South African Journal of Botany 126 (2019) 277–281

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Alkaloids isolated from Haemanthus humilis Jacq., an indigenous South

African Amaryllidaceae: Anticancer activity of coccinine and montanine
M. Masi a,1, S. Van slambrouck b,1, S. Gunawardana b, M.J. van Rensburg c, P.C. James b, J.G. Mochel b, P.S. Heliso b,
A.S. Albalawi b, A. Cimmino a, W.A.L. van Otterlo c, A. Kornienko d, I.R. Green c,⁎, A. Evidente a,⁎
a
Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo,Via Cintia 4, 80126 Napoli, Italy
b
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA
c
Department of Chemistry and Polymer Science, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa
d
Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, USA

a r t i c l e i n f o a b s t r a c t

Article history: Haemanthus humilis Jacq., an indigenous South African Amaryllidaceae, was studied for the first time for its
Received 6 December 2018 alkaloid content. From the acid extract of its bulbs, albomaculine, incartine, coccinine and montanine, belonging
Received in revised form 25 January 2019 to the lycorine-, homolycorine- and isoquinoline-type alkaloids respectively, were isolated. Coccinine was the
Accepted 28 January 2019
main metabolite (1.49 g/kg) and to the best of our knowledge H. humilis is to date the best natural source of
Available online 2 March 2019
this alkaloid. It is noteworthy that this Amaryllidaceae does not synthesize lycorine, the main and most common
Edited by JJ Nair alkaloid produced by this plant genus. All the isolated alkaloids were bio-assayed for their anticancer activity
against a panel of six human cancer cell lines, with coccinine evaluated for the first time. The results showed
Keywords: that coccinine and montanine have significant activity at low micromolar concentrations.
Alkaloids © 2019 Published by Elsevier B.V. on behalf of SAAB.
Anticancer activity
Coccinine and montanine
Haemanthus humilis
South African Amaryllidaceae

1. Introduction inhibit ascorbic acid biosynthesis in potato tubers was evaluated

The Amaryllidaceae are a family of bulbous plants recognized all
over the world, not only for their beautiful flowers and use in folk
medicine, but also as one of the best sources of alkaloids possessing a
wide range of bioactivities, including antitumor, antiviral, antibacterial,
antifungal, antimalarial, analgesic and cytotoxic activities (Nair et al.,
2013; Nair and van Staden, 2013; Nair and Van Staden, 2014; He et al.,
2015; Cimmino et al., 2017). Plants of this family belong to roughly
1600 species, divided into about 75 genera, and are distributed through-
out both the tropical and subtropical world (Christenhusz and Byng,
2016). Lycorine is the best known and studied Amaryllidaceae alkaloid,
which attracted our interest many decades ago when some authors of
this manuscript were involved in a SAR study of lycorine together
with a number of other Amaryllidaceae alkaloids and their
hemisynthetic derivatives (Kornienko and Evidente, 2008). In another
study performed in our laboratories, the ability of these molecules to
Abbreviations: CC, Column chromatography; 13C NMR, Carbon-13 Nuclear Magnetic
Resonance; 1H NMR, Proton Nuclear Magnetic Resonance; ESI MS, Electrospray Mass
Spectrometry; TLC, Thin Layer Chromatography.
⁎ Corresponding authors.
E-mail addresses: irg@sun.ac.za (I.R. Green), evidente@unina.it (A. Evidente).
