Alkaloids isolated from Hippeastrum reticulatum (L’Hér.) Herb. and their acetylcholinesterase inhibitory activities

7Journal of Medicine and Pharmacy, Volume 10, No.7/2020
Alkaloids isolated from Hippeastrum reticulatum (L’Hér.) Herb. and 
their acetylcholinesterase inhibitory activities
Hoang Xuan Huyen Trang, Ho Viet Duc, Nguyen Thi Hoai
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University
Abstract 
Background: Hippeastrum reticulatum (L.Hér.) Herb is a species of the Hippeastrum Herb. genus. 
Screening studies have shown that this species has the ability to inhibit the enzyme acetylcholinesterase. 
So far, research on this species is still very limited. The purpose of this study is to provide some more 
informations about the chemical composition and bioactive of isolated compounds from this species. 
Materials and method: Bulbs of Hippeastrum reticulatum was collected in Thua Thien Hue province in May 
2018. The compounds were isolated by using various chromatographic methods and their structures were 
identified by 1D and 2D-NMR spectroscopic methods in reference to the literature. The acetylcholinesterase 
inhibitory activity was determined by Ellman’s microplate colorimetric method. Results and conclusions: 
Two alkaloids including N-methyltiramine (1), narciclasine-4-O-β-D-xylopyranoside (2) was isolated from 
bulbs of Hippeastrum reticulatum (L’Hér.) Herb. These compounds were isolated from Hippeastrum Herb. 
genus for the first time. Compound 2 showed moderate acetylcholinesterase inhibitory activity, with IC50 
value of 70.06 ± 1.46 µg/mL.
Keywords: Hippeastrum reticulatum (L’Hér.) Herb., alkaloid, N-methyltiramine, narciclasine-4-O-β-D-
xylopyranoside.
Corresponding author: Nguyen Thi Hoai, email: nthoai@huemed-univ.edu.vn DOI: 10.34071/jmp.2020.7.1
Received: 17/12/2019, Accepted: 23/3/2020
1. INTRODUCTION
Hippeastrum Herb. is a large genus of the 
Amaryllidaceae family of more than 90 species 
recorded. The species belong to this genus 
possess several important biological activities, 
such as antibacterial, antioxidant, antiviral, 
acetylcholinesterase inhibitors... In Vietnam, 
this genus has 2 species: Hippeastrum equestre 
and Hippeastrum reticulatum. Screening studies 
indicated that both species have a potent inhibitory 
effects of acetylcholinesterase, particularly 
Hippeastrum reticulatum. However, there are still 
few studies on Hippeastrum reticulatum species in 
Vietnam so far. 
The aim of this study is to contribute knowledge 
to the chemical composition and bioactivity of 
isolated compounds from Hippeastrum reticulatum.
2. MATERIALS AND METHODS
2.1. Plant materials
The bulb of Hippeastrum reticulatum (L’Hér.) 
Herb. was picked up in May 2018 in Thua Thien Hue 
province, Vietnam. Its identify was confirmed by Dr. 
Vu Tien Chinh, Vietnam National Museum of Nature, 
the Vietnam Academy of Science and Technology.
2.2. Extraction and isolation
The bulb of Hippeastrum reticulatum (L’Hér.) 
Herb. was washed, dried at 50oC (12.5 kg) then 
powdered into powder, extracted with methanol 
(20 L × 3 times) by immersion at room temperature 
to yield extract. This extract was subjected to Diaion 
HP-20 column chromatography. Pass water through 
the column to remove water-soluble components, 
then elute the compounds with methanol to obtain 
a methanol extract (150 g).
The methanol extract was acidified with 2% HCl 
to pH 2 and then extracted with ethyl acetate (1 L × 3 
times) to obtain ethyl acetate (EtOAc) fraction (60 g). 
The remaining acid solution was alkalined with NH3 
to pH 10 and then extracted with dichloromethan 
(CH2Cl2) (1L × 3 times) to obtain C fraction (30 g).
The C fraction (30g) was subjected to silica 
gel column chromatography, eluted with CH2Cl2 – 
methanol – H2O (5:1:0.1, v/v/v) to obtain 5 fractions, 
C1-C5. Fraction C1 (4g) was subjected to reverse-
phase RP-18 silica gel column chromatography 
eluted with aceton – H2O (5:1, v/v) to obtain 6 
fractions, C1.1-C1.6. Fraction C1.3 (600mg) was 
subjected to silica gel chromatography, eluted with 
CH2Cl2-methanol-NH3 (10:1:0.1, v/v/v) to obtain 6 
fractions, C1.3.1-C1.3.6. Fraction C1.3.2 (120mg) 
was subjected to reverse-phase RP-18 silica gel 
column chromatography, eluted with methanol-H2O 
(3:1, v/v) to obtain compound 1 (8 mg). 
