Two new sphingolipids from the stem bark of Synsepalum msolo (Sapotaceae)

Article abstract of DOI:10.1016/j.bbrep.2021.101014

Synsepalum msolo commonly known as Bang Bali in Bali-Nguemba, Cameroon is used in traditional medicine against various diseases. The leaves and stem bark extracts were subjected to silica gel and Sephadex LH₂₀ column chromatography to yield pur

Full text of DOI:10.1016/j.bbrep.2021.101014

Biochemistry and Biophysics Reports 27 (2021) 101014
Contents lists available at ScienceDirect
Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep
Two new sphingolipids from the stem bark of Synsepalum
msolo (Sapotaceae)
,
Ache Roland Ndifora *, Njinga Ngaitad Stanislausb, Chi Godloves Fruc, Ferdinand Talontsid,
Turibio Kuiate Tabopdac, Elisabeth Zeuko’o Menkeme, Ngadjui Bonaventure Tchaleuc,
Yeboah Samuel Owusuf
a Higher Technical Teacher Training College, University of Bamenda, Cameroon
b Department of Pharmaceutical and Medicinal Chemistry, University of Ilorin, Ilorin, Nigeria
c Department of Organic Chemistry, Faculty of Science, University of Yaounde I, Cameroon
d Institute of Environmental Research (INFU), Faculty of Chemistry, TU Dortmund, Otto-Hahn-Str, 644221, Dortmund, Germany
e Department of Biochemistry, Faculty of Science, University of Yaounde I, Cameroon
f Department of Chemistry, University of Botswana, Gaborone, Botswana
A R T I C L E I N F O
A B S T R A C T
Keywords:
Synsepalum msolo commonly known as Bang Bali in Bali-Nguemba, Cameroon is used in traditional medicine
against various diseases. The leaves and stem bark extracts were subjected to silica gel and Sephadex LH20
column chromatography to yield pure compounds. The structures of the compounds were determined by detail
analysis of NMR and Mass spectroscopic data and by comparison with data reported in the literature. Amongst
the isolates, were two new sphingolipids: synsepaloside B (1), synsepaloside C (2), and five known compounds:
(+)-catechin (3), (ꢀ )-epicatechin (4), myricitrin (5), triacontanol (6), and aurantiamide acetate (7). Compounds
1–5 were screened for their antibacterial and anti-yeast activities on several microorganisms. All the tested
Sapotaceae
Synsepalum msolo
Phytoconstituents
Antibacterial
Antifungal
compounds exhibited weak antibacterial (MIC ≥ 200
μ
g/mL) and anti-yeast (MIC > 200
μ
g/mL) activities as
g/mL,
compared to standard: ciprofloxacin 0.468
respectively.
<
MIC >0.234 g/mL and fluconazole MIC = 0.05 μ
μ
1. Introduction
active ingredients such as alkaloids, phenols, tannins, cryogenics, gly-
cocides, and terpeniods. These ingredients are used and found effective
as sweeteners, anti-infections and anti-bacterials [7]. For instance, the
stem bark, roots and leaves of Synsepalum msolo contain terpenoids
(taraxeryl acetate, taraxerol, herranone, and betulinic acid), steroids
(spinaterol and spinasterol-3-O-β-D glucopyranoside) and phenols
(catechin, epicatechin and myricitrin) reported in numerous studies to
demonstrate anticancer, anti HIV, antibacterial, antimalarial, analgesic,
anti-inflammatory, antioxidant, anti-viral, and anti-allergenic activities
[8–19]. Other classes of compounds isolated from this plant includes,
saponins (pachystelanosides A and B) and sphingolipid (pachysteloside
A) [13a,b]. Sphingolipids from eukaryotes and higher plant species are
shown to exhibit antiulcerogenic activity [20], antifungal, antitumor,
immunomodulating, antiviral, antitumor, immunostimulatory [21,22];
antiplasmodial, antileismanial, cytotoxic [23], cell proliferation,
apoptosis, fungal pathogenesis and antibacterial activities [24–27].
Therefore, sphingolipids could be considered as active ingredients with
Medicinal plants provide major source of molecules with varying
medicinal properties due to presence of natural compounds. These
plants are useful for curing human diseases and play a vital role in
healing due to presence of phytochemical constituents [1]. Cameroon
has a very rich flora of medicinal plants serving as phytomedicine,
amongst which Synsepalum msolo belonging to the family Sapotaceae
and locally known as ʽʽBang Bali᾿᾿ in Bali of the North West region. The
plant is found also in Bertoua and Nanga Eboko in the East region and in
the East and tropical regions of Africa in Tanzania, Uganda, Kenya,
Gabon, D.R. Congo, Ivory Coast and Ghana [2]. In Cameroon, the stem
bark and roots are used to treat fever, headache, stomach ache and
malaria [3]. The decoction of the dried stem bark of Synsepalum msolo
alone or in combination with sugarcane is taken orally as a galactogogue
in Tanzania [4,5]. In Taiwan the dried roots decoction is taken orally to
treat diabetes mellitus [6]. Nonetheless, many plant species contain
* Corresponding author.