1
These authors contributed equally
(Evidente et al., 1986). Lycorine belongs to one of the 12 Amaryllidaceae
alkaloid subgroups, namely that of the pyrrolo[de]phenanthridines
(Kornienko and Evidente, 2008), and interest in this alkaloid has re-
cently surged due to its strong anticancer activity against various solid
tumors with dismal prognosis (Van Goietsenoven et al., 2013). This
led to the synthesis of a significant number of lycorine analogues with
the potential of displaying anticancer activity (Lamoral-Theys et al.,
2009, 2010; Dasari et al., 2014). Alkaloids belonging to other subgroups,
viz., crinine-, homolycorine- and pretazettine-types have also received
attention due to their promising anticancer activities (Kornienko
and Evidente, 2008; Evidente and Kornienko, 2009; Evidente et al.,
2009; Van Goietsenoven et al., 2010). Among them, haemanthamine
showed significant activity at low micromolar concentrations (Van
Goietsenoven et al., 2010), which prompted further work aimed at
preparation of haemanthamine derivatives possessing new structural
frameworks (Govindaraju et al., 2018a) and a study of their mode of ac-
tion through co-crystallization with a ribosomal receptor (Pellegrino
et al., 2018). Further notable work involved the synthesis of new
alkaloid-like derivatives (Hudlicky et al., 2002; Ghavre et al., 2016;
van Otterlo and Green, 2018) and the determination of their absolute
configuration (Cimmino et al., 2017).
Amaryllidaceae plants are dominant in the Mediterranean basin,
Andean South America and Southern Africa(Ito et al., 1999). Thus, the

https://doi.org/10.1016/j.sajb.2019.01.036
0254-6299/© 2019 Published by Elsevier B.V. on behalf of SAAB.
278 M. Masi et al. / South African Journal of Botany 126 (2019) 277–281
search for new alkaloids from these less scientifically studied species of
these regions has significant potential and remains an aspiration of
many natural product chemists. In this regard, a crinine-type alkaloid
bulbispermine was isolated from Crinum bulbispermum, an indigenous
southern Africa plant, and demonstrated inhibition of proliferation
of glioblastoma cells through a cytostatic non-cytotoxic effect
(Luchetti et al., 2012). More recently, a new alkaloid belonging to the
2-benzopyrano[3,4-c]indole subgroup was isolated from Narcissus
jonquilla quail bulbs and named jonquailine (Masi et al., 2015). Structur-
ally, it is similar to pretazettine and tazettine, which are the most abun-
dant alkaloids of this subgroup. The C8-epimer of jonquailine is also
known and was previously isolated from Eucharis amazonica(Cabezas
et al., 2003). Jonquailine and pretazettine showed significant anticancer
activity. Tazettine, on the other hand proved to be inactive, while the C8-
epimer of jonquailine has not been tested as yet (Masi et al., 2015). Con-
sidering that the functionalization of C8, as well as its stereochemistry
could play a role in the anticancer activity, the absolute configurations
of jonquailine, its C8-epimer and pretazettine were recently determined
by optical (ORD, ECD and VCD) and computational methods in order to
establish a platform to study their modes of action (Vergura et al., 2018).
In addition, from the acid extracts of Nerine sarniensis bulbs, an indig-
enous South African plant, three new alkaloids, belonging to the
mesembrine- and crinine-families, named crinsarnine, sarniensine and
sarniensinol, were isolated, together with the known bowdensine,
hippadine and 1-O-acetyl-lycorine. Of particular interest was the finding
that sarniensine and crinsarnine showed strong adulticidal activity againt
Aedes aegypti, the vector of yellow and dengue fevers and the Zika virus
(Masi et al., 2016, 2017). As an ongoing aspect of a South Africa–Italy bi-
lateral project involving the study of bioactive metabolites from South
African Amaryllidaceae plants, a further four species, viz., Crinum
graminicola, Crinum buphanoides, Cyrtanthus mackenii and Brunsvigia
grandiflora were evaluated to determine their alkaloid contents. This
work resulted in the first isolation of lycorine-, haemanthamine- and
tazettine-type alkaloids, together with pratorimine and hippadine, from
these four species, as reported recently (Masi et al., 2018).
The present manuscript reports for the first time the isolation of
four alkaloids (1–4), belonging to the lycorine-, homolycorine- and
montanine-types, from Haemanthus humilis, a native South African
Amaryllidaceae, which has not been scientifically investigated previ-
ously. Further, we report significant anticancer activity displayed by
coccinine (3) and montanine (4).
