Results in Chemistry 2 (2020) 100052
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Results in Chemistry
j ourna l homepage: www.e lsev ie r .com/ locate / rechemDifferential constituents in roots, stems and leaves of Newbouldia laevis
Thunb. screened by LC/ESI-Q-TOF-MSAffo Dermane a, Kafui Kpegba b, Kodjo Eloh c,⁎, Dorcas Osei-Safo d, Richard K. Amewu d, Pierluigi Caboni c
a Department of Pharmaceutical Sciences, University of Lomé, 01 BP 1515 Lomé 01, Lomé, Togo
b Laboratoire de Chimie Organique et des Substances Naturelles (Lab COSNat), Département de Chimie, Faculté Des Sciences, Université de Lomé, 01 BP 1515 Lomé 01, Lomé, Togo
c Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
d Chemistry Department, University of Ghana, P.O. Box LG 56, Legon, Ghana⁎ Corresponding author at: Via Ospedale 72, 09124 Cag
E-mail address: kodjoeloh@outlook.com (K. Eloh).
https://doi.org/10.1016/j.rechem.2020.100052
2211-7156/© 2020 The Authors. Published by Elsevier B.Va b s t r a c ta r t i c l e i n f oArticle history:
Received 17 March 2020
Accepted 17 May 2020
Keywords:
Newbouldia laevis
Phytochemistry
Antitumor natural substances
Antimalarial compound
LC-MSNewbouldia laevis (P. Beauv.) Seem. or “Boundary Tree” is amedium sized angiosperm in the Bignoniaceae family.
It is native to tropical Africa, especially used inWest Africa tomark property boundaries in rural areas. Despite its
intensive use in folk medicine, the plant chemical composition is under investigated. The study is aimed at iden-
tifying the chemical constituents of N. laevis crude plant extracts by high-performance liquid chromatography
(HPLC) on linewith anuntargeted high-resolutionmass spectrometry. The usedHPLCmethod showed to be suit-
able for the determination of withasomnine, newbouldine, and lapachol derivatives together with other known
bioactive compounds like phytosterols and triterpenoids. The importance of these chemical constituents is
discussed with respect to the role of N. laevis in West African ethnomedicine.
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).1. Introduction
Approximately two-thirds of the world biological diversity is found
in tropical zones, mainly developing countries. Newbouldia laevis (P.
Beauv.) Seem. (Bignoniaceae) is a valued herb in tropical Africa medi-
cine, and as such was used and grown for centuries in Togo (West
Africa). It is a medium sized angiosperm that grows to a height of
about 10 m. From the six most used plant extracts to treat life-
threatening disease malaria in Togo, N. laevis was reported in a study
to be the most active [1]. At 12.6 μg/ml, N. laevis inhibited 50% growth
of the deadliest unicellular protozoan parasite of humans; Plasmodium
falciparum that causes malaria. Furthermore, N. laevis is frequently
cited to treat several diseases including diarrhea, dysentery, some sexu-
ally transmitted diseases or used as anthelmintic [2].
Medicinal value of plants lies in some chemical substances that pro-
duce a definite physiological action on human body. Recently, Kuete
et al. reported ten compounds isolated from the methanolic root bark
extract of N. laevis that demonstrated strong antimicrobial activity
against twenty-one microorganisms bacterial species as well as three
yeasts [3]. Another study identified furanonaphthoquinones, atraric
acid and a benzofuran derivative in the plant stem barks [4].More, alka-
loids, saponins, flavonoids, glycosides, tannins and cyanides are regu-
larly reported to be present in N. laevis extracts [5–7]. Despite liquidliari, Italy.
. This is an open access article underchromatography (LC) and mass spectrometry (MS) have become a
common tool for investigating quantity, quality and chemical diversity
of plant metabolites, data on N. laevis constituents LC-MS profiles are
still lacking. To speed up future studies involving N. laevis, such as me-
tabolite characterizations, updating metabolites database with relative
retention times and mass spectra is crucial.
Over the past few years, liquid chromatography coupled with mass
spectrometry (LC–MS) applications in natural products analysis have
been growing dramatically because of the ever more improved separa-
tion and detection capabilities of LC–MS apparatuses. In particular,
novel high-resolution hybrid instruments linked to high-performance
LC and the hyphenations of LC–MS with other separation or analytical
techniques greatly lead to unequivocal identification and highly sensi-
tive quantification of natural products at trace concentrations in com-
plex matrices [8,9]. LC-MS proved to be a very useful tool and is
largely applied to the characterization of plant secondary metabolite.