E-mail address: achendifor@yahoo.com (A.R. Ndifor).
https://doi.org/10.1016/j.bbrep.2021.101014
Received 4 December 2020; Received in revised form 28 April 2021; Accepted 2 May 2021
Available online 11 June 2021
2405-5808/© 2021 The Authors.
Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

A.R. Ndifor et al.
Biochemistry and Biophysics Reports 27 (2021) 101014
variant structures that might have potential therapeutic activities. In
continuation of our investigation on Synsepalum msolo, we report herein
the elucidation of the structures of two new sphingolipids and evalua-
tion of their antibacterial and anti-yeast effects along with three known
phenolic compounds on microorganisms.
(m, H-17, H-21, H-22), 5.39 (m, H-13), 5.37 (m, H-12), 5.36 (m, H-8),
5.35 (m, H-9), 4.32 (d, 7.8, H-1′′), 4.28 (d, 4.8, 7.8, H-2), 4.07 (dd, 2.1,
10.5, H-1b), 4.05 (d, 6.0, H2′), 3.92 (dd, 3.9, 10.8, H-6a’’), 3.82 (dd, 3.6,
10.5, H-1a), 3.70 (dd, 3.9, 10.8, H-6b’’), 3.62 (dd, 6.0, 12.0, H-3), 3.54
(dt, 6.6, 12.6, H-4), 3.42 (m, H-5′′), 3.38 (m, H-3′′), 3.30 (m, H-4′′), 3.20
(dd, 7.8, 8.7, H2′′), 2.06 (m, H-10), 2.05 (m, H-5), 2.04 (m, H-7, H-11),
2.02 (m, H-6, H-15, H-18, H-21, H-24), 2.01 (m, H-3′), 2.00 (m, H-14, H-
19, H-20, H-25), 1.70 (m, H-4′), 1.36 (m, H-5′), 1.30 (s, H-30), 1.28 (br.s,
H-26 to H-27 and H-6′ to H-15′), 0.93(t, 6.6, H-29, H-16′). 13C-NMR (δ,
150 MHz): 175.6 (C-1′), 130.2 (C-8, C-16, C-22), 130.0 (C-12, C-17),
129.5 (C-9, C-23), 129.4 (C-13), 103.4 (C-1′′), 73.6 (C-2′′), 76.6 (C-3′′),
76.5 (C-5′′), 74.1 (C-3), 71.6 (C-2′), 71.5 (C-4), 70.3 (C-4′′), 68.5 (C-1),
61.3 (C-6′′), 50.2 (C-2), 34.0 (C-5, C-11, C-18 C-24, C-3′), 32.3 (C-6),
32.0 (C-21), 31.6 (C-14, C-15, C-19, C-20, C-25), 29.2 (C-26 to C-27, C-4′
to C-14′), 26.6 (C-7, C-10), 22.4 (C-28, C-15′), 13.1 (C-29, C-16′), see
Supplemental Table S1.
2. Material and methods
2.1. Plant material
The leaves and stem bark of Synsepalum msolo were collected from
Bali at ‟Manchungˮ in Southern Cameroon, in April 2013. Identification
´ ´
was done by Dr BathelemyTchiengue, a botanist of the Cameroon Na-
tional Herberium, Yaounde, where voucher specimen (No 3849/SRFK)
was deposited.
2.2. General experimental procedure
2.3.1.1. Methanolysis of synsepaloside B (1). Synsepaloside B (3.5 mg)
was refluxed with 0.9 mol L-1 HCl in 82% aqueous MeOH (5 mL) for 20 h
at a temperature of 60 ◦C. The resulting solution was extracted three
times with n-hexane. The n-hexane solution was washed with water
(5 mL) and dried over anhydrous Na2SO4 then concentrated to yield the
fatty acid methyl ester (1.5 mg), identified as methyl hexadecanoate by
analysis of GC-MS. Methyl hexadecanoate also known as hexadecanoic
acid, 2-hydroxy- methylester was obtained as colorless oil, GC-MS: GC,
All reagents were purchased from Merck, Darmstadt, Germany and
are analytical grade. TLC was performed on silica gel 60 F254, 0.1 mm
thick (Merck) of size 20 × 20 cm. TLC spots were detected by fluores-
cence 254 nm or 366 nm and sprayed with 10% H2SO4 followed by
heating at 70 ◦C. 1H, 13C, DEPT, COSY, HMQC, HSQC, HMBC spectra
were recorded in deuterated solvent on either a Bruker Avance 600 MHz
spectrometer or on Varian 500 MHz instrument. Chemical shifts are
referenced to internal tetramethylsilane (δ = 0) and coupling constants J
are reported in Hz. The Low-resolution electrospray-ionization mass
spectrometry (ESI-MS) was carried out on a Micromass Quattro Micro
mass spectrometer, HRTOFESI-MS and TOFESI-MS on micrOTOF 10237,
Bruker compass Data Analysis 4.0. HRESI-MS data were obtained with
an LTQ Orbitrap Spectrometer (Thermo Fisher, Waltham, MA, USA)
equipped with an HESI-II source. IR spectra was recorded on a Perkin-
Elmer spectrophotometer. Melting points were recorded using SMP3
melting point apparatus and is uncorrected.
tR 17.416 min, m/z 286 (Cald. for C17H34O3, 286.25), EI-MS: m/z: 227 [
+.