2. Materials and methods
2.1. General procedure
Optical rotations were measured in CHCl3 on a Jasco P-1010 digital
polarimeter. 1H and 13C NMR spectra were recorded at 400 or 500 and
100 or 125 MHz in CDCl3, on Bruker and Varian spectrometers, respec-
tively. ESI MS spectra were recorded on an Agilent 6230 LC MSTOF.
Analytical and preparative TLCs were performed on silica gel plates
(Merck, Kieselgel 60F254, 0.25 and 0.5 mm). CC was performed on silica
gel (Merck, Kieselgel 60, 0.063–0.200 mm).
2.2. Plant material
The bulbs of H. humilis were purchased from the South African Bulb
Company (Hartbeespoort Dam, NW Province, South Africa) and three
live specimens are growing under the curatorship of Mr. S. Kweleta
(voucher No BG-2018-01) at the Botanical Gardens of Stellenbosch Uni-
versity, Stellenbosch, South Africa.
2.3. Extraction and isolation of alkaloids
Dried and powdered H. humilus bulbs (100 g) were treated with 1%
H2SO4(300 mL) and vigorously stirred for 1 h. Thereafter, the mixture
was filtered through muslin cloth and this extraction process was re-
peated twice more. The aqueous acidic extracts were combined, basified
to pH 9–10 using 12 N NaOH and extracted with EtOAc (3 × 300 mL).
The organic layers were combined, dried with Na2SO4 and evaporated
in vacuo to afford 1.74 g of a yellow solid residue. Fractionation of this
residue by CC, using a mixture of CHCl3–EtOAc–MeOH (2:2:1) as eluent
afforded 11 homogeneous fractions H1–H11. The residue of the fourth
fraction H4(55.0 mg) was further purified by TLC and eluted with
EtOAc–MeOH–H2O (85:10:5) yielding an amorphous solid, identified
as incartine (1, 31.4 mg). The residue of H7(105.9 mg) was further puri-
fied by CC using EtOAc–MeOH–H2O (85:10:5) as an eluent affording a
crystalline compound, identified as albomaculine (2, 24.6 mg). The res-
idue of H9(448.1 mg) was further purified by CC using EtOAc–MeOH–
H2O (70:20:10) as eluent yielding two homogeneous compounds, iden-
tified as coccinine (3, 149.9 mg) and montanine (4, 40.3 mg).
2.4. Cell culture
The human breast MCF7 (HTB-22™), Hs578T (HTB-126™), and
MDA-MB-231 (ATCC®HTB-26™), colon HCT-15 (CCL-225™), and lung
A549 (CCL-185™) cancer cell lines as well as SK-MEL-28 (HTB-72™)
melanoma cells were obtained from the American Type Culture Collec-
tion (ATCC®) and grown in the appropriate Dulbecco's modified Eagle's
(DMEM) or RPMI-medium supplemented with 10% fetal bovine serum,
100 IU/mL penicillin and 100 μg/mL streptomycin and for Hs578T with
0.01 mg/mL bovine insulin (Thermo Fisher Scientific, Waltham, MA,
USA) at 37 °C equilibrated with 5% (v/v) CO2 in a humidified
atmosphere.
2.5. Anticancer activity
Incartine (1), albomaculine (2), coccinine (3) and montanine (4)
were tested for their anticancer activity by measuring their mitochon-
drial dehydrogenase activities with the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma–Aldrich, St.
Louis, MO, USA) in accordance with Romijn et al. (1988). Briefly, cells
were seeded in 96-well plates at an initial density of 1.5 × 104 cells in
100 μL culture medium. After overnight incubation, cells were treated
with the alkaloids in final concentrations ranging from 1 to 50 μM.
After 48 h of incubation, 100 μL medium was removed prior to the
addition of the MTT reagent, and the formed formazan crystals were
dissolved in 200 μL dimethyl sulfoxide (DMSO). Four to six independent
experiments were completed to determine the mean absorbance using
a Cytation™ 3 Cell Imaging Multi-mode reader with Gen5 software
(BioTek Instruments, Inc., Winooski, VT, USA). The data were compared
to DMSO-treated control cells and analyzed using Excel for determina-
tion of mean IC50 concentrations (μM) and standard deviation.