In West Africa, medicinal plants recipes are mostly prepared as de-
coction, infusion, maceration in water, ethanol and methanol or sauce
[10]. More, varying amounts and combinations of compounds present
in the different parts of the plant, roots, stems and leaves could explain
the various therapeutic effects. Thus, understanding the bio-distribution
of these secondary metabolites in roots, stems and leaves is important
to understanding pharmacological efficiency. Therefore, it is mandatory
to determinate most used medicinal plants chemical composition from
different organs using different solvent extractions (different polarity).
In our ongoing activities in medicinal plants chemical characterizationsthe CC BY license (http://creativecommons.org/licenses/by/4.0/).
2 A. Dermane et al. / Results in Chemistry 2 (2020) 100052[11,12], this current study was carried out to compare the chemical
composition of Newbouldia laevis leaves, stem and root barks found in
West Africa by an LC-MSmetabolomics platform. Column chromatogra-
phy and fractionation methods were performed to optimize LC-MS
identification of some major components. Identified compounds were
discussed accordingly to reported pharmacological effects of N. laevis.2. Material and methods
2.1. Plant material
N. laevis leaves, roots and stem barks were harvested at Tsévié
(Togo), in December 2014. Plant material was identified by one of the
authors and confirmed by a botanist, Koffi Akpagana (Botany Depart-
ment, University of Lomé). A Voucher specimen was deposited in the
department Herbarium Centre under the number 233.2.2. Chemicals and reagents
Ethanol, methanol, petroleum ether, acetone, formic acid and CDCl3
were of analytical grade and acquired from Sigma Aldrich (Milan, Italy).
Analytical standards were gently provided by the laboratory or syn-
thetic chemists.2.3. Sample extraction
2.3.1. Hydroethanolic plant extracts
In the face of the reports on the most popular extraction method
used, N. laevis hydroethanolic extract was investigated. Different parts
were washed free of dust, dried in laboratory conditions and reduced
to powder with a Thomas Muler mill (4 series, reference 3375 E20).
Plant powders were subsequently macerated with an ethanol-water
(80/20, v/v) mixture for 72 h. Following proportions were used: 450 g
of leaves in 3 l of solvent mixture; 448 g of stem barks in 3 l; and
429 g of root barks in 2.5 l. Supernatants were collected through cotton
beforefiltration using 0.40 μm filter papers. Filtrates were evaporated to
dryness under vacuum at 40 °C and kept in dark falcons at −20 °C for
fractions, supernatants and pellets preparations.
2.3.2. Methanolic plant extracts
For a comprehensive metabolomics phytochemical analysis, 3 g of
each plant part powders previously obtained were extracted with
30 ml of methanol in 50 ml falcons. Extraction was optimized by
ultrasonication for 30 min then kept for 24 h at room temperature.
Methanol supernatant was then filtered by 0.40 μm microfilter after
centrifugation at 2000 rpm for 10 min. Crude extracts concentrations
were calculated by evaporating an aliquot of 200 μl of each extract
under a gentle nitrogen stream. Diluted samples to 5 mg/ml were ana-
lyzed by LC/ESI/QTOF.
2.3.3. Supernatants and pellets preparation: fractionation by precipitation
in cold ethanol
Hydroethanolic extracts ofN. laevis stemand root barkswere used to
obtain two fractions of pellet and supernatant. To 20 g of each extract,
200 ml of ethanol-water 75/25 (v/v) mixture was added and kept at
4 °C. After 24 h, the mixture was centrifugated at 1500 rpm for
15min. The supernatantwas then collected and the resulting pellet dis-
solved in the same volume of solvent ethanol-water mixture, cooled at
4 °C to be treated under the same conditions as above. The two superna-
tants were mixed and evaporated under vacuum at 40 °C as well as the
pellet to obtain the supernatant and pellet fractions. Samples were kept
in freezer at −20 °C until chemical analysis.2.4. Column chromatography fractionation
Hydroethanolic root and stem barks of N. laevis extracts were sepa-
rated by column chromatography. Following solvent systems were
used to elute the different compound classes: petroleum ether, acetone,
ethanol and methanol. Aliquots of 20 g of extracts were applied on the
top of a 60 × 5 cm column, containing silica gel (reference 60GF254,
0.040–0.060 mm, activated at 150 °C for 3 h) packed with petroleum
ether. As eluent, we used approximately 250ml of every mixture of pe-
troleum ether: acetone (2.5:97.5) then raising progressively the gradi-
ent to 75:25 followed by pure ethanol and finally pure methanol. The
collected fractions were submitted to thin-layer chromatography
(TLC), using as mobile phase a mixture of petroleum ether, acetone
and methanol. Similar fractions based on their TLC profile were mixed
for chemical analysis.