M ꢀ C15H31O]+ (23), 71[C5H11
]
(40), 57 [C4H9]+. (90), see Supple-
mental Table S1 and supplementary data file.
2.3.2. Synsepaloside C (2)
White amorphous solid, mp 196.9 ◦C, [α D20
]
+ 13.6 (c 0.1, MeOH); 1H
NMR (DMSO‑d6, 600 MHz) and 13C NMR (DMSO‑d6, 150 MHz).
HRTOFESI-MS (positive-ion mode), m/z 866.6593 [M+Na]+, TOFESI-
MS (positive-ion mode), m/z 866.7 [M+Na]+, TOFESI-MS (negative-
ion mode) m/z 842.7 [M ꢀ H]- (Cald. for C48H93NO10, 843.6799). 1H-
NMR (δ, 600 MHz): 7.42 (d, 9.0, 2-NH), 5.38 (dd, 3.0, 13.2, H3′), 5.30
(m, H-4′), 4.90 (d, 3.9, 2′′-OH), 4.71 (s, 3-OH), 4.47 (t, 5.7, 6′′-OH), 4.28
(d, 5.7, 2′-OH), 4.14 (d, 7.8, H-1′′), 4.09 (m, H-2), 3.87 (m, H-2′), 3.85
(m, H-5), 3.82 (m, H-1a), 3.66 (m, H-1b), 3.66 (dd, 6.0, 11.4, H-6′′b),
3.45 (dd, 5.2, 11.4, H-6′′a), 3.38 (dd, 8.1, 8.7, H-2′′), 3.19 (m, H-5′′), 3.16
(m, H-3′′), 3.05 (m, H-4′′), 2.94 (dt, 3.6, 8.4, H-3), 1.94 (m, H-5′), 1.76
(m, H-6), 1.24 (br.s, H-6 to H-15 and H-6′ to H-23′), 1.23 (s, H-16, H-24′)
2.3. Extraction and isolation
The powdered dry leaves (0.3 kg) and stem bark (3.3 kg) of S. msolo
were extracted twice with CH2Cl2–CH3OH (1:1 v/v) at ambient tem-
perature for 2 days. The stem bark and leaves extract were concentrated
under reduced pressure to yield dark brown viscous syrups (86 g) and
black viscous syrup (100 g) respectively. 80 g of the stem bark extract
was subjected to silica gel column and eluted with mixtures of n-hexane,
ethyl acetate and methanol, in order of increasing polarities to give
about 146 fractions. The similar fractions were combined using TLC
analysis. Synsepaloside B 1 (6.4 mg), was directly obtained from frac-
tions 121–124 (EtOAc-MeOH 20%) and synsepaloside C 2 (8.7 mg) from
fractions 108–120. Aurantiamide acetate 7 (12 mg) and (ꢀ )-epicatechin
4 (30 mg) were directly obtained from fractions 82–87 and 100–106
respectively. 100 g of the leave extract was washed with n-hexane, ethyl
acetate and methanol to yield 16 g, 18 g and 48 g respectively. The ethyl
acetate fraction (18 g) was subjected over silica gel column and eluted
with increasing polarity of n-hexane, ethyl acetate and methanol to give
124 fractions. The combination of similar fractions yielded triacontanol
6 (5 mg) and (+)-catechin 3 (6 mg). Fractions 121–124 from EtOAc-
MeOH (80:20) was purified over Sephadex LH20 (100 MeOH) repeat-
edly to afford myricitrin 5 (19.2 mg). The compounds were identified
using spectroscopic methods (1D and 2D NMR, MS).
13
and 0.85 (t, 6.9, H-17, H-25′). C-NMR (δ, 150 MHz): 173.7 (C-1′),
130.2 (C-3′), 129.8 (C-5′), 103.4 (C-1′′), 74.0 (C-2′′), 73.4 (C-3), 73.4 (C-
3), 70.8 (C-4 and C-2′), 68.8 (C-1), 61.0 (C-6′′), 49.8 (C-2), 31.8 (C-5′),
31.2 (C-5), 29.2 (C-6 to C-15; C-6′ to C-23′), 22.0 (C-16, C-24′) and 13.8
(C-17, C-25′), see Supplemental Table S2.
2.4. Preparation of stock solution
The stock solution of each compound, ciprofloxacin, and fluconazole
were prepared in pure DMSO for a final concentration of 1 mg/mL. The
stock solutions were filtered with a 0.20 μm sterilized syringe and stored
at ꢀ 20 ◦C until use.
