molecules

Article
Neuroprotective Activities of Crossyne flava Bulbs and
Amaryllidaceae Alkaloids: Implications for Parkinson’s Disease
Sylvester I. Omoruyi 1 , Abobaker S. Ibrakaw 2 , Okobi E. Ekpo 3 , James S. Boatwright 2 , Christopher N. Cupido 4
and Ahmed A. Hussein 1, *

1 Department of Chemistry, Cape Peninsula University of Technology, Symphony Road,

Bellville 7535, South Africa; [email protected]
2 Department of Biodiversity and Conservation Biology, University of the Western Cape, Cape Town, Robert
Sobukwe Road, Bellville 7535, South Africa; [email protected] (A.S.I.); [email protected] (J.S.B.)
3 Department of Anatomy and Cellular Biology, College of Medicine and Health Sciences, Khalifa University,
Abu Dhabi P.O. Box 127788, United Arab Emirates; [email protected]
4 Department of Botany, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa;
[email protected]
* Correspondence: [email protected]; Tel.: +27-21-959-6193; Fax: +27-21-959-3055

Abstract: Parkinson’s disease (PD) is one of the most common neurodegenerative diseases and affects
approximately 6.3 million people worldwide. To date, the treatment of PD remains a challenge, as
available treatment options are known to be associated with serious side effects; hence, the search for



new treatment strategies is critical. Extracts from the Amaryllidaceae plant family as well as their


alkaloids have been reported to have neuroprotective potentials. This study, therefore, investigated
Citation: Omoruyi, S.I.; Ibrakaw, the biological activities of Crossyne flava and its isolated alkaloids in an in vitro MPP+ (1-methyl-4-
A.S.; Ekpo, O.E.; Boatwright, J.S.; phenylpyridinium) PD model using SH-SY5Y cells. The effects of the total extract as well as the four
Cupido, C.N.; Hussein, A.A. compounds isolated from Crossyne flava (i.e., pancratinine B (1), bufanidrine (2), buphanisine (3),
Neuroprotective Activities of Crossyne and epibuphanisine (4)) were evaluated for cell viability, neuroprotection, levels of reactive oxygen
flava Bulbs and Amaryllidaceae
species (ROS), adenosine triphosphate activity (ATP), and caspase 3/7 activity in SH-SY5Y cells.
Alkaloids: Implications for
The results obtained showed that pre-treatment with both the extract and the isolated compounds
Parkinson’s Disease. Molecules 2021,
was effective in protecting the SH-SY5Y cells from MPP+ -induced neurotoxicity and inhibited ROS
26, 3990. https://doi.org/10.3390/
generation, ATP depletion as well as apoptosis induction in the SH-SY5Y cells. The results of this
molecules26133990
study show that the Amaryllidaceae plant family may be a source of novel compounds for the
Academic Editors: Luciana Mosca treatment of neurodegenerative diseases, which validates the reported traditional uses.
and Patrizia Russo
Keywords: Amaryllidaceae; alkaloids; Crossyne flava; Parkinson’s disease; 1-methyl-4-phenylpyridinium
Received: 14 May 2021 (MPP+ ); neuroprotection
Accepted: 24 June 2021
Published: 30 June 2021

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in
Parkinson’s disease (PD) is the second most common neurodegenerative disease,
published maps and institutional affil-
affecting approximately 6.3 million people worldwide, and it is characterized by dopamin-
iations.
ergic neuronal loss in the substantia nigra pars compacta part of the mid-brain [1]. Parkin-
sonism is a motor standpoint for the clinical diagnosis of PD, as it encompasses four PD
symptoms including resting tremor, rigidity, bradykinesia, and postural instability [2].
Other than the dopaminergic loss, PD is pathologically marked by the presence of in-
Copyright: © 2021 by the authors. traneuronal proteinaceous cytoplasmic inclusions made of alpha-synuclein proteins that
Licensee MDPI, Basel, Switzerland. accumulate in Lewy neurites and Lewy bodies [3,4]. Inadequate dopamine (a very vital
This article is an open access article
brain monoamine known to function primarily as an inhibitory neurotransmitter) lev-
distributed under the terms and
els, make it hard to regulate the striatal neurons excitability leading to degeneration and
conditions of the Creative Commons
making the ability to control movements very difficult for patients [5].
Attribution (CC BY) license (https://
Evolutionary advances in the past two decades have supported findings that un-
creativecommons.org/licenses/by/
derstanding PD progression is not only compromised by genetic factors but also by the
4.0/).

Molecules 2021, 26, 3990. https://doi.org/10.3390/molecules26133990 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3990 2 of 14