2.5. Liquid chromatography coupled to mass spectrometry LC-MS/ESI/QTOF
Plant extracts were analyzed by reverse-phase liquid chromatogra-
phy on an Agilent 1200 series LC system using a Kinetex EVO C18,
100 Å, 5 μm, 150 × 2.1 mm (Phenomenex, Castel Maggiore, Italy). The
LC conditions were as follows: flow rate: 0.3 ml/min; solvent A: 0.1%
formic acid in bi-distilled water; solvent B: methanol; and gradient
was from 10% to 100% B over 10 min and kept at this level for 10 min.
Four microliters of every sample were then analyzed by Electrospray
ionization in positive mode using an Agilent 6520 Quadrupole (Q)-
Time of Flight (TOF) mass spectrometry (MS). Mass spectral data were
acquired in them/z range of 100–1500 amu with an acquisition rate of
1.35 spectra/s, averaging 10,000 transients. The source parameters
were adjusted as follows: drying gas temperature 250 °C, drying gas
flow rate 5 L/min, nebulizer pressure 45 psi. Based on the original acqui-
sition files, we performed a pre-processing stepwithMetAlign software
used for automated baseline correction and alignment of all extracted
mass peaks across all samples. Results were stored as CSV file. ESI/
QTOF MS data were then analyzed using the molecular feature extrac-
tion algorithm of the MassHunter Workstation software (version B
03.01 Qualitative Analysis, Agilent Technologies, Santa Clara, CA, USA).
The molecular feature extraction algorithm took all ions into account
exceeding 1000 counts with a charge state equal to one. Blank runs
showed maximum 8 features with intensity threshold at 1000 counts.
Isotope grouping was based on the common organic molecules model.
Features were compared to reported compounds from N. laevis and in
the Metlin database [13].
On the Q-TOF-MS/MS scan dependent; MS/MS autoswitch experi-
ments were performed sequentially on the two main ions recorded by
LC/ESI/MS. The combination of quadrupole ion-selectivity with the full
scan sensitivity of the TOF analyzer allowed the on-line recording of
autoswitch MS/MS experiments using an energy of 40 eV. In this
study, up to two different co-eluting precursor ions could be selected
for further MS/MS experiments. Nitrogen was used as collision gas.
For accurate mass measurements in MS/MS, the [M + H]+ precursor
ion or a characteristic fragment ion of some selected compounds were
used as lock mass.
2.6. Nuclear magnetic resonance (NMR)
Identification of a main compound of TLC single spot sample from
the column chromatographic analysis was carried using nuclear mag-
netic resonance (NMR) spectroscopy. One and two-dimensional NMR
spectra were recorded at 298 K using a VARIAN Spectrometer (model
Mercury Plus) operating at 500 MHz for the 1H nucleus and the
125 MHz for the 13C nucleus using the standard pulse sequences avail-
able in the Varian software. NMR spectra were measured as solutions
in CDCl3 in 5 mm o.d. tubes. Chemical shifts (d) were expressed in
parts per million.
A. Dermane et al. / Results in Chemistry 2 (2020) 100052 3
Table 1
Different studied solvent extracts with respective abbreviations.
