Calanthamine (standard)     8.12 [+ or -] 0.61    28.29

Compounds                  Selectivity

                           AChE (a)   BChE (b)

Naucletine(l)               --         --
Angustidine (2)             0.05      21.09
Nauclefine (3)              --         --
Angustine (4)               --         --
Naudine (5)                 --         --
Angustoline (6)             0.10      10.43
Harmane (7)                 0.04      22.81
3,                          --        --
Strictosamide (9)           --         --
Pumiloside (10)             --         --
Calanthamine (standard)    30.10       0.03

Data presented as mean [+ or -] SD (n = 3).

ND = not determined.

(a) Selectivity for AChE is defined as [IC.sub.50] (BChE)/
[IC.sub.50] (AChE).

(b) Selectivity for BChE is defined as [IC.sub.50] (AChE)/
[IC.sub.50] (BChE).

Table 2
Binding interaction data for bioactive alkaloids from N. officinalis
docked into active site gorge of AChE and BChE.

Compound          Enzyme   Binding         Interacting   Residue
                           energy (kcal)   site

Angustidine (2)   TcAChE   -11.53          CBS *         Trp 84
                  htBChE   -10.14          CS **         Ser 198
                                                         His 438
Nauclefine (3)    hBChE    -10.15          CS **         His 438
                                           OH ***        Gly 116

Compound          Type of       Distance       Ligand
                  interaction   ([Angstrom])   interacting

Angustidine (2)   Hydrophobic   3.47           Aromatic ring
                  Hydrogen      1.83           C-19
                                2.76           C-19
Nauclefine (3)    Hydrophobic   3.44           Aromatic ring
                                4.09           Aromatic ring
                                5.21           Aromatic ring
                  Hydrogen      2.25           C-18

* Choline binding site.

** Catalytic site.

*** Oxyanion hole.
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Author:Liew, Sook Yee; Khaw, Kooi Yeong; Murugaiyah, Vikneswaran; Looi, Chung Yeng; Wong, Yi Li; Mustafa, M
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jan 15, 2015
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