3. Results and discussion
The dried bulbs of H. humilis were extracted and purified as de-
scribed in Materials and Methods section. Lycorine, normally the main
alkaloid found within the bulb extracts of Amaryllidaceae plants (Nair,
2014), was surprisingly absent as revealed from TLC experiments car-
ried out by comparison with various reference alkaloids available in
our laboratories. However, four known alkaloids were isolated and
identified by comparing their spectroscopic and optical rotation data
with those previously reported in literature, namely: incartine (1, Fig.
1) (Berkov et al., 2007; Sarikaya et al., 2012), albomaculine (2, Fig. 1)
(Briggs et al., 1956; de Andrade et al., 2014), coccinine (3, Fig. 1) (Bao
et al., 2013; Inubushi et al., 1960), and montanine (4, Fig. 1) (Bao
et al., 2013; Inubushi et al., 1960). To the best of our knowledge, the al-
kaloid content of H. humilis bulbs has never been reported, while some
phylogenetic studies have been conducted (Meerow et al., 1999; Bay-
Smidt et al., 2011). In particular, a phylogenetic framework to select
candidate plants for phytotherapy and drug discovery related to
 M. Masi et al. / South African Journal of Botany 126 (2019) 277–281
Fig. 1. The structures of incartine (1), albomaculine (2), coccinine (3) and montanine (4).
Alzheimer's disease and depression has been recently presented
(Bay-Smidt et al., 2011). Isolation of the alkaloids 1–4 from H. humilis
is described here for the first time.
Incartine (1) belongs to the lycorine subgroup of Amaryllidaceae
alkaloids (Kornienko and Evidente, 2008) and was isolated for the
first time in 1992 from the flowers of Lycoris incarnate(Kihara et al.,
1992). Its structure was elucidated in 1994 (Kihara et al., 1994) and
it was considered to be a biosynthetic intermediate during the bio-
transformation of galanthine to narcissidine. However, 1 was also iso-
lated from Galanthus elwesii by Berkov et al. (2007). The NMR spectral
interpretations were revised employing 2D experiments and the au-
thors considering the downfield shift of the protons surrounding N
atom, in respect to the literature data (Kihara et al., 1994), suggested
that the compound previously described in the literature was most
probably an incartine salt (Berkov et al., 2007). Following on this, it
was found that 1 has been produced by different genera belonging
to the Amaryllidaceae family, such as Lycoris(Zhu et al., 2015),
Galanthus(Emir et al., 2016), Narcissus(Šafratová et al., 2018),
Hippeastrum(Bessa et al., 2017) and Pancratium(Torras-Claveria et al.,
2010), but to the best of our knowledge this is the first report of its
isolation from a Haemanthus species. Interestingly enough, 1 was
found to display anticholinesterase activity (Emir et al., 2016), but
showed no notable effects against the human immunodeficiency
virus (HIV) in a human cell culture system and no cytotoxicity against
human MT-4 cells (Berkov et al., 2007). Furthermore, no activity was
detected in a prolyl oligopeptidase (POP) assay (Šafratová et al.,
2018).
Albomaculine (2) is a homolycorine type alkaloid (Kornienko and
Evidente, 2008) previously isolated from the Amaryllidaceae species,
Haemanthus albiflos(Crouch et al., 2005), Hippeastrum aulicum and
Hippeastrum calyptratum(de Andrade et al., 2014). In addition, it has
been isolated from the perennial herb Hosta plantaginea belonging to
the Liliaceae family (Wang et al., 2007). To the best of our knowledge
the biological activities of 2 have not been comprehensively
investigated. In fact, only its acetylcholinesterase (AChE) inhibitory
effect has been evaluated and no activity was detected (Wang et al.,
2007).
Coccinine (3) and montanine (4), possessing a 5,11-
methanomorphanthridine ring, belong to the montanine-type alkaloids
of which 4 is the main representative alkaloid (He et al., 2015). This is
one of the 12 main skeleton types of the Amaryllidaceae alkaloids and
the various analogues belonging to this subgroup are essentially
Table 1
In vitro growth inhibitory activity of compounds 1–4 in cancer cell lines.