Extraction Abbreviation
SBME Stem bark methanolic extract
SBS Stem bark hydroethanolic extract supernatant
RBP Root bark hydroethanolic extract pellet
RBME Root bark methanolic extract
RBS Root bark hydroethanolic extract supernatant
RBETOH Root bark ethanolic extract
RBWE Root bark water extract
SBP Stem bark hydroethanolic extract pellet
SBWE Stem bark water extract
LTE Leaves hydroethanolic extract
LME Leaves methanolic extract2.7. Melting point
Melting point of the isolated compoundwas determined on a Koffler
melting point apparatus for samples contained in an open capillary tube
in an electrically heated metal block apparatus.(a)
(b)
Fig. 1. LC-MS chromatograms of N. laevis st3. Results and discussions
Mass spectrometry has become a primary tool for metabolite identi-
fication and quantification in plant research. However, both the size and
the complexity of the data produced by this experimental technique im-
pose great computational challenges for data analysis. In recent years,
numerous web-based tools aimed at supporting mass-spectrometry-
based metabolite profiling and data sharing between analytic laborato-
ries [14–16]. This work applied available data analysis tools like online
XCMS [14], Metlin [15] and MetaboAnalyst 3.0 [16] to explore chemical
composition of a widely used tropical medicinal plant,N. laevis different
solvent crude extracts (Table 1) and fractions by mass spectrometry
coupled to quadrupole time of flight detector. Using this method,
high-resolutionmass spectrometry datawas uploaded online for poten-
tial structure identification. Different evidences were used to propose
probable structures herein. Desirable additional evidence such as chro-
matographic retention behavior for available compound standards
were checked. When important, automatic MS/MS experiments for
fragments and ionization behavior were made to confirm candidate
structures [17].em bark pellet (a) and root pellet (b).
4 A. Dermane et al. / Results in Chemistry 2 (2020) 100052Working with water and methanol gradient mobile phase, twelve
crude extracts were analyzed for their potential bioactive secondary
metabolites determination by high-performance chromatography.
Sample chromatograms are reported on Figs. 1–5. In 20 min, more
than fifty metabolites were successfully separated and detected in
each solvent extract after an electrospray ionization. Fig. 1 shows chro-
matograms obtained from N. laevis root and stem barks hydroethanolic
pellets. Similar metabolites may be present in those two extracts with
different levels.N. laevis root and stem barks pellets share similar bioac-
tivity andmay be used alternatively for the treatment of some ailments.
Unlike pellets, stem and root barks hydroethanolic supernatants (Fig. 2)
were different either qualitatively and quantitively in respect to chem-
ical composition. For instance, filtering hydroalcoholic extracts before
uptake might eliminate some metabolites with positive or negative ef-
fects on patients. Chromatograms on Figs. 3 and 4 compare ethanolic
and water extracts of N. laevis root and stem barks. Fewer components(a)
(b)
Fig. 2. LC-MS chromatograms of N. laevis stem bawere detected compare to hydroethanolic extracts. For a better thera-
peutic synergistic effect of the plant, hydroethanolic extraction could
be a better choice over aqueous or ethanolic extract.
After the establishment of technologies for high-throughput protein
analysis (proteomics), gene expression analysis (transcriptomics) and
DNA sequencing (genomics), metabolomics is now evolving. Metabolo-
mics is the term used for essentially comprehensive, nonbiased, high-
throughput analyses of complex metabolite mixtures typical of plant
extracts [18]. Herein, N. laevis leaves chemical composition was
confronted to stem and root barks by a metabolomics study. Methanol
was used because it is a good solvent for a holistic extraction [19]. Ex-
traction was assisted with an ultrasonicator prior to mass spectrometry
analysis. Fig. 5 illustrates chromatograms of the plant leaves, root and
stem barks while Fig. 6 shows the quantitative chemical compositional
variation of the three extracts under study. The first four identified com-
pounds in stem and root barks extracts showed level differences but therk supernatant (a) and root supernatant (b).
A. Dermane et al. / Results in Chemistry 2 (2020) 100052 5
(a)
(b)
Fig. 3. LC-MS chromatograms of N. laevis stem bark ethanolic extract (a) and root ethanolic extract (b).extracts demonstrated similar quantitative composition for the other
components. Leaves extracts showed different levels of the all metabo-
lites respect to stem and root barks. Many studies reported similar sec-
ondary metabolites levels in root and stem showed [20]. We report in
supporting information (Figs. S4–23) mass spectra and extracted ion
chromatograms of the most important constituents detected.
From this preliminary analysis of those chromatograms, we noticed
matrix effect or interference in response due to the presence of
coelution of some main compounds. When using ESI for analyzing ex-
tracts of complicated matrices, although, LC–MS is one of the most sen-
sitive and selective analytical techniques, it suffers from matrix effects.