2.5. Bacterial and yeast strains
Six bacterial strains: Staphylococcus aureus ATCC 43300, Pseudo-
monas aeruginosa NR48582, Klebsiella pneumoneae ATCC 700603,
Escherichia coli ATCC 25922, Shigella flexneri NR518, Streptococcus
pneumoneae HM145 and 3 yeast strains: Candida albicans NR 29445,
Candida albicans NR 29451 and Candida albicans ATCC 29444 were
assayed. Isolates were obtained from Yaounde Centre Hospital,
Cameroon and the reference strains from BEI resources and the Amer-
ican Type Culture Collection Bacteria and yeast strains were cultivated
2.3.1. Synsepaloside B (1)
White amorphous powder, mp 170.5 ◦C, [α D
]
20+19.8 (c 0.1, MeOH);
IR νmax 3614, 3421, 2927, 1735, 1650, 1542, 1373, 1245, 1033 cmꢀ 1
;
1H NMR (CD3OD, 600 MHz) and 13C NMR(CD3OD, 150 MHz); HRESI-
MS (positive-ion mode) at m/z 880.68431 [M+H]þ, (calcd for
C
51H93NO10, 879.67995). 1H-NMR (δ, 600 MHz): 5.44 (m, H-16), 5.43
2

A.R. Ndifor et al.
Biochemistry and Biophysics Reports 27 (2021) 101014
in petri dishes containing Muller Hinton Agar (MHA) and Sabouraud
Dextrose Agar (SDA) respectively, followed by an incubation period of
24 h at 37 ◦C. Each microorganism was sub-cultured in a new Agar plate
and incubated prior to each experiment.
129.5 (C-9, C-23), 130.2 (C-8, C-16, C-22), and 130.0 (C-12, C- 17), an
amido methine (δH 4.28 (d, 4.8, 7.8, H-2), δ 50.2 (C-2)), an oxygenated
methylene (δH 4.07 and δH 3.82; δC 68.5), thCree oxygenated methines (δH
3.62, 3.54, 4.05; and δ 74.1, 71.5, and 71.6), two terminal methyls (δ
0.93, 6H, t, J = 6.6 Hz)Cand two long aliphatic chains appearing as broad
singlets (δH 1.28). Comparing these spectra data with literature studies
indicates that compound 1 is a glycosphingolipid [29–31], (Fig. 1).
The methanolysis, of 1, afforded fatty acid methylester, identified as
hexadecanoic acid, 2-hydroxy- methylester and its molecular formula
was established to be C17H34O3 at m/z 286, in the GC-MS spectrum. See
supplementary material.
H
2.6. Antimicrobial assays
The antimicrobial activity of each compound was assessed as rec-
ommended by the Clinical and Laboratory Standards Institute with
minor modifications by the use of resazurin dye (CLSI, 2012) [28].
Briefly, forty μL of the compounds with concentration of 1 mg/mL was
In the HRESI-MS spectrum, the long-chain fatty acid (LCFA) [30] was
determined to be 2-hydroxy-hexadecanoic acid due to the fragment ion
at m/z 255 [M ꢀ C16H31O2] + (Fig. 2). The position of the 2-hydroxy
introduced in the microtiter plate containing 60
μL of culture medium
for a final volume of 100 L. 2-fold serial dilutions of samples were
μ
performedin 96-well microplates. The bacterial and yeast inoculum
were prepared with sterile saline solution (NaCl 0.9%) using freshly
cultured microorganisms of 24 h. Each suspension was adjusted to 0.5
McFarland standard then diluted in culture medium to yield a final
concentration of 106 CFU/mL for bacteria and 1.5 × 103 CFU/mL for
group in the LCFA was confirmed by the α-cleavage of alcohol at m/z
227 [C15H31O]+, in the HRESI-MS and GC-MS spectra (Fig. 2).
The long-chain base (LCB) [32] of compound 1 was derived as 2-ami-
no-nonacosa-8,12,16,22-tetraen-1,3,4-triol by analysis of the 1H–1H
COSY, HMQC and HRESI-MS, which showed a peak at m/z 463
yeast. Fifty
μ
L of each microbial suspension were added in each well and
+
[M-163-255+2H]+. The ion peaks appearing at m/z 536 [M ꢀ C25H43
]
plates were incubated for 24 h at 37 ◦C. Thereafter, 10
μ
L of resazurin
and 476 [M-C27H47O2]+ indicated the presence of oxymethine carbons
in LCB, assigned to positions C-3 and C-4 respectively, which were
supported by the COSY correlations of H-1 through H-4 (Fig. 3). This was
further supported by the ion peaks at m/z 532 [M-18-C24H41]+, 476
[M-C27H45O]+ and 337 [M-2H2O-163-343]+ resulting from Mc Lafferty
rearrangement and the 1H–1H COSY correlations between the oxy-
(0.15 mg/mL in PBS) was added followed by additional 4 h incubation
and MIC was recorded visually as the lowest concentration of the
compound required to inhibit 50% growth of pathogens. A negative
control experiment was conducted using 0.1% DMSO.
3. Statistical analysis
methine δ 3.62 (dd, 6.0, 12.0 Hz, H-3) and δ 3.54 (dt, 6.6, 12.6 Hz,
H
H
H-4), respectively (Figs. 2 and 3).
