Alkaloids from Hippeastrum papilio.
ABSTRACT Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt (Razadyne®, Reminyl®) for the treatment of Alzheimer's disease (AD), is obtained from Amaryllidaceae plants, especially those belonging to the genera Leucojum, Narcissus, Lycoris and Ungernia. The growing demand for galanthamine has prompted searches for new sources of this compound, as well as other bioactive alkaloids for the treatment of AD. In this paper we report the isolation of the new alkaloid 11β-hydroxygalanthamine, an epimer of the previously isolated alkaloid habranthine, which was identified using NMR techniques. It has been shown that 11β-hydroxygalanthamine has an important in vitro acetylcholinesterase inhibitory activity. Additionally, Hippeastrum papilio yielded substantial quantities of galanthamine.
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ABSTRACT: An ongoing search for alkaloids in the Amaryllidaceae species using GC-MS resulted in the identification of two crinine-type alkaloids, aulicine (1) and 3-O-methyl-epimacowine, (2) from the indigenous Brazilian species Hippeastrum aulicum and Hippeastrum calyptratum, respectively. In addition, two alkaloids, 11-oxohaemanthamine (3) and 7-methoxy-O-methyllycorenine (4) were both isolated from H. aulicum. Furthermore, we provide here complete NMR spectroscopic data for the homolycorine analogues nerinine (5) and albomaculine (6). The absolute stereochemistry of the 5,10b-ethano bridge in the crinine variants was determined by circular dichroism and X-ray crystallographic analysis, thus presenting the first direct evidence for the presence of crinine-type alkaloids in the genus Hippeastrum.Phytochemistry 04/2014; · 3.05 Impact Factor
Molecules 2011, 16, 7097-7104; doi:10.3390/molecules16087097
Alkaloids from Hippeastrum papilio
Jean Paulo de Andrade 1,2, Strahil Berkov 1,3, Francesc Viladomat 1, Carles Codina 1,
José Angelo S. Zuanazzi 2 and Jaume Bastida 1,*
1 Departament de Products Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat
de Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain
2 Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752,
90610-000, Porto Alegre, RS, Brazil
3 AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +34 934020268.
Received: 9 June 2011; in revised form: 20 July 2011 / Accepted: 26 July 2011 /
Published: 18 August 2011
Abstract: Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide
salt (Razadyne®, Reminyl®) for the treatment of Alzheimer’s disease (AD), is obtained
from Amaryllidaceae plants, especially those belonging to the genera Leucojum, Narcissus,
Lycoris and Ungernia. The growing demand for galanthamine has prompted searches for
new sources of this compound, as well as other bioactive alkaloids for the treatment of AD.
In this paper we report the isolation of the new alkaloid 11β-hydroxygalanthamine, an
epimer of the previously isolated alkaloid habranthine, which was identified using NMR
techniques. It has been shown that 11β-hydroxygalanthamine has an important in vitro
acetylcholinesterase inhibitory activity. Additionally, Hippeastrum papilio yielded
substantial quantities of galanthamine.
Keywords: galanthamine; 11β-hydroxygalanthamine; Hippeastrum papilio; 2D NMR
The object of many studies, Amaryllidaceae alkaloids contain a wide range of chemical structures
and interesting biological properties , showing pronounced antimalarial , antitumoral  and
acetylcholinesterase inhibitory activity . The use of galanthamine in palliative therapy for mild-
Molecules 2011, 16
moderate AD  has prompted the search for analogous compounds bearing the galanthamine-type
skeleton. Additionally, as most of the galanthamine used in clinics is supplied from natural sources,
there is considerable interest in finding new Amaryllidaceae species for a sustainable production of
Plants of the genus Hippeastrum, which is endemic to South America, have yielded interesting
bioactive compounds such as montanine, with significant psychopharmacological and acetylcholinesterase
inhibitory activity [7,8] and candimine, active against Trychomonas vaginalis . Recent nrDNA ITS
sequences data have included it within the Hippeastroid subclade and alluded to a probable Brazilian
We have identified six known alkaloids, including significant quantities of galanthamine which
was the main alkaloid isolated, from the bulbs and leaves of Hippeastrum papilio (Ravenna) Van
Scheepen, which grows in Southern Brazil. Furthermore, we have clarified the correct position of the
hydroxyl-substituent in the alkaloid habranthine through the isolation of its epimer, the new alkaloid
11β-hydroxygalanthamine. Habranthine was isolated from Pancratium maritimum without certainty
about the stereochemistry of the hydroxyl substituent at position 11 in the galanthamine-type skeleton.
