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Targeted Isolation of Indole Alkaloids from Streptomyces sp. CT37

Article in Molecules · March 2020

DOI: 10.3390/molecules25051108

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 molecules
Article
Targeted Isolation of Indole Alkaloids from
Streptomyces sp. CT37
Qing Fang 1 , Fleurdeliz Maglangit 1,2 , Morgane Mugat 3 , Caroline Urwald 3 ,
Kwaku Kyeremeh 4 and Hai Deng 1, *
1 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Walk,
Aberdeen AB24 3UE, Scotland, UK; r01qf16@abdn.ac.uk (Q.F.); r01fm16@abdn.ac.uk (F.M.)
2 Department of Biology and Environmental Science, College of Science, University of the Philippines Cebu,
Lahug, Cebu City 6000, Philippines
3 ENSAIA, 2 avenue de la forêt de Haye, 54505 vandœuvre lès Nancy, France;
morgane.mugat@orange.fr (M.M.); carolinurwald@gmail.com (C.U.)
4 Department of Chemistry, University of Ghana, P.O. Box LG56 Legon-Accra, Ghana; kkyeremeh@ug.edu.gh
* Correspondence: h.deng@abdn.ac.uk; Tel.: +44-1224-272953; Fax: +44-1224-272291




Received: 2 February 2020; Accepted: 26 February 2020; Published: 2 March 2020


Abstract: Four compounds (1–4) were isolated from the extracts of Streptomyces sp. CT37 using
bioassay in conjunction with mass spectrometric molecular networking (MN) driven isolation. Their
complete structures were established by high-resolution electrospray ionization mass spectrometry
(HR-ESIMS), and 1D and 2D nuclear magnetic resonance (NMR) data. Legonimide 1 was identified
as a new alkaloid containing a rare linear imide motif in its structure, while compounds 2–4 were
already known and their structures were elucidated as 1H-indole-3-carbaldehyde, actinopolymorphol
B, (2R,3R)-1-phenylbutane-2,3-diol, respectively. The biosynthetic pathways of 1–4 were proposed
based on the reported biogenesis of indole alkaloids in literature. Bioactivity tests for 1 and 2 revealed
moderate growth inhibition activity against Candida albicans ATCC 10231 with MIC95 values of
21.54 µg/mL and 11.47 µg/mL, respectively.

Keywords: legonimide; indole alkaloids; imide; Streptomyces sp. CT37; natural products; antifungal

1. Introduction
Microbial natural products (NPs) encompass several chemical structures that constitute a treasure
trove of high-value molecules, such as antibiotics, anticancer and antioxidant drugs [1]. Streptomyces a
prominent resource for natural product discovery contributing up to 75% of present day clinically used
antibiotics [2,3]. As part of the efforts of our laboratory to investigate microbial NPs with antibiotic
bioactivity from under-investigated environments, we have studied several Streptomyces isolates from
soil samples collected in Legon, Ghana [4–14].
Recently, metabolomics guided microbial NPs discovery processes have been widely used for
the prediction of bioactive metabolite structures and prioritization of extracts for isolations [15,16].
The method offers a route to deliver qualitative and quantitative data analysis of chemical space for
the metabolites present in complex bacteria fermentation extracts [9]. A useful analytical technique
that has been integrated into the metabolomics-guided microbial NPs discovery is HR-ESIMS and
associated MS/MS fragmentation data. However, the complexity of HR-ESIMS/MS data prevents
manual annotation of the presence of metabolites in the extracts. As such, several computing-based
data mining tools have been applied; one of the widely used is the Molecular Networking (MN)
database [17–20]. The MN database is a web-based server that is extremely useful for dereplicating
natural product structures by comparing both experimental HR-ESIMS (MS/MS) spectra with the

Molecules 2020, 25, 1108; doi:10.3390/molecules25051108 www.mdpi.com/journal/molecules

Molecules 2020, 25, 1108 2 of 10

database [17]. The method is useful for the effective dereplication and annotating of specialised
metabolite (SM) families and individual molecules.
The current study establishes mass spectrometric molecular networking (MN) in conjunction with
bioassay tests to guide isolation of the bioactive components from the Ghanaian isolate, Streptomyces sp.
CT37. The targeted isolation afforded four compounds: legonimide 1, 1H-indole-3-carbaldehyde 2,
actinopolymorphol B 3, and (2R,3R)-1-phenylbutane-2,3-diol 4.

