Abstract
Sceletium tortuosum (SCT) has been utilized medicinally by indigenous
Koi-San people purportedly for mood elevation. SCT extracts are
reported to be neuroprotective and have efficacy in improving
cognition. However, it is still unclear which of the pharmacological
mechanisms of SCT contribute to the therapeutic potential for
neurodegenerative disorders. Hence, this study investigated two
aspects–firstly, the abilities of neuroprotective sub-fractions from
SCT on scavenging radicals, inhibiting some usual targets relevant to
Alzheimer’s disease (AD) or Parkinson’s disease (PD), and secondly
utilizing the network pharmacology related methods to search probable
mechanisms using Surflex-Dock program to show the key targets and
corresponding SCT constituents. The results indicated sub-fractions
from SCT could scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical,
inhibit acetylcholinesterase (AChE), monoamine oxidase type B (MAO-B)
and N-methyl-D-aspartic acid receptor (NMDAR). Furthermore, the results
of gene ontology and docking analyses indicated the key targets
involved in the probable treatment of AD or PD might be AChE, MAO-B,
NMDAR subunit2B (GluN2B-NMDAR), adenosine A[2A] receptor and
cannabinoid receptor 2, and the corresponding constituents in Sceletium
tortuosum might be N-trans-feruloyl-3-methyldopamine,
dihydrojoubertiamine and other mesembrine type alkaloids. In summary,
this study has provided new evidence for the therapeutic potential of
SCT in the treatment of AD or PD, as well as the key targets and
notable constituents in SCT. Therefore, we propose SCT could be a
natural chemical resource for lead compounds in the treatment of
neurodegenerative disorders.
Introduction
Sceletium tortuosum (L.) N.E. Br (SCT), a South African herb, with a
long history of use by Koi-San natives, is reported to have various
pharmacological activities such as anti-depressant [[38]1],
anti-anxiety [[39]2], anti-epileptic [[40]3] and analgesic [[41]4]
activities. Its extracts are reported to have shown efficacy in
improving cognition [[42]5, [43]6]. Cognition deficit is a
predominantly general symptom of Alzheimer’s disease (AD) and in some
cases of Parkinson’s diseases (PD)–hence it is postulated that
neurodegenerative disorders that could be treated by compounds that
possess neuroprotective effects [[44]7–[45]12]. Considering that there
are certain neuroprotective constituents in SCT, which are reported to
have the therapeutic potential in the treatment of neurodegenerative
diseases, there is a need to invesigate the probable mechanisms that
contribute to the possible treatment of neurodegenerative disorders,
especially in AD or PD with cognitive impairments.
“Network pharmacology” (NP) methods have been usually applied in this
form of research to access primary mechanisms of certain traditional
Chinese medicines formula according to their traditional indications
[[46]13–[47]16]. Some studies have also used NP to explore the possible
novel indications for complicated Chinese traditional medicines
[[48]17, [49]18]. Application of NP could be further understood based
on the published report by Fang JS et al who proposed and deciphered
mechanism of action for some of the most widely studied medicinal herbs
used in the treatment of AD [[50]19].
Our previous study [[51]20] has showed the neuroprotective
sub-fractions and possible neuroprotective constituents ([52]Fig 1) in
the neuroprotective sub-fractions extracted from SCT. The petroleum
ether and ethyl acetate fractions were confirmed to possess
neuroprotective efficacy, been further separated by silica gel column
to obtain sub-fractions tested by cell experiments. Furthermore,
natural products generally consist of various and diverse active
constituents depending on the extraction process [[53]21], which can
lead to neuroprotective fractions that exert neuroprotective effect of
SCT and probably caused due to multiple constituents. Thus, it makes
such investigations laborious and difficult to decipher the elicited
mechanisms. Hence, investigating the possibility of SCT extract for
treating neurodegenerative disorders, as an integrated system as
applied by traditional Chinese medicine, would provide insight by
utilizing NP methods is a logical and scientific approach.
Fig 1. The constituents of neuroprotective sub-fractions from SCT in previous
study.
[54]Fig 1
[55]Open in a new tab
(tR represents their retention time in UPLC).