Using modern 2D NMR, we report the correct assignment of 11β-hydroxygalanthamine, a new
alkaloid from Hippeastrum papilio, confirming that the previously reported habranthine is in fact 11α-
hydroxygalanthamine. Furthermore, 11β-hydroxygalathamine has demonstrated a good ability to
inhibit the acetylcholinesterase enzyme, with IC50 of 14.5 ± 0.33 µM.
2. Results and Discussion
Bulbs and leaves of the plant showed similar alkaloid profiles by analytical TLC. After Vaccum
Liquid Chromatography (VLC) and a purification process, the new alkaloid, 11β-hydroxygalanthamine
(1), as well as six known alkaloids, namely galanthamine (2), which was found in relatively high
quantities, narwedine (3), haemanthamine (4), 11-hydroxyvittatine (5), 8-O-demethylmaritidine (6)
and vittatine (7), were isolated and identified by NMR, CD and MS spectrometry (Figure 1). Earlier
studies have reported the isolation of galanthamine and other galanthamine-type alkaloids from
Hippeastrum species, including European cultivars, but mainly as minor compounds.
Figure 1. Isolated alkaloids from Hippeastrum papilio.
1. R1=OH, R2=H, R3=OH, R4=H
2. R1=OH, R2=H, R3=H, R4=H
3. R1+R2=O, R3=H, R4=H
4. R1=OMe, R2=H, R3+R4=CH2, R5=OH
5. R1=OH, R2=H, R3+R4=CH2, R5=OH
6. R1=OH, R2=H, R3=H, R4=Me, R5=H
7. R1=OH, R2=H, R3+R4=CH2, R5=H
Molecules 2011, 16
Compound 1, 11β-hydroxygalanthamine, crystallized as white needles. EI-MS showed a molecular
ion peak at m/z 303. The base peak at m/z 230 evidenced the loss of the hydroxyl group at C-3 and the
C-11/C-12/NMe residue, in agreement with other galanthamine-type alkaloids . The 1H-NMR of
compound 1 (Table 1) was very similar to that of habranthine previously isolated from Habranthus
brachyandrum and Pancratium maritimum [12,13], with small differences in the chemical shifts of
H-11 and H-1. The key to the assignation was the large coupling constant J(11α,12β) = 10.8 Hz, observed
in compound 1, which indicates a trans diaxial relationship between H-11α and H-12β, and therefore a
β-position for the hydroxyl substituent. In contrast, the coupling constants observed for the H-11 of
habranthine (J(11β,12β) = 1.6 Hz) and (J(11β,12α) = 4.5 Hz) indicated that the hydroxyl-substituent should
be in the α-position (endo). Moreover, the H-12 protons in compound 1 are clearly separated in a
double doublet, and 2D analysis confirmed the correct assignment, where NOESY correlations
between H-12β and H-4/H-6β and between H-12α and NMe were observed. The complete assignment
of the 11β-hydroxygalanthamine is presented in Table 1.
Alkaloid 1 has also proven to be an inhibitor of acetylcholinesterase like the majority of the
galanthamine-type alkaloids. In an acetylcholinesterase inhibition screening of several Amaryllidaceae
alkaloids, López et al.  found that habranthine, epimer of compound 1, showed similar activity to
galanthamine. The β-configuration of the hydroxyl group at position 11 in 1 could be unfavourable
for its interactions within the active site of the acetylcholinesterase enzyme. The IC50 for 1 was
14.5 ± 0.33 µM, while galanthamine showed an IC50 of 1.18 ± 0.07 µM.
NMR spectra were recovered in a Varian Mercury 400 MHz instrument using CDCl3 (CD3OD for
compound 5) as a solvent and TMS as the internal standard. Chemical shifts were reported in δ units
(ppm) and coupling constants (J) in Hz. EIMS were obtained on a GC-MS Hewlett-Packard 6890+
MSD 5975 operating in EI mode at 70 Ev.