2. Results and Discussion

Chemical profiling of the Streptomyces sp. CT37 strain was carried out in different cultivation
media [21]. Eight different fermentation broths (ISP2-ISP7, modified Bennett’s, starch casein, Table S1)
were selected based on the recommended medium for Streptomyces species, which differ with respect
to carbon source and salt concentration [22]. The extracts obtained in the eight broths were subjected
to disc diffusion bioassay tests using a panel of Gram-positive, Gram-negative, and fungal pathogens
(Table S2). Only the ISP2 extract showed inhibition zone (9.0 mm) against Candida albicans ATCC
10,231 (Figure S1). Subsequently, large-scale fermentation (6.0 L) was carried out in ISP2 medium,
followed by solid phase extraction (SPE) to yield six fractions, S1–S6. HRESIMS and molecular network
analyses of the fractions identified five major compound families: indole alkaloids, phenols, surfactins,
deferoxamines, phthalates, and cyclopeptides (Figure S2). Manual dereplication [23] using MS1 spectra
of compounds against available databases (AntiBase, The Natural Products Atlas [24]) identified
fraction S2 to harbor alkaloid metabolites that were not previously reported in literature (Table S3).
Disc diffusion assay of S2 against C. albicans ATCC 10,231 revealed zone of inhibition that was not
observed in other fractions. Hence, S2 was further purified by high pressure liquid chromatography
(HPLC) and led to the isolation of four compounds, one of which is a new alkaloid bearing a linear
imide motif, legonimide 1 (2.1 mg), and three known compounds: 2 (1.1 mg), 3 (1.0 mg), and 4 (0.8 mg).

2.1. Structure Elucidation

The structures of compounds 1–4 were established by analysis of the HRESIMS, 1D, and 2D NMR
data and by comparison with the reported data in literature [25–27] (Figure 1).

Figure 1. Compounds 1–4 isolated from Streptomyces sp. CT37 ISP2 extract; compound 1 with correlation
spectroscopy (COSY) (—) and key heteronuclear multiple bond correlation (HMBC) (→) correlations.

Compound 1 was isolated as a pale yellow amorphous solid. The HR-ESIMS spectrum of 1
exhibited a prominent peak at m/z 293.1280 [M + H]+ corresponding to the molecular formula of
C18 H16 N2 O2 (calcd for C18 H17 N2 O2 + 293.1285, ∆ = −1.70 ppm) possessing 12 degrees of unsaturation.
The 1 H nuclear magnetic resonance (NMR) spectrum in DMSO-d6 (Table 1) of 1 indicated the
presence of two exchangeable NH protons (δH 7.37, 10.96), two sp3 methylenes (δH 3.35, 3.46), nine
aromatic methines (δH 7.54, 7.34, 7.31, 7.28, 7.28, 7.21, 7.21, 7.06, and 7.02), and one sp2 methine
(δH 7.18). In addition to the signals corresponding to the above carbons, analysis of the 13 C NMR
and heteronuclear single quantum coherence (HSQC) spectra revealed the presence of six quaternary
carbons, including two carbonyls (δC 173.1, 172.3) and four aromatic groups (δC 136.6, 136.5, 127.3,
and 109.2).
Molecules 2020, 25, 1108 3 of 10

Table 1. 1 H and 13 C NMR assignments of legonimide 1 (600MHz, 298K, DMSO-d6 ).

Position 1H (mult., J in Hz) 13 C

1-NH 10.96 (1H, s) -

2 7.18 (1H, s) 123.6
3 - 109.2
4a - 127.3
4 7.54 (1H, d, J = 7.85) 118.7
5 7.02 (1H, t, J = 7.39) 118.5
6 7.06 (1H, t, J = 7.32) 120.8
7 7.34 (1H, d, J = 7.80) 111.3
7a - 136.5
8 3.46(2H, s) 32.4
9 - 173.1
10-NH 7.37 (1H, s) -
11 - 172.3
12 3.35(2H, s) 42.0
13 - 136.6
14,140 7.28 (2H, d, J = 7.67) 128.1
15,150 7.21 (2H, m) 126.4
16 7.31 (1H, m) 126.3