In this study, spectrophotometric assays were performed on SCT
sub-fractions to assess neuroprotective action related efficacies based
on the scavenging radicals, inhibiting acetylcholinesterase (AChE),
monoamine oxidases (MAOs) and N-methyl-D-aspartic acid receptor
(NMDAR). Subsequently, relevant NP methods and molecular docking were
performed to understand the possible mechanisms that provide evidence
to corelate the therapeutic potential of SCT in the treatment of AD or
PD.
Furthermore, it is important to identify the key targets, and the
corresponding constituents in neuroprotective sub-fractions involved in
the probable treatment of AD or PD [[56]2, [57]4, [58]22–[59]33].
Methods
The neuroprotective sub-fractions from SCT and their identified constituents
Based on our previous study [[60]20], SCT plant powder was extracted
with alcohol and which was further extracted with petroleum ether and
ethyl acetate. The petroleum ether and ethyl acetate fractions were
confirmed to possess neuroprotective efficacy on MPP^+-injured N2a
cells or L-glutamate-injured PC12 cells. The active fractions were
further separated by silica gel column to obtain sub-fractions. The
sub-fractions were also tested by cell experiments [[61]34–[62]36] to
give four neuroprotective sub-fractions–P5, P6, E1 and E3 (“P” and “E”
mean the sub-fractions of petroleum ether and ethyl acetate fractions
respectively). The active sub-fractions were once again preliminarily
identified the constituents that were separated and identified from SCT
in the current study. The chemical structures of these constituents are
depicted in [63]Fig 1.
DPPH scavenging assay
The ability of the neuroprotective sub-fractions from SCT to scavenge
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was tested in 96-well
polystyrene microtiter plates (Corning^®). The extraction and
separation methods to obtain the neuroprotective sub-fractions were
performed as described in the previous study [[64]20]. DPPH (TCI,
Japan) was dissolved in methanol to obtain a concentration of 100 μM.
The wells contained 100 μL DPPH and then added 100 μL of sub-fraction
samples in different concentrations. Blank wells contained methanol in
place of DPPH and control wells contained only methanol in place of
samples. After shocking on a microoscillator, the plate was kept in the
dark for 50 minutes. The absorbance was detected at a wavelength of 517
nm using a microplate reader (Bio-Tek Instruments Inc, USA). The
clearance percent of DPPH was expressed as mean ± SEM calculated by
following formula:
[MATH: Clearance(%)=Acontrol−(Asample−Ablank)Acontrol×100% :MATH]
AChE inhibition assay
The experiment to test the AChE inhibiting ability of neuroprotective
sub-fractions of SCT was performed as per procedure described by Ellman
[[65]37, [66]38]. 160 μL of PBS (0.1 M pH = 8), 10 μL of sample and 10
μL of AChE (0.5 U/mL, Solarbio, Beijing) were mixed in 96 wells plate
for 20 min at 4°C, and then the wells were added 10 μL of
2,2’-dithiodibenzoic acid (10 mM, MedChemExpress) and 10 μL of
acetylthiocholine iodide (10 mM, Solarbio, Beijing) for another 30 min
at 37°C. The absorbance was detected at a wavelength of 405 nm. Blank
wells had PBS added in place of AChE and control wells had PBS added in
place of samples.
MAOs inhibition assay
The MAOs inhibition percent of neuroprotective sub-fractions from SCT
was measured by following procedures described by Holt with some
modifications [[67]39].
This study is got pass by Jinan University Laboratory Animal Ethical
Committee.The IACUC issue number is 20220225–03. All studies related to
animals were performed in accordance with the standards set forth in
the eighth edition of Guide for the Care and Use of Laboratory animals,
published by the National Academy of Sciences, the National Academies
Press, Washington D.C (License number: SCXK(粤)2018-0002).We use
Pentobarbital Sodium as anesthesia and reduce the pain of death in rats
by excessive anaesthesia.
Female Sprague—Dawley rat (286 g) was killed by anesthetic, and its
livers were dissected out, washed in ice-cold PBS (0.2 M, pH 7.6).
Liver tissue (7 g) was homogenized 1:20 (w/v) in 0.3 M sucrose with a
mechanical homogenizer. Following centrifugation at 1100g for 12 min,
the supernatant was further centrifuged at 10 000g for 30 min to obtain
a crude mitochondrial pellet, which was resuspended in 40 ml of PBS
used as the source of MAOs.