association of environmental toxins with free radical formation and oxidative stress [6].
These environmental factors include tobacco use and chemical/pesticide exposure [7,8], a
notable chemical being MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which is a
pro-drug to the neurotoxin MPP+ used in this study. MPP+ is a metabolite of MPTP and
causes permanent PD symptoms, especially the death of dopaminergic neurons. MPP+
exerts its neurotoxic activities through the generation of free radicals, which induces the
inhibition of the mitochondrial complex I electron transport chain and, in turn, causes a
depletion of adenosine triphosphate (ATP) that leads to neuronal cell death.
Presently, there have been no successful disease-modifying treatments to halt the
progression of PD, hence, current and upcoming research has been focused on discovering
more therapeutic approaches for alleviating the motor symptoms of PD [9]. These treatment
approaches are often administered based on the severity of the symptoms and side effects
experienced by individual patients [10]. The most common form of treatment is the use
of levodopa which has been shown to also lead to PD-like symptoms over time. This
challenge has given room to more investigations into the search for novel therapies which
include both synthetic and plant-derived natural products that could be used as effective
alternative treatments for PD [11].
For hundreds of years, medicinal plants have been used in many healthcare systems
in different parts of the world as safe, efficacious, acceptable treatments for a wide variety
of disease conditions with little or no side effects reported compared to chemical-based
drugs [12]. Some discoveries in the past decade have increased the reliance on medicinal
plants in both developing and developed countries, which has led to a remarkable surge
in the acceptance of herbal remedies as a primary source of healthcare and medicinal
products [13]. Herbal medicines have been used by specialists as adjuvant treatment for
PD, to lessen the dosage of dopaminergic drugs and to improve the side effects that come
with prolonged usage of these drugs [14,15]. A notable plant family with diverse biological
activities is the Amaryllidaceae family and its species have been used traditionally for
nervous system-related conditions [16].
Amaryllidaceae are largely domiciled in the tropical and temperate regions of the
world and consist of over 800 species and about 60 genera [17]. Plants from this family are
known for their alkaloids, which have been reported to have several beneficial biological ac-
tivities including antiviral, antibacterial, antifungal, antimalarial, analgesic anti-cancer, and
neuroprotective activities [18–21]. Furthermore, the Amaryllidaceae alkaloid galanthamine
received approval from the United States Food and Drug Administration (FDA) for the
treatment of Alzheimer’s disease (AD) [22]; hence, the exploitation of Amaryllidaceae
alkaloids for novel bioactive compounds with neuroprotective activity is plausible [20,21].
The deciduous bulb plant, Crossyne flava (C. flava), is a member of the Amaryllidaceae
family, which can grow up to 50 cm in height, ranging in size from 9 to 13 cm. It is majorly
distributed in the West Coast of South Africa, has been studied previously under the name
of Boophone flava, and is known to contain 14 alkaloids (Viladomat et al., 1995a). To the best
of our knowledge, there is no reported study on the neuroprotective activities of C. flava.
The present study therefore investigated the neuroprotective activities of C. flava and its
bioactive compounds on MPP+ -induced neurotoxicity in SH-SY5Y cells.

2. Results
2.1. Identification of Compounds
Four known alkaloids (1–4; Figure 1) were isolated and identified based on NMR and
GC-MS analysis (Table 1). Compounds 2–4 were identified as bufanidrine (2), buphanisine
(3), and epibuphanisine (4) [23,24]. These compounds belong to crinine-type alkaloids, and
according to the recent review by Berkov et al., so far there are 85 compounds isolated
from the Amaryllidaceae family and with a crinine skeleton [25]. The first compound
(i.e., pancratinine B) belongs to montanine-type alkaloids and was isolated once from
Pancratium canariense [26].
Molecules 2021, 26, 3990 3 of 14

Figure 1. Chemical structures of isolated compounds 1–4 from C. flava.

Table 1. 1 H (400 MHz) and 13 C (100 MHz) NMR data of compounds 1–4 in CDCl3 .

Pancratinine B (1) Bufanidrine (2) Buphanisine (3) Epibuphanisine (4)

No δC δ, J (Hz) δC δ, J (Hz) δC δ, J (Hz) δC δ, J (Hz)
6.03 br d,
1 133.4 d 132.9 d 6.58 d, 10.2 132.9 d, 6.55 d, 9.8 132.1 d 6.58 d, 10.4
10.4
5.70 br d, 5.95 dd, 5.95 dd, 9.8, 5.96 dd,
2 131.7 d 125.3 d 125.4 d, 125.6 d
10.4 10.2, 5.2 5.2 10.4, 5.4
3.94 br dd, 3.80 ddd,
3 72.3 d 72.6 d 3.82 * 77.2 d 3.81 * 72.3 d
5.0, 10.8 5.4, 4.3, 1.7
1.50 ddd,
2.07 ddd,
4 30.9 t 4.6, 11.0, 28.7 t 2.08 m 28.7 t, 28.2 t 2.20 *
13.3, 4.3, 2.0
12.8
2.35 br d, 1.59 dt, 13.7, 1.58 td, 13.3, 1.62 td, 13.4,
12.9 4.0 4.3 4.2
3.31 dd,
4a 67.5 d 2.95 m 62.6 d 63.1 d 3.43 # 63.3 d 3.32 **
13.7, 4.0
6 61.8 t 3.83 d, 16.8 58.6 t 4.24 d, 17.6 62.3 t 4.45 d, 16.7 61.8 t 4.38 d, 16.7
3.81 * d, 3.83 * d,
4.26 d, 16.8 3.75 d, 16.7
17.6 16.7
6a 125.4 s 117.3 s 126.2 s 125.6 s
7 106.8 d 6.51 s 139.4 s 106.9 s 6.45 s 106.9 d 6.46 s
8 145.9 s 133.4 s 145.7 s 145.9 s
9 147.4 s 148.0 s 146.0 s 146.4 s
10 109.9 d 6.63 s 96.6 d 6.81 s 102.9 d 6.82 s 103.0 d 6.81 s
10a 129.4 s 139.3 s 138.4 s 137.7 s
10b 44.3 s 44.3 s 44.5 s
1.89 ddd,
2.15, ddd, 2.15 ddd,
11 (exo) 49.2 t 2.64 d, 2.2 44.0 t 44.2 t 43.6 t 10.6, 10.6,
12.7, 9.8, 4.8 12.4, 9.2, 4.3
5.2
1.90 ddd, 1.90 ddd,
11 (endo) 11.8, 10.6, 12.4, 11.0, 2.20 *
5.8 6.0
11b 82.5 s
12 (exo) 54.7 t 2.82 d, 11.8 53.6 t 3.37 ** 53.4 t 3.45 # 53.3 t 3.46
2.97 dd, 2.2, 2.87, ddd, 2.93 ddd, 2.94 ddd,
12 (endo)
11.8 14.0, 9.0, 6.0 12.0, 9.0, 6.0 14.0, 9.0, 6.0
5.85, 5.83
5.88, 5.87 br 5.86, 5.85 d,
OCH2 O 100.9 t 5.92 s 100.5 t d/each, 100.7 t 100.9 t
s/each 1.3
1.40
OMe-3 56.3 q 3.42 s 56.4 q 3.35 ** s 56.5 q 3.34 s 56.6 q 3.34 ** s
OMe-7 59.1 q 3.96 s
*, ** Overlapped signals in the same column.