Thorough inspection of the 2D NMR spectra disclosed the presence of 4 substructures (A-D)
(Figure S3), including a 3H-subsituted indole, acetyl group (B), acetamide subunit (C), and one
monosubstituted aromatic ring (D) in the structure of 1. The monosubstituted phenyl group and
the ring in the indole moiety comprised two separate spin systems, H-14 through H-140 and H-4
through H-7, respectively, as indicated in the 1 H-1 H correlation spectroscopy (COSY) spectrum.
The heteronuclear multiple bond correlation (HMBC) from H-8 (δH 3.46) to the carbonyl carbon at C-9
(δC 173.1), and NH-10 (δH 7.37) to carbonyl C-11 (δC 172.3) and methylene carbon at C-12 (δC 42.0),
established the presence of the acetyl and acetamide subunits, respectively. The HMBC correlation
from H-12 (δH 3.35) to C-13 (δC 136.6) and C-14 (δC 128.1) established the connectivity of substructure C
to D at C-13. The cross peaks from H-8 to C-2 (δC 123.6), C-3 (δC 109.2), and C-4a (δC 127.3) confirmed
the connection of the indole subunit (substructure A) to the acetyl group (substructure B) at C-3.
The structure of 1 was further corroborated by the long-range HMBC correlation from NH-10 to C-4a
and C-12; thereby, the imide group flanked the 3H-substituted indole moiety and the monosubstituted
benzene ring (Figures S9–S11). Hence, the structure of 1 was elucidated representing a new alkaloid
containing a linear imide motif as N-(2-(1H-indol-3-yl)acetyl)-2-phenylacetamide and named as
legonimide in association with Legon, Ghana.
The free imide group in 1 can adopt three conformations: cis-trans, trans-trans and cis-cis [28]
(Figure S12). There was no HMBC correlation from NH-10 to C-8, while correlation was observed from
NH-10 to C-12 (Figure S13), suggesting that the imide motif in 1 is likely a cis-trans conformer. This
observation is in agreement with those reported for imide-containing natural products that the cis-trans
conformation is the most stable [29–32].
Compound 2 was identified as 1H-indole-3-carbaldehyde previously isolated from plants [33],
marine sponge [34], and several Streptomyces species [35,36] (Figures S14–S18, Table S4). Compound
3 was consistent with the reported data for actinopolymorphol B from actinopolymorpha rutilus [27]
(Figures S19–S23, Table S4). Compound 4 was 1-phenylbutane-2,3-diol found in several plants [37].
A threo isomer of 4 was previously isolated from a actinomycete Williamsia sp. MCCC 1A11233
(Figures S24–S28) [38]. The configuration of 4 was assigned upon careful comparison of the NMR data
with those reported for the four synthetic stereoisomers, two threo (2S,3R and 2R,3S) and two erythro
(2S,2S and 2R,3R) [26,37] (Table S5). The NMR data of 4 was consistent with the reported data for
threo isomers. Furthermore, comparison of the optical rotation of 4 with the synthetic stereoisomers
Molecules 2020, 25, 1108 4 of 10

pointed to (2R,3R)-1-phenylbutane-2,3-diol [26,37] (Tables S5 and S6). However, we could not exclude
the possibility that compound 4 could be a mixture of enantiomers.

2.2. Proposed Biosynthesis Pathway of 1–4

Based on the reported biogenesis of indole alkaloids from bacteria [11,13,35–38], we proposed the
biosynthesis pathways of compounds 1–4 (Figure 2). Inspection of the structure of legonimide 1 led us
to speculate that it is biosynthesized from the condensation reaction between the phenyl-acetyl-CoA,
a common intermediate, and the indole-3-acetamide 12, an oxidative product of l-tryptophan 6
catalysed by tryptophan 2-monooxygenase [39]. Indole-3-carbaldehyde 2 was found as part of
pathogen defense in cruciferous plants. Biochemical studies indicated that 2 is synthesized from
tryptophan 6 via the intermediate indole-3-acetonitrile by a cytochrome P450 enzyme CYP71B6 in
Arabidopsis thaliana [40]. Compound 2 is also a metabolite produced by human gastrointestinal
bacteria, particularly species of the Lactobacillus genus [41] and various Streptomyces species [35,36].
However, the enzyme(s) responsible for the production of 2 from bacterial origins remain poorly
understood. Compounds 3 and 4 may derive from the carboligation reaction between pyruvate and
indole-3-pyruvate 9 or phenylpyruvate 7 catalysed by a thiamin-diphosphate dependent enzyme to
yield α-hydroxyl acyloins 8 and 11, respectively [11,13]. Possible spontaneous decarboxylation of 8
and 11 could lead to the production of racemers 3 and the intermediates 40 . One of the racemic 40
may be further reduced into 4 [11]. Interestingly, analysis of GNPS network allowed the identification
of ions likely correlated to four intermediates (7, 40 , 9, and 12) in indole alkaloids and phenyls
clusters, suggesting the proposed biosynthetic pathways may be plausible. However, the rest of the
proposed intermediates cannot be observed, possibly due to their instable natures with the tendency
of degradation.