40 μL of MAOs and 40 μL of samples were added in the wells for 20 min
at 37°C and then the supplement of the enzyme substrate and chromogenic
reagent were added for 60 min at 37°C. The enzyme substrate was
tyramine (5 mM, Aladdin, Shanghai) and the chromogenic reagent was a
mixture contained vanillic acid (5 mM, Shanghaiyuanye, China),
4-aminoantipyrine (1mM, Shanghaiyuanye, China), peroxidase (5 U/mL,
Solarbio, Beijing) in PBS. The absorbance was detected at a wavelength
of 490 nm. Blank wells had PBS added in place of tyramine and control
wells had PBS added in place of samples.
The inhibition percentages of AChE and MAOs were expressed as mean ±
SEM calculated by following formula:
[MATH: Inhibitionpercent(%)=Acontrol−(Asample−Ablank)Acontrol×100% :MATH]
Primary culture of rat hippocampal neurons
The hippocampus tissue was separated from Sprague-Dawley neonatal rat
and placed in cold phosphate buffer saline under an asepsis condition,
and then was digested with 0.25% trypsin for 20 min at 37°C. After
trypsinization, hippocampal neurons were suspended in DMEM (Gibco)
containing 10% horse serum (Gibco) and cultured in
polyethylenimine-coated coverslips at a density of 105/cm^2 for 4 h at
37°C. The medium was replaced with neurobasal medium (Gibco) containing
B-27 supplement (Gibco) and L-glutamine (Gibco), and the cells were
cultured at 37°C in a humidified environment of 95% air and 5% CO[2]
for 7 days.
Whole cell patch clamp
To investigate the effect of two sub-fractions from SCT on the NMDAR
mediated current, whole cell patch clamp was used to the record of
NMDAR current by an amplifier (EPC-10, HEKA, Germany). Before
recording, a negative pressure was exerted on the hippocampal neuron’s
surface through microelectrode and formed a GΩ seal resistance, then
the membrane potential was kept in -70 mV. The hippocampal neurons were
exposed to NMDA (100 μM), Glycine (10 μM) and samples in different
concentrations or D-2-Amino-5-phosphonovaleric acid (D-AP5) (100 μM).
NMDA (100 μM) and Glycine (10 μM) were used to activate the NMDA
current. D-AP5, a NMDAR inhibitor, was used as a positive control. The
current signals were recorded by the amplifier under a Gap-free mode
and stored in PatchMaster software (HEKA, Germany).
Recording was allowed to start at least 5 min after the rupture of
patch membrane to ensure stabilization of the intracellular milieu.
Neurons that showed unstable or large (more than ∼50 pA) holding
currents were rejected. The series resistance (<15 MΩ) and membrane
capacitance were compensated and checked regularly during the
recording.
The inhibition percentage of NMDAR was calculated according to the
formula: (1-(I[NMDA + Glycine +Compound] / I[NMDA +Glycine])) x 100%.
Data were expressed as mean ± S.E.M.
Extracellular fluid (pH 7.4) contained 140 mM NaCl, 4 mM KCl, 2 mM
CaCl[2]•2H[2]O, 10 mM HEPES, 5 mM D-Glucose, 0.5 μM TTX, 10 μM NBQX, 10
μM Strychnine and 10 μM Bicuculline. Intracellular fluid (pH 7.2)
contained 10 mM NaCl, 110 mM CsMeS, 2 mM MgCl[2]•6H[2]O, 10 mM HEPES,
10 mM EGTA, 2 mM Na[2]-ATP, 0.2 mM Na[2]-GTP.
Network pharmacology methods to decipher possible mechanisms of SCT
Targets of the constituents identified from SCT in our previous study
were obtained from Polypharmacology Browser 2
([68]https://ppb2.gdb.tools/) [[69]40]. Methods: ECfp4 Naive Bayes
Machine Learning model produced on the fly with 2000 nearest neighbors
from extended connectivity fingerprint ECfp4. Targets of
neurodegenerative disorder were elements of the intersection set
obtained from GeneCards [[70]41] ([71]https://www.genecards.org/,
Relevance score ≥ 10) and DisGeNET [[72]42]
([73]https://www.disgenet.org/, Score gda ≥ 0.1) databases using
following keywords: Alzheimer’s disease, Parkinson’s disease,
amyotrophic lateral sclerosis, spinocerebellar ataxia, Lewy bodies,
frontotemporal dementia, Huntington’s disease and epilepsy.