1
Molecules 2021, 26, 3990 4 of 14

2.2. Dose Response of C. flava and Compounds

To determine the optimal dose of C. flava and compounds that will show neuropro-
tection, a cell viability assay was performed in the SH-SY5Y cells treated with 2.5, 5, and
10 µg/mL of either C. flava extract or compounds. The results show that the total extract
induced a dose-dependent reduction in cell viability, which was only significant at the
5 and 10 µg/mL concentrations (Figure 2A). For the compound-treated cells, compound
1 showed a moderate increase in cell viability at concentrations tested albeit not signif-
icant, while compound 2 showed a slightly concentration-dependent reduction in cell
viability, which was also not significant (Figure 2B,C). In addition, compounds 3 and 4
also showed a concentration-dependent reduction in cell viability and was significant at
10 µg/mL for 3 and at both 5 and 10 µg/mL for 4 when compared to the control (cells
treated with a similar concentration of DMSO in the highest concentration of the com-
pounds) (Figure 2D,E). Together, the C. flava extract did not induce any marked changes
in the cell viability of SH-SY5Y cells at the 2.5 µg/mL concentration, and this was similar
Molecules 2021, 26, x FOR PEER REVIEW 5 of 15
for the compounds. Hence, this was selected as the optimum concentration to be used for
further neuroprotection experiments.

Figure 2.
Figure Dose–response of
2. Dose–response C. flava
of C. flava and
and compounds.
compounds. MTT MTT assay
assay cytotoxicity
cytotoxicity on
on SH-SY5Y
SH-SY5Y cells
cells treated
treated with
with increasing
increasing
concentrations (2.5, 5, and 10 µg/mL) of C. flava (A) compounds 1, 2, 3, and 4 (B–E) for 24 h. Each bar represents
concentrations (2.5, 5, and 10 µg/mL) of C. flava (A) compounds 1, 2, 3, and 4 (B–E) for 24 h. Each bar represents mean mean cell
cell
viability expressed as a percentage of the control.
the control. * Indicates significance at p
significance at p << 0.05.

2.3. C. flava and Compounds Mitigated MPP+ -Induced Toxicity

To determine whether C. flava or compounds protect SH-SY5Y cells from the MPP+ -
induced toxicity, 10,000 cells were plated per well and treated with 2.5 µg/mL of either
extract or compounds for 2 h before the addition of 2000 µM MPP+ and, thereafter, an
MTT assay was performed after 24 h. The results show that treatment of SH-SY5Y cells
with MPP+ led to a significant reduction in SH-SY5Y cell viability when compared to the
control. Indeed MPP+ reduced cell viability to approximately 40–50%, while in the cells
that were exposed to C. flava prior to the addition of MPP+ , cell viability was seen to be
improving towards normal, and this was dose dependent. Indeed, cell viability was 96.5%,
69.3%, and 55.3%, respectively, for the 2.5, 5, and 10 µg/mL concentrations of the extract

D
Molecules 2021, 26, 3990 5 of 14

Figure 2. Dose–response of C. flava and compounds. MTT assay cytotoxicity on SH-SY5Y cells treated with increasing
concentrations (2.5, 5, and 10(Figure
µg/mL)3A).
of C. Similarly, all compounds
flava (A) compounds showed
1, 2, 3, and 4 (B–E)neuroprotective
for 24 h. Each bar activities, and this
represents mean cell was
viability expressed as a percentage of theespecially
significant, control. * Indicates
at the 2.5significance at p < 0.05.
µg/mL concentration (Figure 3B–E).

Figure 3.
Figure Crossyne flava
3. Crossyne flavaand
andcompounds
compoundsshowed
showedprotection
protectionininSH-SY5Y
SH-SY5Ycells.
cells.Cells
Cells were
were pre-treated
pre-treated C. C.
with
with flava
flava (2.5,
(2.5, 5,
5, and 10 µg/mL) (A) and compounds 1, 2, 3, and 4 (B–E) before exposure to MPP + for 24 h. Each bar represents the
and 10 µg/mL) (A) and compounds 1, 2, 3, and 4 (B–E) before exposure to MPP for 24 h. Each bar represents the mean
+

mean percentage cell viability relative to the control, and the significance of the difference is indicated by * p < 0.05 when
extract/compounds are compared to MPP+ -treated cells. φ Represents MPP+ vs. control.