Figure 2. Proposed biosynthesis pathway of 1–4.

2.3. Biological Test

The activity of 1–4 was evaluated against Candida albicans ATCC 10,231 (Table 2). Compounds 1
and 2 showed moderate activity with minimum inhibitory concentration (MIC) values of 21.54 µg/mL
and 11.47 µg/mL, respectively (Figure S29), while no activity was observed for 3 and 4 at the highest
concentration tested (50 µg/mL).
Molecules 2020, 25, 1108 5 of 10

Table 2. Minimum inhibitory concentration of compounds 1–4 against C. albicans 10231.

C. albicans ATCC 10231

MIC (µg/mL)
Legonimide 1 21.54
Compound 2 11.47
Compound 3 >50
Compound 4 >50
Ampicillin 3.193
Tetracycline 0.3615

3. Experimental

3.1. General Experimental Procedures

HR-ESIMS data were obtained in positive ESI mode with a mass range of 100–2000 m/z (maximum
resolution 30,000) on a Thermo Scientific MS system (LTQ XL/LTQ Orbitrap Discovery, Waldbronn,
Germany). Reserpine (m/z 609.2807) was used as a lock mass for internal calibrant during data
acquisition. The following instrument parameters were used: capillary voltage 45 V, spray voltage
4.5 kV, capillary temperature 200 ◦ C, auxiliary gas flow rate 10–20 arbitrary units, and sheath gas
flow rate 5 arbitrary units; furthermore, an automated full dependent MS-MS scan was applied.
The injected samples were chromatographically separated in Thermo Instrument HPLC system
(Accela PDA detector (Waldbronn, Germany), Accela PDA autosampler and Accela Pump, Agilent
Technologies, Waldbronn, Germany) using a C18 (Sunfire 150 × 46 mm) column. The gradient elution
for separation was CH3 CN/H2 O with 0.1% trifluoroacetic acid (TFA) (from 0% to 100% for 30 min, flow
rate, 1.0 mL/min, UV detection max 340 nm).
1D and 2D NMR spectra were acquired on a Bruker AVANCE III HD 600 MHz (AscendTM14.1 Tesla,
UK) with Prodigy TCITM cryoprobe at 298 K in CD3 OD and DMSO-d6 (Goss Scientific, Massachusetts,
MA, USA). Trimethylsilane (TMS) was used as an internal standard.
The optical rotation was measured using ADP 410 polarimeter (Bellingham + Stanley Ltd.
2007, Kent, UK) equipped with a light emitting diode and interference filter. A Fourier transform
infrared (FTIR) spectrometer (2013, PerkinElmer, UK) equipped with an Attenuated Total Reflection
(ATR, PerkinElmer, Buckinghamshire, UK) diamond cell for sample loading was used for infrared
spectroscopy experiments.

3.2. Biological Material Collection and Identification

The soil bacterium Streptomyces sp. CT37 was isolated from the rhizosphere sample collected near
the root of a Caesalpinoideae tree (Tamarindus Indica, Africa) growing in the Botanical Gardens of the
University of Ghana, Legon (5◦ 390 32.72” N, 0◦ 110 55.26” W). The pure strain was cultured following the
protocol given by the International Streptomyces Project (ISP) at 28 ◦ C, supplemented with nalidixic
acid and nystatin (25 mg/L) [22].

3.3. Smale Scale Cultivation of Streptomyces sp. CT37

The small scale culture (50 mL) of Streptomyces sp. CT37 was prepared by inoculating a single
colony of the bacteria in eight different liquid culture media (ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, modified
Bennett’s, and starch casein) (Table S1), and incubated at 28 ◦ C and 180 rpm for 7 days (Incu-shake
FL16-2, SciQuip, Shrewsbury, UK). Subsequently, Diaion®HP-20 (3 g/50 mL, Mitsubishi Chemical
Co., Binasco, Italy) was added to the fermentation cultures and incubated for the next 18–24 h at the
same temperature and in the same shaking conditions. The culture broths were filtered under vacuum
(Buchi pump V100, Buchi, Manchester, UK), and the HP-20 resin was rinsed with Milli-Q water and
extracted exhaustively with methanol (MeOH, Fisher Chemical HPLC grade). All the methanol extracts
were combined, and concentrated under reduced pressure (Buchi Rotavapor R200, Buchi, Manchester,
Molecules 2020, 25, 1108 6 of 10

UK) to generate the total crude extract (6.1 g). All eight crude extracts were then subjected to disc
diffusion tests.