All data were performed in triplicate and resulting MIC values
expressed as mean ± standard deviation. The data were processed using
the software SPSS 17.0 for Windows.
The positions of the olefinic double bonds at C-8/C-9 and C-12/C13
were affirmed by the ion peaks at m/z 301 [C22H37]+, 275 [C20H35]+,
221 [C16H29]+, and 207 [C16H22
]
+ in the positive ion mode HRESI-MS
spectrum due to α/β-cleavages of the double bonds. In addition, the
4. Results and discussion
Mc Lafferty rearrangements resulting to the ion peaks at m/z 532 [M-18-
+
+
C
24H41]+, 547 [M-18-C23H39
]
and 451 [M-18-C18H31
]
strongly
Synsepaloside B (1) was isolated as white amorphous powder. Its
molecular formula C51H93NO10 was assigned on the basis of the HRESI-
MS (positive-ion mode) at m/z 880.68431 [M+H]þ, and 1D and 2D
NMR experiments. The IR spectrum showed absorption bands of hy-
droxyl (3341 cmꢀ 1) and amide (1643 cmꢀ 1) groups. The 1H and 13C
NMR spectrum of 1 presented the characteristic signals of a β-D-gluco-
pyranoside moiety (δH 4.11, 1H, d, J = 7.8 Hz, anomeric proton, δC 103.3
(CH), 70.2 (CH), 76.5 (CH), 76.6 (CH), 73.6 (CH), and 61.3 (CH2)), an
amide linkage 175.6 (C-1′), four olefinic methines δH 5.35 (1H, m, H-9),
5.36 (1H, m, H-8), 5.37 (1H, m, H-12), 5.39 (2H, m, H-13, H-23), 5.43
(2H, m, H-17, H-22), 5.44 (1H, m, H-16) with carbons at δC 129.4 (C-13),
confirmed the position of the double bonds at C8/C-9 and C-12/C-13,
respectively (Fig. 2). The ion peak appearing at m/z 207 [C15H27
]
+ and
+
153 [C11H21
]
corroborate a third double bond at C-16/C-17 on the
LCB. The peaks of the cleavages of an allylic bond at m/z 768
[M ꢀ C18H15
]
+ and 794 [M ꢀ C6H13
]
+ affirmed the fourth double bond
position at C-22/C-23, on the LCB.
The E geometry at positions 12, 16 and 22 was supported by the
chemical shift of the carbons next to the double bond at (δC 34.0 (C-11),
δ 32.3 (C-14), δ 32.3 (C-15), δ 32.3 (C-18), δ 32.3 (C-21), δ 34.0 (C-
2C4)), while the CZ geometry at CC-8/C-9 was aCffirmed by theCchemical
Fig. 1. Structure of isolated compounds.
3

A.R. Ndifor et al.
Biochemistry and Biophysics Reports 27 (2021) 101014
Fig. 2. Mass fragmentation pattern of synsepaloside B (1) following the HRESI-MS, GC-MS and ESI-MS.
Fig. 3. Key COSY correlations of synsepaloside B (1). COSY and HMBC correlations of synsepaloside C (2).
shifts of the carbons next to the olefinic double bonds at (δC 26.6 (C-7)
trans (E) double bond appear in the range of δC 32–34 [30]. Thus, the Δ8,
was determined to be Cis (Z) due to the down field chemical shift values
in the range of 26–28 [23], and Δ12, Δ16 and Δ22 double bonds were
and δ 26.6 (C-10)). The chemical shifts for the adjacent carbons to a cis
C
(Z) double bond appear in the range of δC 26–28 [33], while those of a
4

A.R. Ndifor et al.
Biochemistry and Biophysics Reports 27 (2021) 101014
determined to be trans (E), due to their upfield chemical shift values in
the range of 32–34 [29]. Therefore, the structure of compound 1 was
determined as 1-O-β-D-glucopyranosyl-(8Z,12E,16E,22E)-2-[(2′)-2′
-hydroxyhexadecanoylamino]-nonacosa-8,12,16,22-tetraen-1,3,4-triol,
an unreported sphingolipid.
carbon at δC 130.2 (C-3′) and δC 129.8 (C-4′) respectively, in the VLCFA.
The Δ3 double bond of 2 was determined to be trans (E), by comparing
the large vicinal coupling constant of the proton at δH 5.38 (1H, dd,
J = 13.2 Hz, H-3′) and upfield chemical shift value of C-5′ in the range of
31.8, with literature data of flavuside B [27]. The LCB of 2 was deter-
mined to be 2-aminoheptadecan-1,3,4-triol due to fragment ion at m/z
301.2 (Fig. 4). The ion peaks at m/z 183 and 169 were attributed to the
Synsepaloside C (2) was isolated as white amorphous solid. Its mo-
lecular formula C48H93NO10 was deduced from its HRTOFESI-MS
showing a pseudomolecular ion peak [M+Na]+ at m/z 866.6593. The
1H and 13C NMR spectrum of 2 showed similar features to that of syn-
sepaloside B. Compound 2 displayed resonances of a β-D-glucopyrano-
side moiety at (δH 4.15, 1H, d, J = 7.8 Hz, anomeric proton, δC 103.4
(CH), 70.0 (CH), 76.5 (CH), 76.9 (CH), 73.4 (CH), and 61.0 (CH2)), an
amide linkage (δH 7.42, 1H, d, J = 9.0 Hz, δC 173.7 (C-1′), an olefinic
α/β-cleavages that confirmed the location of hydroxyl functions on the
LCB. This was further supported by the COSY correlations for H-1
through H-5 (Fig. 3).