An HP-5 MS column (30 m × 0.25 mm × 0.25 µm) was used. The temperature program was:
100–180 °C at 15 °C min–1, 1 min hold at 180 °C and 180–300 °C at 5 °C min–1 and
10 min hold at 300 °C.Injector temperature was 280 °C. The flow rate of carrier gas (Helium) was
0.8 mL min–1. Split ratio was 1:20. A QSTAR Elite hybrid Quadrupole-Time of Flight (QToF) mass
spectrometer (Applied Biosystems, PE Sciex, Concord, ON, Canada) was used for HR-MS analysis.
ToF MS data were recorded from m/z 70 to 700 amu with an accumulation time of 1 s and a pause
between the mass range of 0.55 ms, operating in the positive mode. Reserpine (1 ρmol/µL) in product
ion scan mode of m/z 609 was used for calibration of the mass spectrometer. Optical rotations were
carried out on a Perkin-Elmer 241 polarimeter. A Jasco-J-810 Spectrophotometer was used to run CD
spectra, all recorded in MeOH. UV spectra were obtained on a DINKO UV2310 instrument and IR
spectra were recorded on a Nicolet Avatar 320 FT-IR spectrophotometer.
Molecules 2011, 16
Table 1. 1H NMR, COSY, NOESY, HSQC and HMBC data of 11β-hydroxygalanthamine (400 MHz, CDCl3).
4.88 br s
2.70 ddd (15.6, 5.2, 2.8)
2.37 br dt (15.6, 1.6)
4.20 br t (4.8)
6.29 dd (10.4, 4.8)
5.96 d (10.0)
3.60 d (15.2)
3.93 d (14.8)
6.61 d (8.0)
6.68 d (8.0)
4.05 dd (10.8, 4.0)
3.02 dd (14.0, 3.2)
3.17 dd (13.6, 10.8)
3.84 s (3H)
2.45 s (3H)
H δ (J in Hz)COSYNOESY HSQC
1 H-2α, H-2β
H-1, H-2α, H-3
H-2α, H-2β, H-11α
H-1, H-2β, H-3
H-1, H-2α, H-3
H-2α, H-2β, H-4
H-4, H-6β, H-12β
H-6β, H-7, NMe
H-4a, H-6α, H-12β
C-3, C-4a, C-11
C-1, C-3, C-4, C-10b
C-1, C-4, C-4a
C-1, C-3, C-10b
C-6a, C-7, C-10a, C-12, NMe
C-6a, C-7, C-10a, C-12, NMe
C-6, C-9, C-10a
H-1, H-12α, H-12β, NMe
H-11α, H-12β, NMe
H-4a, H-6β, H-11α, H-12α
H-6α, H-11α, H-12α
C-6, C-10b, C-11
C-6, C-11, NMe
Molecules 2011, 16
3.2. Plant Material
Hippeastrum papilio was collected during the flowering period (November, 2009) in the South of
Brazil (Caxias do Sul-RS). A voucher specimen (ICN-149428) has been deposited in the Institute of
Botany, Universidade do Rio Grande do Sul (UFRGS), Porto Alegre, and identified by Julie Dutilh
PhD, University of Campinas.