Interestingly, compound 3 showed significant neuroprotection at concentrations tested,

while compound 2 was only significant at the 2 and 5 µg/mL concentrations. Together,
C. flava and compounds conferred neuroprotection in SH-SY5Y cells exposed to MPP+
toxicity, and the 2.5 µg/mL showed the best activity and was selected as the optimum
concentration to be used for further studies.

2.4. C. flava Prevented MPP+ -Induced Alterations in Cell Morphology

Following the neuroprotection experiments, changes in the morphology of MPP+ -
alone exposed cells, as well as cells treated with the extract and compounds post-MPP+
exposure, were evaluated. The results showed, as expected, that MPP+ induced morpholog-
ical changes in cells ranging from loss of neuronal projections, cell shrinkage to roundness
of cells. However, cells pre-treated with C. flava and compounds showed improved cell
morphology with minor changes when compared to the control (Figure 4). These results
tend to indicate a restoration of cell morphology that may suggest a neuroprotection effect
for C. flava.

2.5. C. flava and Compounds Mitigate MPP+ -Induced ROS Generation

To understand the mechanisms of action of C. flava and the compounds in MPP+ -
induced neurotoxicity in SH-SY5Y cells, the effects of the extract and compounds on ROS
generation was next investigated. It is widely established that MPP+ treatment triggers
ROS production; hence, the ability of neuroprotective agents to inhibit ROS production is a
possible neuroprotection mechanism [27,28]. In this study, cells pre-treated with 2.5 µg/mL
 2.4. C. flava Prevented MPP+-Induced Alterations in Cell Morphology
Following the neuroprotection experiments, changes in the morphology of MPP+-
Molecules 2021, 26, 3990 alone exposed cells, as well as cells treated with the extract and compounds6 post-MPP of 14
+

exposure, were evaluated. The results showed, as expected, that MPP+ induced morpho-
logical changes in cells ranging from loss of neuronal projections, cell shrinkage to round-
ness of cells. However, cells pre-treated with C. flava and compounds showed improved
of the extract and the compounds before the addition of MPP+ significantly inhibited
cell morphology
MPP+ -induced ROS with minor changes
generation as seen inwhen
Figurecompared to the control
5. Taken together (Figure
these results 4).that
show These re-
sults tend
C. flava tothe
and indicate a restoration
compounds ofROS
mitigated cell production
morphology that may
induced suggest
by the a neuroprotection
neurotoxin, MPP+ ,
effect for C. flava.
in SH-SY5Y cells.

Figure 4. Crossyne flava inhibited SH-SY5Y morphological changes induced by MPP+ . SH-SY5Y cells
were pre-treated
Figure 4. Crossynewith
flavatotal extractSH-SY5Y
inhibited and compounds at the 2.5 changes
morphological µg/mL before exposure
induced by MPPto +2000 µM
. SH-SY5Y cells
MPP + for 24 h. Cells were visualized, and images were acquired using the light microscope at
were pre-treated with total extract and compounds at the 2.5 µg/mL before exposure to 2000 µM
100× magnification.

2.6. C. flava and Compounds Attenuated MPP+ -Induced Loss of ATP

As a pathology of PD, ATP is depleted following the generation of ROS [29,30].
Considering this, we next investigated the changes in ATP levels to ascertain if C. flava
extract and compounds would inhibit the depletion of ATP in the SH-SY5Y cells. The
results obtained showed that MPP+ treatment significantly reduced ATP levels in the cells
when compared to the control, but pre-treatment with the extract and the compounds
attenuated ATP levels in the cells for all treatment conditions (Figure 6).
 generation was next investigated. It is widely established that MPP treatment triggers
ROS production; hence, the ability of neuroprotective agents to inhibit ROS production is
a possible neuroprotection mechanism [27,28]. In this study, cells pre-treated with 2.5
µg/mL of the extract and the compounds before the addition of MPP+ significantly inhib-
ited MPP+-induced ROS generation as seen in Figure 5. Taken together these results show
Molecules 2021, 26, 3990 7 of 14
that C. flava and the compounds mitigated ROS production induced by the neurotoxin,
MPP+, in SH-SY5Y cells.

Figure 5. Crossyne flava and compounds mitigated ROS production induced by MPP+ . SH-SY5Y +cells were pre-treated with
Figure 5. Crossyne flava and compounds mitigated ROS production induced by MPP . SH-SY5Y cells
2.5 µg/mL of C. flava (A) and 2.5 µg/mL of compounds (B) before exposure to MPP+ for 24 h, and ROS production was
were pre-treated with 2.5 µg/mL of C. flava (A) and 2.5 µg/mL of compounds (B) before exposure to
measured using
MPPDCFDA fluorescent
+ for 24 h, and ROS dye. Each barwas
production represents
measured theusing
mean DCFDA
fluorescent intensity expressed
fluorescent dye. Each as
bara percentage
repre- of the
control. p + -treated
cules 2021, 26, x FOR PEERThe significance
REVIEW of the difference is indicated by * < 0.05 when extract/compounds are compared
sents the mean fluorescent intensity expressed as a percentage of the control. The significance of the to MPP
8 of 15
cells. φ Represents MPP + vs. the control.
difference is indicated by * p < 0.05 when extract/compounds are compared to MPP+-treated cells. ϕ
Represents MPP+ vs. the control.