Protein–protein interaction data were acquired from STRING 11.0
[[74]43] ([75]https://string-db.org/cgi/input.pl) with the species
limited to “Homo sapiens”.
GO and KEGG pathway enrichment analyses were performed by DAVID
Bioinformatics Resources 6.8 [[76]44] ([77]https://david.ncifcrf.gov/).
The targets from the intersection set of targets of the constituents
and diseases were submitted to obtain the terms of biological process,
molecular function, cellular component and Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathways.
All visualized network models were established via Cytoscape 3.8.0. The
topological feature of each node in network model was assessed by
calculating three parameters named “Degree”, “Betweenness Centrality
(BC)” and “Closeness Centrality (CC) by Network Analyze tool in
Cytoscape software.
Preliminary verification for the possible mechanisms by surflex-dock
The constituents were prepared by Sybyl-X 2.0. As docking ligands,
their energy was minimized according following parameter settings:
Powell method, 0.005 kcal/mol·A gradient termination, 1000 max
iteration and Gasteiger-Huckel charges. Other settings were kept
default.
The protein structures were obtained from PDB Protein Data Bank
([78]http://www.rcsb.org/). To make docking pockets, the protein
structures were extracted ligand substructure, repaired sidechains,
added hydrogens and minimized their energy. Protomol generation mode
was selected as “Ligand” and other settings were default. Reference
molecules were set as their original ligands. Results of Total Score
were output as the criterion to comparing the predictive affinities.
Statistical method
Each value was an average of data from 3 independent experiments and
each experiment included 3 replicates. Data were expressed as mean ±
SEM and analyzed using GraphPad Prism V8.0 (GraphPad Software, Inc.,
San Diego, CA, USA). One-way analysis of variance (ANOVA) and Dunnett’s
test were used to evaluate statistical differences.
Results
SCT sub-fractions scavenge DPPH radical
The scavenging ability of DPPH radical of the SCT sub-fractions is
depicted in [79]Fig 2. The clearance percentages of four sub-fractions
could all reach more than 40% at their highest concentration (500
μg/mL). Fraction E3 was the most potent sub-fraction on scavenging DPPH
radical among these four neuroprotective sub-fractions from SCT,
although weaker than the positive compounds–vitamin C.
Fig 2. The DPPH clearance percentages of active sub-fractions.
[80]Fig 2
[81]Open in a new tab
Data were expressed as mean ± S.E.M. from the data obtained from three
independent experiments (n = 3). NS represents the mean of group has no
significant different with the mean of control group.
SCT sub-fractions inhibit AChE
The AChE inhibition percent of four sub-fractions could reach more than
40% at their highest concentration (1000 μg/mL). Since contrast to
Huperzine–a AChE inhibitor, their effects on AChE were considered as
slight efficacy. It also showed that fraction E1 exhibited more than
60% inhibition percent on AChE, which was the most potent sub-fraction
among the extracts ([82]Fig 3).
Fig 3. The inhibition percentages of active sub-fractions on AChE.
[83]Fig 3
[84]Open in a new tab
Data were expressed as mean ± S.E.M. obtained from three independent
experiments (n = 3). NS represents the mean of group has no significant
difference compared to the mean of control group.
SCT sub-fractions inhibit MAOs
The results depicted in [85]Fig 4 showed, MAO-A selective
inhibitor—clorgiline could inhibit the MAOs by about 60% at 50 μM,
while MAO-B selective inhibitor–pargyline could inhibit the MAOs by
close to 100% at 50 μM. Since the enzyme substrate was tyramine, which
could be common enzyme substrate for both MAO-A and MAO-B, the enzyme
activity of the MAOs we used in this study was considered to be
contributed mainly by MAO-B [[86]45].
Fig 4. The inhibition percentages of active sub-fractions on MAOs.
[87]Fig 4
[88]Open in a new tab
Data were expressed as mean ± S.E.M. obtained from three independent
experiments (n = 3). NS represents the mean of group showed no
significant difference with the mean of control group.
Except fraction E3, other three active sub-fractions presented more
than 40% inhibition percent on MAOs at their highest concentration
(1000 μg/mL). The observed inhibition results were regarded as mild.