3.3. Extraction and Isolation of Alkaloids
Fresh bulbs (2 Kg) were crushed and exhaustively extracted with EtOH (96% v/v) at room
temperature for 48 h and the combined macerate was filtered and evaporated to dryness under reduced
pressure. The bulb crude extract (50 g) was acidified to pH 2 with diluted H2SO4 and extracted with
Et2O (4 × 250 mL) to remove neutral material. The aqueous solution was basified with 25% ammonia
up to pH 11 and extracted with n-hexane (8 × 250 mL) to give extract A (0.55 g). Another extraction
using EtOAc (8 × 250 mL) gave extract B (1.2 g) and the last extraction using EtOAc-MeOH (3:1,
3 × 250 mL) gave extract C (3.4 g). Extract A yielded galanthamine (2) by crystallization from
acetone. Extract B was subjected to a VLC column (3 × 6 cm) using silica gel (250 g – Kieselgel –
mesh 0.15/0.30), eluting with n-hexane gradually enriched with EtOAc (0 → 100%) and then with
MeOH (0→50%). Fractions of 100 mL were collected (190 in total) monitored by TLC (Dragendorff’s
reagent, UV light λ 254 nm) and combined according to their TLC profiles, obtaining three fractions:
70–90 (fraction I), 100–124 (fraction II) and 125 – 145 (fraction III). From I, galanthamine (2,
150 mg, 0.0075% of fresh bulbs) was isolated again by crystallization from acetone. Fraction II
(250 mg) was subjected to a VLC column (1,5 × 3,5 cm) using n-hexane gradually enriched with
EtOAc (0–100%) and then with MeOH (0–50%), providing 100 fractions. After combining fractions
55–85, PTLC (20 cm × 20 cm × 0.25 mm, Silica gel F254, EtOAc:CHCl3:n-Hexane:MeOH = 4:2:2:1,
v/v/v/v, in NH3 atmosphere) was used to isolate haemanthamine (4, 80 mg) and 8-O-demethyl-
maritidine (6, 3.5 mg). From III, using PTLC (20 cm × 20 cm × 0.25 mm, Silica gel F254, EtOAc-
CHCl3-MeOH = 4:2:1, in NH3 atmosphere) 11-hydroxyvittatine (5, 10mg) and 11β-hydroxy-
galanthamine (1, 55 mg, 0.00275% of fresh bulbs) were isolated. Fresh leaves (approx. 1Kg) were also
submitted to alkaloid extraction. Their alkaloid profile obtained by TLC and GC-MS was quite similar
to that observed for bulbs, with additional traces of narwedine (3) and vittatine (7), which were
identified by comparing their GC-EI-MS spectra and Kovats retention indices (RI) with our own
library database. All known alkaloids isolated were identified by comparing their physical and
spectroscopic data with those of alkaloids previously isolated and characterized by our group [4,15-18].
3.4. Microplate AChE Assay
The assay for measuring AChE activity was performed as described by López et al. .
Galanthamine hydrobromide was used as a positive control. The IC50 of 11β-hydroxygalanthamine,
galanthamine hydrobromide and galanthamine was measured in triplicate and the results are presented
as a mean ± standard deviation using the software package Prism (Graph Pad Inc., San Diego, USA).
Both isolated alkaloids and the positive control were evaluated at a concentration ranging from 10–3 M
to 10–8 M.
Molecules 2011, 16
11β-Hydroxygalanthamine (1). White needles. UV (MeOH) λmax nm: 212.5, 287. [α]D24 = –20° (c 1.1,
CHCl3); CD [Θ]20λ: [Θ]230 +586, [Θ]247 –1950, [Θ]291 +3782. IR (CHCl3) νmax cm–1: 3360, 2925, 2854,
1730, 1624, 1590, 1508, 1440, 1280, 1096, 1044, 977, 756. 1H-NMR, COSY, NOESY, HSQC, HMBC
(400 MHz, CDCl3) and 13C-NMR (100 MHz, CDCl3) see Table 1. EI-MS 70eV (rel. int.): 303(M+, 32),
302(18), 286(11), 231(22), 230(100), 213(25), 181(12), 174(10), 97(96), 57(12). HR-QTOF-MS [M +
H]+: 304.1550 (cald for C17H22NO4, 304.1549).
Although the chemical synthesis of galanthamine has been achieved, its supply for clinical use still
comes from natural sources. Hippeastrum papilio is able to produce great quantities of galanthamine
but more studies on its genetic improvement, hybridization or in vitro culture are needed. Our search
for new sources of galanthamine and acetylcholinesterase inhibitors has resulted in the isolation and
identification of 11β-hydroxygalanthamine (1), a new acetylcholinesterase inhibitor alkaloid. The
compound was structurally elucidated by 2D NMR, which allowed us to distinguish it from its epimer,
habranthine. Galanthamine-type alkaloids are well-known for their inhibitory activity of the
acetylcholinesterase enzyme. The action of galanthamine as an allosterically potentiating ligand in
nicotinic acetylcholine receptors  and its ability to inhibit β-amyloid aggregation  could also
play a role in successful AD therapy. The discovery of new galanthamine-type candidates is therefore
of real interest for the future management of this disease.
The authors are grateful for the collaboration of SCT-UB technicians and the Generalitat de
Catalunya (2009-SGR-1060) for the financial support. The authors also thank Julie Dutilh for
authentication of plant material. J.P.A. thanks Agencia Española de Cooperación Internacional para el
Desarollo (BECAS-MAEC-AECID) for a doctoral fellowship.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Not available.
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