8Journal of Medicine and Pharmacy, Volume 10, No.7/2020
Fraction C4 (8 g) was subjected to reverse-phase 
RP-18 silica gel column chromatography eluted with 
methanol – H2O (3:1, v/v) to obtain 5 fractions, 
C4.1-C4.5. Fraction C4.3 (1 g) was subjected to 
silica gel column chromatography, eluted with 
CH2Cl2 – methanol (10:1, v/v) to obtain 4 fractions, 
C4.3.1-C4.3.4. Fraction C4.3.3 (60 mg) was further 
purifed by Sephadex LH-20, eluted with methanol to 
obtain compound 2 (15 mg).
2.3. Acetylcholinesterase inhibition assay
The inhibitory activities of acetylcholinesterase 
(AchE) were measured using modified Ellman’s 
method [3]. Principle of the method: ATCI 
(acetylthiocholine iodide) substrate is hydrolyzed 
by the catalysis of AChE to create thiocholine. 
Thiocholine reacts with DTNB solution (5,5′-dithiobis 
(2- nitrobenzoic acid) reagent to release yellow 
compound 5-thio-2-nitrobenzoic acid. Measure the 
absorbance of the solution formed at 405 nm to 
determine the AChE inhibitory activity. The positive 
control used is Galantamine.
The procedure of the test method (Table 1): add 
pH 8 buffered buffer, sample and 0.25 IU/mL AChE 
enzyme solution, successively, to each well of the 96-
well plate. The mixture is well mixed and incubated 
for 15 minutes at room temperature. After that, 
DTNB test 2.4 mM and ATCI substrate solution 2.4 
mM were added to the mixture and mixed well. 
Continue incubating the mixture for 15 minutes at 
room temperature, then the solution is measured 
for absorbance at 405 nm. Absorbance is measured 
on the ELISA Micropate Reader EMR 500 (US). Each 
test sample was repeated three times. The AChE 
inhibitory activity of the sample is calculated by the 
formula:
Where E and S were the enzymatic activities 
with and without the tested sample, respectively. 
The AChE inhibitory activity of each sample was 
expressed in terms of the concentration (in µg/
mL) required to inhibit the hydrolysis of AchE by 
50% (the IC50 value), which was calculated from the 
logarithmic dose-inhibition curve.
I (%)= x100
S
E
Table 1. The composition of the reaction evaluates the AChE inhibitory activity.
No. Compositions Test sample (µl) Control sample (µl)
1 Phosphate buffer pH 8 140 140
2 Test sample 20 0
3 DMSO 10% 0 20
4 AChE 0,25 IU/ml 20 20
5 DTNB 2,4 mM 10 10
6 ATCI 2,4 mM 10 10
Total volume 
(µl) 200 200
3. RESULTS AND DISCUSSION
Compound 1 was isolated as a white amorphous 
powder. The 1H NMR spectrum of 1 in methanol-d
4
showed typical signals of four protons of para-
disubstituted benzene ring at δH 6.79, 7.11 (each, 
2H, d, J = 8.5 Hz), two methylene groups at δH 2.90, 
3.21 (each, 2H, t, J = 7.5 Hz), and a methyl group at 
δH 2.72 (3H, s). Thus, compound 1 was determined 
as N-methyltyramine [4].
Compound 2 was obtained as a creamy needles. 
The 1H NMR spectrum of 2 in methanol-d
4
 showed 
typical signals of an aromatic proton at δH 6.79 (s), 
an olefinic proton at δH 6.22 (m), a methylenedioxy 
group at δH 6.06 (s), and an anomeric proton at δH 
4.41 (d, J = 7.5 Hz). The signals in the range δH 3.2-4.4 
indicated hydrogen attached to carbon containing 
hetero atom (O or N).
The 13C NMR and HSQC spectra showed the 
presence of 19 carbon signals corresponding to two 
methylenes, ten methines and seven quaternary 
carbons. The presence of carbonyl carbon was 
clearly seen via signal at δC 170.5. Meanwhile, the 
HSQC cross-peak between two protons at δH 6.22 
and carbon at δC 103.8 confirmed the appearance 
of methylendioxy group. In addition, the HSQC 
spectrum showed the protons at δH 6.22 (m, H-1), 
4.30 (m, H-2), 4.07 (m, H-3), 4.02 (m, H-4), 4.43 (brd, 
J = 10.0 Hz, H-4a), 6.79 (s, H-10), 4.41 (d, J = 7.5 Hz, 
H-1’), 3.33 (overlapped, H-2’), 3.40 (t, J = 9.0 Hz, 
H-3’), 3.61 (m, H-4’), 4.05 (overlapped, H-5’a), and 
3.33 (overlapped, H-5’b) correlated with carbons δC 
123.80 (C-1), 70.3 (C-2), 72.0 (C-3), 79.6 (C-4), 52.0 
9Journal of Medicine and Pharmacy, Volume 10, No.7/2020
(C-4a), 97.3 (C-10), 104.0 (C-1’), 74.8 (C-2’), 77.6 (C-
3’), 71.0 (C-4’), 67.2 (C-5’), respectively.