Matrix effects could cause either a loss in response (ion suppression)
or an increase in response (ion enhancement). As a matter of fact, we
performed a column chromatography on the hydroethanolic root and
stem barks extracts to eliminate some invasive components. Using
thin-layer chromatography, similar fractions were mixed to optimize
LC-MS compound identification. A pure compoundwas successfully iso-
lated and characterized by: 13C and 1H NMR spectroscopy (Table S1 and
Figs. S1–2) and 2D NMR spectroscopic (Fig. S3), melting point and high-
resolution mass spectrometry. The pure isolated compound was subse-
quently identified as beta-sitosterol. The effect of beta-sitosterol onplasma insulin and glucose levels in normo- and hyperglycemic rats in-
dicated an increase of the fasting plasma insulin levels with a corre-
sponding decrease in fasting glycemia [21]. Beta-sitosterol is also
reported for its multiple anti-tumor activities [22].
Furthermore, Fractions R1-R3 after analysis by LC-MS revealed com-
pounds not detected in previous crude extracts. Principal groups of
compounds identified in analyzed extracts (Table 1) are presented
below. Structures of main specific secondary metabolites to N. laevis
[23,24] are showed on Fig. 7.
3.1. Pyrazole alkaloids
From spectra analysis, we realized that N. laevis extracts contain a
large amount of pyrazole alkaloids. Table 2 shows identified alkaloids
with distribution in different solvent extracts (Table 1). Withasomnine
and newbouldine (Fig. 7) derivatives were the main molecules and
were readily present in every sample. A large number of structurally di-
verse natural compounds containing azole nucleus constitute an impor-
tant class of biologically active heterocycles that are gaining more
attention in the field of medicinal chemistry. Withasomnine was iso-
lated from the roots of the Indian Withania somnifera that has been
6 A. Dermane et al. / Results in Chemistry 2 (2020) 100052
(a)
(b)
Fig. 4. LC-MS chromatograms of N. laevis stem bark water extract (a) and root bark water extract (b).used in traditional Ayurvedic medicine for the treatment of enlarged
spleen, migraines, and many infections and as an aphrodisiac.
Withasomnine displays both Central nervous system and circulatory
system depressant properties as well as being a mild analgesic [25]. Its
analogues isolated from similar plant sources are described in more re-
cent literature. More, from a theoretical study on pyrazole alkaloids of
natural origin, withasomnine exhibited a strong antimalarial activity
along with its derivatives para-hydroxy and -methoxy [26].
3.2. Lapachol derivatives
Table 3 displays detected lapachol derivatives from N. laevis sol-
vent extracts and were more present in root bark extracts. Lapachol
derivatives are naturally occurring naphthoquinones compoundshaving cytotoxic properties that can be advantageous for treating
some types of cancer. These compounds induce oxidative stress
and nucleophilic alkylation. Lapachol antiviral, antimicrobial, anti-
inflammatory, and antimalarial effects, as well as its significant effect
on Trypanosoma cruzi (responsible of sleeping sickness) are re-
ported. More, lapachol was reported to inhibit Onchocerca ochengi
parasites [27]. In addition, more recent investigations have shown
that lapachol is an effective reagent for preparing new bioactive sub-
stances [28]. It is believed that the antitumor activity of lapachol may
be related to its interaction with nucleic acids [29]. The
naphthoparaquinone β-lapachone potential as an anti-
trypanosomal agent was also reported. Studies demonstrated that
β-lapachone can directly target DNA topoisomerases and inhibit
their activity, which results in cytotoxicity [30].
A. Dermane et al. / Results in Chemistry 2 (2020) 100052 7
(a)
(b)
(c)
Fig. 5. LC-MS chromatograms of N. laevis root bark (a), leaves (b), and stem bark (c) methanolic extracts.
Fig. 6.Metabolomics analysis of N. laevis root bark, leaves and stem barkmethanolic extracts. Major components are plotted on Y-axis and normalized chromatographic peak areas to 100
(%) on X-axis.