2.6. C. flava and Compounds Attenuated MPP+-Induced Loss of ATP

As a pathology of PD, ATP is depleted following the generation of ROS [29,30]. Con-
sidering this, we next investigated the changes in ATP levels to ascertain if C. flava extract
and compounds would inhibit the depletion of ATP in the SH-SY5Y cells. The results ob-
tained showed that MPP+ treatment significantly reduced ATP levels in the cells when
compared to the control, but pre-treatment with the extract and the compounds attenu-
ated ATP levels in the cells for all treatment conditions (Figure 6).

Figure 6. Crossyne flava and compounds attenuated MPP+ -induced ATP degeneration. Cells were pre-treated with
Figure 6. Crossyne flava and compounds attenuated MPP+-induced ATP degeneration. Cells were
2.5 µg/mL of C. flava (A) and compounds (B) before exposure to 2000 µM of MPP+ for 24 h and ATP levels assessed. Each
pre-treated with 2.5 µg/mL of C. flava (A) and compounds (B) before exposure to 2000 µM of MPP+
bar representsfor
the24mean percentage level relative to the control, and the significance of the difference is indicated
h and ATP levels assessed. Each bar represents the mean percentage level relative to the con- with *
when extract/compounds are compared to MPP + and φ (MPP+ vs. control).
trol, and the significance of the difference is indicated with * when extract/compounds are compared
to MPP+ and ϕ (MPP+ vs. control).
2.7. C. flava and Compounds Inhibited MPP+ -Induced Apoptosis
2.7. C. flava and Compounds Inhibited
The reduction of ATPMPP +-Induced Apoptosis
levels in PD is known to lead to cell death [31]. Thus, to further
investigate
The reduction of ATPthelevels
mechanism
in PD isofknown
neuroprotection by C.
to lead to cell flava[31].
death andThus,
the compounds,
to further cells were
investigate thetreated prepared
mechanism for the neuroprotection
of neuroprotection experiments
by C. flava as previously
and the compounds, cellsdescribed,
were and the
activities
treated prepared of caspase
for the 3/7 was determined
neuroprotection experiments as as
an previously
indicator fordescribed,
apoptosis.and Caspases
the belong to
a family3/7
activities of caspase of was
cysteine proteases
determined asthat are known
an indicator forto be elevated
apoptosis. duringbelong
Caspases apoptosis,
to with their
activities often linked to cell death [32]. Caspases could
a family of cysteine proteases that are known to be elevated during apoptosis, with theirbe initiators of apoptosis; caspases
8 and 9 are activated during the extrinsic and intrinsic apoptotic
activities often linked to cell death [32]. Caspases could be initiators of apoptosis; caspases pathways, respectively,
and the executioner
8 and 9 are activated during the caspases
extrinsic and(i.e., intrinsic
3 and 7) apoptotic
lie downstream of therespectively,
pathways, apoptotic pathway [33].
The executioner
and the executioner caspases caspases
(i.e., 3 andare7) often used to determine
lie downstream apoptosis
of the apoptotic when the
pathway [33].aim is not to
The executioner caspases are often used to determine apoptosis when the aim is not to The results
distinguish between the intrinsic and extrinsic apoptotic pathways [34,35].
of this study show that + treatment alone, elevated levels of caspase 3/7
distinguish between the intrinsic andfollowing
extrinsic MPPapoptotic pathways [34,35]. The results of
this study showactivities were evident.
that following MPPHowever,
+ treatment pre-treatment
alone, elevated C. flava
withlevels and compounds
of caspase 3/7 activ-mitigated the
increase in the levels of caspase 3/7 towards the
ities were evident. However, pre-treatment with C. flava and compounds mitigatednormal control (Figure 7). the
Together, these
increase in the levels of caspase 3/7 towards the normal control (Figure 7). Together, these
results show that the inhibition of apoptosis is one of the mechanisms of neuroprotection
conferred by C. flava and its bioactive compounds.
 activities often linked to cell death [32]. Caspases could be initiators of apoptosis; caspases
8 and 9 are activated during the extrinsic and intrinsic apoptotic pathways, respectively,
and the executioner caspases (i.e., 3 and 7) lie downstream of the apoptotic pathway [33].
The executioner caspases are often used to determine apoptosis when the aim is not to
distinguish between the intrinsic and extrinsic apoptotic pathways [34,35]. The results of
Molecules 2021, 26, 3990 8 of 14
this study show that following MPP+ treatment alone, elevated levels of caspase 3/7 activ-
ities were evident. However, pre-treatment with C. flava and compounds mitigated the
increase in the levels of caspase 3/7 towards the normal control (Figure 7). Together, these
results
resultsshow
showthat
thatthe
theinhibition
inhibitionof
ofapoptosis
apoptosisisisone
oneof
ofthe
themechanisms
mechanisms of of neuroprotection
neuroprotection
conferred by C. flava and its bioactive compounds.
conferred by C. flava and its bioactive compounds.

Figure 7. C. flava and compounds inhibited MPP+ -induced caspase 3/7 activity. Cells were pre-
Figure 7. C. flava and compounds inhibited MPP+-induced caspase 3/7 activity. Cells were pre-
treated with 2.5 µg/mL of extract (A) and compounds (B) before exposure to 2000 µM of MPP+
treated with 2.5 µg/mL of extract (A) and compounds (B) before exposure to 2000 µM of MPP+ for
for 24 h and activity of caspase 3/7 was determined. Each bar represents the level of caspase 3/7
expressed as a fold of the control, and the significance of the difference is indicated with * when
extract/compounds are compared to MPP+ and φ (MPP+ vs. control).