SCT sub-fractions inhibit NMDAR
Compared to Zembrin^®, the more potent neuroprotective P5 and E1
fractions (compared with P6 and E3 in our previous study [[89]20])
showed potent inhibiting effect on NMDAR-mediated current ([90]Fig 5).
However, this effect is not significant enough to be considered as main
mechanism that elicits antidepressant action of SCT.
Fig 5. The inhibition percentages of active sub-fractions on NMDAR-mediated
current.
[91]Fig 5
[92]Open in a new tab
Data were expressed as mean ± S.E.M. D-AP5 group: n = 4, other groups:
n = 3. NS represents the mean of group has no significant different
with the mean of control group.
Common targets of constituents and neurodegenerative diseases
As indicated in the previous study, the neuroprotective sub-fractions
and underlying neuroprotective constituents (structures were shown in
[93]Fig 1) in SCT [[94]20]. Using Polypharmacology Browser 2, the
predictive targets of the constituents from neuroprotective
sub-fractions were compared with the targets of neurodegenerative
diseases collected from GeneCards and DisGeNET databases. The results
of their intersections were showed as [95]Fig 6. Although the percent
of overlapping targets in targets of HD was the maximum value (16.67%)
among these neurodegenerative diseases, there were only 5 overlapping
targets from the intersection. Therefore, AD or PD was selected as
adaptable disease because of the larger number and percentage of common
targets ([96]Table 1) than other disease conditions.
Fig 6. The intersection of targets from constituents and diseases.
[97]Fig 6
[98]Open in a new tab
AD: Alzheimer‘s Disease; PD: Parkinson’s Disease; ALS: Amyotrophic
Lateral Sclerosis; SCA: Spinocerebellar Ataxia; LBD: Lewy Body
Dementia; FTD: Frontotemporal Dementia; HD: Huntington’s Disease.
Table 1. Overlapping targets of constituents and AD/PD.
Gene Common name
ESR2 Estrogen receptor beta
MAOB Monoamine oxidase type B
HTR6 5-hydroxytryptamine receptor 6
CYP2D6 Cytochrome P450 2D6
ACHE Acetylcholinesterase
SLC6A4 Sodium-dependent serotonin transporter
SLC6A3 Sodium-dependent dopamine transporter
BACE1 Beta-secretase 1
HTR2A 5-hydroxytryptamine receptor 2A
CNR2 Cannabinoid receptor 2
BCHE Cholinesterase
ALOX5 Polyunsaturated fatty acid 5-lipoxygenase
APP Amyloid-beta precursor protein
TSPO Translocator protein
GSK3B Glycogen synthase kinase-3 beta
PTGS2 Prostaglandin G/H synthase 2
ADAM17 Disintegrin and metalloproteinase domain-containing protein 17
BACE2 Beta-secretase 2
GRIN2B N-methyl D-aspartate receptor subtype 2B
CTSD Cathepsin D
TNF Tumor necrosis factor
HTR1A 5-hydroxytryptamine receptor 1A
DRD3 Dopamine D3 receptor
DRD2 Dopamine D2 receptor
DRD1 Dopamine D1 receptor
ADORA2A Adenosine receptor A2a
HTT Huntingtin
[99]Open in a new tab
GO and KEGG pathway enrichment analysis
The overlapping targets of constituents and AD/PD could enrich in more
than 20 terms of biological processes (the terms of which P value <
0.001 were showed as [100]Fig 7), which mainly involved response to
drug (GO:0042493), chemical synaptic transmission (GO:0007268),
locomotory behavior (GO:0007626), memory (GO:0007613), learning
(GO:0007612、GO:0008542), response to amphetamine (GO:0001975),
behavioral response to cocaine (GO:0048148), dopaminergic synaptic
transmission (GO:0001963), prepulse inhibition (GO:0060134), etc. These
biological processes indicate that the extracts of SCT could exert
neurological activities that are helpful to treat cognition deficit and
behavioral disorders. The analysis of cellular functions ([101]Fig 8)
showed that these targets mainly included dopamine binding
(GO:0035240), dopamine neurotransmitter receptor activity (GO:0004952),
beta-amyloid binding (GO:0001540), drug binding (GO:0008144), enzyme
binding (GO:0019899), etc. Moreover, these overlapping targets are
mainly integral component of plasma membrane (GO:0005887), locate on
plasma membrane (GO:0016021) and cell surface (GO:0009986), distribute
on dendrite (GO:0030425) and axon (GO:0030424) ([102]Fig 8). KEGG
pathway analysis of these targets suggested that they play a role in
neuroactive ligand-receptor interaction (hsa04080), serotonergic
synapse (hsa04726), dopaminergic synapse (hsa04728), Alzheimer’s
disease (hsa05010), alcoholism (hsa05034), cAMP signaling pathway
(hsa04024), Parkinson’s disease (hsa05012), calcium signaling pathway
(hsa04020), amphetamine addiction (hsa05031) ([103]Fig 9).