8 A. Dermane et al. / Results in Chemistry 2 (2020) 100052
Fig. 7. Structures of main identified components of N. laevis different solvent extracts.3.3. Triterpenoids
Table 4 presents the multiple triterpenoids and phytosterols identi-
fied in some N. laevis extracts. Triterpenoids were among the most
abundant compounds in N. laevis plant extracts. Triterpenoids are
widely distributed in the vegetable kingdom. Because of their ability
to modulate the activity of several signaling networks, triterpenoids
and phytosterols seem to be particularly promising for the prevention
or treatment of various pathological states in terms of cardiovascular
complications, tumor and cell proliferation, inflammation or hepatotox-
icity. They are highlymultifunctional and the antitumor activity of these
compounds is measured by their ability to block nuclear factor-κB acti-
vation, induce apoptosis, inhibit signal transducer, and activate tran-
scription and angiogenesis [31]. According to the available evidence,Table 2
Retention time, M + H adduct precise mass and extracts in which pyrazole alkaloids were det
No Compound name RT
1 4′-Hydroxywithasomnine 9.12
2 4′-Methoxywithasomnine 10.61
3 Withasomnine 10.73
4 4′-Hydroxynewbouldine 6.87
5 4′-Methoxynewbouldine 10.08triterpenoids provide an excellent base from which to develop new
agents that aremarkedlymore potent. The fact that humans have safely
been ingesting significant amounts of structurally related triterpenoids
compounds as long as they have been consuming for instance olives
(rich sources of triterpenoids) suggests that triterpenoid platform
might be a relatively safe one for the design of new drugs [32].
Ursolic acid present here in N. laevis (Table 4) demonstrated antitu-
mor and anti-inflammatory properties and has been investigated for its
hepatoprotective effects. The mechanism of effect includes suppression
of enzymes that play a role in liver damage such as cytochrome P450,
cytochrome b5, CYP1A and CYP2A, and an increase in antioxidant sub-
stances such as glutathione, metalothionein, zinc, glutathione-S-
transferase and glucuronosyltransferase, with simultaneous protective
effects on liver mitochondria [33]. From Ghosh et al. work, stigmasterolected.
M + H Extracts
201.0998 SBP, SBME, SBS, RBP, RBME, RBS, RBETOH, RBWE
215.1183 RBME, RBS, RBP
185.1073 SBP, SBME, SBS, RBP, RBME, RBS, RBETOH, RBWE
203.1212 RBETOH, SBWE, RBETOH, RBWE
217.1284 RBETOH, SBWE, RBETOH, RBWE
A. Dermane et al. / Results in Chemistry 2 (2020) 100052 9
Table 3
Retention time, M + H adduct precise mass and extracts in which lapachol derivatives were detected.
No Compound name RT M + H Extracts
6 3-Hydroxydehydroiso-α-lapachone 7.77 257.0829 SBP, SBME, SBS, LTE, LME
7 5-Hydroxydehydro-iso- α-lapachone 7.78 257.0832 SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS
8 3,8-Dihydroxydehydroiso-α-lapachone 9.326 273.0757 R1
9 5,7-Dihydroxydehydroiso-α-lapachone 11.23 273.0766 R1
10 Lapachol 11.82 243.1041 R1
11 Beta-lapachone 8.63 243.1020 R1
12 Napabucasin 10.22 288.2887 SBP, SBME, LME, RBP, RBME, RBETOH, SBWE, RBETOH, RBWE
13 2-(1′-Methylethenyl)-5-hydroxynaphtho[2,3-b]furan-4,9-dione 11.48 255.0626 SBP, SBME, SBS, LTE, LME, RBP, RBME, RBSdecreased Ehrlich Ascites Carcinoma tumor volume in mice and in-
creased life span of tumor bearing mice [34]. Canthic acid (Fig. 7) was
first isolated from Canthium dicoccum [35] at a very poor yield. Our
work determinate this acid in N. laeviswith a relatively high concentra-
tion inmethanol leaves extract. This will aid to isolate canthic acid from
N. laevis leaves and evaluate its biological activity including anticancer
activities.3.4. Other compounds
Other compounds identified are shown in Table 5. Sphingolipids
(SLs) were the main compounds and potentially contribute to N. laevis
bioactivity. In recent years, there is more and more evidence that SLs
function as key components inmodulating cell responses and act as sig-
naling and regulatory molecules. Sphingolipids represent a major class
of lipids that are ubiquitous constituents of membranes in eukaryotes.
Intensive research on SL metabolism and function has revealed mem-
bers of the SL family as bioactive molecules playing roles from regula-
tion of signal transduction pathways, through direction of protein
sorting to the mediation of cell-to-cell interactions and recognition.
SLs have also been reported to dynamically cluster with sterols to
form lipid microdomains or rafts, which function as hubs for effective
signal transduction and protein sorting [36].