3. Discussion
Parkinson’s’ disease continues to pose a challenge to quality of life, and despite the
many years that have passed since this condition was first reported, treatment options
are still lacking [36,37]. The most widely used treatment for PD is levodopa, which
addresses only some of the symptoms and may lead to serious side effects if used for
too long [38–40]. The present study investigated the neuroprotective effects of C. flava
and its bioactive alkaloids in an in vitro PD model. We provide the first evidence of the
neuroprotective activities of bioactive compounds derived from C. flava in a PD model
and these compounds included pancratinine B (1), bufanidrine (2), buphanisine (3), and
epibuphanisine. The findings show that both C. flava and its compounds attenuated the
neurotoxicity induced by MPP+ . These findings are consistent with the traditional uses
of the Amaryllidaceae plant family, as a previous study reported that plants from this
family were being used traditionally for the treatment of neurological disorders [41]. Some
biological and pharmacological studies have also validated the claim of previous traditional
uses [42–45].
Furthermore, the Amaryllidaceae plant family also has a unique family of alkaloids
that have been shown to offer neuroprotective effects [46–48]. In this study, the four
alkaloids isolated and identified demonstrated neuroprotective activities in the PD model.
Interestingly, bufanidrine (2) and buphanisine (3) have previously been shown to have a
strong affinity for the serotonin reuptake transport protein (SERT), suspected to be linked to
the presence of the 1,3-dioxole moiety in the Amaryllidaceae alkaloids, which was considered
to be responsible for their neuroprotective effects in AD [49–51]. Whether this moiety is also
responsible for the anti-PD effects seen in this study is not yet known, but there is similarity in
the pathways of the progression of both PD and AD which may be linked to oxidative stress
and mitochondrial dysfunction [29,52,53]. Similarly, epibuphanisine (4) has also been shown
to bind to SERT and the GABAA -benzodiazepine receptor and to inhibit acetylcholinesterase
as a mechanism of action within the central nervous system, which can also be linked to
anti-anxiety and anti-Alzheimer’s effects [50,54].
Mitochondrial oxidative stress is a mechanism in the pathology of PD in which there is
impairment of the mitochondrial complex I and, subsequently, a depletion in ATP levels in
brain cells [55]. Following MPP+ toxicity, a decrease in ATP as well as an increase in the lev-
els of ROS production were observed that, in turn, mitigate other mitochondrial complexes,
Molecules 2021, 26, 3990 9 of 14

such as III and IV, as well as suppress mitochondrial function including mitochondrial
gene expression, protein expression, and oxidative phosphorylation proteins [56–58]. In
line with this, the findings from this study showed that C. flava and compounds prevented
the accumulation of ROS in the cells following exposure to MPP+ . In addition, dopamine
and calcium signaling in the cells was also affected by ATP loss, all of which lead to PD [59].
Thus, the level of ATP generation in the cells is a critical test for mitochondrial integrity.
The findings from the current study showed that C. flava and compounds attenuated
MPP+ -induced ATP depletion in cells, which provides an indication of improved mito-
chondrial function and integrity. Consistent with our findings, Boophone disticha belonging
to the Amaryllidaceae family has been shown to repeal the effect of 6-hydroxydopamine
(6-OHDA)-induced ATP loss in SH-SY5Y cells [60].
Additionally, ATP depletion in cells following neurotoxicity induced by MPP+ leads
to cell death (either in the form of apoptosis or necrosis) [59,61]. Apoptosis is a form of
programmed cell death that involves a cascade of events that either go via the intrinsic (or
mitochondrial) pathway or the extrinsic pathway [33]. However, critical to the downstream
of both pathways are the executioner caspases, such as caspase 3/7, which are effector
caspases that promote cleavage of cellular content and eventual cell death [62]. The
inhibition of apoptosis in neurodegenerative diseases has been suggested to be a therapeutic
option [63], which is critical for the normal functioning of cells in PD progression. Findings
from the current study showed that C. flava and compounds were able to rescue cells from
apoptosis induced by MPP+ treatment. Importantly, it is difficult to infer from the current
study whether the necrosis pathway was also activated, which will be worth investigating
in future studies.

4. Materials and Methods

4.1. Chemical and Reagents
Organic solvents, such as methanol (HPLC grade), ethanol, ethyl acetate, and hexane,
were supplied by Merck (Cape Town, South Africa). Thin layer chromatography (TLC)
was performed on normal-phase (Merck) silica gel 60 PF254 pre-coated aluminum plates.
Column chromatography was conducted on silica gel 60 H (0.040–0.063 mm particle
size, Merck, Cape Town, South Africa) and Sephadex LH-20 (Sigma–Aldrich, Cape Town,
South Africa).
The NMR spectra were recorded on an Avance 400 MHz NMR spectrometer (Bruker,
Rheinstetten, Germany) in deuterated chloroform, using the solvent signals as the internal
reference. The GC-MS analysis was performed utilizing an Agilent Technologies 7820A
coupled with an MSD5977E. Samples of 1.0 mg were dissolved in 1.0 mL of CH2 Cl2 , and
1.0 µL was injected directly into the GC-MS operating in the electron ionization (EI) mode
at 70 Ev and utilizing an HP5 MS column (30 m, 0.25 mm i.d., film thickness 0.25 m). The
temperature gradient performed was adjusted as 40–80 ◦ C (8 min), 80–220 ◦ C (10 ◦ C/min),
hold at 220 ◦ C for 5 min, 220–300 ◦ C (20 ◦ C/min), and 10 min hold at 300 ◦ C. The injector
and detector temperatures were both at 250 ◦ C, with source and MS Quad at 230 ◦ C and
150 ◦ C, respectively, and the flowrate of the carrier gas (He) was 1.5 mL/min. A split ratio
of 1:3 was applied.