Fig 7. Enrichment analyses for constituents-AD/PD common targets: Biological
process.
[104]Fig 7
[105]Open in a new tab
Fig 8. Enrichment analyses for constituents-AD/PD common targets: Molecular
function and cellular component.
[106]Fig 8
[107]Open in a new tab
Fig 9. Enrichment analyses for constituents-AD/PD common targets: KEGG
pathway.
[108]Fig 9
[109]Open in a new tab
Constituents–targets–disease network diagram
The interactions of the overlapping targets, constituents and their
possible targets and targets of AD or PD were fed into cytoscape 3.8.0
software to obtain a constituents-targets-disease network diagram
([110]Fig 10). In this network diagram, there were 59 nodes and 345
edges, including 23 constituents, 30 targets, 2 diseases, 4
sub-fractions and 1 plant. The result of network analysis ([111]Table
2) showed that degrees of the targets, of which gene names are SLC6A4,
DRD2, ACHE, HTR1A, SLC6A3, HTT, APP, HTR2A, MAOB, BACE1, DRD3, TNF,
CNR2, BCHE, DRD1 and GRIN2B, are more than 13 with betweenness
centralities more than 0.005 and closeness centralities more than 0.5.
The degrees of all constituents in this diagram are equal or greater
than 6.
Fig 10. SCT-sub-fraction-constituents-targets-disease network diagram.
[112]Fig 10
[113]Open in a new tab
Table 2. The results of topological analysis for the network.
Node Name Degree Betweenness Centrality Closeness Centrality
SLC6A4 32 0.06260 0.62766
DRD2 31 0.05874 0.64130
ACHE 30 0.10778 0.65556
HTR1A 30 0.07519 0.64130
SLC6A3 26 0.03614 0.59596
HTT 23 0.03893 0.57282
APP 20 0.04156 0.57843
HTR2A 20 0.02568 0.56731
MAOB 20 0.02614 0.55140
BACE1 19 0.05334 0.57282
DRD3 17 0.01538 0.55660
TNF 15 0.08981 0.52679
CNR2 15 0.02514 0.55140
BCHE 14 0.01292 0.53636
DRD1 13 0.00678 0.52212
GRIN2B 13 0.00617 0.52212
PTGS2 12 0.02749 0.51754
CYP2D6 12 0.00583 0.53153
GSK3B 10 0.01317 0.47967
ADAM17 7 0.00234 0.45385
ADORA2A 7 0.00621 0.50000
ALOX5 7 0.00475 0.45385
HTR6 7 0.00442 0.49167
BACE2 6 0.00107 0.43704
CTSD 5 0.00113 0.42143
ESR2 5 0.00722 0.42143
TSPO 5 0.00034 0.41259
TNFRSF1A 4 0.00171 0.36646
RIPK1 3 0 0.35119
TRAF2 3 0 0.35119
1.19 12 0.02202 0.51304
4.97 11 0.00727 0.50000
3.45 10 0.00977 0.50000
3.86 10 0.01547 0.50000
5.26 10 0.03077 0.50427
1.44 9 0.00425 0.49580
3.28 9 0.00307 0.50000
1.24 9 0.00949 0.49580
2.67 9 0.00869 0.48361
2.12 8 0.00342 0.48361
2.731 8 0.00244 0.49167
3.50 8 0.00250 0.47967
1.93 8 0.00212 0.45385
2.73 7 0.02801 0.45385
4.03 7 0.00167 0.47581
4.14 7 0.00193 0.47581
2.11 7 0.00152 0.45038
2.97 7 0.00471 0.47967
3.35 7 0.01321 0.50427
4.07 7 0.00184 0.48361
5.261 7 0.00153 0.47200
3.71 6 0.00130 0.46825
4.22 6 0.00612 0.45385
[114]Open in a new tab
Key targets in the possible mechanisms of SCT in the treatment of AD or PD
Targets with a greater degree value (more than 13) or enriched in AD or
PD KEGG pathway were selected to be docked with constituents from
neuroprotective sub-fractions by Surflex-Dock (Total Score results
showed as [115]Fig 11).