The effect of evocarpine (EVO), a quinolone alkaloid isolated from
Evodiae fructus, on Ca2+-blocking activity has been examined. In the iso-
lated rat thoracic aorta, evocarpine significantly inhibited the contrac-Table 4
Retention time, M + H adduct precise mass and extracts in which triterpenoids and phytoster
No Compound name RT M + H
14 Canthic acid 12.82 473.3674
15 Ursolic acid 13.57 457.3619
16 Stigmasterol 16.86 413.3779
17 b-Sitosterol-3-O-bdglucopyranoside 16.93 577.4500
18 Beta-sitostenone 15.58 413.3778
19 Stigmasterol glucoside 17.02 592.4566 (M + NH
Table 5
Retention time, M + H adduct precise mass and extracts in which the other compounds were
No Compound name RT M + H
20 Hexadecadihydrosphingosine 10.11 274.2741
21 Evocarpine 10.33 340.2635
22 4,5-Di-O-methyl-8-prenylafzelechin-4beta-ol 11.42 387.1787
23 Chrysoeriol 10.76 301.0712
24 Apigenin 10.82 271.0593
25 Phytosphingosine 11.11 318.2995
26 Chrysarobin 14.53 241.0847
27 4′-Methoxybenzophenone-2-carboxylic acid 9.42 257.0808
28 Bis(2-ethylhexyl) phthalate 11.32 391.2852
29 Diisodecyl phthalate 13.44 447.3469
30 Harmalol 8.35 201.1022
31 Newbouldiamide 18.59 670.6351tion induced by K+ and that induced by external Ca2+ in the
depolarized muscle [37]. Apigenin, a naturally occurring plant flavone,
abundantly present N. laevis, is recognized as a bioactive flavonoid
shown to possess anti-inflammatory, antioxidant and anticancer prop-
erties. Epidemiologic studies suggest that a diet rich in flavones is re-
lated to a decreased risk of certain cancers, particularly cancers of the
breast, digestive tract, skin, prostate and certain hematological malig-
nancies. It has been suggested that apigenin may be protective in
other diseases that are affected by oxidative process, such as cardiovas-
cular and neurological disorders [38]. Harmalol present here in fraction
R1 (Table 5), was reported to decrease heart rate and increased pulse
pressure, peak aortic flow, and myocardial contractile force in dogs
and could be useful to relieve cardiovascular pains.4. Conclusion
In thiswork, the results of thefirst chemical investigation on second-
ary metabolites from all organs of the leaves, stem and root barks of
Newbouldia laevis are reported. Beta-sitosterol was successfully isolated
and identified using spectroscopymethods. More, high resolution mass
spectrometry coupled with high pressure liquid chromatography was
used to detect bioactive compounds from different chemical classes:
pyrazole alkaloids, lapachol derivatives, triterpenoids, phytosterol and
other chemical class compounds. Identified compounds were reported
to be strong anticancer and antimalaria agents among other properties.
This work is the first report combining a wholistic chemical study of allols were detected.
Extracts
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS, RBETOH, SBWE, RBETOH, RBWE
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS, RBETOH
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS, RBETOH
4) RBETOH, SBWE, THWE, RBETOH, RBWE
detected.
Extracts
RBETOH, SBWE, RBETOH, RBWE
RBETOH, SBWE, RBETOH, RBWE
RBETOH, SBWE, THWE, RBETOH, RBWE
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS
SBP, SBME, SBS, LME, RBP, RBME, RBS
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS, RBETOH, SBWE, RBETOH, RBWE
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS
SBP, SBME, SBS, LTE, RBP, RBME, RBS
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS, RBETOH, SBWE, THWE, RBETOH, RBWE
R2, RBETOH, SBWE, THWE, RBETOH, RBWE
R3
SBP, SBME, SBS, LTE, LME, RBP, RBME, RBS
10 A. Dermane et al. / Results in Chemistry 2 (2020) 100052N. laevis plant organs using soft and laboratory like extractions. Future
studies could focus on isolation and bioactivity evaluation of newly de-
tected components in N. laevis.
Author contributions
Affo Dermane: Conceptualization; Data curation and Formal analy-
sis Kafui KPEGBA: Supervision and Conceptualization Kodjo Eloh:
Writing - Original draft, Data curation and Formal analysis Dorcas
Osei-Safo: Supervision andMethodology Richard Amewu: Supervision
and Visualization Pierluigi Caboni: Resources and Writing - review &
editing.
Declaration of competing interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.rechem.2020.100052.
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