4.2. Collection and Identification of the Plant Material

Bulbs of C. flava were collected in November 2018 in the Karoo National Gardens,
Worcester, South Africa. The identity of the species was authenticated by one of the co-
authors (CNC) and voucher specimens (UFH 20 April 2020) were deposited in the Giffen
Herbarium of the University of Fort Hare (UFH), South Africa.

4.3. Preparation of Plant Extract and Isolation of Compounds

Fresh bulbs (150 g) of C. flava were blended and extracted with methanol for 2 days.
The total extracts were combined and evaporated under reduced pressure at 40 ◦ C to give
a yield of 21 g. For isolation of compounds, 20 g of the total extract was loaded on a silica
Molecules 2021, 26, 3990 10 of 14

gel column (5 × 35 cm) and eluted with a gradient mixture of hexane and ethyl acetate of
increasing polarity. Similar fractions were pooled together according to their TLC profile to
give 25 main fractions. Fraction XI (63.7 mg) was chromatographed on Sephadex using
isocratic 5% aqueous ethanol, then Prep-TLC to yield compound 1 (5.2 mg), while Fraction
XV (108.2 mg) was chromatographed under the same conditions to yield compound 166.6
(7.2 mg). In addition, Fraction XIV (100.3 mg) was chromatographed on a silica gel column
(using an isocratic elution of 5% DCM/MeOH) to yield compounds labeled as (170-1,
7.3 mg) and (170-2, 5.3 mg), respectively.
Pancratinine B (1) GC-MS: Rt 22.478 min; MS; m/z: 301.3 (C17 H19 NO4 ), 286.2, 245.2,
203.2, 174.1, 131.1, 103.1, 77.1. [α]D 1 13
25 = +69.0 (c, 0.05, CH2 Cl2 ). H/ C NMR (400/100 MHz,
CDCl3 ), see Table 1.
Bufanidrine (2), GC-MS: Rt 23.138 min; MS; m/z: 315.3 (C18 H21 NO4 ), 300.3, 260.2,
245.2, 231.2, 202.2, 115.1. [α]D 1 13
25 = −138.1 (c, 0.04, CH2 Cl2 ). H/ C NMR (400/100 MHz,
CDCl3 ), see Table 1.
Buphanisine (3), GC-MS: Rt 20.350 min; MS; m/z: 285.3 (C17 H19 NO3 ), 254.2, 215.2,
157.2, 115.1, 77.1. [α]D 1 13
25 = −78.8 (c, 0.04, CH2 Cl2 ). H/ C-NMR (400/100 MHz, CDCl3 ),
see Table 1.
Epibuphanisine (4), GC-MS: Rt 20.285 min; MS; m/z: 285.3 (C17 H19 NO3 ), 254.2, 215.2,
157.2, 115.1, 77.1. [α]D 1 13
25 = +8.8 (c, 0.2, CH2 Cl2 ). H/ C NMR (400/100 MHz, CDCl3 ), see
Table 1.

4.4. Cell Culture and Maintenance

The human neuroblastoma SH-SY5Y cells were generously donated by the Blackburn
Laboratory, University of Cape Town. Cells were grown in Dulbecco’s modified Eagle’s
Mmedium (DMEM, Gibco, Life Technologies Corporation, Paisley, UK), supplemented
with 10% fetal bovine serum, (FBS, Gibco, Life Technologies Corporation, Paisley, UK),
100 U/mL penicillin, and 100 µg/mL streptomycin (Lonza Group Ltd., Verviers, Belgium).
Cultures were incubated at 37 ◦ C in humidified air with 5% CO2 with a medium change
every three days. Cells were sub-cultured when they attained 70 to 80 percent confluency
using a solution of 0.25% trypsin EDTA (Lonza Group Ltd., Verviers, Belgium).

4.5. Treatments
Stock solutions of 40 mg/mL of C. flava extract as well as compounds were prepared
in dimethyl sulfoxide (DMSO) (Sigma–Aldrich, St. Louis, MO, USA) from which final
concentrations were made in cell growth medium. To determine the optimum concentration
of C. flava and compounds to be used for neuroprotection studies, SH-SY5Y cells were plated
at a density of 10,000 cells/well and treated with concentrations (2.5, 5, and 10 µg/mL) of
the extract of C. flava and compounds (1, 2, 3, and 4) (Table 1). The vehicle-treated cells
(cells treated with the same concentration of DMSO like that of the highest concentration
of extract or compounds) were used as the control. All treatments lasted for 24 h, and the
2.5 µg/mL concentration was selected for the neuroprotection studies. Cells were plated as
described above and pre-treated with 2.5 µg/mL of the C. flava extract and compounds for
2 h prior to the addition of 2000 µM MPP+ to the extract, and compounds containing wells
and treatments were incubated for 24 h [64,65]. The concentration of MPP+ used for this
study was informed by a previous study from our laboratory [45] and the untreated cells
served as the control.