Fig 11. Surflex-dock results of SCT constituents with key targets in total
score.
[116]Fig 11
[117]Open in a new tab
The Total Score results indicated that many vital targets involved in
AD or PD, for example AChE (ACHE), MAO-B (MAOB), GluN2B-NMDAR (GRIN2B),
adenosine A2A receptor (A2AR, ADORA2A) and cannabinoid receptor 2
(CB2R, CNR2), have potent predicted binding activity with several SCT
constituents. Moreover, SCT constituents as [118]Fig 12 showed have
higher Total Score with corresponding targets, which indicated that
they are more possible to affect the targets to exert neuroprotective
efficacy for the treatment of AD or PD.
Fig 12. Constituents from SCT with their possible targets predicted by total
score in surflex-dock.
[119]Fig 12
[120]Open in a new tab
Discussion
The outcomes of this study demonstrated the efficacies of SCT
neuroprotective sub-fractions in scavenging DPPH radical, inhibiting
AChE, MAOs and NMDAR by experiments performed using in vitro models.
The clearance percent of four sub-fractions could reach more than 40%
at 500 μg/mL. In contrast to the radical scavenging efficacy of SCT
extract in the previous study, E3 could present comparative performance
on scavenging DPPH radical [[121]46], which indicated the constituents
with antioxidant effect of SCT was enriched in the ethyl acetate
sub-fraction. Antioxidative effect is a known mechanism of certain
compounds eliciting neuroprotective action [[122]7, [123]47–[124]49].
The results further suggest that SCT has potential to treat
neurodegenerative disorders through its antioxidative effect.
The study also showed moderate inhibiting effect of SCT neuroprotective
sub-fractions on AChE, which was more potent than the effect of SCT
extract in previous study based on comparing their test concentrations
[[125]50]. The reduction of acetylcholine level in AD patient may cause
cognitive and memory impairments [[126]51]. Hence, AChE may accelerate
the progression of AD though promoting the fibration of β-amyloid
[[127]52]. Scopolamine, a muscarinic receptor antagonist, produces a
blocking of the activity of the muscarinic acetylcholine receptor, and
the concomitant appearance of transient cognitive amnesia and
electrophysiological changes, which resemble those observed in AD
[[128]53, [129]54]. There are certain AChE inhibitors approved for AD,
for example, donepezil and galanthamine. In fact, some studies had
described the neuroprotective effect of AChE inhibitor [[130]22,
[131]23]. Hence, inhibiting AChE appears to be an underlying mechanism
of the neuroprotective action of SCT.
The results of MAOs inhibiting assay showed, except E3, other three
active sub-fractions (P5, P6 and E1) all presented more than 40%
inhibition percent on MAOs at 1000 μg/mL, which is still more potent
than the inhibiting effect of SCT extract on MAO-A by comparing their
concentrations [[132]50]. Since MAO-A selective inhibitor (clorgyline)
could not inhibit the crude MAOs close to 100% at 50 μM, while MAO-B
selective inhibitor (pargyline) could inhibit it close to 100% at at 50
μM. This result indicated that enzyme activity of the crude MAOs used
in this study mainly contributed by MAO-B [[133]39, [134]45]. Excess
MAOs catalyze oxidation of amino substance causing the generation of
oxidative stress [[135]55, [136]56]. Moreover, a MAO-B
inhibitor–selegiline approved for PD was reported to suppress excess
GABA produced from astrocytes and restores the impaired spike
probability, synaptic plasticity, and learning and memory in the mice
[[137]24]. However, some clinical trials showed that the cognitive
function of the placebo group had no significant difference compared to
the group treated with selegiline for a long term therapy [[138]25,
[139]26]. Instead of irreversible inhibitor like selegiline, a
reversible MAO-B inhibitor (KDS2010) does not induce compensatory
mechanisms in a long term therapy, which further attenuated increased
astrocytic GABA levels and astrogliosis, enhanced synaptic
transmission, rescued learning and memory impairments in APP/PS1 mice
[[140]27]. Thereby, MAO-B is considered as a key target of SCT in the
treatment of AD or PD.