Cell lines: in vitro IC50 growth inhibitory values (μM)a
Alkaloid No A549 HCT-15
Incartine 1 N50 N50
Albomaculine 2 N50 N50
Coccinine 3 5.9 ± 0.8 16.8 ± 1.8
Montanine 4 1.9 ± 0.4 6.8 ± 0.5
a
IC50 after 48 h of treatment with 1–4 relative to DMSO ± SD from several independent experiments, each performed in eight replicates, determined by MTT assay.
279
distinguished by the different functionalization of the cyclohexene
ring (Bao et al., 2013). Alkaloids 3 and 4 were first isolated in 1955
from three unidentified species of Haemanthus(Wildman and
Kaufman, 1955) and their congeners have been found in subsequent de-
cades (Bao et al., 2013). However, while it has been reported that 4 was
isolated from different Amaryllidaceae species, very few reports on the
natural sources of 3 are available (He et al., 2015). Recently, alkaloids 3
and 4 were found to be the major ones isolated from Haemanthus
coccineus L., Haemanthus sanguineus Jacq. and Haemanthus montanus
Baker and almost constituted the totality of the acid extracts (Stafford
et al., 2013). Their relative abundance was evaluated by GC–MS and
the last two Haemanthus species, vide supra, showed relatively good
production of coccinine (3) in respect to montanine (4) (Stafford
et al., 2013). In the present study on H. humilis, we obtained a similar re-
sult and can thus report for the first time that 3 is produced in a very
high yield (1.49 g/kg). The ESI MS spectrum of 4 showed the protonated
form [M + H]+ at m/z 302 as the base peak while the optical rotation
([α]25D: − 87.3 (c = 0.5 CHCl3)) was very similar to that reported by
Bao et al., 2013 ([α]17D: −89.0 (c = 0.6 CHCl3)). Furthermore, the 1H
and 13C NMR data (Table S1 of Supporting Information) were in agree-
ment with those reported in literature for 4(Bao et al., 2013; Jin and
Weinreb, 1997). A series of synthetic studies have been published on
the total synthesis of 4 and its related alkaloids (Bao et al., 2013) and
an interesting skeletal rearrangement of the alkaloid haemanthamine
was recently utilized to generate a series of compounds possessing the
montanine alkaloidal ring system (Govindaraju et al., 2018b).
Montanine 4 showed interesting biological activities, such as cytotoxic-
ity (Silva et al., 2008), AChE inhibitory effects (Pagliosa et al., 2010),
anti-arthritic effects (Farinon et al., 2017) and activity against
apoptosis-resistant cancer cells (Govindaraju et al., 2018b). The SERT
and P-gp binding activity were also demonstrated by 3, but to the best
of our knowledge no information has been reported on its anticancer ac-
tivity (Stafford et al., 2013).