The connections through C1–O–C1′′ and C2–NH–C1′ in compound 2
were strengthened using HMBC connectivity from H-2 to C-1 and C-3;
from 2-NH to C-1’; from H-3 to C-4; from H-2′ to C-1’; from H-3′ to C-1′
and C-2’; from H-4′ to C-1′, C-5′ and C-6’; from 2′-OH to C-2’; from 3-OH
to C-3 and C-4; from H-1 to C-1′′, respectively. Therefore, the structure of
methine at δ 5.38 (dd, J = 3.0, 13.2 Hz, H-3′) and δ 5.30 (m, H-4′),
H
assigned to carbon at δC 130.2 (C-3′) and δC 129.8 (C-4H′), an oxygenated
methylene (δH 3.66 and δH 3.82; δ 68.8), an amido methine (δH 4.09, m,
H-2); δC 49.8), three oxygenated Cmethines (δH 2.94, 3.85, 3.87; and δC
73.4, 70.8, and 70.8), two terminal methyls (δH 0.87, 6H, t, J = 6.9 Hz)
and two long aliphatic chains appearing as broad singlets (δH 1.24). The
comparison of these spectra data with literature values suggest that
compound 2 is a glycosphingolipid [29–31], (Fig. 1). Compound 2 dif-
fers from 1 in that, 2 have a very long-chain fatty acid (VLCFA) [32]. The
VLCFA was determined to be 2′-hydroxypentacos-3-enoic acid due to the
characteristic fragment ion at m/z 381.3 (Fig. 4).
2
was established as 1-O-β-D-glucopyranosyl-2-[(2′,3′E)-2′-hydrox-
ypentacos-3′-enoylamino]-heptadecan-1,3,4-triol, reported for the first
time (Fig. 1).
Compounds 1–5, were screened for their antibacterial and anti-yeast
potency against 6 bacterial strains including Staphylococcus aureus ATCC
43300, Pseudomonas aeruginosa NR48582, Klebsiella pneumoneae ATCC
700603, Escherichia coli ATCC 25922, Shigella flexneri NR518, Strepto-
coccus pneumoneae HM145 and 3 yeast strains Candida albicans NR
29445 Candida albicans NR 29451 and Candida albicans ATCC 29444.
See Supplementary Table S3.
The molecular ion peak at m/z 295 was attributed to the α-cleavage
that supported the location of the olefinic double bond at H-3’/H4′ in
the VLCFA (Fig. 4). Furthermore, the fragment ion at m/z 335.1
resulting from the Mc Lafferty fragmentation processes strongly sup-
ports the position of the double bond and hydroxy function at H-2’
(Fig. 4). The signal of the olefinic double bond was observed at proton δ
All the tested phytoconstituents exhibited weak activity on the bac-
teria and yeast strains with minimum inhibitory concentration (MIC) ≥
200 μg/mL. However, as compared to the reference drugs ciprofloxacin
and fluconazole, the tested products where much less active. These data
suggest that the tested sphingolipids and phenolics of Synsepalum msolo
were almost inactive in this experiment against the evaluated
5.38 (dd, J = 3.0, 13.2 Hz, H-3′) and δ 5.30 (m, H-4′), attributed tHo
H
Fig. 4. TOFESI-MS mass fragmentation pattern of synsepaloside C (2).
5

A.R. Ndifor et al.
Biochemistry and Biophysics Reports 27 (2021) 101014
[7] O. Akinyemi, S.O. Oyewole, K.A. Jimoh, Medicinal plants and sustainable human
health: a review, Hortic. Int. J. 2 (4) (2018) 194–195.
microorganisms.
[8] J.-F. Hong, Y.-F. Song, Z. Liu, Z.-C. Zheng, H.-J. Chen, S.-S. Wang, Anticancer
activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft
model via induction of autophagy and apoptotic cell death, cell cycle arrest and
inhibition of cell migration, Mol. Med. Rep. 13 (2016) 4541–4548.
[9] J. Liao, F. Wu, W. Lin, Z. Huang, Taraxerol exerts potent anticancer effects via
induction of apoptosis and inhibition of Nf-kB signalling pathway in human middle
ear epithelial cholesteatoma cells, Trop. J. Pharmaceut. Res. June 17 (6) (2018)
1011–1017.