Furthermore, P5 and E1 fractions SCT presented a non-significant
inhibition of NMDAR-mediated current in hippocampal neurons of
Sprague-Dawley neonatal rats, which was more potent than the effect of
Zembrin^® on NMDAR-mediated current and consistent with previous
results [[141]57]. NMDAR, an ionotropic glutamate receptor, which
constitute a calcium-permeable component of fast excitatory
neurotransmission, have been verified to participate
neuro-physiologically in many cell signaling pathways resulting in
several neurological diseases. An NMDAR inhibitor–esketamine was
approved for depressive disorder ought to his rapid antidepressant
action. The previous studies showed the potential of SCT on treating
depressive disorder [[142]2, [143]4, [144]28–[145]33]. However, the
results of this study indicated that the influence on NMDAR of these
two fractions may be a subsequent effect resulted from affecting other
targets but not NMDAR. Thus leading to a possibility that the
anti-depressive action of SCT extract can be due to inhibition efficacy
of mesembrine and mesembrenone on phosphodiesterase-4 and serotonin
transporter [[146]58].
Therefore, results of the in vitro experiments indicated that there are
neuroprotective constituents in SCT could protect neurons to treat
neurodegenerative disorders by scavenging radicals, inhibiting AChE,
MAOs and NMDAR. Different sub-fractions represented different degrees
of influence on AChE, MAOs and NMDAR.
Moreover, the neuroprotective sub-fractions of SCT used to assess the
potential use to treat AD or PD, was further supported by network
pharmacology related methods applied in this study, which was also
supported by the observed influence of SCT extract on cognition
[[147]5, [148]6]. Among several neurodegenerative disorders, the
targets of AD or PD from database have most overlapping numbers with
the targets predicted by Polypharmacology Browser 2. It is understood
that the overlapping targets could be involved in memory, learning and
behavior related biological process and enrich in AD and PD
corresponding KEGG pathway. The network analysis and Surflex-Dock
results have indicated that some key targets, AChE, MAO-B,
GluN2B-NMDAR, A2AR and CB2R, can be influenced by SCT in the probable
treatment of AD or PD, and other constituents SCT or similar moieties
of close chemical structures, such as egonie, sceletium A4,
dihydrojoubertiamine, N-trans-feruloyl-3-methyldopamine,
N-methyldihydrojoubertinamine and so on, should be concerned to have
potential in affecting on corresponding targets ([149]Fig 12).
In this study, the primary purpose was to explore possible targets of
the neuroprotective SCT on neurodegenerative disorders by network
pharmacology. According to the identified constituents from SCT in our
previous study, the results of network pharmacology studies indicated
some potential targets (AChE、MAOs and NMDAR) for SCT. Therefore, the
neuroprotective SCT sub-factions were further tested in vitro for their
efficacy on the potential targets. Encouragingly, the results of the
fraction-targets in vitro experiments actually supported the network
pharmacology results in this study. However, different sub-factions
contained different natural products, the content of natural products
were also various, which resulted in the different effects of different
sub-factions to these potential target in this study. Certainly, in the
next stage, the further studies would carry out to explain the
bioactivity mechanism of different sub-fractions on their specific
targets.
Conclusion
SCT neuroprotective sub-fractions have moderate potency of scavenging
radicals, inhibiting AChE, MAOs and NMDAR, which are the possible
mechanisms of its neuroprotective effect. The identified and other
related constituents in SCT may have affects on biological systems to
alter AChE, MAO-B, GluN2B-NMDAR, A2AR and CB2R, to exert their
therapeutic potential in the probable treatment of AD or PD.
Abbreviations
SCT
Sceletium tortuosum
MPP^+
1-methyl-4-phenylpyridinium
AChE
Acetylcholinesterase
MAO
monoamine oxidase
NMDAR
N-methyl-D-aspartic acid receptor
AD
Alzheimer’s disease
PD
Parkinson’s disease
DPPH
1,1-diphenyl-2-picrylhydrazyl
A2AR
adenosine A2A receptor
CB2R
cannabinoid receptor 2
KEGG
Kyoto Encyclopedia of Genes and Genomes
Data Availability
All relevant data are within the paper.
Funding Statement
The author(s) received no specific funding for this work.
References