4.6. Cell Viability Assays

The MTT (Sigma–Aldrich, St. Louis, MO, USA) cell viability assay was used to
determine the viability of cells following treatment with both plant extracts and MPP+ .
Cells were seeded in 96-well plates and treated as stated above after which the MTT
assay was performed. Briefly, after treatment, 10 or 20 µL (depending on well volume)
of 5 mg/mL MTT solution in PBS (Lonza Group Ltd., Verviers, Belgium) was added to
each well and left to incubate in the dark at 37 ◦ C for 4 h. After incubation, the medium
Molecules 2021, 26, 3990 11 of 14

containing the MTT dye was discarded, and the MTT formazan was solubilized with
100 µL of DMSO for absorbance reading using a microplate reader (BMG Labtech Omega®
POLARStar) at a wavelength of 570 nm. Cell viability was calculated and expressed as
percentage of control.

4.7. Cell Morphology

To visualize changes in cell morphology of the SH-SY5Y cells following the respec-
tive treatments, cells were seeded in 96-well plates at a density of 10,000 cells per well
and pre-treated with 2.5 µg/mL of C. flava extract and compounds for 2 h prior to the
addition of 2000 µM of MPP+ . After the 24 h treatment, changes in morphology for the
various treatment conditions were observed using the Zeiss inverted light microscope with
10× objective lens. Images were captured using the Zeiss software version 2.3.

4.8. Reactive Oxygen Species (ROS) Assay

To ascertain whether C. flava and compounds impacted on MPP+ -induced ROS produc-
tion, SH-SY5Y cells were plated in black 96-well plates and following overnight attachment,
the cells were exposed to C. flava and compounds before introduction of MPP+ . At the
termination of the experiment after 24 h, cells were washed with PBS and stained with
20 µM of 20 ,70 -dichlorofluorescin diacetate (DCFDA, Sigma–Aldrich, St. Louis, MO, USA)
fluorescent dye diluted in un-supplemented DMEM for 1 h. Thereafter, the dye-containing
medium was aspirated, and cells were washed again with PBS and the fluorescence inten-
sity of the DCFDA dye in the cells was read in PBS using a microplate reader (BMG Labtech
Omega® POLARStar), and values obtained were expressed as percentages of control.

4.9. Adenosine Triphosphate Assay

The Mitochondrial ToxGlo ATP assay kit (Promega, Madison, WI, USA) was used to
investigate ATP levels in the cells. Briefly, cells were plated at a density of 10,000 cells per
well in a white 96-well plate and after attachment, cells were treated as per neuroprotection
assay above. After treatment, cells were processed according to the manufacturer’s protocol,
the luminescence intensity was read using the microplate reader (BMG Labtech Omega®
POLARStar), and readings were expressed as percentages of the control.

4.10. Caspase 3/7 Apoptosis Assay

To investigate apoptosis in the cells, the caspase 3/7 assay kit (Promega, Madison, WI,
USA) was used to estimate levels of apoptosis in the cells according to the manufacturer’s
instructions. Briefly, cells were plated in a white 96-well plate at a density of 10,000 cells
per well and allowed to attach overnight, after which cells were pre-treated with C. flava
and compounds before the addition of 2000 µM MPP+ . Treatments lasted for 24 h and
at the end of the experiments, equal volumes of the caspase 3/7 assay mix were added
to each well, and the luminescence intensity was read with a microplate reader (BMG
Labtech Omega® POLARStar). The luminescence intensity of treated cells was expressed
as percentages of the control.

4.11. Statistical Analysis

Data generated from this study were expressed as means ± standard error of means
of at least three independent experiments analyzed using GraphPad Prism version 6.
Significance between groups was determined using one-way analysis of variance (ANOVA).
A value of p < 0.05 was considered significant.

5. Conclusions
This study investigated the neuroprotective effects of C. flava and its isolated bioactive
compounds in an in vitro model of PD. Four alkaloids were isolated, and we presented the
first evidence of the activities of these compounds and the C. flava extract in a PD model. As
a mechanism of action, it was found that both the extract and the compounds attenuated
Molecules 2021, 26, 3990 12 of 14

ATP levels in the cells and inhibited MPP+ -induced apoptosis. This further lends credence
to the acclaimed traditional use of the Amaryllidaceae family to treat nervous system
disorders as well as shows the potential of exploiting Amaryllidaceae alkaloids for novel
drug candidates.

Author Contributions: Conceptualization, A.A.H.; methodology, S.I.O., A.S.I.; formal analysis,

A.A.H., S.I.O.; investigation, S.I.O., A.S.I.; resources, A.A.H.; data curation, A.A.H., S.I.O.; writing—
original draft preparation, S.I.O.; writing—review and editing, S.I.O., O.E.E., and A.A.H.; supervision,
J.S.B., C.N.C., and A.A.H.; project administration, A.A.H., O.E.E.; funding acquisition, A.A.H., O.E.E.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Research Foundation, grant number 106055-2016.
Data Availability Statement: Not Applicable.
Acknowledgments: We would like to thank the Hantam National Botanical Garden in the Northern
Cape for providing the plant material, and the Libyan Embassy for providing Ph.D. bursary to A.S.I.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1, 2, 3, and 4 are available from the authors
on request.

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