5. Conclusion
Two new sphingolipids [synsepaloside B (1) and synsepaloside C
(2)], three phenolics [catechin (3), epicatechin (4) and myricitrin (5)], a
fatty alcohol [triacontanol] and a peptide derivative [aurantiamide ac-
etate] were isolated from the leaves and stem bark of a folk medicine
Synsepalum msolo. Compounds 3–7 have known biological activities, and
have not been reported previously as constituents of Synsepalum msolo.
Analysis of the new sphingolipids and known compounds were done
using NMR and mass spectra data and by comparison to those publish in
the literature. The screening of the sphingolipids, catechin, epicatechin
and myricitrin with several microorganisms demonstrated weak anti-
bacterial and antifungal action. However, based on the weak activity
observed, we recommend that other classes of compounds isolated from
this plant in the future, should be subjected for investigations to identify
the components with potent antibacterial and antifungal action.
[10] C. Chandramu, R.D. Manohar, D.G. Krupadanam, R.V. Dashavantha, Isolation,
characterization and biological activity of betulinic acid and ursolic acid from
Vitex negundo L, Phytother Res. 17 (2) (2003) 129–134.
[11] T.Y. Fujioka, R. E Kashiwada, L.M. Kilkuskie, L.M. Cosentino, J.B. Ballas, W. P
Jiang, I.S. Janzen, K.H. Chen Lee, Anti-AIDS Agents, 11. Betulinic acid and platanic
acid as anti-HIV principles from syzygium claviflorum, and the anti-HIV activity of
structurally related triterpenoids, J. Nat. Prod. 57 (2) (1994) 243–247.
[12] S. Arora, G. Kumar, S. Meena, Gas chromatography-mass spectroscopy analysis of
root of an economically important plant, cenchrus ciliaris L. From tar desert,
Rajasthan (India), Asian J. Pharmaceut. Clin. Res. 10 (64) (2017).
[13] (a) K.T. Ache, F. Turibio, M.F. Talontsi, O.S. Tala, Yeboah, B.T. Ngadjui,
Characterization of constituents from the roots of pachystela msolo engler
(Sapotaceae), IJCPS 3 (6) (2015) 1431–1435;
(b) R.N. Ache, K.T. Turibio, O.S. Yeboah, B.T. Ngadjui, Two new triterpenoidal
saponins from roots of pachystela msolo, NPC 10 (11) (2015) 1933–1936.
[14] J.N. Nyemb, A.T. Tchinda, E. Talla, E. B Nanga, D. T Ngoudjou, C. Henoumont,
S. Laurent, J. Iqbal, J.T. Mbafor, Vitellaroside, A new cerebroside from Vitellaria
paradoxa (Sapotaceae) and its bioactivities, Nat. Prod. Chem. Res. 6 (1) (2018)
1–9.
CRediT authorship contribution statement
Ache Roland Ndifor: Conceptualization, methodology, data cura-
tion, investigation, writing original draft. Njinga Ngaitad Stanislaus:
Data curation, writing original draft. Chi Godloves Fru: Data curation,
writing original draft. Ferdinand Talontsi: Data curation, writing
original draft. Turibio Kuiate Tabopda: Co-Supervision, project
administration, data curation, writing original draft. Elisabeth Zeuko’o
Menkem: Data curation, writing of original draft. Ngadjui Bona-
venture Tchaleu: Supervision, project administration, funding acqui-
sition. Yeboah Samuel Owusu: Project administration, funding
acquisition, Investigation.
[15] J. Shay, H.A. Elbaz, I. Lee, S.P. Zielske, M.H. Malek, M. Hüttemann, Review article:
molecular mechanisms and therapeutic effects of (ꢀ ) epicatechin and other
polyphenols in cancer, inflammation, diabetes, and neurodegeneration, Oxid. Med.
Cell. Longevity (2015) 1–13. ID 181260.
[16] J. Bae, N. Kim, Y. Shin, S.-Y. Kim, Y.-J. Kim, Activity of catechins and their
applications, Biomed. Dermatol. 4 (8) (2020) 1–10.
[17] P.W. Taylor, J.M.T. Hamilton-Miller, P.D. Stapleton, Antimicrobial properties of
green tea catechins, Food Sci. Technol. Bull. 2 (2005) 71–81.
´
´
[18] A. Ricardo, M.C. EscandOn, T. Hector, Antibacterial effect of kaempherol and
(-)-epicatechin on Helicobacter pylor, Eur. Food Res. Technol. 242 (2016)
1495–1502.
[19] A. Dorothy, H.Y. Okoth, N. Chena, A. Koorbanally, Antibacterial and antioxidant
activities of flavonoids from Lannea alta (Engl.) Engl, Aanacardiaceae,
Phytochemistry Letters 6 (3) (2013) 476–481.
Declaration of competing interest
None.
[20] E. Okuyama, M. Yamazaki, The principles of Tetragonia tetragonoides having
antiulcerogenic activity. II. Isolation and structure of cerebrosides, Chem. Pharm.
Bull. 31 (1983) 2209–2219.