3.4. Disc Diffusion Assay

Disc diffusion assay was carried out following Kirby-Bauer disc diffusion susceptibility test
Protocol [42]. Agar plates were inoculated with a standardized inoculum of pathogens (Candida albicans
ATCC 10231, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus
ATCC 25923, Streptococcus B. ATCC 12386, Staphylococcus epidermidis ATCC 35984, Enterococcus faecalis
ATCC 29212, Table S2). Thereafter, filter paper discs (6 mm) impregnated with the test extracts
(1 mg/mL) were placed on the agar surface. Oxytetracycline (30 µg/mL, Oxoid, Winchester, UK) was
used as antibiotic control, while the growth media and Milli-Q water were used as negative control.
The petri dishes were incubated at 37 ◦ C for 18 h (Memmert INB200, Buchenbach, Germany), and the
diameters of inhibition growth zones were measured. The ISP2 culture medium showed inhibition
zone against C. albicans; hence it was scaled up.

3.5. Large-Scale Fermentation of Streptomyces sp. CT37

The seed culture of Streptomyces sp. CT37 (50 mL) was prepared following the same inoculation
procedures as the small-scale cultivation. The seed culture was then used to inoculate (1:100)
twenty-four 2.0-L baffled flasks (polycarbonate CorningTM , Flintshire, UK) containing 250 mL ISP2
broth each. The flasks were plugged with foam stoppers (polyurethane Fisherbrand™, Loughborough,
UK). Fermentation, incubation, and extraction of the cultures were carried out as described above.
The methanol extracts were combined, and evaporated to dryness under reduced pressure to yield
6.1 g of total crude extract, which was then fractionated using Strata®C18-E solid phase extraction
(SPE) (55 µm, 70 Å, 20 g/60 mL) cartridges. The SPE column was equilibrated with four column
volumes (CV) of methanol and Milli-Q water prior to loading the crude sample onto the column.
The column was then eluted stepwise with solvent mixtures of decreasing polarity (8 CV/solvent
mixture): Milli-Q water, 25% MeOH, 50% MeOH, 75% MeOH, 100% MeOH, and 100% MeOH with
0.05% TFA (Acros Organics, Morris, NJ, USA). The eluents were collected separately and labeled as
fractions S1–S6. All the fractions (S1–S6) were concentrated under reduced pressure and subjected to
HR-ESIMS analysis (0.1 mg/mL).

3.6. Feature Based Molecular Networking

The MS/MS data of S1–S6 fractions were converted from .RAW to .mzXML files using the
ProteoWizard MSconvert software [43]. A molecular network was generated using Feature-Based
Molecular Networking (FBMN) workflow [44] on Global Natural Product Social networking (GNPS) [17]
(https://gnps.ucsd.edu). The mass spectrometry data were preprocessed with MZMINE v2.38 [45] and
exported to GNPS for FBMN analysis. The data were filtered to remove all MS/MS fragment ions within
±17 Da of the precursor m/z. MS/MS spectra were window filtered by choosing only the top 6 fragment
ions in the ±50 Da window throughout the spectrum. The precursor ion mass tolerance was set to
0.02 Da with an MS/MS fragment ion tolerance of 0.02 Da to create consensus spectra. The consensus
spectra that contained fewer than four spectra were discarded. The edges were filtered to ensure a
cosine score above 0.65 and more than four matched peaks. The edges between two nodes were kept
in the network if each of the nodes appeared in each other’s respective top 15 most similar nodes. The
spectra in the network were then searched against GNPS spectral libraries [46] and annotated by the
DEREPLICATOR [23]. The library spectra were filtered in the same manner as the input data, where
a score above 0.65 and at least 4 matched peaks are required. The created molecular network was
visualized using Cytoscape software v3.4.0 (Seattle, WA, US).
Molecules 2020, 25, 1108 7 of 10