In the present study, 1–4 were evaluated for their potential antican-
cer activity against a panel of six human cancer cell lines, including sev-
eral breast (MCF7, Hs578T, MDA-MB-231), colon (HCT-15), lung
(A549) and melanoma (SK-MEL-28) (Table 1). Coccinine (3) and
montanine (4) were found to significantly affect the cell viability of al-
most all the tested cancer cell lines after 48 h and no such effects were
observed for incartine (1) and albomaculine (2) tested at concentra-
tions of up to 50 μM. The IC50 values of both coccinine (3) and
montanine (4) for all these cell lines were determined to be in the low
SK-MEL-28 MCF7 MDA-MB-231 Hs578T
N50 N50 N50 N50
N50 N50 N50 N50
N50 7.9 ± 0.9 13.8 ± 0.8 5.3 ± 0.4
23.2 ± 1.9 4.4 ± 0.4 3.4 ± 0.9 3.6 ± 1.7
280 M. Masi et al. / South African Journal of Botany 126 (2019) 277–281
μM concentration range, except for the highly metastatic SK-MEL-28
melanoma cells. While the effect of montanine (4) on cancer cells has
previously been suggested (Silva et al., 2008), this is the first study
reporting on the anticancer activity of coccinine (3) and demonstrating
that montanine (4) is more potent than coccinine (3) in cancer. Inter-
estingly, a higher activity for montanine (4), as compared to coccinine
(3), was also detected for binding the serotonin transporter protein
SERT(Stafford et al., 2013). SERT has primarily been associated with
anti-depression by inhibiting the reuptake of serotonin (Stahl, 1998),
however there is emerging evidence that serotonin acts as a growth fac-
tor and affects the growth and survival of human cancer cells (Sarrouilhe
and Mesnil, 2018). These indications are linked to the higher expression
levels of serotonin or 5-hydroxytryptamine (5-HT) and its synthesizing
enzyme, tryptophan hydroxylase (TPH) 1 or 2, respectively in the body
and brain, found in numerous cancer cell lines and tissues (Shinka
et al., 2011). Since several studies indicate that the cell lines used in
this report express SERT and TPH-1(Gautam et al., 2016; Pai et al.,
2009; Cheng et al., 2012), the observed anticancer activity could be ex-
plained by the binding of montanine (4), and to a lesser extent of
coccinine (3), to SERT, thereby dysregulating the 5-HT system and
inhibiting the growth of these different cancer cell lines. This idea is fur-
ther supported by the fact that montanine had no significant effect on the
cell viability of T-lymphocytes isolated from lymph nodes from healthy
mouse in concentrations up to 10 μM (Farinon et al., 2017) and that rest-
ing T-lymphocytes do not express SERT(Ahern, 2011). While this rea-
soning is plausible, additional studies are required to explore this
hypothetical mechanism of action in cancer cells and to elucidate the di-
rect effects of montanine and coccinine on normal and immune cells to
evaluate their potential application as novel anticancer drugs.
4. Conclusions
The alkaloids present in the indigenous South African Amaryllidaceae
species H. humilis were investigated for the first time. From the acid
extract of its bulbs, coccinine (3) was isolated as the main metabolite to-
gether with albomaculine (2), incartine (1) and montanine (4). To the
best of our knowledge, H. humilis is the best natural source of coccinine
(3), while it is the first time that incartine (1) has been isolated from a
Haemanthus species. All the isolated alkaloids were bio-assayed for
their anticancer activity against a panel of six human cancer cell lines,
with coccinine (3) being evaluated for the first time. The results illus-
trated that coccinine (3) and montanine (4) have significant anticancer
activity against several human cancer cell lines at low micromolar con-
centrations. Future studies could focus on understanding their mode of
action and on their potential use as anticancer agents.
Acknowledgements
The work was supported in part by grants from a bilateral project be-
tween the Italian and South-Africa Ministry of Foreign Affairs Area:
South Africa, Medicine and Health Project number: ZA14MO06, titled:
Bioactive metabolites from South-African plants with anticancer activ-
ity. It was also supported by the Programme STAR 2017, UniNA and
Compagnia di San Paolo. Additionally, this study was supported by the
National Science Foundation/EPSCoR Cooperative Agreement #IIA-
1355423, the South Dakota Research and Innovation Center, BioSNTR,
and by the State of South Dakota. Any opinions, findings, and conclu-
sions or recommendations expressed in this material are those of the au-
thor(s) and do not necessarily reflect the views of the National Science
Foundation. WvO and IRG thank the South African National Research
Foundation (NRF) (Incentive and CPRR funding), and Stellenbosch
University (Faculty and Departmental support). WvO and IRG acknowl-
edge this work is supported in part by the National Research Foundation
of South Africa (Grant Number 113322). An application for a NEMBA
bioprospecting permit has been submitted. AE is associated with the
Istituto of Chimica Biomolecolare del CNR, Pozzuoli, Italy.
Appendix A. Supplementary data
NMR spectra of 1-4 are available online. Supplementary data to
this article can be found online at https://doi.org/10.1016/j.sajb.2019.
01.036.
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