[21] P. Muralidhar, P. Radhika, N. Krishna, R.D. Venkata, R.C. Bheemasankara, Sphin-
golipids from marine organisms: a review, Nat. Prod. Sci. 9 (2003) 117–142.
[22] C.R. Emura, T. Higuchi, Miyamoto, A.-F. Amphimelibiosides, Six new ceramide di-
hexosides isolated from a Japanese marine sponge amphimedon sp, J. Org. Chem.
70 (2005) 3031–3038.
Acknowledgements
The authors acknowledge the contributions of Dr Tala Michel Feussi
and Dr Nana Federick for the accurate mass measurements. This work
was partially funded by the Network for Analytical and Bio-assay Ser-
vices in Africa (NABSA). Professor Fabrice Fekam Boyom Head of
AntiMicrobial and Biocontrol Agents Unit focusing on Drug discovery
against pathogenic protozoa, fungi, and bacteria, in the department of
Biochemistry Faculty of Science, University of Yaounde I, Cameroon is
acknowledged for performing the biological activity.
[23] M.A. Tantry, A. Idris, I.A. Khan, Glycosylsphingolipids from euonymus japonicus
thunb, Fitoterapia 89 (2013) 58–67.
[24] L.M. Obeid, C.M. Linardic, L.A. Karolak, Y.A. Hannun, Programmed cell death
induced by ceramide, Science 259 (1993) 1769–1771.
[25] J. Cheng, T.S. Park, L.C. Chio, A.S. Fischl, X.S. Ye, Induction of apoptosis by
sphingoid long-chain bases in Aspergillus nidulans, Mol. Cell Biol. 23 (2003)
163–177.
[26] C.D.L. Luberto, E.A. Toffaletti, S.C. Wills, A. Tucker, J.R. Casadevall, Y.A. Perfect,
Hannun, M. Del-Poeta, Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in
pathogenesis of Cryptococcus neoformans, Genes Dev. 15 (2001) 201–212.
[27] Y. Gouhua, S. Louis, Y. Keumja, S.L. Alain, K. Gun-Do, D.C. Hong, S.K. Jung,
H. Jongki, W.S. Byeng, Flavusides A and B, Antibacterial cerebrosides from the
marine-derived fungus Aspergillus flavus, Chem. Pharm. Bull. 59 (2011)
1174–1177.
Appendix A. Supplementary data
[28] Clinical and Laboratory Standards Institute, Methods for dilution of antimicrobial
susceptibility tests for bacteria that grow aerobically; approved standard — ninth
edition. CLSI document M07–A9. https://doi.org/10.4103/0976-237X.91790,
2012.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbrep.2021.101014.
[29] Z. Duo, H.B.X. Chen, T. Kai, G. Jun-Ming, H. Xiang-Zhong, J. Zhi-Yong, Two new
sphingolipids from the leaves of piper betle L, Molecules 18 (2013) 11241–11249.
[30] A.-Q. Jia, X. Yang, W.-X. Wang, Y.-H. Jia, Glycocerebroside bearing a novel long-
chain base from Sagina japonica (Caryophyllaceae), Fitoterapia 81 (2010)
540–545.
References
[1] A.K. Garg, M. Faheem, S. Singh, Role of medicinal plant in human health disease,
Asian J. Plant Sci. Res. 11 (1) (2021) 19–21.
´
[2] A. Aubreville, Sapotaceae, second ed., Flore du Cameroun, 1964, pp. 86–87.
[31] F. Cateni, J. Zilic, G. Falsone, G. Scialino, E. Banfi, New cerebrosides from
Euphorbia peplis L.: antimicrobial activity evaluation, Bioorg. Med. Chem. Lett 13
(2003) 4345–4350.
[3] R.N. Ache, B.A. Keugni, L.A. Fotio, T. Dimo, O.S. Yeboah, B.T. Ngadjui, Analgesic
and anti-inflammatory activities of herranone, spinasterol and crude extract of
Pachystela msolo in rat and mice, WJPB 2 (2) (2015), 09–15.
[32] V.M. Louise, A.N. Johnathan, D.M. Diana, F.D. Jean, Plant sphingolipids: their
importance in cellular organization and adaption, Biochim. Biophys. Acta 1861
(2016) 1329–1335.
¨
[4] C.K. Ruffo, A. Birnie, B. Tengnas, Edible Wild Plants of Tanzania, Regional L and
Management Unit/Sida Technical Handbook, 2002, pp. 634–635.
[5] W. D Newmark, Ecological Studies 155, Conserving Biodiversity in East African
Forests: A Study of the Eastern Arch Mountains, 2002, pp. 42–43.
[6] A.R. Ivan, Medicinal Plants of the World. Chemical Constituents, Traditional and
Modern Medicinal Uses, 2005, pp. 438–439.
[33] D. Tazoo, K. Krohn, H. Hussain, S.F. Kouam, E.Z. Dongo, Laportoside A and
Laportomide A: A new cerebroside and a new ceramide from leaves of Laportea
ovalifolia, Zeitschrift für Naturforschung, Naturforsh. 62b (2007) 1208–1212.
6