3.7. HPLC Isolation

The compounds of interest were identified in S2 fraction, hence further fractionation was carried out
in this fraction using High Pressure Liquid Chromatography (HPLC, Agilent Technologies 1260 infinity,
Waldbronn, Germany). The purification was performed using a linear gradient from 10% H2O:MeOH
(95:5) to 100% MeOH for 40 min with a solvent flow rate of 1.5 mL/min (C-18 ACE 10 µM 10 × 250 mm
column). As a result, 1 (2.1 mg), 2 (1.1 mg), 3 (1.0 mg), and 4 (0.8 mg) were isolated.
Legonimide 1: pale yellow amorphous solid; UV (CH3 OH): 250 nm; 1 H, 13 C NMR data, see Table 1;
HR-ESIMS (+) m/z 293.1280 [M + H]+ (calcd. for C18 H17 N2 O2 + , 293.1285; ∆ = −1.70 ppm).
1 H-indole-3-carboxaldehyde 2: pale yellow solid; UV(CH3 OH) 240, 260, 280 nm; 1 H, 13 C NMR data,
see Table S4; HR-ESIMS (+) [M + H]+ = 146.0603 (calcd. for C9 H8 NO+ 146.060, ∆ = 1.37 ppm).
Actinopolymorphol B 3: yellow solid; UV (MeOH) max nm 220, 280; [α]25 +27.3 (c = 0.5, CH3 OH); 1 H,
13 CNMR data, see Table S4; HR-ESIMS (+) [M + H]+ = 204.1023 (calcd. for C12 H14 NO2 + , 204.1019,
∆ = 1.96 ppm).
(2R,3R)-1-phenylbutane-2,3-diol 4. yellow oil; [α]25 D +12.1 (c 0.5, MeOH); UV (MeOH) max nm 250; IR
νmax (cm−1 ) 3410, 2931, 2874, 1712, 1645, 1409, 1207, 1012, 802, 754; 1 H, 13 C NMR data, see Table S5;
HR-ESIMS (+) [M + H]+ = 167.1070 (calcd. for C10 H15 O2 + , 167.1067, ∆ = −1.80 ppm).

3.8. Minimum Inhibitory Concentration

Minimum inhibitory concentrations (MIC) of 1–4 against C. albicans ATCC 10,231 were determined
using a conventional broth dilution assay in accordance with standards recommended by the
National Committee for Clinical Laboratory Standards (NCCLS) [47] and as described previously [7].
The antibiotics ampicillin (Sigma) and tetracycline (Sigma) were used as standards. The absorbance
was recorded after 24 h (OD600 ) in a SpectraMax ABS Plus (Molecular Device) plate reader. The MIC
was defined as the lowest concentration of compound that inhibited ≥ 95% of the growth of C. albicans
after overnight incubation.

4. Conclusions
The bioassay and molecular network-assisted isolation afforded four alkaloids 1-4 from the
Ghanaian soil bacterium, Streptomyces sp. CT37. Their structures were deduced by analysis of the
HRESIMS, 1D, and 2D NMR. Legonimide 1 was identified as a new alkaloid bearing the imide motif in
its structure, while 2-4 were known and their structures were elucidated as 1H-indole-3-carbaldehyde,
3-hydroxy-4-(1H-indol-3-yl) butan-2-one, and (2R,3R)-1-phenylbutane-2,3-diol, respectively. Alkaloids
1 and 2 exhibited moderate inhibition against C. albicans ATCC 10,231 with MIC values of 21.54 µg/mL
and 11.47 µg/mL, respectively.

Supplementary Materials: The supplementary materials are available online.

Author Contributions: Q.F., M.M., C.U., and F.M. formal analysis and investigation. H.D. and K.K. funding
acquisition and methodology. Q.F., and H.D. writing original draft. K.K., F.M., and H.D. review and editing.
H.D. supervision and project administration. All authors have read and agreed to the published version of
the manuscript.
Funding: Q.F. is grateful to the University of Aberdeen Elphinstone Scholarship and Scottish Funding
Council/ScotCHEM for financial support through the PEER/PERCE Funding. F.M. thanks the University
of the Philippines for the Faculty, Reps, and Staff Development Program (FRAS DP) for the PhD grant fellowship.
H.D. and K.K. thank the financial supports of Leverhulme Trust-Royal Society Africa award (AA090088) and
the jointly funded UK Medical Research Council-UK Department for International Development (MRC/DFID)
Concordat agreement African Research Leaders Award (MR/S00520X/1).
Conflicts of Interest: The authors declare no conflict of interest.
Molecules 2020, 25, 1108 8 of 10

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