In the HMBC spectrum, the correlations of 
H-2 to C-1/C-4/C-10b (δC 133.3), of H-3 to C-1/C-
4a, of H-4 to C-4a, of H-4a to C-1/C-3/C-4/C-10a 
(δC 132.1)/C-10b, H-10 to C-8 (δC 135.9)/C-9 (δC 
154.5)/C-6a (δC 107.0)/C-10a, and of methylendioxy 
protons to C-8/C-9 were observed (Figure 2). These 
evidences led to the construction of narciclasine 
skeleton. 
The correlation of H-2’ to C-3’; of H-3’ to C-2’/C-4’; 
of H-4’ to C-3’; of H-5’ to C-4’/C-3’ confirmed the 
structure of pentose moiety. The coupling constant 
of H-1’ (J = 7.5 Hz) indicated the β-configuration 
of sugar unit. The series of carbon signals at δC 
104.0, 74.8, 77.6, 71.0, 67.2 were indicative of 
β-D-xylopyranoside moiety [5]. The pentose sugar 
was further suggested to be xylopyranoside by 
comparison with the reported values in literature 
[10]. Notably, the position of D-xylopyranosyl at C-4 
was confirmed by the HMBC cross-peaks H-1’/C-4, 
H-4/C-1’. Spectral data of compound 2 was compared 
with reference [2]. Based on the above evidences, 
compound 2 was confirmed as narciclasine-4-O-β-
D-xylopyranoside. 
Figure 1. Structures of 1, 2 isolated from Hippeastrum reticulatum.
Figure 2. Key HMBC (1H→13C, arrows) correlations of 2.
O
O NH
OH
OH
O
O
OH
OH
OH
OOH
1
2
3
44a 1'
2'
4'
66a
7
9
10
10a 10b
8
H
N
HO
2
O
O NH
OH
OH
O
O
OH
OH
OH
OOH
1
2
3
44a 1'
2'
4'
66a
7
9
10
10a 10b
8
1
10
Journal of Medicine and Pharmacy, Volume 10, No.7/2020
Table 2. 1H (500 MHz) and 13C (125 MHz) NMR data of 2 in methanol-d4 [δ (ppm), J (Hz)]
Position δC δH
1 123.8 6.22 t (4.0, 6.5)
2 70.3 4.30 m
3 72.0 4.07 m
4 79.6 4.02 m
4a 52.1 4.43 m
6 170.5 -
6a 107.0 -
7 146.0 -
8 135.9 -
9 154.5 -
10a 132.1 -
10b 133.3 -
10 97.3 6.79 s
1’ 104.0 4.41 (d, 7.5)
2’ 74.8 3.33 m
3’ 77.7 3.39 m
4’ 71.1 3.61 m
5’ 67.3 4.05 m
-OCH2O- 103.8 6.06 brs
The AChE inhibitory activities of the isolated 
compounds were assessed at various concentrations. 
Galanthamine was used as a positive control. The 
results are summarized in Table 3. Results showed 
that compound 2 did not show the ability to inhibit 
AChE at the test concentration (500 µg / mL). 
Compound 1 have a moderate AChE inhibiting effect 
with IC50 values of 70,06 ± 1,46. N-Methyltyramine 
(1) has been isolated from a variety of plant species 
such as Coryphantha missouriensis (Cactaceae) 
[6], Pilosocereus maxonii (Rose) [7], and Acacia 
schweinfurthii (Leguminosae) [9]. Previous studies 
have shown that N-methyltyramine (1) increases 
blood pressure in the anaesthetized rat, relaxes 
guinea pig ileum and increases both the force and 
rate of contraction of guinea-pig right atrium by 
inducing the release of noradrenaline [1]. However, 
this is the first report of AChE inhibition activity of 
N-methyltyramine (1).
Table 3. In vitro AChE inhibitory activities of compounds 1 and 2
Compounds IC50 (µg/mL)
N-methyltyramine (1) 70.06 ± 1.46
Narciclasine-4-O-β-D-xylopyranoside (2) > 500
Galantamine 0.33 ± 0.01
4. CONCLUSION
Two alkaloids, N-methyltiramine (1), narciclasine-
4-O-β-D-xylopyranoside (2), were isolated from the 
bulb of Hippeastrum reticulatum and identified by 
comparison of their 1D and 2D NMR spectroscopic 
with those reported in the literature. These 
compounds were isolated from Hippeastrum Herb. 
genus for the first time. Compound 1 showed 
moderate inhibitory activities against AChE, with 
IC50 values of 70.06 ± 1.46 µg/mL.
11
Journal of Medicine and Pharmacy, Volume 10, No.7/2020
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